@article {70093, title = {iGABASnFR2 is an improved genetically encoded protein sensor of GABA}, journal = {eLife}, year = {2026}, month = {2026 Mar 18}, abstract = {

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However, the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20\% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance when compared directly to the original sensor. iGABASnFR2 enabled the first measurements of direction-selective GABA release in the retina. In vivo imaging in somatosensory cortex revealed that iGABASnFR2 can report volume-transmitted GABA release following whisker stimulation. Overall, the improved sensitivity and kinetics of iGABASnFR2 make it a more effective tool for imaging GABAergic transmission in intact neural circuits.

}, doi = {10.7554/eLife.108319.3}, url = {https://doi.org/10.7554/eLife.108319.3}, author = {Kolb, Ilya and Hasseman, Jeremy P and Matsumoto, Akihiro and Jensen, Thomas P and Kopach, Olga and Arthur, Benjamin J and Zhang, Yan and Tsang, Arthur and Reep, Daniel and Tsegaye, Getahun and Zheng, Jihong and Patel, Ronak H and Looger, Loren L and Marvin, Jonathan S and Korff, Wyatt L and Rusakov, Dmitri A and Yonehara, Keisuke and Turner, Glenn C} } @article {70322, title = {Image analysis tools for Electron Microscopy}, journal = {bioRxiv}, year = {2026}, month = {2026-03-14}, abstract = {

Electron Microscopy (EM) is widely used in many scientific fields, particularly in life sciences, offering high-resolution information on the ultrastructure of biological organisms. Accurate characterization of EM image quality is important for assessing the EM tool performance, in addition to sample preparation protocol, imaging conditions, etc.This paper provides an overview of tools we developed as plugins for the popular image processing package Fiji (ImageJ) (1). These tools include signal-to-noise ratio analysis, contrast evaluation, and resolution analysis, as well as the capability to import images acquired on custom FIB-SEM instruments (2). We have also made these tools available in Python, with both versions available on GitHub.

}, doi = {10.64898/2026.03.11.711125}, url = {https://www.biorxiv.org/content/early/2026/03/14/2026.03.11.711125}, author = {Shtengel, David H. and Shtengel, Gleb and Xu, C. Shan and Hess, Harald F.} } @article {70283, title = {Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis}, journal = {eLife}, year = {2026}, month = {2026 Feb 06}, abstract = {

Cortical neurogenesis proceeds through a precise temporal program in which radial glia sequentially generate distinct neuronal subtypes and later glia, yet how post-transcriptional regulators coordinate these transitions remain poorly understood. We previously identified that a decreasing temporal gradient of the RNA-binding protein Imp encodes neural stem cell age in\ Drosophila. In this work, we extend our investigation to Imp1, a mammalian homologue of Imp, and its role in murine neocortical development. Using TEMPO to track birth-order dynamics, we demonstrate that sustained Imp1 overexpression during early neurogenesis arrests temporal fate progression, shifting neuronal populations toward deeper cortical layers V-VI. Immunostaining with layer-specific transcription factors Cux1 and Ctip2 confirmed that laminar repositioning results from genuine changes in neuronal identity rather than migratory defects, with neurons adopting molecular identities matching their final positions. Temporal window-specific manipulations reveal distinct stage-specific effects where early-stage Imp1 induction produces cascading effects on fate specification and moderately delays the neuronal-to-gliogenic transition, while mid-stage induction induces neuronal accumulation in the subplate region. Live imaging of organotypic cultures reveals continuous neuronal recruitment within intermediate and ventricular zones, with mid-stage-born neurons accumulating at significantly faster rates than earlier cohorts. Strikingly, mid-stage Imp1 overexpression also induces ectopic glial-like foci distributed throughout the cortical plate, featuring dramatic cellular expansion and morphological heterogeneity. These findings establish Imp1 as a dosage- and stage-dependent temporal rheostat orchestrating developmental transitions in radial glial progenitors, controlling neuronal fate decisions and spatial organization. This work advances our understanding of molecular timing mechanisms governing neuronal diversity in the mammalian cortex.

}, doi = {10.7554/elife.109847.1}, url = {http://dx.doi.org/10.7554/eLife.109847.1}, author = {Azur, Romie Angelo G and Feliciano, Daniel and Espinosa-Medina, Isabel and Adhikari, Raghabendra and Lilao-Garz{\'o}n, Joaquin and Jansen, Ella and Yang, Ching-Po and Lee, Tzumin} } @article {69831, title = {iGABASnFR2: Improved genetically encoded protein sensors of GABA}, journal = {bioRxiv}, year = {2025}, month = {2025 Mar 25}, abstract = {

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis, and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20\% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance, enabling us to make the first measurements of direction selective GABA release in the retina and confirm a longstanding hypothesis for how sensitivity to motion arises in the visual system.

}, doi = {10.1101/2025.03.25.644953}, url = {https://www.biorxiv.org/content/early/2025/03/26/2025.03.25.644953}, author = {Kolb, Ilya and Hasseman, Jeremy P. and Matsumoto, Akihiro and Arthur, Benjamin J. and Zhang, Yan and Tsang, Arthur and Reep, Daniel and Tsegaye, Getahun and Zheng, Jihong and Patel, Ronak and Looger, Loren L. and Marvin, Jonathan S. and Korff, Wyatt L. and Yonehara, Keisuke and Turner, Glenn C.} } @article {70182, title = {iGlucoSnFR2: A genetically encoded fluorescent sensor for measuring intracellular or extracellular glucose in vivo in mouse brain.}, journal = {Sci Adv}, volume = {11}, year = {2025}, month = {2025 Nov 14}, pages = {eadz3889}, abstract = {

Continuous glucose monitors have proven invaluable for monitoring blood glucose levels for diabetics, but they are of limited use for observing glucose dynamics at the cellular (or subcellular) level. We have developed a second generation, genetically encoded intensity-based glucose sensing fluorescent reporter (iGlucoSnFR2). We show that when it is targeted to the cytosol, it reports intracellular glucose consumption and gluconeogenesis in cell culture, along with efflux from the endoplasmic reticulum. It outperforms the original iGlucoSnFR in vivo when observed by fiber photometry in mouse brain and reports transient increase in glucose concentration when stimulated by noradrenaline or electrical stimulation. Last, we demonstrate that membrane localized iGlucoSnFR2 can be calibrated in vivo to indicate absolute changes in extracellular glucose concentration in awake mice. We anticipate iGlucoSnFR2 facilitating previously unobservable measurements of glucose dynamics with high spatial and temporal resolution in living mammals and other experimental organisms.

}, issn = {2375-2548}, doi = {10.1126/sciadv.adz3889}, url = {https://doi.org/10.1126/sciadv.adz3889}, author = {Marvin, Jonathan S and M{\"a}chler, Philipp and Meng, Chengbo and Ates, Tayfun and Patel, Ronak H and Adhikari, Raghabendra and Makurath, Monika A and Ku, Zaneta and Feliciano, Daniel and Atasoy, Deniz and Cui, Guohong and Kleinfeld, David and Brown, Timothy A} } @article {70147, title = {Illuminating renal proximal tubule architecture through high-resolution volume em and machine learning analysis.}, journal = {J Am Soc Nephrol}, year = {2025}, month = {2025 Oct 06}, abstract = {

BACKGROUND: Kidney epithelial cells perform complex vectorial fluid and solute transport at high volumes and rapid rates. Their structural organization both reflects and enables these sophisticated physiological functions. However, our understanding of the nanoscale spatial organization and intracellular ultrastructure that underlies these crucial cellular functions remains limited.

METHODS: To address this knowledge gap, we generated and reconstructed an extensive electron microscopic dataset of renal proximal tubule (PT) epithelial cells at isotropic resolutions down to 4nm. We employed artificial intelligence-based segmentation tools to identify, trace, and measure all major subcellular components. We complemented this analysis with immunofluorescence microscopy to connect subcellular architecture to biochemical function.

RESULTS: Our ultrastructural analysis revealed complex organization of membrane-bound compartments in proximal tubule cells. The apical endocytic system featured deep invaginations connected to an anastomosing meshwork of dense apical tubules, rather than discrete structures. The endoplasmic reticulum displayed distinct structural domains: fenestrated sheets in the basolateral region and smaller, disconnected clusters in the subapical region. We identified, quantified, and visualized membrane contact sites between endoplasmic reticulum, plasma membrane, mitochondria, and apical endocytic compartments. Immunofluorescence microscopy demonstrated distinct localization patterns for endoplasmic reticulum resident proteins at mitochondrial and plasma membrane interfaces.

CONCLUSIONS: This study provides novel insights into proximal tubule cell organization, revealing specialized compartmentalization and unexpected connections between membrane-bound organelles. We identified previously uncharacterized structures, including mitochondria-plasma membrane bridges and an interconnected endocytic meshwork, suggesting mechanisms for efficient energy distribution, cargo processing and structural support. Morphological differences between 4nm and 8nm datasets indicate subsegment-specific specializations within the proximal tubule. This comprehensive open-source dataset provides a foundation for understanding how subcellular architecture supports specialized epithelial function in health and disease.

}, issn = {1533-3450}, doi = {10.1681/ASN.0000000884}, url = {https://doi.org/10.1681/asn.0000000884}, author = {Pandya, Raj D and Lackner, Emily M and Xu, C Shan and Zugates, Christopher and Burdyniuk, Mariia and Reyna-Neyra, Andrea and Pandya, Vraj D and Li, Wei-Ping and Pang, Song and Weisz, Ora A and Caplan, Michael J} } @article {70084, title = {Imaging cellular activity simultaneously across all organs of a vertebrate reveals body-wide circuits}, journal = {bioRxiv}, year = {2025}, month = {2025 August 22}, abstract = {

All cells in an animal collectively ensure, moment-to-moment, the survival of the whole organism in the face of environmental stressors1,2. Physiology seeks to elucidate the intricate network of interactions that sustain life, which often span multiple organs, cell types, and timescales, but a major challenge lies in the inability to simultaneously record time-varying cellular activity throughout the entire body.We developed WHOLISTIC, a method to image second-timescale, time-varying intracellular dynamics across cell-types of the vertebrate body. By advancing and integrating volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors in transparent young Danio rerio (zebrafish) and adult Danionella, the method enables real-time recording of cellular dynamics across the organism. Calcium is a universal intracellular messenger, with a large array of cellular processes depending on changes in calcium concentration across varying time-scales, making it an ideal proxy of cellular activity3.Using this platform to screen the dynamics of all cells in the body, we discovered unexpected responses of specific cell types to stimuli, such as chondrocyte reactions to cold, meningeal responses to ketamine, and state-dependent activity, such as oscillatory ependymal-cell activity during periods of extended motor quiescence. At the organ scale, the method uncovered pulsating traveling waves along the kidney nephron. At the multi-organ scale, we uncovered muscle synergies and independencies, as well as muscle-organ interactions. Integration with optogenetics allowed us to all-optically determine the causal direction of brain-body interactions. At the whole-organism scale, the method captured the rapid brainstem-controlled redistribution of blood flow across the body.Finally, we advanced Whole-Body Expansion Microscopy4 to provide ground-truth molecular and ultrastructural anatomical context, explaining the spatiotemporal structure of activity captured by WHOLISTIC. Together, these innovations establish a new paradigm for systems biology, bridging cellular and organismal physiology, with broad implications for both fundamental research and drug discovery.

}, doi = {10.1101/2025.08.20.670374}, url = {https://www.biorxiv.org/content/early/2025/08/22/2025.08.20.670374.1}, author = {Ruetten, Virginia M. S. and Zheng, Wei and Siwanowicz, Igor and Mensh, Brett D. and Eddison, Mark and Hu, Amy and Chi, Yunfeng and Lemire, Andrew L. and Guo, Caiying and Kadobianskyi, Mykola and Renz, Marc and Lelek-Greskovic, Sara and He, Yisheng and Close, Kari and Ihrke, Gudrun and Dev, Aparna and Petruncio, Alyson and Wan, Yinan and Engert, Florian and Fishman, Mark C. and Judkewitz, Benjamin and Rubinov, Mikail and Keller, Philipp J. and Satou, Chie and Yu, Guoqiang and Tillberg, Paul W. and Sahani, Maneesh and Ahrens, Misha B.} } @article {69110, title = {Imaging neuronal voltage beyond the scattering limit}, journal = {Nat Methods}, year = {2025}, month = {2025 May 19}, abstract = {

Voltage imaging is a promising technique for high-speed recording of neuronal population activity. However, tissue scattering severely limits its application in dense neuronal populations. Here, we adopted the principle of localization microscopy, a technique that enables super-resolution imaging of single-molecules, to resolve dense neuronal activities\ in vivo. Leveraging the sparse activation of neurons during action potentials (APs), we precisely localize the fluorescence change associated with each AP, creating a super-resolution image of neuronal activities. This approach, termed\ Activity\ Localization\ Imaging (ALI), identifies overlapping neurons and separates their activities with over 10-fold greater precision than what tissue scattering permits. Using ALI, we simultaneously recorded over a hundred densely-labeled CA1 neurons, creating a map of hippocampal theta oscillation at single-cell and single-cycle resolution.

Preprint:\ https://doi.org/10.1101/2023.12.03.56840

}, doi = {10.1038/s41592-025-02692-5}, url = {https://doi.org/10.1038/s41592-025-02692-5}, author = {Tsai-Wen Chen and Xian-Bin Huang and Sarah E. Plutkis and Katie L. Holland and Luke D. Lavis and Bei-Jung Lin} } @article {70190, title = {Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis}, journal = {bioRxiv}, year = {2025}, month = {2025 Nov 18}, abstract = {

Cortical neurogenesis proceeds through a precise temporal program in which radial glia sequentially generate distinct neuronal subtypes and later glia, yet how post-transcriptional regulators coordinate these transitions remain poorly understood. We previously identified that a decreasing temporal gradient of the RNA-binding protein Imp encodes neural stem cell age in Drosophila. In this work, we extend our investigation to Imp1, a mammalian homologue of Imp, and its role in murine neocortical development. Using TEMPO to track birth-order dynamics, we demonstrate that sustained Imp1 overexpression during early neurogenesis arrests temporal fate progression, shifting neuronal populations toward deeper cortical layers V-VI. Immunostaining with layer-specific transcription factors Cux1 and Ctip2 confirmed that laminar repositioning results from genuine changes in neuronal identity rather than migratory defects, with neurons adopting molecular identities matching their final positions. Temporal window-specific manipulations reveal distinct stage-specific effects where early-stage Imp1 induction produces cascading effects on fate specification and moderately delays the neuronal-to-gliogenic transition, while mid-stage induction induces neuronal accumulation in the subplate region. Live imaging of organotypic cultures reveals continuous neuronal recruitment within intermediate and ventricular zones, with mid-stage-born neurons accumulating at significantly faster rates than earlier cohorts. Strikingly, mid-stage Imp1 overexpression also induces ectopic glial-like foci distributed throughout the cortical plate, featuring dramatic cellular expansion and morphological heterogeneity. These findings establish Imp1 as a dosage- and stage-dependent temporal rheostat orchestrating developmental transitions in radial glial progenitors, controlling neuronal fate decisions and spatial organization. This work advances our understanding of molecular timing mechanisms governing neuronal diversity in the mammalian cortex.

}, doi = {10.1101/2025.11.18.688993}, url = {https://www.biorxiv.org/content/early/2025/11/18/2025.11.18.688993}, author = {Azur, Romie Angelo G. and Feliciano, Daniel and Espinosa-Medina, Isabel and Adhikari, Raghabendra and Lilao-Garz{\'o}n, Joaquin and Jensen, Ella and Yang, Ching-Po and Lee, Tzumin} } @article {69709, title = {In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors}, journal = {Science}, year = {2025}, month = {2025 Jun 05}, abstract = {

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

}, doi = {10.1126/science.adt7705}, url = {https://doi.org/10.1126/science.adt7705}, author = {Zheng, Yu and Cai, Ruyi and Wang, Kui and Zhang, Junwei and Zhuo, Yizhou and Dong, Hui and Zhang, Yuqi and Wang, Yifan and Deng, Fei and Ji, En and Cui, Yiwen and Fang, Shilin and Zhang, Xinxin and Zhang, Kecheng and Wang, Jinxu and Li, Guochuan and Miao, Xiaolei and Wang, Zhenghua and Yang, Yuqing and Li, Shaochuang and Grimm, Jonathan and Johnsson, Kai and Schreiter, Eric and Lavis, Luke and Chen, Zhixing and Mu, Yu and Li, Yulong} } @article {70207, title = {Innovative In Vivo Imaging and Single Cell Expression from Tumor Bulk and Corpus Callosum Reveal Glioma Stem Cells with Unique Regulatory Programs}, journal = {Cancers}, volume = {17}, year = {2025}, month = {2025 Nov 30}, pages = {3851}, abstract = {

Background/Objectives: High-grade gliomas (HGGs), including glioblastomas, are among the most aggressive brain tumors{\r a} due to their high intratumoral heterogeneity and extensive infiltration. Glioma stem-like cells (GSCs) frequently invade along white matter tracts such as the corpus callosum, but the molecular programs driving this region-specific invasion remain poorly defined. The aim of this study was to identify transcriptional signatures associated with GSC infiltration into the corpus callosum. Methods: We established an orthotopic xenograft model by implanting fluorescently labeled human GSCs into nude mouse brains. Tumor growth and invasion patterns were assessed using tissue clearing, light-sheet fluorescence microscopy, and histological analyses. To characterize region-specific molecular profiles, we performed microfluidic-based single-cell RNA expression analysis of 48 invasion- and stemness-related genes in cells isolated from the tumor bulk (TB) and corpus callosum (CC). Results: By six weeks post-implantation, GSCs displayed marked tropism for the corpus callosum, with distinct infiltration patterns captured by three-dimensional imaging. Single-cell gene expression profiling revealed significant differences in 7 of the 48 genes (14.6\%) between TB- and CC-derived GSCs. These genes{\textemdash}NES, CCND1, GUSB, NOTCH1, E2F1, EGFR, and TGFB1{\textemdash}collectively defined a {\textquotedblleft}corpus callosum invasion signature{\textquotedblright} (CC-Iv). CC-derived cells showed a unimodal, high-expression profile of CC-Iv genes, whereas TB cells exhibited bimodal distributions, suggesting heterogeneous transcriptional states. Importantly, higher CC-Iv expression correlated with worse survival in patients with low-grade gliomas. Conclusions: This multimodal approach identified a corpus callosum-specific invasion signature in glioma stem-like cells, revealing how local microenvironmental cues shape transcriptional reprogramming during infiltration. These findings provide new insights into the spatial heterogeneity of gliomas and highlight potential molecular targets for therapies designed to limit tumor spread through white matter tracts.

}, issn = {2072-6694}, doi = {10.3390/cancers17233851}, url = {https://www.mdpi.com/2072-6694/17/23/3851}, author = {Dos Santos, Nat{\'a}lia and Aquino, Aline and Preu{\ss}er, Friedrich and Rusak, Fabio Rojas and Jandrey, Elisa Helena Farias and Uno, Miyuki and Furuya, Tatiane Katsue and Lancellotti, Carmen Lucia Penteado and Maldaun, Marcos Vinicius Calfat and Chammas, Roger and Preibisch, Stephan and Camargo, Anamaria Aranha and Masotti, Cibele and Costa, {\'E}rico Tosoni} } @article {70214, title = {Innovative In Vivo Imaging and Single Cell Expression from Tumor Bulk and Corpus Callosum Reveal Glioma Stem Cells with Unique Regulatory Programs}, journal = {Cancers}, volume = {17}, year = {2025}, month = {2025 Nov 30}, pages = {3851}, abstract = {

Background/Objectives: High-grade gliomas (HGGs), including glioblastomas, are among the most aggressive brain tumors due to their high intratumoral heterogeneity and extensive infiltration. Glioma stem-like cells (GSCs) frequently invade along white matter tracts such as the corpus callosum, but the molecular programs driving this region-specific invasion remain poorly defined. The aim of this study was to identify transcriptional signatures associated with GSC infiltration into the corpus callosum. Methods: We established an orthotopic xenograft model by implanting fluorescently labeled human GSCs into nude mouse brains. Tumor growth and invasion patterns were assessed using tissue clearing, light-sheet fluorescence microscopy, and histological analyses. To characterize region-specific molecular profiles, we performed microfluidic-based single-cell RNA expression analysis of 48 invasion- and stemness-related genes in cells isolated from the tumor bulk (TB) and corpus callosum (CC). Results: By six weeks post-implantation, GSCs displayed marked tropism for the corpus callosum, with distinct infiltration patterns captured by three-dimensional imaging. Single-cell gene expression profiling revealed significant differences in 7 of the 48 genes (14.6\%) between TB- and CC-derived GSCs. These genes{\textemdash}NES, CCND1, GUSB, NOTCH1, E2F1, EGFR, and TGFB1{\textemdash}collectively defined a {\textquotedblleft}corpus callosum invasion signature{\textquotedblright} (CC-Iv). CC-derived cells showed a unimodal, high-expression profile of CC-Iv genes, whereas TB cells exhibited bimodal distributions, suggesting heterogeneous transcriptional states. Importantly, higher CC-Iv expression correlated with worse survival in patients with low-grade gliomas. Conclusions: This multimodal approach identified a corpus callosum-specific invasion signature in glioma stem-like cells, revealing how local microenvironmental cues shape transcriptional reprogramming during infiltration. These findings provide new insights into the spatial heterogeneity of gliomas and highlight potential molecular targets for therapies designed to limit tumor spread through white matter tracts.

}, issn = {2072-6694}, doi = {10.3390/cancers17233851}, url = {https://www.mdpi.com/2072-6694/17/23/3851}, author = {Dos Santos, Nat{\'a}lia and Aquino, Aline and Preu{\ss}er, Friedrich and Rusak, Fabio Rojas and Jandrey, Elisa Helena Farias and Uno, Miyuki and Furuya, Tatiane Katsue and Lancellotti, Carmen Lucia Penteado and Maldaun, Marcos Vinicius Calfat and Chammas, Roger and Preibisch, Stephan and Camargo, Anamaria Aranha and Masotti, Cibele and Costa, {\'E}rico Tosoni} } @article {70139, title = {In-situ glial cell-surface proteomics identifies pro-longevity factors in Drosophila}, journal = {eLife}, year = {2025}, month = {2025 Dec 12}, abstract = {

Much focus has shifted towards understanding how glial dysfunction contributes to age-related neurodegeneration due to the critical roles glial cells play in maintaining healthy brain function. Cell-cell interactions, which are largely mediated by cell-surface proteins, control many critical aspects of development and physiology; as such, dysregulation of glial cell-surface proteins in particular is hypothesized to play an important role in age-related neurodegeneration. However, it remains technically difficult to profile glial cell-surface proteins in intact brains. Here, we applied a cell-surface proteomic profiling method to glial cells from intact brains in\ Drosophila,\ which enabled us to fully profile cell-surface proteomes\ in-situ, preserving native cell-cell interactions that would otherwise be omitted using traditional proteomics methods. Applying this platform to young and old flies, we investigated how glial cell-surface proteomes change during aging. We identified candidate genes predicted to be involved in brain aging, including several associated with neural development and synapse wiring molecules not previously thought to be particularly active in glia. Through a functional genetic screen, we identified one surface protein, DIP-β, which is down-regulated in old flies and can increase fly lifespan when overexpressed in adult glial cells. We further performed whole-head single-nucleus RNA-seq, and revealed that DIP-β overexpression mainly impacts glial and fat cells. We also found that glial DIP-β overexpression was associated with improved cell-cell communication, which may contribute to the observed lifespan extension. Our study is the first to apply\ in-situ\ cell-surface proteomics to glial cells in\ Drosophila, and to identify DIP-β as a potential glial regulator of brain aging.

}, doi = {10.7554/eLife.109422.1}, url = {https://doi.org/10.7554/eLife.109422.1}, author = {Marques, Madeline P. and Sun, Bo and Park, Ye-Jin and Jackson, Tyler and Lu, Tzu-Chiao and Qi, Yanyan and Harrison, Erin and Wang, Miranda C. and Venkatachalam, Kartik and Pasha, Omar Moussa and Varanasi, Amogh and Carey, Dominique Kiki and Mani, D.R. and Zirin, Jonathan and Qadiri, Mujeeb and Hu, Yanhui and Perrimon, Norbert and Carr, Steven A. and Udeshi, Namrata D. and Luo, Liqun and Li, Jiefu and Li, Hongjie} } @conference {70301, title = {An investigation of unsupervised cell tracking and interactive fine-tuning}, booktitle = {2025 IEEE/CVF International Conference on Computer Vision Workshops (ICCVW)}, year = {2025}, month = {2026 Feb 23}, abstract = {

Most existing deep learning-based cell tracking methods rely on supervised learning, requiring large-scale annotated datasets that are often unavailable in real-world scenarios. Moreover, many approaches lack tools and methods for correcting mispredicted links or incorporating corrections through fine-tuning. These limitations contribute to the limited adoption of deep learning-based tracking methods in the life sciences, where manual tracking remains the predominant approach. To reduce the annotation burden and enable model training without extensive labeled data, we introduce a loss function for unsupervised training. Our method leverages the predictable dynamics inherent in many biological processes, providing an initialization that does not require an annotated dataset. We further investigate how minimal user-provided annotations can refine tracking accuracy. To this end, we propose an active learning framework that selectively identifies uncertain decisions within the tracking graph, allowing for efficient annotation of the most informative data points. We evaluate our approach on two microscopy datasets, demonstrating the effectiveness of both our unsupervised training strategy and active learning scheme in improving tracking performance. Our implementation and reproducible experiments are available at github.com/funkelab/attrackt and github.com/funkelab/attrackt_experiments, respectively.

}, keywords = {Active learning, Annotations, Cell Tracking, Computer vision, Conferences, Data models, Fine-Tuning, Life sciences, Manuals, Microscopy, Supervised learning, Training, Unsupervised Deep Learning}, doi = {10.1109/ICCVW69036.2025.00610}, url = {https://doi.org/10.1109/ICCVW69036.2025.00610}, author = {Lalit, Manan and Funke, Jan} } @article {70055, title = {(Invited) Nanoscale Insights into Dopamine Release: Single-Strand DNA Functionalized SWCNTs in Neuroscience}, journal = {ECS Meeting Abstracts}, volume = {MA2025-01}, year = {2025}, month = {2025 July 01}, pages = {933}, abstract = {

Single-stranded DNA (ssDNA)-functionalized single-wall carbon nanotubes (SWCNTs) exhibit exceptional optical sensitivity to catecholamines, including dopamine and norepinephrine{\textemdash}key signaling molecules that play vital roles in brain function. This unique capability positions SWCNTs as powerful tools for advancing our understanding of neurochemical processes involving dopaminergic and noradrenergic neurons. In this presentation, I will highlight how our lab has leveraged SWCNT nanosensors to push the boundaries of dopamine neuroscience. For studies in cultured neurons, we developed a composite nanofilm strategy that enabled us to visualize dopamine release with exceptional resolution, capturing single bouton activity with quantal sensitivity while monitoring thousands of release sites simultaneously in large imaging fields of view. By combining SWCNT-based activity imaging with immunofluorescence, electron microscopy, and cutting-edge molecular, cellular and genetic techniques, we have gained new insights into neurobiological properties of dopamine release sites in dopaminergic neurons that had heretofore been inaccessible with conventional methods of inquiry. Building on these advances, I will discuss recent progress in the development of in vivo-compatible dopamine nanosensors. These innovations have allowed us to monitor dopamine dynamics in awake and behaving mice, bridging the gap between molecular-scale imaging and real-time behavior analysis. Furthermore, I will discuss methodological developments that enabled the deployment of these nanosensors in vivo. Looking ahead, these SWCNT-enabled technological advancements hold potential for the study of neurochemical signaling, offering deeper insights into both normal brain function and the pathophysiology of disorders involving catecholamines. Future work aims to expand the applications of these nanosensors to other neural circuits and neuromodulators, ultimately advancing our ability to decode the brain{\textquoteright}s chemical language.

}, doi = {10.1149/MA2025-0111933mtgabs}, url = {https://dx.doi.org/10.1149/MA2025-0111933mtgabs}, author = {Beyene, Abraham G.} } @article {70116, title = {stk32a links sleep homeostasis to suppression of sensory and motor systems}, journal = {bioRxiv}, year = {2025}, month = {2025 Sep 11}, abstract = {

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood. To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies. We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain. Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

}, doi = {10.1101/2025.09.09.675098}, url = {https://www.biorxiv.org/content/early/2025/09/11/2025.09.09.675098}, author = {Tran, Steven and Emtage, Jasmine and Zhang, Chaodong and Liu, Xiaoyao and Lecoeuche, Marina and Andreev, Andrey and Oikonomou, Grigorios and Narayan, Sujatha and Garcia, Brianna and Cammidge, Tasha and Gonzales, Cristina and Hurley, Hannah and Yap, Misha and Li, Shan and Wang, Feng and Wang, Ting-Yu and Ahrens, Misha B. and Chou, Tsui-Fen and Xu, Min and Liu, Qinghua and Prober, David A.} } @article {68969, title = {iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP}, journal = {Proc Natl Acad Sci U S A}, year = {2024}, month = {2024 May 21}, abstract = {

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the\ ε-subunit of a bacterial F0-F1\ ATPase. Optimizing the linkers joining the two domains resulted in a \~{} 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 μM to 500 μM. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

}, doi = {10.1073/pnas.2314604121 }, author = {Jonathan S. Marvin and Alexandros C. Kokotos and Mukesh Kumar and Camila Pulido and Ariana N. Tkachuk and Jocelyn Shuxin Yao and Timothy A. Brown and Timothy A. Ryan} } @article {69576, title = {An Image Processing Tool for Automated Quantification of Bacterial Burdens in Zebrafish Larvae}, journal = {bioRxiv}, year = {2024}, month = {2024 Aug 19}, abstract = {

Zebrafish larvae are used to model the pathogenesis of multiple bacteria. This transparent model offers the unique advantage of allowing quantification of fluorescent bacterial burdens (fluorescent pixel counts: FPC) in vivo by facile microscopical methods, replacing enumeration of bacteria using time-intensive plating of lysates on bacteriological media. Accurate FPC measurements require laborious manual image processing to mark the outside borders of the animals so as to delineate the bacteria inside the animals from those in the culture medium that they are in. Here, we have developed an automated ImageJ/Fiji-based macro that accurately detect the outside borders of Mycobacterium marinum-infected larvae.

}, doi = {10.1101/2024.08.16.608298}, url = {hhtps://doi.org/10.1101/2024.08.16.608298}, author = {Yamaguchi, Naoya and Otsuna, Hideo and Eisenberg-Bord, Michal and Ramakrishnan, Lalita} } @article {69708, title = {An Image Processing Tool for Automated Quantification of Bacterial Burdens in Zebrafish Larvae.}, journal = {Zebrafish}, year = {2024}, month = {12/2024}, abstract = {

Zebrafish larvae are used to model the pathogenesis of multiple bacteria. This transparent model offers the unique advantage of allowing quantification of fluorescent bacterial burdens (fluorescent pixel counts [FPC]) by facile microscopical methods, replacing enumeration of bacteria using time-intensive plating of lysates on bacteriological media. Accurate FPC measurements require laborious manual image processing to mark the outside borders of the animals so as to delineate the bacteria inside the animals from those in the culture medium that they are in. Here, we have developed an automated ImageJ/Fiji-based macro that accurately detects the outside borders of -infected larvae.

}, issn = {1557-8542}, doi = {10.1089/zeb.2024.0170}, author = {Yamaguchi, Naoya and Otsuna, Hideo and Eisenberg-Bord, Michal and Ramakrishnan, Lalita} } @article {69142, title = {Image processing tools for petabyte-scale light sheet microscopy data.}, journal = {Nat. Methods}, year = {2024}, month = {2024 Oct 17}, abstract = {

Light sheet microscopy is a powerful technique for high-speed three-dimensional imaging of subcellular dynamics and large biological specimens. However, it often generates datasets ranging from hundreds of gigabytes to petabytes in size for a single experiment. Conventional computational tools process such images far slower than the time to acquire them and often fail outright due to memory limitations. To address these challenges, we present PetaKit5D, a scalable software solution for efficient petabyte-scale light sheet image processing. This software incorporates a suite of commonly used processing tools that are optimized for memory and performance. Notable advancements include rapid image readers and writers, fast and memory-efficient geometric transformations, high-performance Richardson-Lucy deconvolution and scalable Zarr-based stitching. These features outperform state-of-the-art methods by over one order of magnitude, enabling the processing of petabyte-scale image data at the full teravoxel rates of modern imaging cameras. The software opens new avenues for biological discoveries through large-scale imaging experiments.

}, doi = {10.1038/s41592-024-02475-4}, url = {https://doi.org/10.1038/s41592-024-02475-4}, author = {Xiongtao Ruan and Matthew Mueller and Gaoxiang Liu and Frederik G{\"o}rlitz and Tian-Ming Fu and Daniel E. Milkie and Joshua Lillvis and Alison Killilea and Eric Betzig and Srigokul Upadhyayula} } @article {69440, title = {Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore}, journal = {bioRxiv}, year = {2024}, month = {2024 May 10}, abstract = {

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

}, doi = {10.1101/2024.05.09.593460}, url = {https://www.biorxiv.org/content/early/2024/05/10/2024.05.09.593460}, author = {Fiore, Antonio and Yu, Guoqiang and Northey, Jason J. and Patel, Ronak and Ravenscroft, Thomas A. and Ikegami, Richard and Kolkman, Wiert and Kumar, Pratik and Grimm, Jonathan B. and Dilan, Tanya L. and Ruetten, Virginia M.S. and Ahrens, Misha B. and Shroff, Hari and Lavis, Luke D. and Wang, Shaohe and Weaver, Valerie M. and Pedram, Kayvon} } @article {69215, title = {Integrating across behaviors and timescales to understand the neural control of movement.}, journal = {Current Opinion in Neurobiology}, volume = {85}, year = {2024}, month = {2024 Feb 13}, pages = {102843}, abstract = {

The nervous system evolved to enable navigation throughout the environment in the pursuit of resources. Evolutionarily newer structures allowed increasingly complex adaptations but necessarily added redundancy. A dominant view of movement neuroscientists is that there is a one-to-one mapping between brain region and function. However, recent experimental data is hard to reconcile with the most conservative interpretation of this framework, suggesting a degree of functional redundancy during the performance of well-learned, constrained behaviors. This apparent redundancy likely stems from the bidirectional interactions between the various cortical and subcortical structures involved in motor control. We posit that these bidirectional connections enable flexible interactions across structures that change depending upon behavioral demands, such as during acquisition, execution or adaptation of a skill. Observing the system across both multiple actions and behavioral timescales can help isolate the functional contributions of individual structures, leading to an integrated understanding of the neural control of movement.

}, issn = {1873-6882}, doi = {10.1016/j.conb.2024.102843}, author = {Gmaz, Jimmie M and Keller, Jason A and Dudman, Joshua T and Gallego, Juan A} } @article {69609, title = {Integration of estimated regional gene expression with neuroimaging and clinical phenotypes at biobank scale.}, journal = {PLoS Biol}, volume = {22}, year = {2024}, month = {2024 Sep 13}, pages = {e3002782}, abstract = {

An understanding of human brain individuality requires the integration of data on brain organization across people and brain regions, molecular and systems scales, as well as healthy and clinical states. Here, we help advance this understanding by leveraging methods from computational genomics to integrate large-scale genomic, transcriptomic, neuroimaging, and electronic-health record data sets. We estimated genetically regulated gene expression (gr-expression) of 18,647 genes, across 10 cortical and subcortical regions of 45,549 people from the UK Biobank. First, we showed that patterns of estimated gr-expression reflect known genetic-ancestry relationships, regional identities, as well as inter-regional correlation structure of directly assayed gene expression. Second, we performed transcriptome-wide association studies (TWAS) to discover 1,065 associations between individual variation in gr-expression and gray-matter volumes across people and brain regions. We benchmarked these associations against results from genome-wide association studies (GWAS) of the same sample and found hundreds of novel associations relative to these GWAS. Third, we integrated our results with clinical associations of gr-expression from the Vanderbilt Biobank. This integration allowed us to link genes, via gr-expression, to neuroimaging and clinical phenotypes. Fourth, we identified associations of polygenic gr-expression with structural and functional MRI phenotypes in the Human Connectome Project (HCP), a small neuroimaging-genomic data set with high-quality functional imaging data. Finally, we showed that estimates of gr-expression and magnitudes of TWAS were generally replicable and that the p-values of TWAS were replicable in large samples. Collectively, our results provide a powerful new resource for integrating gr-expression with population genetics of brain organization and disease.

}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3002782}, url = {https://doi.org/10.1371/journal.pbio.3002782}, author = {Hoang, Nhung and Sardaripour, Neda and Ramey, Grace D and Schilling, Kurt and Liao, Emily and Chen, Yiting and Park, Jee Hyun and Bledsoe, Xavier and Landman, Bennett A and Gamazon, Eric R and Benton, Mary Lauren and Capra, John A and Rubinov, Mikail} } @article {69141, title = {Interactive simulation and visualization of point spread functions in single molecule imaging.}, journal = {Optics Communications}, year = {2024}, month = {2024 Mar 20}, abstract = {

The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. Optical aberrations and fixed fluorophore dipoles often result in non-isotropic and distorted PSFs, impairing and biasing conventional fitting approaches. Further, PSF shapes are deliberately modified in PSF engineering approaches for providing improved sensitivity, e.g., for 3D localization or determination of dipole orientation. As this can lead to highly complex PSF shapes, a tool for visualizing expected PSFs would facilitate the interpretation of obtained data and the design of experimental approaches. To this end, we introduce a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. Our tool incorporates a wide range of microscope and fluorophore parameters, including orientationally constrained fluorophores, as well as custom aberrations, transmission and phase masks, thus enabling an accurate representation of various imaging conditions. An additional feature is the simulation of crowded molecular environments with overlapping PSFs. Further, our app directly provides the Cram{\'e}r{\textendash}Rao bound for assessing the best achievable localization precision under given conditions. Finally, our software allows for the fitting of custom aberrations directly from experimental data, as well as the generation of a large dataset with randomized simulation parameters, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.

}, doi = {10.1016/j.optcom.2024.130463}, url = {https://www.sciencedirect.com/science/article/pii/S0030401824002001?via\%3Dihub}, author = {Magdalena C. Schneider and Fabian Hinterer and Alexander Jesacher and Gerhard J. Sch{\"u}tz} } @article {69420, title = {An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 May 07}, pages = {3793}, abstract = {

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.

}, keywords = {Actins, Animals, Formins, Hela Cells, Homeostasis, Humans, Interphase, Microtubules, Mitochondria, Mitochondrial Dynamics, Mitochondrial Proteins, Reactive Oxygen Species}, issn = {2041-1723}, doi = {10.1038/s41467-024-48189-1}, author = {Coscia, Stephen M and Moore, Andrew S and Thompson, Cameron P and Tirrito, Christian F and Ostap, E Michael and Holzbaur, Erika L F} } @article {69643, title = {IRE1α silences dsRNA to prevent taxane-induced pyroptosis in triple-negative breast cancer.}, journal = {Cell}, year = {2024}, month = {2024 Oct 14}, abstract = {

Chemotherapy is often combined with immune checkpoint inhibitor (ICIs) to enhance immunotherapy responses. Despite the approval of chemo-immunotherapy in multiple human cancers, many immunologically cold tumors remain unresponsive. The mechanisms determining the immunogenicity of chemotherapy are elusive. Here, we identify the ER stress sensor IRE1α as a critical checkpoint that restricts the immunostimulatory effects of taxane chemotherapy and prevents the innate immune recognition of immunologically cold triple-negative breast cancer (TNBC). IRE1α RNase silences taxane-induced double-stranded RNA (dsRNA) through regulated IRE1-dependent decay (RIDD) to prevent NLRP3 inflammasome-dependent pyroptosis. Inhibition of IRE1α in Trp53 TNBC allows taxane to induce extensive dsRNAs that are sensed by ZBP1, which in turn activates NLRP3-GSDMD-mediated pyroptosis. Consequently, IRE1α RNase inhibitor plus taxane converts PD-L1-negative, ICI-unresponsive TNBC tumors into PD-L1 immunogenic tumors that are hyper-sensitive to ICI. We reveal IRE1α as a cancer cell defense mechanism that prevents taxane-induced danger signal accumulation and pyroptotic cell death.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.09.032}, url = {https://doi.org/10.1016/j.cell.2024.09.032}, author = {Xu, Longyong and Peng, Fanglue and Luo, Qin and Ding, Yao and Yuan, Fei and Zheng, Liting and He, Wei and Zhang, Sophie S and Fu, Xin and Liu, Jin and Mutlu, Ayse Sena and Wang, Shuyue and Nehring, Ralf Bernd and Li, Xingyu and Tang, Qianzi and Li, Catherine and Lv, Xiangdong and Dobrolecki, Lacey E and Zhang, Weijie and Han, Dong and Zhao, Na and Jaehnig, Eric and Wang, Jingyi and Wu, Weiche and Graham, Davis A and Li, Yumei and Chen, Rui and Peng, Weiyi and Chen, Yiwen and Catic, Andre and Zhang, Zhibin and Zhang, Bing and Mustoe, Anthony M and Koong, Albert C and Miles, George and Lewis, Michael T and Wang, Meng C and Rosenberg, Susan M and O{\textquoteright}Malley, Bert W and Westbrook, Thomas F and Xu, Han and Zhang, Xiang H-F and Osborne, C Kent and Li, Jin Billy and Ellis, Matthew J and Rimawi, Mothaffar F and Rosen, Jeffrey M and Chen, Xi} } @article {69483, title = {It only takes seconds for a human monoclonal autoantibody to inhibit N-methyl-D-aspartate receptors}, journal = {bioRxiv}, year = {2024}, month = {2024 Jun 01}, abstract = {

Transfer of autoantibodies targeting ionotropic N-methyl-D-aspartate receptors in autoimmune encephalitis patients into mice leads to typical disease signs. Long-term effects of the pathogenic antibodies consist of immunoglobulin G-induced crosslinking and receptor internalization. We focused on the direct and immediate impact of a specific pathogenic patient-derived monoclonal autoantibody (immunoglobulin G $\#$003-102) on receptor function.We performed cell-attached recordings in cells transfected with the GluN1 and GluN2A subunit of the N-methyl-D-aspartate receptor. Immunoglobulin G $\#$003-102 binds to the amino-terminal domain of the glycine-binding GluN1 subunit. It reduced simultaneous receptor openings significantly compared to controls at both low and high glutamate and glycine concentrations. Closer examination of our data in 50-second to 2-second intervals revealed, that Immunoglobulin G $\#$003-102 rapidly decreases the number of open receptors. However, antigen-binding fragments of immunoglobulin G $\#$003-102 did not reduce the receptor openings.In conclusion, patient-derived immunoglobulin G $\#$003-102 inhibits N-methyl-D-aspartate receptors rapidly and directly before receptor internalization occurs and the entire immunoglobulin G is necessary for this acute inhibitory effect. This suggests an application of the antigen-binding fragment-like constructs of $\#$003-102 as a potential new treatment strategy for shielding the pathogenic epitopes on the N-methyl-D-aspartate receptors.

}, doi = {10.1101/2024.05.28.595700}, url = {https://www.biorxiv.org/content/early/2024/06/01/2024.05.28.595700}, author = {Yang, Shang and Heckmann, Johanna and Taha, Abdulla and Gao, Shiqiang and Steinke, Stephan and Hust, Michael and Pr{\"u}{\ss}, Harald and Furukawa, Hiro and Geis, Christian and Heckmann, Manfred and Yu-Strzelczyk, Jing} } @article {69525, title = {iTome Volumetric Serial Sectioning Apparatus for TEM}, journal = {bioRxiv}, year = {2024}, month = {2024 Jul 07}, abstract = {

An automated ultra-microtome capable of sectioning thousands of ultrathin sections onto standard TEM slot grids was developed and used to section: a complete Drosophila melanogaster first-instar larva, three sections per grid, into 4,866 34-nm-thick sections with a cutting and pickup success rate of 99.74\%; 30 microns of mouse cortex measuring roughly 400 um x 2000 um at 40 nm per slice; and a full adult Drosophila brain and ventral nerve column into 9,300 sections with a pickup success rate of 99.95\%. The apparatus uses optical interferometers to monitor a reference distance between the cutting knife and multiple sample blocks. Cut sections are picked up from the knife-boat water surface while they are still anchored to the cutting knife. Blocks without embedded tissue are used to displace tissue-containing sections away from the knife edge so that the tissue regions end up in the grid slot instead of on the grid rim.

}, doi = {10.1101/2024.07.02.601671}, url = {https://doi.org/10.1101/2024.07.02.601671 }, author = {Peale, David R. and Hess, Harald and Lee, P. R. and Cardona, Albert and Bock, Davi D. and Schneider-Mizell, Casey and Fetter, Richard D. and Lee, Wei-Ping and Robinson, Camenzind G. and Iyer, Nirmala and Managan, Claire} } @article {68697, title = {Identifying determinants of synaptic specificity by integrating connectomes and transcriptomes}, journal = {bioRxiv}, year = {2023}, month = {2023 Apr 04}, abstract = {

How do developing neurons select their synaptic partners? To identify molecules matching synaptic partners, we integrated the synapse-level connectome of neural circuits with the developmental expression patterns and binding specificities of cell adhesion molecules (CAMs) on pre- and postsynaptic neurons. We focused on parallel synaptic pathways in the Drosophila visual system, in which closely related neurons form synapses onto closely related target neurons. We show that the choice of synaptic partners correlates with the matching expression of receptor-ligand pairs of Beat and Side proteins of the immunoglobulin superfamily (IgSF) CAMs. Genetic analysis demonstrates that these proteins determine the choice between alternative synaptic targets. Combining transcriptomes, connectomes, and protein interactome maps provides a framework to uncover the molecular logic of synaptic connectivity.

}, doi = {10.1101/2023.04.03.534791}, url = {https://doi.org/10.1101/2023.04.03.534791}, author = {Juyoun Yoo and Mark Dombrovski and Parmis Mirshahidi and Aljoscha Nern and Samuel A. LoCascio and S. Lawrence Zipursky and Yerbol Z. Kurmangaliyev} } @article {69055, title = {Imagining the future of optical microscopy: everything, everywhere, all at once.}, journal = {Communications Biology}, volume = {6}, year = {2023}, month = {2023 Oct 28}, pages = {1096}, abstract = {

The optical microscope has revolutionized biology since at least the 17 Century. Since then, it has progressed from a largely observational tool to a powerful bioanalytical platform. However, realizing its full potential to study live specimens is hindered by a daunting array of technical challenges. Here, we delve into the current state of live imaging to explore the barriers that must be overcome and the possibilities that lie ahead. We venture to envision a future where we can visualize and study everything, everywhere, all at once - from the intricate inner workings of a single cell to the dynamic interplay across entire organisms, and a world where scientists could access the necessary microscopy technologies anywhere.

}, keywords = {Microscopy}, issn = {2399-3642}, doi = {10.1038/s42003-023-05468-9}, author = {Balasubramanian, Harikrushnan and Hobson, Chad M and Chew, Teng-Leong and Aaron, Jesse S} } @article {68562, title = {In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes.}, journal = {Nature Structural \& Molecular Biology}, volume = {30}, year = {2023}, month = {2023 Mar 01}, pages = {360-9}, abstract = {

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

}, issn = {1545-9985}, doi = {10.1038/s41594-022-00861-0}, author = {Chen, Zhen and Greenan, Garrett A and Shiozaki, Momoko and Liu, Yanxin and Skinner, Will M and Zhao, Xiaowei and Zhao, Shumei and Yan, Rui and Yu, Zhiheng and Lishko, Polina V and Agard, David A and Vale, Ronald D} } @article {68837, title = {Input density tunes Kenyon cell sensory responses in the Drosophila mushroom body.}, journal = {Current Biology}, year = {2023}, month = {2023 Jun 20}, abstract = {

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2023.05.064}, author = {Ahmed, Maria and Rajagopalan, Adithya E and Pan, Yijie and Li, Ye and Williams, Donnell L and Pedersen, Erik A and Thakral, Manav and Previero, Angelica and Close, Kari C and Christoforou, Christina P and Cai, Dawen and Turner, Glenn C and Clowney, E Josephine} } @article {68886, title = {Input density tunes Kenyon cell sensory responses in the Drosophila mushroom body.}, journal = {Current Biology}, volume = {33}, year = {2023}, month = {2023 Jul 10}, pages = {2742-2760.e12}, abstract = {

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

}, keywords = {Animals, Drosophila, Drosophila melanogaster, Drosophila Proteins, Mushroom Bodies, Neurons, Odorants}, issn = {1879-0445}, doi = {10.1016/j.cub.2023.05.064}, author = {Ahmed, Maria and Rajagopalan, Adithya E and Pan, Yijie and Li, Ye and Williams, Donnell L and Pedersen, Erik A and Thakral, Manav and Previero, Angelica and Close, Kari C and Christoforou, Christina P and Cai, Dawen and Turner, Glenn C and Clowney, E Josephine} } @article {68934, title = {Investigating the composition and recruitment of the mycobacterial ImuA{\textquoteright}-ImuB-DnaE2 mutasome.}, journal = {eLife}, volume = {12}, year = {2023}, month = {2023 Aug 02}, abstract = {

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded {\textquoteright}mycobacterial mutasome{\textquoteright} - minimally comprising DnaE2 polymerase and ImuA{\textquoteright} and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

}, keywords = {Antitubercular Agents, Bacterial Proteins, DNA Repair, Mutagenesis, Mycobacterium tuberculosis}, issn = {2050-084X}, doi = {10.7554/eLife.75628}, author = {Gessner, Sophia and Martin, Zela Alexandria-Mae and Reiche, Michael A and Santos, Joana A and Dinkele, Ryan and Ramudzuli, Atondaho and Dhar, Neeraj and de Wet, Timothy J and Anoosheh, Saber and Lang, Dirk M and Aaron, Jesse and Chew, Teng-Leong and Herrmann, Jennifer and M{\"u}ller, Rolf and McKinney, John D and Woodgate, Roger and Mizrahi, Valerie and Venclovas, {\v C}eslovas and Lamers, Meindert H and Warner, Digby F} } @article {67931, title = {Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses.}, journal = {Viruses}, volume = {14}, year = {2022}, month = {2022 Jan 18}, abstract = {

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.

}, issn = {1999-4915}, doi = {10.3390/v14020177}, author = {Munshi, Sneha and Neupane, Krishna and Ileperuma, Sandaru M and Halma, Matthew T J and Kelly, Jamie A and Halpern, Clarissa F and Dinman, Jonathan D and Loerch, Sarah and Woodside, Michael T} } @article {67891, title = {Idiosyncratic learning performance in flies.}, journal = {Biology Letters}, volume = {18}, year = {2022}, month = {2022 Feb 01}, pages = {20210424}, abstract = {

Individuals vary in their innate behaviours, even when they have the same genome and have been reared in the same environment. The extent of individuality in plastic behaviours, like learning, is less well characterized. Also unknown is the extent to which intragenotypic differences in learning generalize: if an individual performs well in one assay, will it perform well in other assays? We investigated this using the fruit fly , an organism long-used to study the mechanistic basis of learning and memory. We found that isogenic flies, reared in identical laboratory conditions, and subject to classical conditioning that associated odorants with electric shock, exhibit clear individuality in their learning responses. Flies that performed well when an odour was paired with shock tended to perform well when the odour was paired with bitter taste or when other odours were paired with shock. Thus, individuality in learning performance appears to be prominent in isogenic animals reared identically, and individual differences in learning performance generalize across some aversive sensory modalities. Establishing these results in flies opens up the possibility of studying the genetic and neural circuit basis of individual differences in learning in a highly suitable model organism.

}, issn = {1744-957X}, doi = {10.1098/rsbl.2021.0424}, author = {Smith, Matthew A-Y and Honegger, Kyle S and Turner, Glenn and de Bivort, Benjamin} } @article {67858, title = {The importance of accounting for movement when relating neuronal activity to sensory and cognitive processes.}, journal = {Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {42}, year = {2022}, month = {2022 Feb 23}, pages = {1375-1382}, abstract = {

A surprising finding of recent studies in mouse is the dominance of widespread\ movement-related\ activity\ throughout the brain, including in early\ sensory\ areas. In awake subjects, failing to\ account\ for\ movement\ risks misattributing\ movement-related\ activity\ to other (e.g.,\ sensory\ or\ cognitive)\ processes. In this article, we 1) review task designs for separating task-related\ and\ movement-related\ activity, 2) review three {\textquoteright}case studies{\textquoteright} in which\ not\ considering\ movement\ would have resulted in critically different interpretations of\ neuronal\ function, and 3) discuss functional couplings that may prevent us from ever fully isolating\ sensory,\ motor, and\ cognitive-related\ activity. Our main thesis is that neural signals\ related\ to\ movement\ are ubiquitous, and therefore ought to be considered first and foremost when attempting to correlate\ neuronal\ activity\ with task-related\ processes.

}, doi = {10.1523/JNEUROSCI.1919-21.2021}, author = {Edward Zagha and Jeffrey C Erlich and Soohyun Lee and Gyorgy Lur and Daniel H O{\textquoteright}Connor and Nicholas A Steinmetz and Carsen Stringer and Hongdian Yang} } @article {68418, title = {In situ cell-type-specific cell-surface proteomic profiling in mice.}, journal = {Neuron}, year = {2022}, month = {10/2022}, abstract = {

Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in\ vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.09.025}, author = {Shuster, S Andrew and Li, Jiefu and Chon, URee and Sinantha-Hu, Miley C and Luginbuhl, David J and Udeshi, Namrata D and Carey, Dominique Kiki and Takeo, Yukari H and Xie, Qijing and Xu, Chuanyun and Mani, D R and Han, Shuo and Ting, Alice Y and Carr, Steven A and Luo, Liqun} } @article {68082, title = {In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes}, journal = {bioRxiv}, year = {2022}, month = {2022 May 16}, abstract = {

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform\ in situ\ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

}, doi = {10.1101/2022.05.15.492011}, url = {https://doi.org/10.1101/2022.05.15.492011}, author = {Zhen Chen and Garrett A. Greenan and Momoko Shiozaki and Yanxin Liu and Will M. Skinner and Xiaowei Zhao and Shumei Zhao and Rui Yan and Caiying Guo and Zhiheng Yu and Polina V. Lishko and David A. Agard and Ronald D. Vale} } @article {68010, title = {In situ single particle classification reveals distinct 60S maturation intermediates in cells.}, journal = {eLife}, year = {2022}, month = {2022 Aug 25}, abstract = {

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure.\ In situ\ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of\ in situ\ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the\ Saccharomyces cerevisiae\ cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

}, doi = {10.7554/eLife.79272}, url = {https://doi.org/10.7554/eLife.79272}, author = {Bronwyn A. Lucas and Kexin Zhang and Sarah Loerch and Nikolaus Grigorieff} } @article {68314, title = {In vivo visualization of nitrate dynamics using a genetically encoded biosensor}, journal = {Science Advances}, volume = {8}, year = {2022}, month = {2022 Oct 19}, abstract = {

Nitrate (NO3-) uptake and distribution are critical to plant life. Although the upstream regulation of nitrate uptake and downstream responses to nitrate in a variety of cells have been well-studied, it is still not possible to directly visualize the spatial and temporal distribution of nitrate with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded biosensor, nlsNitraMeter3.0, for the quantitative visualization of nitrate distribution in\ Arabidopsis thaliana. The biosensor tracked the spatiotemporal distribution of nitrate along the primary root axis and disruptions by genetic mutation of transport (low nitrate uptake) and assimilation (high nitrate accumulation). The developed biosensor effectively monitors nitrate concentrations at cellular level in real time and spatiotemporal changes during the plant life cycle.

}, doi = {10.1126/sciadv.abq4915}, url = {https://doi.org/10.1126/sciadv.abq4915}, author = {Yen-Ning Chen and Heather Cartwright and Cheng-Hsun Ho} } @article {68556, title = {Inhibition of coronavirus HCoV-OC43 by targeting the eIF4F complex.}, journal = {Frontiers in Pharmacology}, volume = {13}, year = {2022}, month = {2022 Dec 01}, pages = {1029093}, abstract = {

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.

}, issn = {1663-9812}, doi = {10.3389/fphar.2022.1029093}, author = {Feng, Yongmei and Grotegut, Stefan and Jovanovic, Predrag and Gandin, Valentina and Olson, Steven H and Murad, Rabi and Beall, Anne and Colayco, Sharon and De-Jesus, Paul and Chanda, Sumit and English, Brian P and Singer, Robert H and Jackson, Michael and Topisirovic, Ivan and Ronai, Ze{\textquoteright}ev A} } @article {67814, title = {Internal Models in Control, Bioengineering, and Neuroscience}, journal = {Annual Review of Control, Robotics, and Autonomous Systems}, volume = {5}, year = {2022}, month = {2021 Nov 29}, abstract = {

Internal models are nowadays customarily used in different domains of science and engineering to describe how living organisms or artificial computational units embed their acquired knowledge about recurring events taking place in the surrounding environment. This article reviews the internal model principle in control theory, bioengineering, and neuroscience, illustrating the fundamental concepts and theoretical developments of the few last decades of research.

}, issn = {2573-5144}, doi = {10.1146/control.2022.5.issue-110.1146/annurev-control-042920-102205}, url = {https://www.annualreviews.org/toc/control/5/1https://www.annualreviews.org/doi/10.1146/annurev-control-042920-102205https://www.annualreviews.org/doi/pdf/10.1146/annurev-control-042920-102205}, author = {Bin, Michelangelo and Huang, Jie and Isidori, Alberto and Marconi, Lorenzo and Mischiati, Matteo and Sontag, Eduardo} } @article {68293, title = {Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.}, journal = {PLoS One}, volume = {17}, year = {2022}, month = {2022 Aug 12}, pages = {e0271799}, abstract = {

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

}, keywords = {Animals, Cryoelectron Microscopy, Electron Microscope Tomography, Lasers, Mice, Microscopy, Confocal, Synapses, Synaptosomes}, issn = {1932-6203}, doi = {10.1371/journal.pone.0271799}, author = {Gogoi, Prerana and Shiozaki, Momoko and Gouaux, Eric} } @article {66020, title = {Idiosyncratic learning performance in flies generalizes across modalities.}, journal = {bioRxiv}, year = {2021}, month = {2021 Jan 25}, abstract = {

Individuals vary in their innate behaviors, even when they have the same genome and have been reared in the same environment. The extent of individuality in plastic behaviors, like learning, is less well characterized. Also unknown is the extent to which intragenotypic differences in learning generalize: if an individual performs well in one assay, will it perform well in other assays? We investigated this using the fruit fly\ Drosophila melanogaster, an organism long-used to study the mechanistic basis of learning and memory. We found that isogenic flies, reared in identical lab conditions, and subject to classical conditioning that associated odorants with electric shock, exhibit clear individuality in their learning responses. Flies that performed well when an odor was paired with shock tended to perform well when other odors were paired with shock, or when the original odor was paired with bitter taste. Thus, individuality in learning performance appears to be prominent in isogenic animals reared identically, and individual differences in learning performance generalize across stimulus modalities. Establishing these results in flies opens up the possibility of studying the genetic and neural circuit basis of individual differences in learning in a highly suitable model organism.

}, author = {Matthew Smith and Kyle S. Honegger and Glenn Turner and Benjamin de Bivort} } @article {66002, title = {Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.}, journal = {Journal of Cell Biology}, volume = {220}, year = {2021}, month = {2021 Feb 01}, abstract = {

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

}, issn = {1540-8140}, doi = {10.1083/jcb.202006180}, author = {Kanfer, Gil and Sarraf, Shireen A and Maman, Yaakov and Baldwin, Heather and Dominguez-Martin, Eunice and Johnson, Kory R and Ward, Michael E and Kampmann, Martin and Lippincott-Schwartz, Jennifer and Youle, Richard J} } @article {67871, title = {Image-based representation of massive spatial transcriptomics datasets.}, journal = {bioRxiv}, year = {2021}, month = {2021 Dec 07}, abstract = {

We present STIM, an imaging-based computational framework for exploring, visualizing, and processing high-throughput spatial sequencing datasets. STIM is built on the powerful ImgLib2, N5 and BigDataViewer (BDV) frameworks enabling transfer of computer vision techniques to datasets with irregular measurement-spacing and arbitrary spatial resolution, such as spatial transcriptomics data generated by multiplexed targeted hybridization or spatial sequencing technologies. We illustrate STIM{\textquoteright}s capabilities by representing, visualizing, and automatically registering publicly available spatial sequencing data from 14 serial sections of mouse brain tissue.

}, doi = {10.1101/2021.12.07.471629}, url = {https://doi.org/10.1101/2021.12.07.471629}, author = {Stephan Preibisch and Nikos Karaiskos and Nikolaus Rajewsky} } @article {67670, title = {Imaging Africa: a strategic approach to optical microscopy training in Africa.}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {2021 Aug 20}, pages = {847-855}, issn = {1548-7105}, doi = {10.1038/s41592-021-01227-y}, author = {Reiche, Michael A and Warner, Digby F and Aaron, Jesse and Khuon, Satya and Fletcher, Daniel A and Hahn, Klaus and Rogers, Kelly L and Mhlanga, Musa and Koch, Anastasia and Quaye, Wendye and Chew, Teng-Leong} } @article {67627, title = {In~vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Jun 22}, pages = {109284}, abstract = {

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1\ μM to 10\ mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109284}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {65815, title = {In vivo optogenetics with stimulus calibration.}, journal = {Methods in Molecular Biology}, volume = {2188}, year = {2021}, month = {2020 Nov 06}, pages = {273-283}, abstract = {

Optogenetic reagents allow for depolarization and hyperpolarization of cells with light. This provides unprecedented spatial and temporal resolution to the control of neuronal activity both in vitro and in vivo. In the intact animal this requires strategies to deliver light deep into the highly scattering tissue of the brain. A general approach that we describe here is to implant optical fibers just above brain regions targeted for light delivery. In part due to the fact that expression of optogenetic proteins is accomplished by techniques with inherent variability (e.g., viral expression\ levels), it also requires strategies to measure and calibrate the effect of stimulation. Here we describe general procedures that allow one to simultaneously stimulate neurons and use photometry with genetically encoded activity indicators to precisely calibrate stimulation.

}, issn = {1940-6029}, doi = {10.1007/978-1-0716-0818-0_14}, author = {Coddington, Luke T and Dudman, Joshua T} } @article {67658, title = {An inexpensive, high-precision, modular spherical treadmill setup optimized for experiments.}, journal = {Frontiers in Behavioral Neuroscience}, volume = {15}, year = {2021}, month = {2021 Jul 16}, pages = {689573}, abstract = {

To pursue a more mechanistic understanding of the neural control of behavior, many neuroethologists study animal behavior in controlled laboratory environments. One popular approach is to measure the movements of restrained animals while presenting controlled sensory stimulation. This approach is especially powerful when applied to genetic model organisms, such as , where modern genetic tools enable unprecedented access to the nervous system for activity monitoring or targeted manipulation. While there is a long history of measuring the behavior of body- and head-fixed insects walking on an air-supported ball, the methods typically require complex setups with many custom components. Here we present a compact, simplified setup for these experiments that achieves high-performance at low cost. The simplified setup integrates existing hardware and software solutions with new component designs. We replaced expensive optomechanical and custom machined components with off-the-shelf and 3D-printed parts, and built the system around a low-cost camera that achieves 180 Hz imaging and an inexpensive tablet computer to present view-angle-corrected stimuli updated through a local network. We quantify the performance of the integrated system and characterize the visually guided behavior of flies in response to a range of visual stimuli. In this paper, we thoroughly document the improved system; the accompanying repository incorporates CAD files, parts lists, source code, and detailed instructions. We detail a complete ~$300 system, including a cold-anesthesia tethering stage, that is ideal for hands-on teaching laboratories. This represents a nearly 50-fold cost reduction as compared to a typical system used in research laboratories, yet is fully featured and yields excellent performance. We report the current state of this system, which started with a 1-day teaching lab for which we built seven parallel setups and continues toward a setup in our lab for larger-scale analysis of visual-motor behavior in flies. Because of the simplicity, compactness, and low cost of this system, we believe that high-performance measurements of tethered insect behavior should now be widely accessible and suitable for integration into many systems. This access enables broad opportunities for comparative work across labs, species, and behavioral paradigms.

}, issn = {1662-5153}, doi = {10.3389/fnbeh.2021.689573}, author = {Loesche, Frank and Reiser, Michael B} } @article {67303, title = {Information flow, cell types and stereotypy in a full olfactory connectome.}, journal = {eLife}, volume = {10}, year = {2021}, month = {2021 May 25}, abstract = {

The connectome provides large scale connectivity and morphology information for the majority of the central brain of . Using this data set, we provide a complete description of the olfactory system, covering all first, second and lateral horn-associated third-order neurons. We develop a generally applicable strategy to extract information flow and layered organisation from connectome graphs, mapping olfactory input to descending interneurons. This identifies a range of motifs including highly lateralised circuits in the antennal lobe and patterns of convergence downstream of the mushroom body and lateral horn. Leveraging a second data set we provide a first quantitative assessment of inter- versus intra-individual stereotypy. Comparing neurons across two brains (three hemispheres) reveals striking similarity in neuronal morphology across brains. Connectivity correlates with morphology and neurons of the same morphological type show similar connection variability within the same brain as across two brains.

}, issn = {2050-084X}, doi = {10.7554/eLife.66018}, author = {Schlegel, Philipp and Bates, Alexander Shakeel and St{\"u}rner, Tomke and Jagannathan, Sridhar R and Drummond, Nikolas and Hsu, Joseph and Serratosa Capdevila, Laia and Javier, Alexandre and Marin, Elizabeth C and Barth-Maron, Asa and Tamimi, Imaan Fm and Li, Feng and Rubin, Gerald M and Plaza, Stephen M and Costa, Marta and Jefferis, Gregory Sxe} } @article {67656, title = {Intercellular Arc Signaling Regulates Vasodilation.}, journal = {Journal of Neuroscience}, year = {2021}, month = {2021 Jul 29}, abstract = {

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation and Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0440-21.2021}, author = {de la Pe{\~n}a, June Bryan and Barragan-Iglesias, Paulino and Lou, Tzu-Fang and Kunder, Nikesh and Loerch, Sarah and Shukla, Tarjani and Basavarajappa, Lokesh and Song, Jane and James, Dominique N and Megat, Salim and Moy, Jamie K and Wanghzou, Andi and Ray, Pradipta R and Hoyt, Kenneth and Steward, Oswald and Price, Theodore J and Shepherd, Jason and Campbell, Zachary T} } @article {66025, title = {Internal state configures olfactory behavior and early sensory processing in larvae.}, journal = {Science Advances}, volume = {7}, year = {2021}, month = {2021 Jan 01}, abstract = {

Animals exhibit different behavioral responses to the same sensory cue depending on their internal state at a given moment. How and where in the brain are sensory inputs combined with state information to select an appropriate behavior? Here, we investigate how food deprivation affects olfactory behavior in larvae. We find that certain odors repel well-fed animals but attract food-deprived animals and that feeding state flexibly alters neural processing in the first olfactory center, the antennal lobe. Hunger differentially modulates two output pathways required for opposing behavioral responses. Upon food deprivation, attraction-mediating uniglomerular projection neurons show elevated odor-evoked activity, whereas an aversion-mediating multiglomerular projection neuron receives odor-evoked inhibition. The switch between these two pathways is regulated by the lone serotonergic neuron in the antennal lobe, CSD. Our findings demonstrate how flexible behaviors can arise from state-dependent circuit dynamics in an early sensory processing center.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abd6900}, author = {Vogt, Katrin and Zimmerman, David M and Schlichting, Matthias and Hernandez-Nunez, Luis and Qin, Shanshan and Malacon, Karen and Rosbash, Michael and Pehlevan, Cengiz and Cardona, Albert and Samuel, Aravinthan D T} } @article {67210, title = {Intracellular mRNA transport and localized translation.}, journal = {Nature Reviews Molecular Cell Biology}, volume = {22}, year = {2021}, month = {2021 Jul 1}, pages = {483-504}, abstract = {

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

}, issn = {1471-0080}, doi = {10.1038/s41580-021-00356-8}, author = {Das, Sulagna and Vera, Maria and Gandin, Valentina and Singer, Robert H and Tutucci, Evelina} } @article {65637, title = {Identification of dopaminergic neurons that can both establish associative memory and acutely terminate its behavioral expression.}, journal = {Journal of Neuroscience}, volume = {40}, year = {2020}, month = {2020 Jul 29}, pages = {5990-6006}, abstract = {

An adaptive transition from exploring the environment in search of vital resources to exploiting these resources once the search is successful is important to all animals. Here we study the neuronal circuitry that allows larval of either sex to negotiate this exploration-exploitation transition. We do so by combining Pavlovian conditioning with high-resolution behavioral tracking, optogenetic manipulation of individually identified neurons, and EM-data-based analyses of synaptic organization. We find that optogenetic activation of the dopaminergic neuron DAN-i1 can both establish memory during training, and acutely terminate learned search behavior in a subsequent recall test. Its activation leaves innate behavior unaffected, however. Specifically, DAN-i1 activation can establish associative memories of opposite valence upon paired and unpaired training with odor, and its activation during the recall test can terminate the search behavior resulting from either of these memories. Our results further suggest that in its behavioral significance DAN-i1 activation resembles but does not equal sugar reward. Dendrogram analyses of all the synaptic connections between DAN-i1 and its two main targets, the Kenyon cells and the mushroom body output neuron MBON-i1, further suggest that the DAN-i1 signals during training and during the recall test could be delivered to the Kenyon cells and to MBON-i1, respectively, within previously unrecognized, locally confined branching structures. This would provide an elegant circuit motif to terminate search upon its successful completion.In the struggle for survival animals have to explore their environment in search of food. Once food is found, however, it is adaptive to prioritize exploiting it over continuing a search that would now be as pointless as searching for the glasses you are wearing. This exploration-exploitation trade-off is important for animals and humans, as well as for technical search devices. We investigate which of the only 10,000 neurons of a fruit fly larva can tip the balance in this trade-off, and identify a single dopamine neuron called DAN-i1 that can do so. Given the similarities in dopamine neuron function across the animal kingdom, this may reflect a general principle of how search is terminated once it is successful.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0290-20.2020}, author = {Schleyer, Michael and Weiglein, Ali{\'c}e and Thoener, Juliane and Strauch, Martin and Hartenstein, Volker and Kantar Weigelt, Melisa and Schuller, Sarah and Saumweber, Timo and Eichler, Katharina and Rohwedder, Astrid and Merhof, Dorit and Zlatic, Marta and Thum, Andreas S and Gerber, Bertram} } @article {65301, title = {Identifying neural substrates of competitive interactions and sequence transitions during mechanosensory responses in Drosophila.}, journal = {PLoS Genetics}, volume = {16}, year = {2020}, month = {2020 Feb 14}, pages = {e1008589}, abstract = {

Nervous systems have the ability to select appropriate actions and action sequences in response to sensory cues. The circuit mechanisms by which nervous systems achieve choice, stability and transitions between behaviors are still incompletely understood. To identify neurons and brain areas involved in controlling these processes, we combined a large-scale neuronal inactivation screen with automated action detection in response to a mechanosensory cue in Drosophila larva. We analyzed behaviors from 2.9x105 larvae and identified 66 candidate lines for mechanosensory responses out of which 25 for competitive interactions between actions. We further characterize in detail the neurons in these lines and analyzed their connectivity using electron microscopy. We found the neurons in the mechanosensory network are located in different regions of the nervous system consistent with a distributed model of sensorimotor decision-making. These findings provide the basis for understanding how selection and transition between behaviors are controlled by the nervous system.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1008589}, author = {Masson, Jean-Baptiste and Laurent, Francois and Cardona, Albert and Barre, Chloe and Skatchkovsky, Nicolas and Zlatic, Marta and Jovanic, Tihana} } @article {65130, title = {Idiosyncratic neural coding and neuromodulation of olfactory individuality in Drosophila.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, year = {2020}, month = {2020 Sep 22}, pages = {23292-23297}, abstract = {

Innate behavioral biases and preferences can vary significantly among individuals of the same genotype. Though individuality is a fundamental property of behavior, it is not currently understood how individual differences in brain structure and physiology produce idiosyncratic behaviors. Here we present evidence for idiosyncrasy in olfactory behavior and neural responses in We show that individual female from a highly inbred laboratory strain exhibit idiosyncratic odor preferences that persist for days. We used in vivo calcium imaging of neural responses to compare projection neuron (second-order neurons that convey odor information from the sensory periphery to the central brain) responses to the same odors across animals. We found that, while odor responses appear grossly stereotyped, upon closer inspection, many individual differences are apparent across antennal lobe (AL) glomeruli (compact microcircuits corresponding to different odor channels). Moreover, we show that neuromodulation, environmental stress in the form of altered nutrition, and activity of certain AL local interneurons affect the magnitude of interfly behavioral variability. Taken together, this work demonstrates that individual exhibit idiosyncratic olfactory preferences and idiosyncratic neural responses to odors, and that behavioral idiosyncrasies are subject to neuromodulation and regulation by neurons in the AL.

}, issn = {1091-6490}, doi = {10.1073/pnas.1901623116}, author = {Honegger, Kyle S and Smith, Matthew A-Y and Churgin, Matthew A and Turner, Glenn C and de Bivort, Benjamin L} } @inbook {65285, title = {Imaging Cellular Proteins and Structures}, booktitle = {The Liver : Biology and Pathobiology}, year = {2020}, pages = {965 - 978}, publisher = {Wiley}, organization = {Wiley}, edition = {1}, abstract = {

This chapter describes many of the technologies, which have the potential to provide new insights into fundamental aspects of liver biology. Imaging live liver tissue in an animal with multiphoton microscopy coupled with photoactivatable fluorescent proteins and/or additional fluorescent proteins could be used to follow the lineage and fates of individual transplanted stem cells or developing transgenic cells in liver. Proteins or other molecules are labeled with a dye that can be excited with light source. Cells and proteins are generally too small to detect with the naked eye, relatively transparent when imaged by light microscopy, and are highly dynamic. With the increased signal to noise, isotropic and volumetric imaging and high speeds lattice light sheet allows for 3D super-resolution microscopy, as well. Photomultiplier tubes, while capable of detecting and counting single photons, are less useful for high-speed imaging because they normally only detect a single pixel at a time.

}, isbn = {9781119436829}, doi = {10.1002/978111943681210.1002/9781119436812.ch72}, url = {https://onlinelibrary.wiley.com/doi/pdf/10.1002/9781119436812.ch72}, author = {Weigel, Aubrey V. and Snapp, Erik Lee}, editor = {Arias, Irwin M. and Alter, Harvey J. and Boyer, James L. and Cohen, David E. and Shafritz, David A. and Thorgeirsson, Snorri S. and Wolkoff, Allan W.} } @article {65854, title = {Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging.}, journal = {PLoS Biology}, volume = {18}, year = {2020}, month = {2020 Nov 24}, pages = {e3000965}, abstract = {

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3000965}, author = {Qian, Yong and Cosio, Danielle M Orozco and Piatkevich, Kiryl D and Aufmkolk, Sarah and Su, Wan-Chi and Celiker, Orhan T and Schohl, Anne and Murdock, Mitchell H and Abhi Aggarwal and Chang, Yu-Fen and Wiseman, Paul W and Ruthazer, Edward S and Boyden, Edward S and Campbell, Robert E} } @article {65275, title = {Improved HaloTag Ligand Enables BRET Imaging With NanoLuc}, journal = {Frontiers in Chemistry}, volume = {7}, year = {2020}, month = {2020 Jan 14}, doi = {10.3389/fchem.2019.0093810.3389/fchem.2019.00938.s001}, url = {https://www.frontiersin.org/article/10.3389/fchem.2019.00938/fullhttps://www.frontiersin.org/article/10.3389/fchem.2019.00938/fullhttps://www.frontiersin.org/articles/10.3389/fchem.2019.00938/supplementary-material/10.3389/fchem.2019.00938.s001}, author = {Thirukkumaran, Ovia Margaret and Wang, Congrong and Asouzu, Nnamdi Joseph and Fron, Eduard and Rocha, Susana and Hofkens, Johan and Lavis, Luke D. and Mizuno, Hideaki} } @article {65896, title = {In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning.}, journal = {Nature Communications}, volume = {11}, year = {2020}, month = {2020 Dec 15}, pages = {6391}, abstract = {

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish{\textquoteright}s color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

}, keywords = {Animals, Cell Differentiation, Cell Proliferation, Chromatophores, Microphthalmia-Associated Transcription Factor, Mutagenesis, Skin, Skin Pigmentation, Transcriptome, X-Ray Diffraction, Zebrafish, Zebrafish Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-020-20088-1}, author = {Gur, Dvir and Bain, Emily J and Johnson, Kory R and Aman, Andy J and Pasoili, H Amalia and Flynn, Jessica D and Allen, Michael C and Deheyn, Dimitri D and Lee, Jennifer C and Lippincott-Schwartz, Jennifer and Parichy, David M} } @article {65235, title = {In situ structure determination at nanometer resolution using TYGRESS.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Feb 01}, pages = {201-08}, abstract = {

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called {\textquoteright}tomography-guided 3D reconstruction of subcellular structures{\textquoteright} (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 {\r A}. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0651-0}, author = {Song, Kangkang and Shang, Zhiguo and Fu, Xiaofeng and Lou, Xiaochu and Grigorieff, Nikolaus and Nicastro, Daniela} } @conference {65835, title = {Inpainting Networks Learn to Separate Cells in Microscopy Images}, booktitle = {British Machine Vision Conference}, year = {2020}, month = {2020 Sep}, abstract = {

Deep neural networks trained to inpaint partially occluded images show a deep understanding of image composition and have even been shown to remove objects from images convincingly. In this work, we investigate how this implicit knowledge of image composition can be be used to separate cells in densely populated microscopy images. We propose a measure for the independence of two image regions given a fully self-supervised inpainting network and separate objects by maximizing this independence. We evaluate our method on two cell segmentation datasets and show that cells can be separated completely unsupervised. Furthermore, combined with simple foreground detection, our method yields instance segmentation of similar quality to fully supervised methods.

}, url = {https://www.bmvc2020-conference.com/assets/papers/0528.pdf}, author = {Wolf, Steffen and Hamprecht, Fred A and Funke, Jan} } @article {65314, title = {Input connectivity reveals additional heterogeneity of dopaminergic reinforcement in Drosophila}, journal = {Current Biology}, volume = {30}, year = {2020}, month = {2020 Aug 17}, pages = {3200-11}, abstract = {

Different types of\ Drosophila\ dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [1]. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions [2,\ 3]. Here we used a recent electron microscope volume of the fly brain [4] to reconstruct the fine anatomy of individual DANs within three MB compartments. We find the 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype (γ5fb) receives direct recurrent input from γ5β{\textquoteright}2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from the MB. We similarly reconstructed the single aversively reinforcing PPL1-γ1pedc DAN. The γ1pedc DAN inputs are mostly different to those of γ5 DANs and are clustered onto distinct branches of its dendritic tree, presumably separating its established roles in aversive reinforcement and appetitive motivation [5,\ 6]. Additional tracing identified neurons that provide broad input to γ5, β{\textquoteright}2a and γ1pedc DANs suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal additional complexity of dopaminergic reinforcement circuits between and within MB compartments.

}, author = {Otto, Nils and Pleijzier, Markus W. and Morgan, Isabel C. and Edmondson-Stait, Amelia J and Heinz, Konrad J. and Stark, Ildiko and Dempsey, Georgia and Ito, Masayoshi and Kapoor, Ishaan and Hsu, Joseph and Schlegel, Philipp M. and Bates, Alexander S. and Costa, Marta and Ito, Kei and Bock, Davi D. and Rubin, Gerald M. and Jefferis, Gregory S.X.E. and Waddell, Scott} } @article {65762, title = {Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins}, journal = {bioRxiv}, year = {2020}, month = {09/2020}, pages = {2020.09.25.313528}, abstract = {

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

}, doi = {10.1101/2020.09.25.313528}, author = {Zitter, Elke De and Hugelier, Siewert and Duw{\'e}, Sam and Vandenberg, Wim and Tebo, Alison G. and Meervelt, Luc Van and Dedecker, Peter} } @article {68478, title = {Intracellular signaling dynamics and their role in coordinating tissue repair.}, journal = {Wiley Interdiscip Rev Syst Biol Med}, volume = {12}, year = {2020}, month = {05/2020}, pages = {e1479}, abstract = {

Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal-regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs. This article is categorized under: Models of Systems Properties and Processes \> Mechanistic Models Biological Mechanisms \> Cell Signaling Laboratory Methods and Technologies \> Imaging Models of Systems Properties and Processes \> Organ, Tissue, and Physiological Models.

}, keywords = {Animals, Calcium, Cell Movement, Epithelial Cells, Extracellular Signal-Regulated MAP Kinases, Humans, Models, Biological, NF-kappa B, Signal Transduction, Wound Healing}, issn = {1939-005X}, doi = {10.1002/wsbm.1479}, author = {Ghilardi, Samuel J and O{\textquoteright}Reilly, Breanna M and Sgro, Allyson E} } @article {65177, title = {Identification of cell types from single-cell transcriptomic data.}, journal = {Methods in Molecular Biology}, volume = {1935}, year = {2019}, month = {2019 Oct 16}, pages = {45-77}, abstract = {

Unprecedented technological advances in single-cell RNA-sequencing (scRNA-seq) technology have now made it possible to profile genome-wide expression in single cells at low cost and high throughput. There is substantial ongoing effort to use scRNA-seq measurements to identify the "cell types" that form components of a complex tissue, akin to taxonomizing species in ecology. Cell type classification from scRNA-seq data involves the application of computational tools rooted in dimensionality reduction and clustering, and statistical analysis to identify molecular signatures that are unique to each type. As datasets continue to grow in size and complexity, computational challenges abound, requiring analytical methods to be scalable, flexible, and robust. Moreover, careful consideration needs to be paid to experimental biases and statistical challenges that are unique to these measurements to avoid artifacts. This chapter introduces these topics in the context of cell-type identification, and outlines an instructive step-by-step example bioinformatic pipeline for researchers entering this field.

}, keywords = {Animals, Cluster Analysis, Computational Biology, Gene Expression Profiling, Genome, High-Throughput Nucleotide Sequencing, Humans, Mice, RNA, Sequence Analysis, RNA, Single-Cell Analysis, Transcriptome}, issn = {1940-6029}, doi = {10.1007/978-1-4939-9057-3_4}, author = {Shekhar, Karthik and Menon, Vilas} } @article {49449, title = {Idiosyncratic choice bias in decision tasks naturally emerges from neuronal network dynamics.}, journal = {Nature Human Behavior}, volume = {3}, year = {2019}, month = {2019 Sep 02}, pages = {1190-1202}, abstract = {

Idiosyncratic tendency to choose one alternative over others in the absence of an identified reason, is a common observation in two-alternative forced-choice experiments. It is tempting to account for it as resulting from the (unknown) participant-specific history and thus treat it as a measurement noise. Indeed, idiosyncratic choice biases are typically considered as nuisance. Care is taken to account for them by adding an ad-hoc bias parameter or by counterbalancing the choices to average them out. Here we quantify idiosyncratic choice biases in a perceptual discrimination task and a motor task. We report substantial and significant biases in both cases. Then, we present theoretical evidence that even in idealized experiments, in which the settings are symmetric, idiosyncratic choice bias is expected to emerge from the dynamics of competing neuronal networks. We thus argue that idiosyncratic choice bias reflects the microscopic dynamics of choice and therefore is virtually inevitable in any comparison or decision task.

}, doi = {10.1101/284877}, author = {Lebovich, Lior and Darshan, Ran and Lavi, Yoni and Hansel, David and Loewenstein, Yonatan} } @article {65178, title = {ilastik: interactive machine learning for (bio)image analysis.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Sep 30}, pages = {1226-32}, abstract = {

We present ilastik, an easy-to-use interactive tool that brings machine-learning-based (bio)image analysis to end users without substantial computational expertise. It contains pre-defined workflows for image segmentation, object classification, counting and tracking. Users adapt the workflows to the problem at hand by interactively providing sparse training annotations for a nonlinear classifier. ilastik can process data in up to five dimensions (3D, time and number of channels). Its computational back end runs operations on-demand wherever possible, allowing for interactive prediction on data larger than RAM. Once the classifiers are trained, ilastik workflows can be applied to new data from the command line without further user interaction. We describe all ilastik workflows in detail, including three case studies and a discussion on the expected performance.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0582-9}, author = {Berg, Stuart and Kutra, Dominik and Kroeger, Thorben and Straehle, Christoph N and Kausler, Bernhard X and Haubold, Carsten and Schiegg, Martin and Ales, Janez and Beier, Thorsten and Rudy, Markus and Eren, Kemal and Cervantes, Jaime I and Xu, Buote and Beuttenmueller, Fynn and Wolny, Adrian and Zhang, Chong and Koethe, Ullrich and Hamprecht, Fred A and Kreshuk, Anna} } @article {65092, title = {Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor.}, journal = {Science Advances}, volume = {5}, year = {2019}, month = {2019 Jul 10}, pages = {eaaw3108}, abstract = {

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000{\textendash}1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 {\textmu}m. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

}, doi = {10.1126/sciadv.aaw3108}, url = {http://advances.sciencemag.org/lookup/doi/10.1126/sciadv.aaw3108https://syndication.highwire.org/content/doi/10.1126/sciadv.aaw3108}, author = {Beyene, Abraham G. and Delevich, Kristen and Del Bonis-O{\textquoteright}Donnell, Jackson Travis and Piekarski, David J. and Lin, Wan Chen and Thomas, A. Wren and Yang, Sarah J. and Kosillo, Polina and Yang, Darwin and Prounis, George S. and Wilbrecht, Linda and Landry, Markita P.} } @article {67223, title = {Importance Weighted Adversarial Variational Autoencoders for Spike Inference from Calcium Imaging Data}, journal = {CoRR}, volume = {abs/1906.03214}, year = {2019}, month = {10/2019}, abstract = {

The Importance Weighted Auto Encoder (IWAE) objective has been shown to improve the training of generative models over the standard Variational Auto Encoder (VAE) objective. Here, we derive importance weighted extensions to Adversarial Variational Bayes (AVB) and Adversarial Autoencoder (AAE). These latent variable models use implicitly defined inference networks whose approximate posterior density qφ(z|x) cannot be directly evaluated, an essential ingredient for importance weighting. We show improved training and inference in latent variable models with our adversarially trained importance weighting method, and derive new theoretical connections between adversarial generative model training criteria and marginal likelihood based methods. We apply these methods to the important problem of inferring spiking neural activity from calcium imaging data, a challenging posterior inference problem in neuroscience, and show that posterior samples from the adversarial methods outperform factorized posteriors used in VAEs.

}, url = {http://arxiv.org/abs/1906.03214}, author = {Daniel Jiwoong Im and Sridhama Prakhya and Jinyao Yan and Srinivas C. Turaga and Kristin Branson} } @article {65024, title = {In situ structure of rotavirus VP1 RNA-dependent RNA polymerase.}, journal = {Journal of Molecular Biology}, volume = {431}, year = {2019}, month = {2019 Jun 21}, pages = {3124-38}, abstract = {

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the {\textquotedblleft}double-layer particle{\textquotedblright} or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp\ in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions ({\textquotedblleft}triple-layer particles{\textquotedblright} or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA,\ in vitro\ as well as\ in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

}, doi = {10.1016/j.jmb.2019.06.016}, author = {Jenni, Simon and Salgado, Eric N. and Herrmann, Tobias and Li, Zongli and Grant, Timothy and Grigorieff, Nikolaus and Trapani, Stefano and Estrozi, Leandro F. and Harrison, Stephen C.} } @article {64866, title = {In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {bioRxiv}, year = {2019}, month = {2019 Mar 8}, abstract = {Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {\textmu}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.}, doi = {10.1101/571422}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {64957, title = {Inaccurate secondary structure predictions often indicate protein fold switching.}, journal = {Protein Science}, volume = {28}, year = {2019}, month = {2019 Aug}, pages = {1487-93}, abstract = {

Although most proteins conform to the classical one-structure/one-function paradigm, an increasing number of proteins with dual structures and functions have been discovered. In response to cellular stimuli, such proteins undergo structural changes sufficiently dramatic to remodel even their secondary structures and domain organization. This "fold-switching" capability fosters protein multi-functionality, enabling cells to establish tight control over various biochemical processes. Accurate predictions of fold-switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Recently, we developed a prediction method for fold-switching proteins using structure-based thermodynamic calculations and discrepancies between predicted and experimentally determined protein secondary structure. Here we seek to leverage the negative information found in these secondary structure prediction discrepancies. To do this, we quantified secondary structure prediction accuracies of 192 known fold-switching regions (FSRs) within solved protein structures found in the Protein Data Bank (PDB). We find that the secondary structure prediction accuracies for these FSRs vary widely. Inaccurate secondary structure predictions are strongly associated with fold-switching proteins compared to equally long segments of non-fold-switching proteins selected at random. These inaccurate predictions are enriched in helix-to-strand and strand-to-coil discrepancies. Finally, we find that most proteins with inaccurate secondary structure predictions are underrepresented in the PDB compared with their alternatively folded cognates, suggesting that unequal representation of fold-switching conformers within the PDB could be an important cause of inaccurate secondary structure predictions. These results demonstrate that inconsistent secondary structure predictions can serve as a useful preliminary marker of fold switching. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.3664}, author = {Mishra, Soumya and Looger, Loren L and Porter, Lauren L} } @article {69338, title = {Innexin expression in electrically coupled motor circuits}, journal = {Neuroscience Letters}, volume = {695}, year = {2019}, month = {2019 Mar 16}, pages = {19-24}, abstract = {

The many roles of innexins, the molecules that form gap junctions in invertebrates, have been explored in numerous species. Here, we present a summary of innexin expression and function in two small, central pattern generating circuits found in crustaceans: the stomatogastric ganglion and the cardiac ganglion. The two ganglia express multiple innexin genes, exhibit varying combinations of symmetrical and rectifying gap junctions, as well as gap junctions within and across different cell types. Past studies have revealed correlations in ion channel and innexin expression in coupled neurons, as well as intriguing functional relationships between ion channel conductances and electrical coupling. Together, these studies suggest a putative role for innexins in correlating activity between coupled neurons at the levels of gene expression and physiological activity during development and in the adult animal.

}, keywords = {Cardiac ganglion, Central pattern generators, Crustacean, Neuronal homeostasis, Stomatogastric ganglion}, issn = {0304-3940}, doi = {https://doi.org/10.1016/j.neulet.2017.07.016}, url = {https://www.sciencedirect.com/science/article/pii/S0304394017305785}, author = {Adriane G. Otopalik and Brian Lane and David J. Schulz and Eve Marder} } @article {65212, title = {Interactions between Dpr11 and DIP-γ control selection of amacrine neurons in color vision circuits.}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Nov 06}, abstract = {

R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11{\textquoteright}s binding partner DIP-g, while pR7 PRs synapse onto DIP-g-negative pDm8. Dpr11 and DIP-g expression patterns define {\textquoteright}yellow{\textquoteright} and {\textquoteright}pale{\textquoteright} color vision circuits. We examined Dm8 neurons in these circuits by electron microscopic reconstruction and expansion microscopy. and mutations affect the morphologies of yDm8 distal ({\textquoteright}home column{\textquoteright}) dendrites. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 PRs, and interactions between Dpr11 and DIP-g are required for yDm8 survival. These interactions also allow yDm8 neurons to select yR7 PRs as their appropriate home column partners. yDm8 and pDm8 neurons do not normally compete for survival signals or R7 partners, but can be forced to do so by manipulation of R7 subtype fate.

}, issn = {2050-084X}, doi = {10.7554/eLife.48935}, author = {Menon, Kaushiki P and Kulkarni, Vivek and Takemura, Shin-ya and Anaya, Michael and Zinn, Kai} } @article {64851, title = {Interfacial actin protrusions mechanically enhance killing by cytotoxic T cells.}, journal = {Science Immunology}, volume = {4}, year = {2019}, month = {2019 Mar 22}, abstract = {

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.

}, issn = {2470-9468}, doi = {10.1126/sciimmunol.aav5445}, author = {Tamzalit, Fella and Wang, Mitchell S and Jin, Weiyang and Tello-Lafoz, Maria and Boyko, Vitaly and Heddleston, John M and Black, Charles T and Kam, Lance C and Huse, Morgan} } @conference {56205, title = {Internal models in control, biology and neuroscience.}, booktitle = {2018 IEEE Conference on Decision and Control (CDC)}, year = {2019}, month = {2019 Jan 21}, abstract = {

This tutorial paper deals with the Internal Model Principle (IMP) from different perspectives. The goal is to start from the principle as introduced and commonly used in the control theory and then enlarge the vision to other fields where {\textquotedblleft}internal models{\textquotedblright} play a role. The biology and neuroscience fields are specifically targeted in the paper. The paper ends by presenting an {\textquotedblleft}abstract{\textquotedblright} theory of IMP applicable to a large class of systems.

}, doi = {10.1109/CDC.2018.8619624}, author = {Huang, J and Isidori, A and Marconi, L and Mischiati, Matteo and Sontag, E and Wonham, W M} } @article {65150, title = {Isomeric tuning yields bright and targetable red Ca indicators.}, journal = {Journal of the American Chemical Society}, volume = {141}, year = {2019}, month = {2019 Sep 04}, pages = {13734-13738}, abstract = {

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca fluxes in the primary cilium.

}, issn = {1520-5126}, doi = {10.1021/jacs.9b06092}, author = {Deo, Claire and Sheu, Shu-Hsien and Seo, Jinyoung and Clapham, David E and Lavis, Luke D} } @article {49222, title = {Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonates.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 May 10}, abstract = {

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

}, issn = {2050-084X}, doi = {10.7554/eLife.36620}, author = {Yu, Zhou and Surface, Lauren E and Park, Chong Yon and Horlbeck, Max A and Wyant, Gregory A and Abu-Remaileh, Monther and Peterson, Timothy R and Sabatini, David M and Weissman, Jonathan S and O{\textquoteright}Shea, Erin K} } @article {49043, title = {Image co-localization - co-occurrence versus correlation.}, journal = {Journal of Cell Science}, volume = {131}, year = {2018}, month = {2018 Feb 08}, abstract = {

Fluorescence image co-localization analysis is widely utilized to suggest biomolecular interaction. However, there exists some confusion as to its correct implementation and interpretation. In reality, co-localization analysis consists of at least two distinct sets of methods, termed co-occurrence and correlation. Each approach has inherent and often contrasting strengths and weaknesses. Yet, neither one can be considered to always be preferable for any given application. Rather, each method is most appropriate for answering different types of biological question. This Review discusses the main factors affecting multicolor image co-occurrence and correlation analysis, while giving insight into the types of biological behavior that are better suited to one approach or the other. Further, the limits of pixel-based co-localization analysis are discussed in the context of increasingly popular super-resolution imaging techniques.

}, issn = {1477-9137}, doi = {10.1242/jcs.211847}, author = {Aaron, Jesse S and Taylor, Aaron B and Chew, Teng-Leong} } @article {49545, title = {Imaging cortical dynamics in GCaMP transgenic rats with a head-mounted widefield macroscope.}, journal = {Neuron}, year = {2018}, month = {2018 Oct 22}, abstract = {

Widefield imaging of calcium dynamics is an emerging method for mapping regional neural activity but is currently limited to restrained animals. Here we describe cScope, a head-mounted widefield macroscope\ developed to image large-scale cortical dynamics in rats during natural behavior. cScope provides a 7.8\ {\texttimes} 4\ mm field of view and dual illumination paths for both fluorescence and hemodynamic correction and can be fabricated at low cost using readily attainable components. We also report the development of Thy-1 transgenic rat strains with widespread neuronal expression of the calcium indicator GCaMP6f. We combined these two technologies to image large-scale calcium dynamics in the dorsal neocortex during a visual evidence accumulation\ task. Quantitative analysis of task-related dynamics revealed multiple regions having neural signals that encode behavioral choice and sensory evidence. Our results provide a new transgenic resource for calcium imaging in rats and extend the domain of head-mounted microscopes to larger-scale cortical dynamics.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2018.09.050}, author = {Scott, Benjamin B and Thiberge, Stephan Y and Guo, Caiying and Tervo, D Gowanlock R and Brody, Carlos D and Karpova, Alla Y and Tank, David W} } @article {49293, title = {Imaging dynamic and selective low-complexity domain interactions that control gene transcription.}, journal = {Science (New York, N.Y.)}, volume = {361}, year = {2018}, month = {2018 Jun 21}, pages = {eaar2555}, abstract = {

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs), but how they drive transactivation remains unclear. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for novel drugs targeting gene regulatory interactions implicated in disease.

}, issn = {1095-9203}, doi = {10.1126/science.aar2555}, author = {Chong, Shasha and Dugast-Darzacq, Claire and Liu, Zhe and Dong, Peng and Dailey, Gina M and Cattoglio, Claudia and Heckert, Alec and Banala, Sambashiva and Lavis, Luke and Darzacq, Xavier and Tjian, Robert} } @article {49245, title = {Imaging mRNA in vivo, from birth to death.}, journal = {Annual Review of Biophysics}, volume = {47}, year = {2018}, month = {2018 May 20}, pages = {85-106}, abstract = {

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

}, issn = {1936-1238}, doi = {10.1146/annurev-biophys-070317-033037}, author = {Tutucci, Evelina and Livingston, Nathan M and Singer, Robert H and Wu, Bin} } @article {65769, title = {Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy}, journal = {Biochemistry}, volume = {57}, year = {2018}, month = {11/2018}, pages = {5648 {\textendash} 5653}, abstract = {

Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.

}, doi = {10.1021/acs.biochem.8b00649}, url = {https://pubs-acs-org.accesdistant.sorbonne-universite.fr/doi/pdf/10.1021/acs.biochem.8b00649}, author = {Tebo, Alison G and Pimenta, Frederico M and Zhang, Yu and Gautier, Arnaud} } @article {49494, title = {Improved methods for marking active neuron populations.}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {2018 Oct 25}, pages = {4440}, abstract = {

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-06935-2}, author = {Moeyaert, Benjamien and Holt, Graham and Madangopal, Rajtarun and Perez-Alvarez, Alberto and Fearey, Brenna C and Trojanowski, Nicholas F and Ledderose, Julia and Zolnik, Timothy A and Das, Aniruddha and Patel, Davina and Brown, Timothy A and Sachdev, Robert N S and Eickholt, Britta J and Larkum, Matthew E and Turrigiano, Gina G and Dana, Hod and Gee, Christine E and Oertner, Thomas G and Hope, Bruce T and Schreiter, Eric R} } @article {49466, title = {In toto imaging and reconstruction of post-implantation mouse development at the single-cell level.}, journal = {Cell}, volume = {175}, year = {2018}, month = {2018 Oct 10}, pages = {859-876}, abstract = {

The mouse embryo has long been central to the study of mammalian development; however, elucidating the cell behaviors governing gastrulation and the formation of tissues and organs remains a fundamental challenge. A major obstacle is the lack of live imaging and image analysis technologies capable of systematically following cellular dynamics across the developing embryo. We developed a light-sheet microscope that adapts itself to the dramatic changes in size, shape, and optical properties of the post-implantation\ mouse embryo and captures its development from gastrulation to early organogenesis at\ the cellular level. We furthermore developed a computational framework for reconstructing long-term cell tracks, cell divisions, dynamic fate maps, and maps of tissue morphogenesis across the entire embryo. By jointly analyzing cellular dynamics in multiple embryos registered in space and time, we built a dynamic atlas of post-implantation mouse development that, together with our microscopy and computational methods, is provided as a resource.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2018.09.031}, author = {McDole, Katie and Guignard, Leo and Amat, Fernando and Berger, Andrew and Malandain, Gr{\'e}goire and Royer, Lo{\"\i}c A and Turaga, Srinivas C and Branson, Kristin and Keller, Philipp J} } @article {49367, title = {In vivo measurement of afferent activity with axon-specific calcium imaging.}, journal = {Nature Neuroscience}, year = {2018}, month = {2018 Aug 20}, abstract = {

In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity-both electrically and optically-has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions.

}, issn = {1546-1726}, doi = {10.1038/s41593-018-0211-4}, author = {Broussard, Gerard Joey and Liang, Yajie and Fridman, Marina and Unger, Elizabeth K and Meng, Guanghan and Xiao, Xian and Ji, Na and Petreanu, Leopoldo and Tian, Lin} } @article {49369, title = {Inhibitory control of prefrontal cortex by the claustrum.}, journal = {Neuron}, volume = {99}, year = {2018}, month = {2018 Aug 07}, pages = {1029-39}, abstract = {

The claustrum is a small subcortical nucleus that has extensive excitatory connections with many cortical areas. While the anatomical connectivity from the claustrum to the cortex has been studied intensively, the physiological effect and underlying circuit mechanisms of claustrocortical communication remain elusive. Here we show that the claustrum provides strong, widespread, and long-lasting feedforward inhibition of the prefrontal cortex (PFC) sufficient to silence ongoing neural activity. This claustrocortical feedforward inhibition was predominantly mediated by interneurons containing neuropeptide Y, and to a lesser extent those containing parvalbumin. Therefore, in contrast to other long-range excitatory inputs to the PFC, the claustrocortical pathway is designed to provide overall inhibition of cortical activity. This unique circuit organization allows the claustrum to rapidly and powerfully suppress cortical networks and suggests a distinct role for the claustrum in regulating cognitive processes in prefrontal circuits.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2018.07.031}, author = {Jackson, Jesse and Karnani, Mahesh M and Zemelman, Boris V and Burdakov, Denis and Lee, Albert K} } @conference {49333, title = {Insights from Biology: low power circuits in the fruit fly.}, booktitle = {International Symposium on Low Power Electronics and Design}, year = {2018}, month = {2018 Jul 23-25}, publisher = {ACM}, organization = {ACM}, address = {Seattle, WA}, abstract = {

Fruit flies (Drosophila melanogaster) are small insects, with correspondingly small power budgets. Despite this, they perform sophisticated neural computations in real time. Careful study of these insects is revealing how some of these circuits work. Insights from these systems might be helpful in designing other low power circuits.

}, url = {https://dl.acm.org/citation.cfm?id=3241337}, author = {Scheffer, Louis K} } @article {49435, title = {Integration of parallel opposing memories underlies memory extinction.}, journal = {Cell}, volume = {175}, year = {2018}, month = {2018 Sep 06}, pages = {709-22}, abstract = {

Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment,\ a process called memory extinction. Here, we show that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2018.08.021}, author = {Felsenberg, Johannes and Jacob, Pedro F and Walker, Thomas and Barnstedt, Oliver and Edmondson-Stait, Amelia J and Pleijzier, Markus W and Otto, Nils and Schlegel, Philipp and Sharifi, Nadiya and Perisse, Emmanuel and Smith, Carlas S and Lauritzen, J Scott and Costa, Marta and Jefferis, Gregory S X E and Bock, Davi D and Waddell, Scott} } @article {49094, title = {Integrative structure and functional anatomy of a nuclear pore complex.}, journal = {Nature}, volume = {555}, year = {2018}, month = {2018 Mar 14}, pages = {475-82}, abstract = {

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

}, issn = {1476-4687}, doi = {10.1038/nature26003}, author = {Kim, Seung Joong and Fernandez-Martinez, Javier and Nudelman, Ilona and Shi, Yi and Zhang, Wenzhu and Raveh, Barak and Herricks, Thurston and Slaughter, Brian D and Hogan, Joanna A and Upla, Paula and Chemmama, Ilan E and Pellarin, Riccardo and Echeverria, Ignacia and Shivaraju, Manjunatha and Chaudhury, Azraa S and Wang, Junjie and Williams, Rosemary and Unruh, Jay R and Greenberg, Charles H and Jacobs, Erica Y and Yu, Zhiheng and de la Cruz, M Jason and Mironska, Roxana and Stokes, David L and Aitchison, John D and Jarrold, Martin F and Gerton, Jennifer L and Ludtke, Steven J and Akey, Christopher W and Chait, Brian T and Sali, Andrej and Rout, Michael P} } @article {49059, title = {Integrative whole-brain neuroscience in larval zebrafish.}, journal = {Current Opinion in Neurobiology}, volume = {50}, year = {2018}, month = {2018 Feb 24}, pages = {136-145}, abstract = {

Due to their small size and transparency, zebrafish larvae are amenable to a range of fluorescence microscopy techniques. With the development of sensitive genetically encoded calcium indicators, this has extended to the whole-brain imaging of neural activity with cellular resolution. This technique has been used to study brain-wide population dynamics accompanying sensory processing and sensorimotor transformations, and has spurred the development of innovative closed-loop behavioral paradigms in which stimulus-response relationships can be studied. More recently, microscopes have been developed that allow whole-brain calcium imaging in freely swimming and behaving larvae. In this review, we highlight the technologies underlying whole-brain functional imaging in zebrafish, provide examples of the sensory and motor processes that have been studied with this technique, and discuss the need to merge data from whole-brain functional imaging studies with neurochemical and anatomical information to develop holistic models of functional neural circuits.

}, issn = {1873-6882}, doi = {10.1016/j.conb.2018.02.004}, author = {Vanwalleghem, Gilles C and Ahrens, Misha B and Scott, Ethan K} } @article {49451, title = {Integrin-mediated attachment of the blastoderm to the vitelline envelope impacts gastrulation of insects.}, journal = {bioRxiv}, year = {2018}, month = {2018 Oct 2}, abstract = {

During gastrulation, physical forces reshape the simple embryonic tissue to form a complex body plan of multicellular organisms. These forces often cause large-scale asymmetric movements of the embryonic tissue. In many embryos, the tissue undergoing gastrulation movements is surrounded by a rigid protective shell. While it is well recognized that gastrulation movements depend on forces generated by tissue-intrinsic contractility, it is not known if interactions between the tissue and the protective shell provide additional forces that impact gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle Tribolium castaneum tightly adheres in a temporally coordinated manner to the vitelline envelope surrounding the embryo. This attachment generates an additional force that counteracts the tissue-intrinsic contractile forces to create asymmetric tissue movements. Furthermore, this localized attachment is mediated by a specific integrin, and its knock-down leads to a gastrulation phenotype consistent with complete loss of attachment. Moreover, analysis of another integrin in the fruit fly Drosophila melanogaster suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline. Together, our findings reveal a conserved mechanism whereby the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable counter-forces that shape gastrulation movements in insects.

}, doi = {10.1101/421701}, author = {Muenster, Stefan and Jain, Akanksha and Mietke, Alexander and Pavlopoulos, Anastasios and Grill, Stephan W and Tomancak, Pavel} } @article {49314, title = {Interacting organelles.}, journal = {Current Opinion in Cell Biology}, volume = {53}, year = {2018}, month = {2018 Aug}, pages = {84-91}, abstract = {

Eukaryotic cells are organized into membrane-bound organelles. These organelles communicate with one another through vesicular trafficking pathways and membrane contact sites (MCSs). MCSs are sites of close apposition between two or more organelles that play diverse roles in the exchange of metabolites, lipids and proteins. Organelle interactions at MCSs also are important for organelle division and biogenesis. For example, the division of several organelles, including mitochondria and endosomes, seem to be regulated by contacts with the endoplasmic reticulum (ER). Moreover, the biogenesis of autophagosomes and peroxisomes involves contributions from the ER and multiple other cellular compartments. Thus, organelle-organelle interactions allow cells to alter the shape and activities of their membrane-bound compartments, allowing them to cope with different developmental and environmental conditions.

}, doi = {10.1016/j.ceb.2018.06.003}, author = {Cohen, Sarah and Valm, Alex M and Lippincott-Schwartz, Jennifer} } @article {65091, title = {Interleukin 2 modulates thymic-derived regulatory T cell epigenetic landscape.}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {2018 Dec 18}, pages = {5368}, abstract = {

Foxp3CD4 regulatory T (T) cells are essential for preventing fatal autoimmunity and safeguard immune homeostasis in vivo. While expression of the transcription factor Foxp3 and IL-2 signals are both required for the development and function of T cells, the commitment to the T cell lineage occurs during thymic selection upon T cell receptor (TCR) triggering, and precedes the expression of Foxp3. Whether signals beside TCR contribute to establish T cell epigenetic and functional identity is still unknown. Here, using a mouse model with reduced IL-2 signaling, we show that IL-2 regulates the positioning of the pioneer factor SATB1 in CD4 thymocytes and controls genome wide chromatin accessibility of thymic-derived T cells. We also show that T cells receiving only low IL-2 signals can suppress endogenous but not WT autoreactive T cell responses in vitro and in vivo. Our findings have broad implications for potential therapeutic strategies to reprogram T cells in vivo.

}, keywords = {Animals, Autoimmunity, Cell Differentiation, Cellular Reprogramming, Disease Models, Animal, Epigenesis, Genetic, Female, Forkhead Transcription Factors, HEK293 Cells, Humans, Interleukin-2, Interleukin-2 Receptor alpha Subunit, Listeriosis, Male, Matrix Attachment Region Binding Proteins, Mice, Mice, Inbred C57BL, Mice, Transgenic, Signal Transduction, T-Lymphocytes, Regulatory, Thymocytes, Thymus Gland}, issn = {2041-1723}, doi = {10.1038/s41467-018-07806-6}, author = {Chorro, Laurent and Suzuki, Masako and Chin, Shu Shien and Williams, Tere M and Snapp, Erik L and Odagiu, Livia and Labrecque, Nathalie and Lauvau, Gr{\'e}goire} } @article {49343, title = {Inverse-response Ca2+ indicators for optogenetic visualization of neuronal inhibition.}, journal = {Scientific Reports}, volume = {8}, year = {2018}, month = {2018 Aug 06}, pages = {11758}, abstract = {

We have developed a series of yellow genetically encoded Ca indicators for optical imaging (Y-GECOs) with inverted responses to Ca and apparent dissociation constants (K{\textquoteright}) ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca indicators, we expressed the four highest affinity variants (K{\textquoteright}s = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower K{\textquoteright} was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-30080-x}, author = {Zhao, Yufeng and Bushey, Daniel and Zhao, Yongxin and Schreiter, Eric R and Harrison, D Jed and Wong, Allan M and Campbell, Robert E} } @article {48318, title = {Imaging mRNA and protein interactions within neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Mar 07}, pages = {E1875-E1884}, abstract = {

RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.

}, issn = {1091-6490}, doi = {10.1073/pnas.1621440114}, author = {Eliscovich, Carolina and Shenoy, Shailesh M and Singer, Robert H} } @article {48939, title = {Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Nov 07}, pages = {E10056-65}, abstract = {

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

}, issn = {1091-6490}, doi = {10.1073/pnas.1706600114}, author = {Pak, Alexander J and Grime, John M A and Sengupta, Prabuddha and Chen, Antony K and Durumeric, Aleksander E P and Srivastava, Anand and Yeager, Mark and Briggs, John A G and Lippincott-Schwartz, Jennifer and Voth, Gregory A} } @article {48766, title = {Imp and Syp RNA-binding proteins govern decommissioning of Drosophila neural stem cells.}, journal = {Development (Cambridge, England)}, volume = {144}, year = {2017}, month = {2017 Aug 29}, pages = {3454-64}, abstract = {

The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a "decommissioning" phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the Mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages.

}, issn = {1477-9129}, doi = {10.1242/dev.149500}, author = {Yang, Ching-Po and Samuels, Tamsin J and Huang, Yaling and Yang, Lu and Ish-Horowicz, David and Davis, Ilan and Lee, Tzumin} } @article {68995, title = {Improved Proliferative Capacity of NP-Like Cells Derived from Human Mesenchymal Stromal Cells and Neuronal Transdifferentiation by Small Molecules.}, journal = {Neurochem Res}, volume = {42}, year = {2017}, month = {02/2017}, pages = {415-427}, abstract = {

Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

}, keywords = {Adolescent, Adult, Amides, Cell Proliferation, Cell Transdifferentiation, Cells, Cultured, Colforsin, Drug Combinations, Heterocyclic Compounds, 4 or More Rings, Humans, Male, Mesenchymal Stem Cells, Neural Stem Cells, Neurons, Pyridines, Young Adult}, issn = {1573-6903}, doi = {10.1007/s11064-016-2086-7}, author = {Aguilera-Castrejon, Alejandro and Pasantes-Morales, Herminia and Montesinos, Juan Jose and Cort{\'e}s-Medina, Lorena V and Castro-Manrreza, Marta E and Mayani, H{\'e}ctor and Ramos-Mandujano, Gerardo} } @article {48368, title = {In vivo patch-clamp recording in awake head-fixed rodents.}, journal = {Cold Spring Harbor Protocols}, volume = {2017}, year = {2017}, month = {2017 Apr 03}, pages = {pdb.prot095802}, abstract = {

Whole-cell recording has been used to measure and manipulate a neuron{\textquoteright}s spiking and subthreshold membrane potential, allowing assessment of the cell{\textquoteright}s inputs and outputs as well as its intrinsic membrane properties. This technique has also been combined with pharmacology and optogenetics as well as morphological reconstruction to address critical questions concerning neuronal integration, plasticity, and connectivity. This protocol describes a technique for obtaining whole-cell recordings in awake head-fixed animals, allowing such questions to be investigated within the context of an intact network and natural behavioral states. First, animals are habituated to sit quietly with their heads fixed in place. Then, a whole-cell recording is obtained using an efficient, blind patching protocol. We have successfully applied this technique to rats and mice.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot095802}, author = {Lee, Doyun and Lee, Albert K} } @article {48253, title = {Inhibitory suppression of heterogeneously tuned excitation enhances spatial coding in CA1 place cells.}, journal = {Nature Neuroscience}, volume = {20}, year = {2017}, month = {2017 Jan 23}, pages = {417-26}, abstract = {

Place cells in the CA1 region of the hippocampus express location-specific firing despite receiving a steady barrage of heterogeneously tuned excitatory inputs that should compromise output dynamic range and timing. We examined the role of synaptic inhibition in countering the deleterious effects of off-target excitation. Intracellular recordings in behaving mice demonstrate that bimodal excitation drives place cells, while unimodal excitation drives weaker or no spatial tuning in interneurons. Optogenetic hyperpolarization of interneurons had spatially uniform effects on place cell membrane potential dynamics, substantially reducing spatial selectivity. These data and a computational model suggest that spatially uniform inhibitory conductance enhances rate coding in place cells by suppressing out-of-field excitation and by limiting dendritic amplification. Similarly, we observed that inhibitory suppression of phasic noise generated by out-of-field excitation enhances temporal coding by expanding the range of theta phase precession. Thus, spatially uniform inhibition allows proficient and flexible coding in hippocampal CA1 by suppressing heterogeneously tuned excitation.

}, issn = {1546-1726}, doi = {10.1038/nn.4486}, author = {Grienberger, Christine and Milstein, Aaron D and Bittner, Katie C and Romani, Sandro and Magee, Jeffrey C} } @article {48492, title = {Integrating Results across Methodologies Is Essential for Producing Robust Neuronal Taxonomies.}, journal = {Neuron}, volume = {94}, year = {2017}, month = {2017 May 17}, pages = {747-751.e1}, abstract = {

Elucidating the diversity and spatial organization of cell types in the brain is an essential goal of neuroscience, with many emerging technologies helping to advance this endeavor. Using a new in\ situ hybridization method that can measure the expression of hundreds of genes in a given mouse brain section (amplified seqFISH), Shah et\ al. (2016) describe a spatial organization of hippocampal cell types that differs from previous reports. In seeking to understand this discrepancy, we find that many of the barcoded genes used by seqFISH to characterize this spatial organization, when cross-validated by other sensitive methodologies, exhibit negligible expression in the hippocampus. Additionally, the results of Shah et\ al. (2016) do not recapitulate canonical cellular hierarchies and improperly classify major neuronal cell types. We suggest that, when describing the spatial organization of brain regions, cross-validation using multiple techniques should be used to yield robust and informative cellular classification. This Matters Arising paper is in response to Shah et\ al. (2016), published in Neuron. See also the response by Shah et\ al. (2017), published in this issue.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2017.04.023}, author = {Cembrowski, Mark S and Spruston, Nelson} } @article {48907, title = {Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Oct 24}, pages = {E9873-E9882}, abstract = {

RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse β-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host-parasite or symbiotic interactions.

}, issn = {1091-6490}, doi = {10.1073/pnas.1706365114}, author = {Haimovich, Gal and Ecker, Christopher M and Dunagin, Margaret C and Eggan, Elliott and Raj, Arjun and Gerst, Jeffrey E and Singer, Robert H} } @article {65773, title = {Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein}, journal = {ChemPhotoChem}, volume = {1}, year = {2017}, month = {02/2017}, pages = {89 {\textendash} 92}, abstract = {

Long-distance biological electron transfer occurs through a hopping mechanism and often involves tyrosine as a high potential intermediate, for example in the early charge separation steps during photosynthesis. Protein design allows for the development of minimal systems to study the underlying principles of complex systems. Herein, we report the development of the first ruthenium-linked designed protein for the photogeneration of a tyrosine radical by intramolecular electron transfer.

}, doi = {10.1002/cptc.201600044}, url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cptc.201600044}, author = {Tebo, Alison G and Quaranta, Annamaria and Herrero, Christian and Pecoraro, Vincent L and Aukauloo, Ally} } @article {48643, title = {Ion Channels: History, Diversity, and Impact.}, journal = {Cold Spring Harbor Protocols}, volume = {2017}, year = {2017}, month = {2017 Jul 05}, pages = {pdb.top092288}, abstract = {

From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.

}, issn = {1559-6095}, doi = {10.1101/pdb.top092288}, author = {Brenowitz, Stephan and Duguid, Ian and Kammermeier, Paul J} } @article {47728, title = {Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion.}, journal = {Scientific Reports}, volume = {6}, year = {2016}, month = {2016}, pages = {30806}, abstract = {

We use Drosophila larval locomotion as a model to elucidate the working principles of motor circuits. Larval locomotion is generated by rhythmic and sequential contractions of body-wall muscles from the posterior to anterior segments, which in turn are regulated by motor neurons present in the corresponding neuromeres. Motor neurons are known to receive both excitatory and inhibitory inputs, combined action of which likely regulates patterned motor activity during locomotion. Although recent studies identified candidate inhibitory premotor interneurons, the identity of premotor interneurons that provide excitatory drive to motor neurons during locomotion remains unknown. In this study, we searched for and identified two putative excitatory premotor interneurons in this system, termed CLI1 and CLI2 (cholinergic lateral interneuron 1 and 2). These neurons were segmentally arrayed and activated sequentially from the posterior to anterior segments during peristalsis. Consistent with their being excitatory premotor interneurons, the CLIs formed GRASP- and ChAT-positive putative synapses with motoneurons and were active just prior to motoneuronal firing in each segment. Moreover, local activation of CLI1s induced contraction of muscles in the corresponding body segments. Taken together, our results suggest that the CLIs directly activate motoneurons sequentially along the segments during larval locomotion.

}, issn = {2045-2322}, doi = {10.1038/srep30806}, author = {Hasegawa, Eri and Truman, James W and Nose, Akinao} } @article {48008, title = {Illuminating the neuronal architecture underlying context in fear memory.}, journal = {Cell}, volume = {167}, year = {2016}, month = {2016 Nov 3}, pages = {888-9}, abstract = {

Context plays a foundational role in determining how to interpret potentially fear-producing stimuli, yet the precise neurobiological substrates of context are poorly understood. In this issue of Cell, Xu et\ al. elegantly show that parallel neuronal circuits are necessary for two distinct roles of context in fear conditioning.

}, author = {Cembrowski, Mark S and Spruston, N} } @article {48296, title = {Image-based correction of continuous and discontinuous non-planar axial distortion in serial section microscopy.}, journal = {Bioinformatics (Oxford, England)}, year = {2016}, month = {2016 Dec 23}, abstract = {

MOTIVATION: Serial section microscopy is an established method for detailed anatomy reconstruction of biological specimen. During the last decade, high resolution electron microscopy (EM) of serial sections has become the de-facto standard for reconstruction of neural connectivity at ever increasing scales (EM connectomics). In serial section microscopy, the axial dimension of the volume is sampled by physically removing thin sections from the embedded specimen and subsequently imaging either the block-face or the section series. This process has limited precision leading to inhomogeneous non-planar sampling of the axial dimension of the volume which, in turn, results in distorted image volumes. This includes that section series may be collected and imaged in unknown order.

RESULTS: We developed methods to identify and correct these distortions through image-based signal analysis without any additional physical apparatus or measurements. We demonstrate the efficacy of our methods in proof of principle experiments and application to real world problems.

AVAILABILITY AND IMPLEMENTATION: We made our work available as libraries for the ImageJ distribution Fiji and for deployment in a high performance parallel computing environment. Our sources are open and available at http://github.com/saalfeldlab/section-sort, http://github.com/saalfeldlab/z-spacing and http://github.com/saalfeldlab/z-spacing-spark CONTACT: : saalfelds@janelia.hhmi.orgSupplementary information: Supplementary data are available at Bioinformatics online.

}, issn = {1367-4811}, doi = {10.1093/bioinformatics/btw794}, author = {Hanslovsky, Philipp and Bogovic, John A and Saalfeld, Stephan} } @article {47877, title = {Imaging far and wide.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Sep 23}, pages = {e21072}, abstract = {

A custom-built objective lens called the Mesolens allows relatively large biological specimens to be imaged with cellular resolution.

}, doi = {10.7554/eLife.18659}, author = {Chhetri, Raghav K and Keller, Philipp J} } @article {47812, title = {Improving data quality in neuronal population recordings.}, journal = {Nature Neuroscience}, volume = {19}, year = {2016}, month = {2016 Aug 26}, pages = {1165-74}, abstract = {

Understanding how the brain operates requires understanding how large sets of neurons function together. Modern recording technology makes it possible to simultaneously record the activity of hundreds of neurons, and technological developments will soon allow recording of thousands or tens of thousands. As with all experimental techniques, these methods are subject to confounds that complicate the interpretation of such recordings, and could lead to erroneous scientific conclusions. Here we discuss methods for assessing and improving the quality of data from these techniques and outline likely future directions in this field.

}, issn = {1546-1726}, doi = {10.1038/nn.4365}, author = {Harris, Kenneth D and Quiroga, Rodrigo Quian and Freeman, Jeremy and Smith, Spencer L} } @article {47548, title = {in vivo brain imaging with adaptive optical microscope.}, journal = {Conference on Lasers and Electro-Optics (CLEO): Applications and Technology}, year = {2016}, month = {2016 Jun }, pages = {AM40.1}, abstract = {

The diffraction limited resolution of two photon and confocal microscope can be recovered using adaptive optics to explore the detailed neuronal network in the brains of zebrafish and mouse in vivo.

}, doi = {10.1364/CLEO_AT.2016.AM4O.1}, author = {Wang, Kai and Sun, Wenzhi and Ji, Na and Betzig, Eric} } @article {47969, title = {Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.}, journal = {Science (New York, N.Y.)}, volume = {354}, year = {2016}, month = {2016 Oct 28}, pages = {433-46}, abstract = {

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER {\textquotedblleft}sheet{\textquotedblright} proliferation after overexpression of tubular junction{\textendash}forming proteins.

}, doi = {10.1126/science.aaf3928}, author = {Nixon-Abell, Jonathon and Obara, Christopher J and Weigel, Aubrey V and Li, Dong and Legant, Wesley R and Xu, CShan and Pasolli, H Amalia and Harvey, Kirsten and Hess, Harald F and Betzig, Eric and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {48842, title = {Inhibitory control of correlated intrinsic variability in cortical networks.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Dec 07}, abstract = {

Cortical networks exhibit intrinsic dynamics that drive coordinated, large-scale fluctuations across neuronal populations and create noise correlations that impact sensory coding. To investigate the network-level mechanisms that underlie these dynamics, we developed novel computational techniques to fit a deterministic spiking network model directly to multi-neuron recordings from different rodent species, sensory modalities, and behavioral states. The model generated correlated variability without external noise and accurately reproduced the diverse activity patterns in our recordings. Analysis of the model parameters suggested that differences in noise correlations across recordings were due primarily to differences in the strength of feedback inhibition. Further analysis of our recordings confirmed that putative inhibitory neurons were indeed more active during desynchronized cortical states with weak noise correlations. Our results demonstrate that network models with intrinsically-generated variability can accurately reproduce the activity patterns observed in multi-neuron recordings and suggest that inhibition modulates the interactions between intrinsic dynamics and sensory inputs to control the strength of noise correlations.

}, issn = {2050-084X}, doi = {10.7554/eLife.19695}, author = {Stringer, Carsen and Pachitariu, Marius and Steinmetz, Nicholas A and Okun, Michael and Bartho, Peter and Harris, Kenneth D and Sahani, Maneesh and Lesica, Nicholas A} } @article {65751, title = {Inhibitory control of correlated intrinsic variability in cortical networks}, journal = {Elife}, volume = {5}, year = {2016}, month = {12/2016}, pages = {e19695}, abstract = {

Cortical networks exhibit intrinsic dynamics that drive coordinated, large-scale fluctuations across neuronal populations and create noise correlations that impact sensory coding. To investigate the network-level mechanisms that underlie these dynamics, we developed novel computational techniques to fit a deterministic spiking network model directly to multi-neuron recordings from different rodent species, sensory modalities, and behavioral states. The model generated correlated variability without external noise and accurately reproduced the diverse activity patterns in our recordings. Analysis of the model parameters suggested that differences in noise correlations across recordings were due primarily to differences in the strength of feedback inhibition. Further analysis of our recordings confirmed that putative inhibitory neurons were indeed more active during desynchronized cortical states with weak noise correlations. Our results demonstrate that network models with intrinsically-generated variability can accurately reproduce the activity patterns observed in multi-neuron recordings and suggest that inhibition modulates the interactions between intrinsic dynamics and sensory inputs to control the strength of noise correlations.

}, doi = {https://doi.org/10.7554/eLife.19695}, author = {Stringer, Carsen and Pachitariu, Marius and Steinmetz, Nicholas A and Okun, Michael and Bartho, Peter and Harris, Kenneth D and Sahani, Maneesh and Lesica, Nicholas A} } @article {48990, title = {The insect central complex.}, journal = {Current Biology : CB}, volume = {26}, year = {2016}, month = {2016 Jun 06}, pages = {R453-7}, abstract = {

Hordes of tourists flock to Washington, D.C. every spring to see the cherry trees blossom. Once in the city, they must find their way to the Tidal Basin where the Japanese trees grow. Fortunately, a number of visual landmarks can help them to navigate. In 1910, the United States Congress passed The Height of Buildings Act, limiting the elevation of commercial and residential structures in D.C. to 130 feet. Thus, the 555-foot-tall Washington Monument often looms large against the horizon, serving as an anchor point to help set the tourists{\textquoteright} sense of direction. Once their heading is set, they can lose sight of the monument behind buildings or groups of tall Scandinavian visitors and still use their internal compass to navigate to the Basin. This compass keeps track of their paces and turns and updates their sense of where they are and where they need to go. Yet while their heading informs their actions, it does not dictate them. Tourists who have been to D.C. in the past can, for example, use remembered views to alter their routes to avoid crowds. On an even finer scale, their leg movements also depend on their current state - they might increase the frequency and length of their strides if hunger pangs compete with their desire to see cherry blossoms, for example. The way in which these disparate cues and motivations influence exploration is a neuroscience mystery across creatures large and small.

}, keywords = {Animals, Brain, Cues, Insecta, Spatial Navigation, Vision, Ocular}, issn = {1879-0445}, doi = {10.1016/j.cub.2016.04.006}, author = {Turner-Evans, Daniel B and Jayaraman, Vivek} } @article {46522, title = {Identification of genes that promote or inhibit olfactory memory formation in Drosophila.}, journal = {Genetics}, volume = {199}, year = {2015}, month = {2015 Apr}, pages = {1173-82}, abstract = {

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified \>500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified \>40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.

}, issn = {1943-2631}, doi = {10.1534/genetics.114.173575}, author = {Walkinshaw, Erica and Gai, Yunchao and Farkas, Caitlin and Richter, Daniel and Nicholas, Eric and Keleman, Krystyna and Davis, Ronald L} } @article {47037, title = {Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion.}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015 Sep 03}, pages = {e0136660}, abstract = {

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs{\textquoteright} wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0136660}, author = {Itakura, Yuki and Kohsaka, Hiroshi and Ohyama, Tomoko and Zlatic, Marta and Pulver, Stefan R and Nose, Akinao} } @article {47237, title = {Ig superfamily ligand and receptor pairs expressed in synaptic partners in Drosophila.}, journal = {Cell}, volume = {163}, year = {2015}, month = {2015 Dec 17}, pages = {1756-69}, abstract = {

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.11.021}, author = {Tan, Liming and Zhang, Kelvin Xi and Pecot, Matthew Y and Nagarkar-Jaiswal, Sonal and Lee, Pei-Tseng and Takemura, Shin-ya and McEwen, Jason M and Nern, Aljoscha and Xu, Shuwa and Tadros, Wael and Chen, Zhenqing and Zinn, Kai and Bellen, Hugo J and Morey, Marta and Zipursky, S Lawrence} } @article {47161, title = {Imaging fictive locomotor patterns in larval Drosophila.}, journal = {Journal of Neurophysiology}, volume = {114}, year = {2015}, month = {2015 Nov 06}, pages = {2564-77}, abstract = {

We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being \~{}10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques.

}, issn = {1522-1598}, doi = {10.1152/jn.00731.2015}, author = {Pulver, Stefan R and Bayley, Timothy G and Taylor, Adam L and Berni, Jimena and Bate, Michael and Hedwig, Berthold} } @article {46771, title = {Imaging live-cell dynamics and structure at the single-molecule level.}, journal = {Molecular Cell}, volume = {58}, year = {2015}, month = {2015 May 21}, pages = {644-59}, abstract = {

Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen.

}, doi = {10.1016/j.molcel.2015.02.033}, author = {Liu, Zhe and Lavis, Luke D and Betzig, Eric} } @article {47127, title = {Imaging the neural basis of locomotion.}, journal = {Cell}, volume = {163}, year = {2015}, month = {2015 Oct 22}, pages = {541-2}, abstract = {

To investigate the fundamental question of how nervous systems encode, organize, and sequence behaviors, Kato et\ al. imaged neural activity with cellular resolution across the brain of the worm Caenorhabditis elegans. Locomotion behavior seems to be continuously represented by cyclical patterns of distributed neural activity that are present even in immobilized animals.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.10.014}, author = {Branson, Kristin and Freeman, Jeremy} } @article {47734, title = {Imaging transcription: past, present, and future.}, journal = {Cold Spring Harbor Symposia on Quantitative Biology}, volume = {80}, year = {2015}, month = {2015}, pages = {1-8}, abstract = {

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

}, issn = {1943-4456}, doi = {10.1101/sqb.2015.80.027201}, author = {Coleman, Robert A and Liu, Zhe and Darzacq, Xavier and Tjian, Robert and Singer, Robert H and Lionnet, Timoth{\'e}e} } @article {48389, title = {Impermanence of dendritic spines in live adult CA1 hippocampus.}, journal = {Nature}, volume = {523}, year = {2015}, month = {2015 Jul 30}, pages = {592-6}, abstract = {

The mammalian hippocampus is crucial for episodic memory formation and transiently retains information for about 3-4 weeks in adult mice and longer in humans. Although neuroscientists widely believe that neural synapses are elemental sites of information storage, there has been no direct evidence that hippocampal synapses persist for time intervals commensurate with the duration of hippocampal-dependent memory. Here we tested the prediction that the lifetimes of hippocampal synapses match the longevity of hippocampal memory. By using time-lapse two-photon microendoscopy in the CA1 hippocampal area of live mice, we monitored the turnover dynamics of the pyramidal neurons{\textquoteright} basal dendritic spines, postsynaptic structures whose turnover dynamics are thought to reflect those of excitatory synaptic connections. Strikingly, CA1 spine turnover dynamics differed sharply from those seen previously in the neocortex. Mathematical modelling revealed that the data best matched kinetic models with a single population of spines with a mean lifetime of approximately 1-2 weeks. This implies \~{}100\% turnover in \~{}2-3 times this interval, a near full erasure of the synaptic connectivity pattern. Although N-methyl-d-aspartate (NMDA) receptor blockade stabilizes spines in the neocortex, in CA1 it transiently increased the rate of spine loss and thus lowered spine density. These results reveal that adult neocortical and hippocampal pyramidal neurons have divergent patterns of spine regulation and quantitatively support the idea that the transience of hippocampal-dependent memory directly reflects the turnover dynamics of hippocampal synapses.

}, keywords = {Animals, CA1 Region, Hippocampal, Dendritic Spines, Endoscopy, Kinetics, Male, Memory, Episodic, Mice, Neocortex, Neuronal Plasticity, Photons, Pyramidal Cells, Receptors, N-Methyl-D-Aspartate, Synapses, Time Factors}, issn = {1476-4687}, doi = {10.1038/nature14467}, author = {Attardo, Alessio and Fitzgerald, James E and Schnitzer, Mark J} } @article {46701, title = {In the right place at the right time: visualizing and understanding mRNA localization.}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, year = {2015}, month = {2015 Feb}, pages = {95-109}, abstract = {

The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

}, keywords = {Animals, Biological Transport, Humans, Neurons, Protein Biosynthesis, Protein Transport, RNA, Messenger}, issn = {1471-0080}, doi = {10.1038/nrm3918}, author = {Buxbaum, Adina R and Haimovich, Gal and Singer, Robert H} } @article {46534, title = {In vivo neuroimaging through the highly scattering tissue via iterative multi-photon adaptive compensation technique.}, journal = {Optics Express}, volume = {23}, year = {2015}, month = {2015 Mar 9}, pages = {6145-50}, abstract = {

For in vivo deep tissue imaging, high order wavefront measurement and correction is needed for handling the severe wavefront distortion. Towards such a goal, we have developed the iterative multi-photon adaptive compensation technique (IMPACT). In this work, we explore using IMPACT to perform calcium imaging of neocortex through the intact skull of adult mice, and to image through the highly scattering white matter on the hippocampus surface.

}, issn = {1094-4087}, doi = {10.1364/OE.23.006145}, author = {Kong, Lingjie and Cui, Meng} } @article {46885, title = {InferenceMAP: mapping of single-molecule dynamics with Bayesian inference.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jun 30}, pages = {594-5}, issn = {1548-7105}, doi = {10.1038/nmeth.3441}, author = {Beheiry, Mohamed El and Dahan, Maxime and Masson, Jean-Baptiste} } @article {46929, title = {Inferring transient particle transport dynamics in live cells.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jul 20}, pages = {838-40}, abstract = {

Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3483}, author = {Monnier, Nilah and Barry, Zachary and Park, Hye Yoon and Su, Kuan-Chung and Katz, Zachary and English, Brian P and Dey, Arkajit and Pan, Keyao and Cheeseman, Iain M and Singer, Robert H and Bathe, Mark} } @article {47087, title = {Inhibitory gating of input comparison in the CA1 microcircuit.}, journal = {Neuron}, volume = {87}, year = {2015}, month = {2015 Sep 23}, pages = {1274-89}, abstract = {

Spatial and temporal features of synaptic inputs engage integration mechanisms on multiple scales, including presynaptic release sites, postsynaptic dendrites, and networks of inhibitory interneurons. Here we investigate how these mechanisms cooperate to filter synaptic input in hippocampal area CA1. Dendritic recordings from CA1 pyramidal neurons reveal that proximal inputs from CA3 as well as distal inputs from entorhinal cortex layer III (ECIII) sum sublinearly or linearly at low firing rates due to feedforward inhibition, but sum supralinearly at high firing rates due to synaptic facilitation, producing a high-pass filter. However, during ECIII and CA3 input comparison, supralinear dendritic integration is dynamically balanced by feedforward and\ feedback inhibition, resulting in suppression of dendritic complex spiking. We find that a particular subpopulation of CA1 interneurons expressing neuropeptide Y (NPY) contributes prominently to this dynamic filter by integrating both ECIII and CA3 input pathways and potently inhibiting CA1 pyramidal neuron dendrites.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2015.08.025}, author = {Milstein, Aaron D and Bloss, Erik B and Apostolides, Pierre F and Vaidya, Sachin P and Dilly, Geoffrey A and Zemelman, Boris V and Magee, Jeffrey C} } @article {47284, title = {The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity.}, journal = {Neuron}, volume = {88}, year = {2015}, month = {2015 Dec 16}, pages = {1157-64}, abstract = {

It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. Here we show that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2015.10.049}, author = {Harris, Nathan and Braiser, Daniel J and Dickman, Dion K and Fetter, Richard D and Tong, Amy and Davis, Graeme W} } @conference {47229, title = {Interactive exemplar-based segmentation toolkit for biomedical image analysis.}, booktitle = {International Symposium on Biomedical Imaging}, year = {2015}, month = {2015 Apr}, abstract = {

In the field of biomedical imaging analysis on single-cell level, reliable and fast segmentation of the cell nuclei from the background on three-dimensional images is highly needed for the further analysis. In this work we propose an interactive cell segmentation toolkit that first establishes a set of exemplar regions from user input through an easy and intuitive interface in both 2D and 3D in real-time, then
extracts the shape and intensity features from those exemplars. Based on a local contrast-constrained region growing scheme, each connected component in the whole image would be filtered by the features from exemplars, forming an {\textquotedblleft}exemplar-matching{\textquotedblright} group which passed the filtering and would be part of the final segmentation result, and a {\textquotedblleft}non-exemplar-matching{\textquotedblright} group in which components
would be further segmented by the gradient vector field based algorithm. The results of the filtering process are visualized back to the user in near real-time, thus enhancing the experience in exemplar selecting and parameter tuning. The toolkit is distributed as a plugin within the open source Vaa3D system (http://vaa3d.org).

}, author = {Li, Xiang and Zhou, Zhi and Keller, Philipp J and Zeng, Hongkui and Liu, Tianming and Peng, Hanchuan} } @article {47619, title = {Intracellular and extracellular forces drive primary cilia movement.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Feb 3}, pages = {1410-5}, abstract = {

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

}, keywords = {Animals, Centrosome, Cilia, Dogs, Madin Darby Canine Kidney Cells, Microscopy, Electron, Movement}, issn = {1091-6490}, doi = {10.1073/pnas.1421845112}, author = {Battle, Christopher and Ott, Carolyn M and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Schmidt, Christoph F} } @article {45695, title = {Identification of loci that cause phenotypic variation in diverse species with the reciprocal hemizygosity test.}, journal = {Trends in Genetics}, volume = {30}, year = {2014}, month = {2014 Dec}, pages = {547-554}, abstract = {

The reciprocal hemizygosity test is a straightforward genetic test that can positively identify genes that have evolved to contribute to a phenotypic difference between strains or between species. The test involves a comparison between hybrids that are genetically identical throughout the genome except at the test locus, which is rendered hemizygous for alternative alleles from the two parental strains. If the two reciprocal hemizygotes display different phenotypes, then the two parental alleles must have evolved. New methods for targeted mutagenesis will allow application of the reciprocal hemizygosity test in many organisms. This review discusses the principles, advantages, and limitations of the test.

}, issn = {0168-9525}, doi = {10.1016/j.tig.2014.09.006}, author = {Stern, David L} } @article {46231, title = {Identifying synapses using deep and wide multiscale recursive networks.}, journal = {arXiv}, year = {2014}, month = {2014 Sep 5}, pages = {arXiv:1409.1789 [cs.CV]}, abstract = {

In this work, we propose a learning framework for identifying synapses using a deep and wide multi-scale recursive (DAWMR) network, previously considered in image segmentation applications. We apply this approach on electron microscopy data from invertebrate fly brain tissue. By learning features directly from the data, we are able to achieve considerable improvements over existing techniques that rely on a small set of hand-designed features. We show that this system can reduce the amount of manual annotation required, in both acquisition of training data as well as verification of inferred detections.

}, url = {http://arxiv.org/abs/1409.1789}, author = {Huang, Gary and Plaza, Stephen M} } @article {45032, title = {Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits.}, journal = {Frontiers in Neural Circuits}, volume = {8}, year = {2014}, month = {2014 Jun 27}, pages = {68}, abstract = {

The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly-the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments.

}, issn = {1662-5110}, doi = {10.3389/fncir.2014.00068}, author = {Hayworth, Kenneth J and Morgan, Josh L and Schalek, Richard and Berger, Daniel R and Hildebrand, David G C and Lichtman, Jeff W} } @article {45060, title = {Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.}, journal = {Methods in Cell Biology}, volume = {123}, year = {2014}, month = {2014}, pages = {273-94}, abstract = {

Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250nm laterally and \>500nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.

}, issn = {0091-679X}, doi = {10.1016/B978-0-12-420138-5.00015-X}, author = {Shtengel, Gleb and Wang, Yilin and Zhang, Zhen and Goh, Wah Ing and Hess, Harald F and Kanchanawong, Pakorn} } @article {45166, title = {Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 May 20}, pages = {e02203}, abstract = {

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.02203}, author = {Wisniewski, Jan and Hajj, Bassam and Chen, Jiji and Mizuguchi, Gaku and Xiao, Hua and Wei, Debbie and Dahan, Maxime and Wu, Carl} } @article {47898, title = {Improving reliability and absolute quantification of human brain microarray data by filtering and scaling probes using RNA-Seq.}, journal = {BMC genomics}, volume = {15}, year = {2014}, month = {2014}, pages = {154}, abstract = {

BACKGROUND: High-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel. However, existing microarray data from clinical, pharmaceutical, and academic settings represent valuable and often underappreciated resources, and methods for assessing and improving the quality of these data are lacking.

RESULTS: To quantitatively assess the quality of microarray probes, we directly compare RNA-Seq to Agilent microarrays by processing 231 unique samples from the Allen Human Brain Atlas using RNA-Seq. Both techniques provide highly consistent, highly reproducible gene expression measurements in adult human brain, with RNA-Seq slightly outperforming microarray results overall. We show that RNA-Seq can be used as ground truth to assess the reliability of most microarray probes, remove probes with off-target effects, and scale probe intensities to match the expression levels identified by RNA-Seq. These sequencing scaled microarray intensities (SSMIs) provide more reliable, quantitative estimates of absolute expression levels for many genes when compared with unscaled intensities. Finally, we validate this result in two human cell lines, showing that linear scaling factors can be applied across experiments using the same microarray platform.

CONCLUSIONS: Microarrays provide consistent, reproducible gene expression measurements, which are improved using RNA-Seq as ground truth. We expect that our strategy could be used to improve probe quality for many data sets from major existing repositories.

}, keywords = {Brain, Cluster Analysis, Computational Biology, Gene Expression, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, Humans, Neocortex, Oligonucleotide Array Sequence Analysis, Reproducibility of Results, Sequence Analysis, RNA, Transcriptome}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-154}, author = {Miller, Jeremy A and Menon, Vilas and Goldy, Jeff and Kaykas, Ajamete and Lee, Chang-Kyu and Smith, Kimberly A and Shen, Elaine H and Phillips, John W and Lein, Ed S and Hawrylycz, Mike J} } @article {45786, title = {In vivo fluorescence microscopy via iterative multi-photon adaptive compensation technique.}, journal = {Optical Express}, volume = {22}, year = {2014}, month = {2014 Oct 6}, pages = {23786-94}, abstract = {

Iterative multi-photon adaptive compensation technique (IMPACT) has been developed for wavefront measurement and compensation in highly scattering tissues. Our previous report was largely based on the measurements of fixed tissue. Here we demonstrate the advantages of IMPACT for in vivo imaging and report the latest results. In particular, we show that IMPACT can be used for functional imaging of awake mice, and greatly improve the in vivo neuron imaging in mouse cortex at large depth (~660 microns). Moreover, IMPACT enables neuron imaging through the intact skull of adult mice, which promises noninvasive optical measurements in mouse brain.

}, issn = {1094-4087}, doi = {10.1364/OE.22.023786}, author = {Kong, Lingjie and Cui, Meng} } @article {6719, title = {Independent optical excitation of distinct neural populations.}, journal = {Nature Methods}, volume = {11}, year = {2014}, month = {2014 Feb 9}, pages = {338-46}, abstract = {

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson{\textquoteright}s excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2836}, author = {Klapoetke, Nathan C and Murata, Yasunobu and Kim, Sung Soo and Pulver, Stefan R and Birdsey-Benson, Amanda and Cho, Yong Ku and Morimoto, Tania K and Chuong, Amy S and Carpenter, Eric J and Tian, Zhijian and Wang, Jun and Xie, Yinlong and Yan, Zhixiang and Zhang, Yong and Chow, Brian Y and Surek, Barbara and Melkonian, Michael and Jayaraman, Vivek and Constantine-Paton, Martha and Wong, Gane Ka-Shu and Boyden, Edward S} } @article {45178, title = {The inhibitory microcircuit of the substantia nigra provides feedback gain control of the basal ganglia output.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 May 21}, pages = {e02397}, abstract = {

Dysfunction of the basal ganglia produces severe deficits in the timing, initiation, and vigor of movement. These diverse impairments suggest a control system gone awry. In engineered systems, feedback is critical for control. By contrast, models of the basal ganglia highlight feedforward circuitry and ignore intrinsic feedback circuits. In this study, we show that feedback via axon collaterals of substantia nigra projection neurons control the gain of the basal ganglia output. Through a combination of physiology, optogenetics, anatomy, and circuit mapping, we elaborate a general circuit mechanism for gain control in a microcircuit lacking interneurons. Our data suggest that diverse tonic firing rates, weak unitary connections and a spatially diffuse collateral circuit with distinct topography and kinetics from feedforward input is sufficient to implement divisive feedback inhibition. The importance of feedback for engineered systems implies that the intranigral microcircuit, despite its absence from canonical models, could be essential to basal ganglia function. DOI: http://dx.doi.org/10.7554/eLife.02397.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.02397}, author = {Brown, Jennifer and Pan, Wei-Xing and Dudman, Joshua Tate} } @article {45371, title = {Integrated action of pheromone signals in promoting courtship behavior in male mice.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jul 29}, pages = {e03025}, abstract = {

The mammalian vomeronasal organ encodes pheromone information about gender, reproductive status, genetic background and individual differences. It remains unknown how pheromone information interacts to trigger innate behaviors. In this study, we identify vomeronasal receptors responsible for detecting female pheromones. A sub-group of V1re clade members recognizes gender-identifying cues in female urine. Multiple members of the V1rj clade are cognate receptors for urinary estrus signals, as well as for sulfated estrogen (SE) compounds. In both cases, the same cue activates multiple homologous receptors, suggesting redundancy in encoding female pheromone cues. Neither gender-specific cues nor SEs alone are sufficient to promote courtship behavior in male mice, whereas robust courtship behavior can be induced when the two cues are applied together. Thus, integrated action of different female cues is required in pheromone-triggered mating behavior. These results suggest a gating mechanism in the vomeronasal circuit in promoting specific innate behavior.DOI: http://dx.doi.org/10.7554/eLife.03025.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.03025}, author = {Haga-Yamanaka, Sachiko and Ma, Limei and He, Jie and Qiu, Qiang and Lavis, Luke D and Looger, Loren L and Yu, C Ron} } @article {49004, title = {Interference and shaping in sensorimotor adaptations with rewards.}, journal = {PLoS Computational B2014-01-09iology}, volume = {10}, year = {2014}, month = {2014 Jan}, pages = {e1003377}, abstract = {

When a perturbation is applied in a sensorimotor transformation task, subjects can adapt and maintain performance by either relying on sensory feedback, or, in the absence of such feedback, on information provided by rewards. For example, in a classical rotation task where movement endpoints must be rotated to reach a fixed target, human subjects can successfully adapt their reaching movements solely on the basis of binary rewards, although this proves much more difficult than with visual feedback. Here, we investigate such a reward-driven sensorimotor adaptation process in a minimal computational model of the task. The key assumption of the model is that synaptic plasticity is gated by the reward. We study how the learning dynamics depend on the target size, the movement variability, the rotation angle and the number of targets. We show that when the movement is perturbed for multiple targets, the adaptation process for the different targets can interfere destructively or constructively depending on the similarities between the sensory stimuli (the targets) and the overlap in their neuronal representations. Destructive interferences can result in a drastic slowdown of the adaptation. As a result of interference, the time to adapt varies non-linearly with the number of targets. Our analysis shows that these interferences are weaker if the reward varies smoothly with the subject{\textquoteright}s performance instead of being binary. We demonstrate how shaping the reward or shaping the task can accelerate the adaptation dramatically by reducing the destructive interferences. We argue that experimentally investigating the dynamics of reward-driven sensorimotor adaptation for more than one sensory stimulus can shed light on the underlying learning rules.

}, keywords = {Adaptation, Physiological, Algorithms, Biomechanical Phenomena, Brain, Computational Biology, Computer Simulation, Feedback, Sensory, Humans, Learning, Models, Neurological, Movement, Neuronal Plasticity, Neurons, Psychomotor Performance, Reproducibility of Results, Reward, Rotation, Synapses}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.1003377}, author = {Darshan, Ran and Leblois, Arthur and Hansel, David} } @article {45720, title = {Internal models direct dragonfly interception steering.}, journal = {Nature}, year = {2014}, month = {2014 Dec 10}, abstract = {

Sensorimotor control in vertebrates relies on internal models. When extending an arm to reach for an object, the brain uses predictive models of both limb dynamics and target properties. Whether invertebrates use such models remains unclear. Here we examine to what extent prey interception by dragonflies (Plathemis lydia), a behaviour analogous to targeted reaching, requires internal models. By simultaneously tracking the position and orientation of a dragonfly{\textquoteright}s head and body during flight, we provide evidence that interception steering is driven by forward and inverse models of dragonfly body dynamics and by models of prey motion. Predictive rotations of the dragonfly{\textquoteright}s head continuously track the prey{\textquoteright}s angular position. The head-body angles established by prey tracking appear to guide systematic rotations of the dragonfly{\textquoteright}s body to align it with the prey{\textquoteright}s flight path. Model-driven control thus underlies the bulk of interception steering manoeuvres, while vision is used for reactions to unexpected prey movements. These findings illuminate the computational sophistication with which insects construct behaviour.

}, issn = {1476-4687}, doi = {10.1038/nature14045}, author = {Mischiati, Matteo and Lin, Huai-Ti and Herold, Paul and Imler, Elliot and Olberg, Robert and Leonardo, Anthony} } @article {45459, title = {Intrinsic bursting of aII amacrine cells underlies oscillations in the rd1 mouse retina.}, journal = {Journal of Neurophysiology}, volume = {112}, year = {2014}, month = {2014 Sep 15}, pages = {1491-504}, abstract = {

In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell{\textquoteright}s intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell.

}, issn = {1522-1598}, doi = {10.1152/jn.00437.2014}, author = {Choi, Hannah and Zhang, Lei and Cembrowski, Mark S and Sabottke, Carl F and Markowitz, Alexander L and Butts, Daniel A and Kath, William L and Singer, Joshua H and Riecke, Hermann} } @article {6887, title = {iPfam: a database of protein family and domain interactions found in the Protein Data Bank.}, journal = {Nucleic acids research}, volume = {42}, year = {2014}, month = {2014 Jan}, pages = {D364-73}, abstract = {The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.}, keywords = {Databases, Internet, Ligands, Protein, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Interaction Maps, Proteins, Software}, issn = {1362-4962}, doi = {10.1093/nar/gkt1210}, author = {Finn, Robert D and Miller, Benjamin L and Clements, Jody and Bateman, Alex} } @article {45782, title = {Isolation of mitochondria from animal tissue.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.prot080010}, abstract = {

Rat or mouse liver is the most frequently used tissue for mitochondrial preparations because it is readily available, easy to homogenize, and replete with mitochondria. A motor-driven Teflon and glass Potter-Elvehjem homogenizer is the best choice for homogenizing liver, but if one is not available, this tissue is soft enough that a Dounce homogenizer with a loose (A) pestle can also be used. The yield and purity of the mitochondrial preparation will be influenced by the method and speed of preparation and the age and physiological condition of the animal.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot080010}, author = {Clayton, David A and Shadel, Gerald S} } @article {47884, title = {Isolation of mitochondria from cells and tissues.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.top074542}, abstract = {

Mitochondria are complex organelles at the center of cellular metabolism, apoptosis, and signaling. They continue to be the subject of intense basic investigation to understand their composition and function, but they have also captivated the attention of clinical researchers because of the growing knowledge of the (sometimes unexpected) roles of mitochondria in human diseases and aging. A full understanding of these intriguing organelles often requires their purification from cells or tissues under specific physiological or pathological conditions. Here we provide some introductory considerations for those interested in purifying mitochondria for subsequent downstream biophysical, structural, and functional analysis.

}, keywords = {Animals, Cell Fractionation, Cells, Cultured, Humans, Mitochondria, Tissue Culture Techniques}, issn = {1559-6095}, doi = {10.1101/pdb.top074542}, author = {Clayton, David A and Shadel, Gerald S} } @article {45783, title = {Isolation of mitochondria from tissue culture cells.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.prot080002}, abstract = {

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from \~{}10$^{9}$ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot080002}, author = {Clayton, David A and Shadel, Gerald S} } @article {5840, title = {Identification of nonvisual photomotor response cells in the vertebrate hindbrain.}, journal = {The Journal of Neuroscience}, volume = {33}, year = {2013}, month = {2013 Feb 27}, pages = {3834-43}, abstract = {

Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light but does not require the eyes, pineal gland, or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical nonvisual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Using in vivo calcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3689-12.2013}, author = {Kokel, David and Dunn, Timothy W. and Ahrens, Misha B and Alshut, R{\"u}diger and Cheung, Chung Yan J. and Saint-Amant, Louis and Bruni, Giancarlo and Mateus, Rita and van Ham, Tjakko J. and Shiraki, Tomoya and Fukada, Yoshitaka and Kojima, Daisuke and Yeh, Jing-Ruey J. and Mikut, Ralf and von Lintig, Johannes and Engert, Florian and Peters, Randall T.} } @article {47220, title = {Imaging a population code for odor identity in the Drosophila mushroom body.}, journal = {The Journal of Neuroscience : the official journal of the Society for Neuroscience}, volume = {33}, year = {2013}, month = {2013 Jun 19}, pages = {10568-81}, abstract = {

The brain represents sensory information in the coordinated activity of neuronal ensembles. Although the microcircuits underlying olfactory processing are well characterized in Drosophila, no studies to date have examined the encoding of odor identity by populations of neurons and related it to the odor specificity of olfactory behavior. Here we used two-photon Ca(2+) imaging to record odor-evoked responses from \>100 neurons simultaneously in the Drosophila mushroom body (MB). For the first time, we demonstrate quantitatively that MB population responses contain substantial information on odor identity. Using a series of increasingly similar odor blends, we identified conditions in which odor discrimination is difficult behaviorally. We found that MB ensemble responses accounted well for olfactory acuity in this task. Kenyon cell ensembles with as few as 25 cells were sufficient to match behavioral discrimination accuracy. Using a generalization task, we demonstrated that the MB population code could predict the flies{\textquoteright} responses to novel odors. The degree to which flies generalized a learned aversive association to unfamiliar test odors depended upon the relative similarity between the odors{\textquoteright} evoked MB activity patterns. Discrimination and generalization place different demands on the animal, yet the flies{\textquoteright} choices in these tasks were reliably predicted based on the amount of overlap between MB activity patterns. Therefore, these different behaviors can be understood in the context of a single physiological framework.

}, keywords = {Animals, Calcium, Discrimination (Psychology), Drosophila, Generalization (Psychology), Image Processing, Computer-Assisted, Learning, Linear Models, Mushroom Bodies, Neuroimaging, Odors, Olfactory Pathways, Olfactory Perception, Psychomotor Performance, Synaptic Transmission}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0682-12.2013}, author = {Campbell, Robert A A and Honegger, Kyle S and Qin, Hongtao and Li, Wanhe and Demir, Ebru and Turner, Glenn C} } @article {5982, title = {Imaging morphogenesis: technological advances and biological insights.}, journal = {Science}, volume = {340}, year = {2013}, month = {2013 Jun 7}, pages = {1234168}, abstract = {

Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

Science Profile: A Research Career in Focus

}, issn = {1095-9203}, doi = {10.1126/science.1234168}, author = {Keller, Philipp J} } @article {6670, title = {Imaging neuronal populations in behaving rodents: paradigms for studying neural circuits underlying behavior in the mammalian cortex.}, journal = {The Journal of Neuroscience : the Official Journal of the Society for Neuroscience}, volume = {33}, year = {2013}, month = {2013 Nov 6}, pages = {17631-40}, abstract = {

Understanding the neural correlates of behavior in the mammalian cortex requires measurements of activity in awake, behaving animals. Rodents have emerged as a powerful model for dissecting the cortical circuits underlying behavior attributable to the convergence of several methods. Genetically encoded calcium indicators combined with viral-mediated or transgenic tools enable chronic monitoring of calcium signals in neuronal populations and subcellular structures of identified cell types. Stable one- and two-photon imaging of neuronal activity in awake, behaving animals is now possible using new behavioral paradigms in head-fixed animals, or using novel miniature head-mounted microscopes in freely moving animals. This mini-symposium will highlight recent applications of these methods for studying sensorimotor integration, decision making, learning, and memory in cortical and subcortical brain areas. We will outline future prospects and challenges for identifying the neural underpinnings of task-dependent behavior using cellular imaging in rodents.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3255-13.2013}, author = {Chen, Jerry L and Andermann, Mark L and Keck, Tara and Xu, Ning-long and Ziv, Yaniv} } @article {6655, title = {Imaging the transcriptome.}, journal = {Molecular Systems Biology}, volume = {9}, year = {2013}, month = {2013 Nov 26}, pages = {710}, issn = {1744-4292}, doi = {10.1038/msb.2013.67}, author = {Lionnet, Timoth{\'e}e} } @article {5884, title = {In vivo imaging of zebrafish embryogenesis.}, journal = {Methods}, volume = {62}, year = {2013}, month = {2013 Aug 15}, pages = {268-78}, abstract = {

The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis. Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key properties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms.

}, issn = {1095-9130}, doi = {10.1016/j.ymeth.2013.03.015}, author = {Keller, Philipp J} } @article {47634, title = {Incisive imaging and computation for cellular mysteries: lessons from abscission.}, journal = {Cell}, volume = {155}, year = {2013}, month = {2013 Dec 5}, pages = {1220-31}, abstract = {

The final cleavage event that terminates cell division, abscission of the small, dense intercellular bridge, has been particularly challenging to resolve. Here, we describe imaging innovations that helped answer long-standing questions about the mechanism of abscission. We further explain how computational modeling of high-resolution data was employed to test hypotheses and generate additional insights. We present the model that emerges from application of these complimentary approaches. Similar experimental strategies will undoubtedly reveal exciting details about other underresolved cellular structures.

}, keywords = {Animals, Cell Physiological Processes, Cytokinesis, Humans, Models, Biological, Plant Cells}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.11.011}, author = {Elia, Natalie and Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47636, title = {Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures.}, journal = {Autophagy}, volume = {9}, year = {2013}, month = {2013 Dec}, pages = {2154-5}, abstract = {

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for ?-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.

}, keywords = {Animals, Autophagy, Cell Culture Techniques, Cell Polarity, Cell Separation, Cells, Cultured, Collagen, Hepatocytes, Mitochondrial Dynamics, Primary Cell Culture, Rats, Tissue Scaffolds}, issn = {1554-8635}, doi = {10.4161/auto.26167}, author = {Fu, Dong and Lippincott-Schwartz, Jennifer and Arias, Irwin M} } @article {6453, title = {Infernal 1.1: 100-fold faster RNA homology searches.}, journal = {Bioinformatics}, volume = {29}, year = {2013}, month = {2013 Sep 19}, pages = {2933-5}, abstract = {

SUMMARY: Infernal builds probabilistic profiles of the sequence and secondary structure of an RNA family called covariance models (CMs) from structurally annotated multiple sequence alignments given as input. Infernal uses CMs to search for new family members in sequence databases and to create potentially large multiple sequence alignments. Version 1.1 of Infernal introduces a new filter pipeline for RNA homology search based on accelerated profile hidden Markov model (HMM) methods and HMM-banded CM alignment methods. This enables \~{}100-fold acceleration over the previous version and \~{}10 000-fold acceleration over exhaustive non-filtered CM searches. AVAILABILITY: Source code, documentation and the benchmark are downloadable from http://infernal.janelia.org. Infernal is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X. Documentation includes a user{\textquoteright}s guide with a tutorial, a discussion of file formats and user options and additional details on methods implemented in the software. CONTACT: nawrockie@janelia.hhmi.org.

}, issn = {1367-4811}, doi = {10.1093/bioinformatics/btt509}, author = {Nawrocki, Eric P and Eddy, Sean R.} } @article {46575, title = {Inferring neural population dynamics from multiple partial recordings of the same neural circuit.}, journal = {Advances in Neural Information Processing Systems (NIPS) }, volume = {26}, year = {2013}, month = {2013}, abstract = {

Simultaneous recordings of the activity of large neural populations are extremely valuable as they can be used to infer the dynamics and interactions of neurons in a local circuit, shedding light on the computations performed. It is now possible to measure the activity of hundreds of neurons using 2-photon calcium imaging. However, many computations are thought to involve circuits consisting of thousands of neurons, such as cortical barrels in rodent somatosensory cortex. Here we contribute a statistical method for stitching" together sequentially imaged sets of neurons into one model by phrasing the problem as fitting a latent dynamical system with missing observations. This method allows us to substantially expand the population-sizes for which population dynamics can be characterized---beyond the number of simultaneously imaged neurons. In particular, we demonstrate using recordings in mouse somatosensory cortex that this method makes it possible to predict noise correlations between non-simultaneously recorded neuron pairs.

}, url = {http://papers.nips.cc/paper/4874-inferring-neural-population-dynamics-from-multiple-partial-recordings-of-the-same-neural-circuit}, author = {Turaga, Srinivas C and Buesing, Lars and Packer, Adam M. and Dalgleish, Henry and Pettit, Noah and H{\"a}usser, Michael and Macke, Jakob H.} } @article {47899, title = {The influence of synaptic weight distribution on neuronal population dynamics.}, journal = {PLoS computational biology}, volume = {9}, year = {2013}, month = {2013 Oct}, pages = {e1003248}, abstract = {

The manner in which different distributions of synaptic weights onto cortical neurons shape their spiking activity remains open. To characterize a homogeneous neuronal population, we use the master equation for generalized leaky integrate-and-fire neurons with shot-noise synapses. We develop fast semi-analytic numerical methods to solve this equation for either current or conductance synapses, with and without synaptic depression. We show that its solutions match simulations of equivalent neuronal networks better than those of the Fokker-Planck equation and we compute bounds on the network response to non-instantaneous synapses. We apply these methods to study different synaptic weight distributions in feed-forward networks. We characterize the synaptic amplitude distributions using a set of measures, called tail weight numbers, designed to quantify the preponderance of very strong synapses. Even if synaptic amplitude distributions are equated for both the total current and average synaptic weight, distributions with sparse but strong synapses produce higher responses for small inputs, leading to a larger operating range. Furthermore, despite their small number, such synapses enable the network to respond faster and with more stability in the face of external fluctuations.

}, keywords = {Action Potentials, Computer Simulation, Models, Neurological, Neurons, Synapses}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.1003248}, author = {Iyer, Ramakrishnan and Menon, Vilas and Buice, Michael and Koch, Christof and Mihalas, Stefan} } @article {47638, title = {Insulin triggers surface-directed trafficking of sequestered GLUT4 storage vesicles marked by Rab10.}, journal = {Small GTPases}, volume = {4}, year = {2013}, month = {2013 Jul-Sep}, pages = {193-7}, abstract = {

Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don{\textquoteright}t interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

}, keywords = {Adipocytes, Animals, Cell Line, Cell Membrane, Endosomes, Glucose Transporter Type 4, Insulin, Mice, rab GTP-Binding Proteins, RNA Interference, RNA, Small Interfering}, issn = {2154-1256}, doi = {10.4161/sgtp.26471}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47219, title = {Integration of the olfactory code across dendritic claws of single mushroom body neurons.}, journal = {Nature Neuroscience}, volume = {16}, year = {2013}, month = {2013 Dec}, pages = {1821-9}, abstract = {

In the olfactory system, sensory inputs are arranged in different glomerular channels, which respond in combinatorial ensembles to the various chemical features of an odor. We investigated where and how this combinatorial code is read out deeper in the brain. We exploited the unique morphology of neurons in the Drosophila mushroom body, which receive input on large dendritic claws. Imaging odor responses of these dendritic claws revealed that input channels with distinct odor tuning converge on individual mushroom body neurons. We determined how these inputs interact to drive the cell to spike threshold using intracellular recordings to examine mushroom body responses to optogenetically controlled input. Our results provide an elegant explanation for the characteristic selectivity of mushroom body neurons: these cells receive different types of input and require those inputs to be coactive to spike. These results establish the mushroom body as an important site of integration in the fly olfactory system.

}, keywords = {Animals, Animals, Genetically Modified, Calcium, Dendrites, Drosophila, Drosophila Proteins, Electric Stimulation, Female, Luminescent Proteins, Membrane Potentials, Models, Neurological, Mushroom Bodies, Neurons, Odors, Olfactory Pathways, Patch-Clamp Techniques, Photic Stimulation, Rhodopsin, Synapses}, issn = {1546-1726}, doi = {10.1038/nn.3547}, author = {Gruntman, Eyal and Turner, Glenn C} } @article {47746, title = {Integration of the olfactory code across dendritic claws of single mushroom body neurons.}, journal = {Nature Neuroscience}, volume = {16}, year = {2013}, month = {2013 Oct 20}, pages = {1821-9}, abstract = {

In the olfactory system, sensory inputs are arranged in different glomerular channels, which respond in combinatorial ensembles to the various chemical features of an odor. Here we investigate where and how this combinatorial code is read out deeper in the brain. We exploit the unique morphology of neurons in the mushroom body (MB), which receive input on large dendritic claws. Imaging odor responses of these dendritic claws shows that input channels with distinct odor tuning converge on individual MB neurons. We determined how these inputs interact to drive the cell to spike threshold using intracellular recordings to examine MB responses to optogenetically controlled input. Our results provide an elegant explanation for the characteristic selectivity of MB neurons: these cells receive different types of input, and require those inputs to be coactive in order to spike. These results establish the MB as an important site of integration in the fly olfactory system.

}, doi = {10.1038/nn.3547}, author = {Gruntman, Eyal and Turner, Glenn C} } @article {6528, title = {Intensity discrimination deficits cause habituation changes in middle-aged Caenorhabditis elegans.}, journal = {Neurobiology of Aging}, volume = {34}, year = {2013}, month = {2013 Feb}, pages = {621-31}, abstract = {

The ability to learn and remember is critical for all animals to survive in the ever-changing environment. As we age, many of our biological faculties decay and of these, decline in learning and memory can be the most distressing. To carefully define age-dependent changes in learning during reproductive age in the nematode Caenorhabditis elegans, we performed a parametric behavioral study of habituation to nonlocalized mechanical stimuli (petri plate taps) over a range of intensities in middle-aged worms. We found that as worms age (from the onset of reproduction to the end of egg laying), response probability habituation increases (at both 10- and 60-second interstimulus intervals) and that these age-related changes were associated with a decrease in the discrimination between stimuli of different intensities. We also used optogenetics to investigate where these age-dependent changes occur. Our data suggest that the changes occur upstream of mechanosensory neuron depolarization. These data support the idea that declines in stimulus intensity discrimination abilities during aging may be one variable underlying age-related cognitive deficits.

}, keywords = {Age Factors, Aging, Animal, Animals, Behavior, Caenorhabditis elegans, Discrimination (Psychology), Habituation, Memory, Physical Stimulation, Psychophysiologic, Short-Term}, issn = {1558-1497}, doi = {10.1016/j.neurobiolaging.2012.03.016}, author = {Timbers, Tiffany A and Giles, Andrew C and Ardiel, Evan L and Kerr, Rex A and Rankin, Catharine H} } @article {6578, title = {Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Nov 5}, pages = {e01319}, abstract = {

Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an \~{}16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01319}, author = {Smith, F Donelson and Reichow, Steve L and Esseltine, Jessica L and Shi, Dan and Langeberg, Lorene K and Scott, John D and Gonen, Tamir} } @article {46726, title = {IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Jan 15}, pages = {176-89}, abstract = {

In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted β-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP(3)-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization.

}, keywords = {Actins, Cell Line, Tumor, Cell Movement, Cell Polarity, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases, HSP27 Heat-Shock Proteins, Humans, Intracellular Signaling Peptides and Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, PTEN Phosphohydrolase, RNA, Messenger, Signal Transduction}, issn = {1549-5477}, doi = {10.1101/gad.177642.111}, author = {St{\"o}hr, Nadine and K{\"o}hn, Marcel and Lederer, Marcell and Glass, Markus and Reinke, Claudia and Singer, Robert H and H{\"u}ttelmaier, Stefan} } @article {6511, title = {Illuminating vertebrate olfactory processing.}, journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {32}, year = {2012}, month = {2012 Oct 10}, pages = {14102-8}, abstract = {

The olfactory system encodes information about molecules by spatiotemporal patterns of activity across distributed populations of neurons and extracts information from these patterns to control specific behaviors. Recent studies used in vivo recordings, optogenetics, and other methods to analyze the mechanisms by which odor information is encoded and processed in the olfactory system, the functional connectivity within and between olfactory brain areas, and the impact of spatiotemporal patterning of neuronal activity on higher-order neurons and behavioral outputs. The results give rise to a faceted picture of olfactory processing and provide insights into fundamental mechanisms underlying neuronal computations. This review focuses on some of this work presented in a Mini-Symposium at the Annual Meeting of the Society for Neuroscience in 2012.

}, keywords = {Animals, Humans, Odors, Olfactory Bulb, Olfactory Pathways, Olfactory Receptor Neurons, Optogenetics}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3328-12.2012}, author = {Spors, Hartwig and Albeanu, Dinu Florin and Murthy, Venkatesh N and Rinberg, Dmitry and Uchida, Naoshige and Wachowiak, Matt and Friedrich, Rainer W} } @article {5163, title = {Imaging neural activity using Thy1-GCaMP transgenic mice.}, journal = {Neuron}, volume = {76}, year = {2012}, month = {2012 Oct 18}, pages = {297-308}, abstract = {

The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent protein based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as in awake behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain, and will greatly facilitate dissecting complex structural and functional relationships of neural networks.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2012.07.011}, author = {Chen, Qian and Cichon, Joseph and Wang, Wenting and Qiu, LI and Lee, Seok-Jin R. and Campbell, Nolan R. and DeStefino, Nicholas and Fu, Zhanyan and Yasuda, Ryohei and Looger, Loren L and Arenkiel, Benjamin R. and Gan, Wenbiao and Feng, Guoping} } @article {3933, title = {Imaging neural activity with genetically encoded calcium indicator.}, journal = {Cold Spring Harbor Protocols}, volume = {2012}, year = {2012}, month = {2012 Jun 1}, pages = {647-56}, abstract = {

Genetically encoded calcium indicators (GECIs), which are based on chimeric fluorescent proteins, can be used to monitor calcium transients in living cells and organisms. Because they are encoded by DNA, GECIs can be delivered to the intact brain noninvasively and targeted to defined populations of neurons and specific subcellular compartments for long-term, repeated measurements in vivo. GECIs have improved iteratively and are becoming useful for imaging neural activity in vivo. Here we summarize extrinsic and intrinsic factors that influence a GECI{\textquoteright}s performance and provides guidelines for selecting the appropriate GECI for a given application. We also review recent progress in GECI design, optimization, and standardized testing protocols.

}, author = {Tian, Lin and Hires, A and Looger, Loren L} } @article {5851, title = {Imaging the post-fusion release and capture of a vesicle membrane protein.}, journal = {Nature Communications}, volume = {3}, year = {2012}, month = {2012}, pages = {1154}, abstract = {

The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter{\textquoteright}s capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

}, issn = {2041-1723}, doi = {10.1038/ncomms2158}, author = {Sochacki, Kem A and Larson, Ben T and Sengupta, Deepali C and Daniels, Mathew P and Shtengel, Gleb and Hess, Harald F and Taraska, Justin W} } @article {46720, title = {Imaging translation in single cells using fluorescent microscopy.}, journal = {Cold Spring Harbor Perspectives in Biology}, volume = {4}, year = {2012}, month = {2012 Nov}, abstract = {

The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.

}, keywords = {Animals, Gene Expression Regulation, Humans, Microscopy, Fluorescence, Neurons, Protein Biosynthesis, Puromycin, Synapses}, issn = {1943-0264}, doi = {10.1101/cshperspect.a012310}, author = {Chao, Jeffrey A and Yoon, Young J and Singer, Robert H} } @article {46160, title = {ImgLib2--generic image processing in Java.}, journal = {Bioinformatics}, volume = {28}, year = {2012}, month = {2012 Nov 15}, pages = {3009-11}, abstract = {

SUMMARY: ImgLib2 is an open-source Java library for n-dimensional data representation and manipulation with focus on image processing. It aims at minimizing code duplication by cleanly separating pixel-algebra, data access and data representation in memory. Algorithms can be implemented for classes of pixel types and generic access patterns by which they become independent of the specific dimensionality, pixel type and data representation. ImgLib2 illustrates that an elegant high-level programming interface can be achieved without sacrificing performance. It provides efficient implementations of common data types, storage layouts and algorithms. It is the data model underlying ImageJ2, the KNIME Image Processing toolbox and an increasing number of Fiji-Plugins.

AVAILABILITY: ImgLib2 is licensed under BSD. Documentation and source code are available at http://imglib2.net and in a public repository at https://github.com/imagej/imglib.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online.

CONTACT: saalfeld@mpi-cbg.de

}, keywords = {Algorithms, Image Processing, Computer-Assisted, Programming Languages, Software}, issn = {1367-4811}, doi = {10.1093/bioinformatics/bts543}, author = {Pietzsch, Tobias and Preibisch, Stephan and Tomancak, Pavel and Saalfeld, Stephan} } @proceedings {46789, title = {ImgLib2{\textemdash}Generic Image Processing in Java}, year = {2012}, month = {2012 Oct 24}, address = {Mondorf-les-Bains, Luxembourg}, author = {Saalfeld, Stephan and Pietzsch, Tobias and Tomancak, Pavel and Preibisch, Stephan} } @conference {6924, title = {Implantable blood pressure monitoring cuff for small laboratory animal.}, booktitle = {Microsystems for Measurement and Instrumentation (MAMNA), 2012}, year = {2012}, month = {2012 Mar}, publisher = {IEEE}, organization = {IEEE}, address = {Annapolis, MD}, abstract = {

Continuous monitoring of blood pressure in laboratory animals is necessary to understand the effect of treatments for cardiovascular related conditions, such as hypertension. Current methods to measure laboratory rat blood pressure require the animal to be constrained. Our proposed method is a small implantable device which fits around the carotid artery of the rat. Initial data from a mock rat artery setup, with equivalent artery pressure as found in the rat, show that the cuff design effectively detects the pressure change inside the mock artery.

}, isbn = {978-1-4673-1781-8}, doi = {10.1109/MAMNA.2012.6195099}, author = {Pais, R. and Duttaroy, A. and Wolever, J. and Dobbs, M. and Pastalkova, E} } @article {4595, title = {An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea.}, journal = {The ISME Journal}, volume = {6}, year = {2012}, month = {2012 Mar}, pages = {610-8}, abstract = {

Reference phylogenies are crucial for providing a taxonomic framework for interpretation of marker gene and metagenomic surveys, which continue to reveal novel species at a remarkable rate. Greengenes is a dedicated full-length 16S rRNA gene database that provides users with a curated taxonomy based on de novo tree inference. We developed a {\textquoteright}taxonomy to tree{\textquoteright} approach for transferring group names from an existing taxonomy to a tree topology, and used it to apply the Greengenes, National Center for Biotechnology Information (NCBI) and cyanoDB (Cyanobacteria only) taxonomies to a de novo tree comprising 408 315 sequences. We also incorporated explicit rank information provided by the NCBI taxonomy to group names (by prefixing rank designations) for better user orientation and classification consistency. The resulting merged taxonomy improved the classification of 75\% of the sequences by one or more ranks relative to the original NCBI taxonomy with the most pronounced improvements occurring in under-classified environmental sequences. We also assessed candidate phyla (divisions) currently defined by NCBI and present recommendations for consolidation of 34 redundantly named groups. All intermediate results from the pipeline, which includes tree inference, jackknifing and transfer of a donor taxonomy to a recipient tree (tax2tree) are available for download. The improved Greengenes taxonomy should provide important infrastructure for a wide range of megasequencing projects studying ecosystems on scales ranging from our own bodies (the Human Microbiome Project) to the entire planet (the Earth Microbiome Project). The implementation of the software can be obtained from http://sourceforge.net/projects/tax2tree/.

}, issn = {1751-7370}, doi = {10.1038/ismej.2011.139}, author = {McDonald, Daniel and Price, Morgan N and Goodrich, Julia and Nawrocki, Eric P and Desantis, Todd Z and Probst, Alexander and Andersen, Gary L and Knight, Rob and Hugenholtz, Philip} } @article {4921, title = {The influence of lipids on voltage-gated ion channels.}, journal = {Current Opinion in Structural Biology}, volume = {22}, year = {2012}, month = {2012 Apr 5}, pages = {529-36}, abstract = {

Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs.

}, issn = {1879-033X}, doi = {10.1016/j.sbi.2012.03.009}, author = {Jiang, Qiu-Xing and Gonen, Tamir} } @article {5323, title = {Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras.}, journal = {Journal of Microscopy}, volume = {246}, year = {2012}, month = {2012 Jun}, pages = {229-36}, abstract = {

In an interferometer-based fluorescence microscope, a beam splitter is often used to combine two emission wavefronts interferometrically. There are two perpendicular paths along which the interference fringes can propagate and normally only one is used for imaging. However, the other path also contains useful information. Here we introduced a second camera to our interferometer-based three-dimensional structured-illumination microscope (I(5)S) to capture the fringes along the normally unused path, which are out of phase by π relative to the fringes along the other path. Based on this complementary phase relationship and the well-defined phase interrelationships among the I(5)S data components, we can deduce and then computationally eliminate the path length errors within the interferometer loop using the simultaneously recorded fringes along the two imaging paths. This self-correction capability can greatly relax the requirement for eliminating the path length differences before and maintaining that status during each imaging session, which are practically challenging tasks. Experimental data is shown to support the theory.

}, issn = {1365-2818}, doi = {10.1111/j.1365-2818.2012.03604.x}, author = {Shao, L and Winoto, L and Agard, D A and Gustafsson, M G L and Sedat, J W} } @article {6327, title = {Interplay of DNA repair with transcription: from structures to mechanisms.}, journal = {Trends in Biochemical Sciences}, volume = {37}, year = {2012}, month = {2012 Dec}, pages = {543-52}, abstract = {

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions.

}, issn = {0968-0004}, doi = {10.1016/j.tibs.2012.09.002}, author = {Deaconescu, Alexandra M and Artsimovitch, Irina and Grigorieff, Nikolaus} } @article {46324, title = {Intracellular recording in behaving animals.}, journal = {Current Opinion in Neurobiology}, volume = {22}, year = {2012}, month = {2012 Feb}, pages = {34-44}, abstract = {

Electrophysiological recordings from behaving animals provide an unparalleled view into the functional role of individual neurons. Intracellular approaches can be especially revealing as they provide information about a neuron{\textquoteright}s inputs and intrinsic cellular properties, which together determine its spiking output. Recent technical developments have made intracellular recording possible during an ever-increasing range of behaviors in both head-fixed and freely moving animals. These recordings have yielded fundamental insights into the cellular and circuit mechanisms underlying neural activity during natural behaviors in such areas as sensory perception, motor sequence generation, and spatial navigation, forging a direct link between cellular and systems neuroscience.

}, keywords = {Animals, Behavior, Animal, Brain, Electrophysiology, Intracellular Space, Neurophysiology}, issn = {1873-6882}, doi = {10.1016/j.conb.2011.10.013}, author = {Long, Michael A and Lee, Albert K} } @article {47653, title = {Intracellular spatial localization regulated by the microtubule network.}, journal = {PloS one}, volume = {7}, year = {2012}, month = {2012}, pages = {e34919}, abstract = {

The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a "structured cytoplasm", in contrast to a free and fluid environment.

}, keywords = {Animals, Cell Cycle, Centrosome, Cytoplasm, Drosophila, Microtubules, Models, Biological, Spindle Apparatus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0034919}, author = {Chen, Jing and Lippincott-Schwartz, Jennifer and Liu, Jian} } @article {65029, title = {An intra-cerebral drug delivery system for freely moving animals.}, journal = {Biomed Microdevices}, volume = {14}, year = {2012}, month = {2012 Oct 01}, pages = {799-809}, abstract = {

Microinfusions of drugs directly into the central nervous system of awake animals represent a widely used means of unravelling brain functions related to behaviour. However, current approaches generally use tethered liquid infusion systems and a syringe pump to deliver drugs into the brain, which often interfere with behaviour. We address this shortfall with a miniaturised electronically-controlled drug delivery system (20 {\texttimes} 17.5 {\texttimes} 5 mm{\textthreesuperior}) designed to be skull-mounted in rats. The device features a micropump connected to two 8-mm-long silicon microprobes with a cross section of 250 {\texttimes} 250 μm{\texttwosuperior} and integrated fluid microchannels. Using an external electronic control unit, the device allows infusion of 16 metered doses (0.25 μL each, 8 per silicon shaft). Each dosage requires 3.375 Ws of electrical power making the device additionally compatible with state-of-the-art wireless headstages. A dosage precision of 0.25 {\textpm} 0.01 μL was determined in vitro before in vivo tests were carried out in awake rats. No passive leakage from the loaded devices into the brain could be detected using methylene blue dye. Finally, the device was used to investigate the effects of the NMDA-receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid, (R)-CPP, administered directly into the prefrontal cortex of rats during performance on a task to assess visual attention and impulsivity. In agreement with previous findings using conventional tethered infusion systems, acute (R)-CPP administration produced a marked increase in impulsivity.

}, keywords = {Animals, Attention, Brain, Drug Delivery Systems, Equipment Design, Impulsive Behavior, Microinjections, Piperazines, Rats, Receptors, N-Methyl-D-Aspartate}, issn = {1572-8781}, doi = {10.1007/s10544-012-9659-2}, author = {Spieth, Sven and Schumacher, Axel and Holtzman, Tahl and Rich, P Dylan and Theobald, David E and Dalley, Jeffrey W and Nouna, Rachid and Messner, Stephan and Zengerle, Roland} } @article {5325, title = {Intra-nuclear mobility and target search mechanisms of transcription factors: a single-molecule perspective on gene expression.}, journal = {Biochimica et Biophysica Acta}, volume = {1819}, year = {2012}, month = {2012 Jun}, pages = {482-93}, abstract = {

Precise expression of specific genes in time and space is at the basis of cellular viability as well as correct development of organisms. Understanding the mechanisms of gene regulation is fundamental and still one of the great challenges for biology. Gene expression is regulated also by specific transcription factors that recognize and bind to specific DNA sequences. Transcription factors dynamics, and especially the way they sample the nucleoplasmic space during the search for their specific target in the genome, are a key aspect for regulation and it has been puzzling researchers for forty years. The scope of this review is to give a state-of-the-art perspective over the intra-nuclear mobility and the target search mechanisms of specific transcription factors at the molecular level. Going through the seminal biochemical experiments that have raised the first questions about target localization and the theoretical grounds concerning target search processes, we describe the most recent experimental achievements and current challenges in understanding transcription factors dynamics and interactions with DNA using in vitro assays as well as in live prokaryotic and eukaryotic cells. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

}, issn = {0006-3002}, doi = {10.1016/j.bbagrm.2012.02.001}, author = {Normanno, Davide and Dahan, Maxime and Darzacq, Xavier} } @article {4923, title = {Intrinsic tethering activity of endosomal Rab proteins.}, journal = {Nature Structural \& Molecular Biology}, volume = {19}, year = {2012}, month = {2012 Jan}, pages = {40-7}, abstract = {

Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion.

}, keywords = {Electron, Endocytosis, Endosomes, GTP-Binding Proteins, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Guanosine Diphosphate, Guanosine Triphosphate, Liposomes, Microscopy, Mutation, Protein Binding, rab GTP-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, SNARE Proteins, Transmission, Two-Hybrid System Techniques, Vesicular Transport Proteins}, issn = {1545-9985}, doi = {10.1038/nsmb.2162}, author = {Lo, Sheng-Ying and Brett, Christopher L and Plemel, Rachael L and Vignali, Marissa and Fields, Stanley and Gonen, Tamir and Merz, Alexey J} } @proceedings {46791, title = {Introduction to ImgLib2}, year = {2012}, month = {2012 Oct 24}, address = {Mondorf-les-Bains, Luxembourg}, author = {Preibisch, Stephan and Pietzsch, Tobias and Myers, Eugene and Tomancak, Pavel and Saalfeld, Stephan} } @proceedings {45795, title = {Investigation of the role of Aubergine RNA-binding proteins in the reproductive plasticity of the pea aphid, Acyrthosiphon pisum}, volume = {52}, year = {2012}, month = {01/2012}, pages = {E202-E202}, publisher = {OXFORD UNIV PRESS INC}, abstract = {

Environmental changes can elicit alterations in the form, behavior and/or physiology of all species, and this developmental response to environment is known as phenotypic plasticity. Despite its ubiquity, the molecular basis for phenotypic plasticity is not fully understood. The pea aphid, Acyrthosiphon pisum, serves as a model for an extreme form of phenotypic plasticity, known as polyphenism. Changes in photoperiod stimulate a switch in female aphid reproductive mode from asexual to sexual reproduction over the course of one generation without changes in genotype. This reproductive polyphenism results in female aphids with ovaries of one of two types: sexual ovaries (producing haploid oocytes via meiosis), or asexual ovaries (producing identical diploid aphid clones via parthenogenesis). To better understand how aphid ovaries could produce different outputs, we surveyed the transcriptomes of sexual and asexual ovaries using RNA-seq. Among genes that exhibited greater than two-fold differences in gene expression between sexual and asexual ovaries, we identified several aubergine paralogs, which encode for germline-specific members of the Argonaute small RNA-binding protein family. The A. pisum genome contains eight aubergine paralogs and at least two piwi paralogs. We are currently comparing the expression patterns of these aphid aubergine paralogs between asexual and sexual aphid ovaries. Aubergine proteins in other species are thought to help suppress the activity of transposable elements, which are found in high quantities throughout the A. pisum genome. Together, these experiments will help elucidate a potential relationship between aubergine paralogs and aphid reproductive plasticity.

}, url = {http://sicb.org/meetings/2012/schedule/abstractdetails.php?id=959}, author = {A Abdelhady and R Cortes and S Musumeci and D Srinivasan and S Shigenobu and D Stern and S Kobayashi} } @article {5923, title = {Ion channel gradients in the apical tuft region of CA1 pyramidal neurons.}, journal = {PLoS one}, volume = {7}, year = {2012}, month = {2012}, pages = {e46652}, abstract = {

Dendritic ion channels play a critical role in shaping synaptic input and are fundamentally important for synaptic integration and plasticity. In the hippocampal region CA1, somato-dendritic gradients of AMPA receptors and the hyperpolarization-activated cation conductance (I(h)) counteract the effects of dendritic filtering on the amplitude, time-course, and temporal integration of distal Schaffer collateral (SC) synaptic inputs within stratum radiatum (SR). While ion channel gradients in CA1 distal apical trunk dendrites within SR have been well characterized, little is known about the patterns of ion channel expression in the distal apical tuft dendrites within stratum lacunosum moleculare (SLM) that receive distinct input from the entorhinal cortex via perforant path (PP) axons. Here, we measured local ion channels densities within these distal apical tuft dendrites to determine if the somato-dendritic gradients of I(h) and AMPA receptors extend into distal tuft dendrites. We also determined the densities of voltage-gated sodium channels and NMDA receptors. We found that the densities of AMPA receptors, I(h,) and voltage-gated sodium channels are similar in tuft dendrites in SLM when compared with distal apical dendrites in SR, while the ratio of NMDA receptors to AMPA receptors increases in tuft dendrites relative to distal apical dendrites within SR. These data indicate that the somato-dendritic gradients of I(h) and AMPA receptors in apical dendrites do not extend into the distal tuft, and the relative densities of voltage-gated sodium channels and NMDA receptors are poised to support nonlinear integration of correlated SC and PP input.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0046652}, author = {Bittner, Katie C and Andrasfalvy, Bertalan K and Magee, Jeffrey C} } @article {3930, title = {Imaging light responses of targeted neuron populations in the rodent retina.}, journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {31}, year = {2011}, month = {2011 Feb 23}, pages = {2855-67}, abstract = {

Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates \>10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.

}, keywords = {Action Potentials, Animals, Female, Inbred C57BL, Male, Mice, Neurons, Ocular, Organ Culture Techniques, Retina, Vision, Voltage-Sensitive Dye Imaging}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.6064-10.2011}, author = {Borghuis, Bart G and Tian, Lin and Xu, Ying and Nikonov, Sergei S and Vardi, Noga and Zemelman, Boris V and Looger, Loren L} } @article {3522, title = {In search of the structure of human olfactory space.}, journal = {Frontiers in Systems Neuroscience}, volume = {5}, year = {2011}, month = {2011 Jun 2}, pages = {65}, abstract = {

We analyze the responses of human observers to an ensemble of monomolecular odorants. Each odorant is characterized by a set of 146 perceptual descriptors obtained from a database of odor character profiles. Each odorant is therefore represented by a point in a highly multidimensional sensory space. In this work we study the arrangement of odorants in this perceptual space. We argue that odorants densely sample a two-dimensional curved surface embedded in the multidimensional sensory space. This surface can account for more than half of the variance of the perceptual data. We also show that only 12\% of experimental variance cannot be explained by curved surfaces of substantially small dimensionality (\<10). We suggest that these curved manifolds represent the relevant spaces sampled by the human olfactory system, thereby providing surrogates for olfactory sensory space. For the case of 2D approximation, we relate the two parameters on the curved surface to the physico-chemical parameters of odorant molecules. We show that one of the dimensions is related to eigenvalues of molecules{\textquoteright} connectivity matrix, while the other is correlated with measures of molecules{\textquoteright} polarity. We discuss the behavioral significance of these findings.

}, author = {Koulakov, Alexei and Kolterman, Brian E. and Enikolopov, Armen and Rinberg, Dmitry} } @article {2311, title = {Inputs to the dorsal striatum of the mouse reflect the parallel circuit architecture of the forebrain.}, journal = {Frontiers in Neuroanatomy}, volume = {4}, year = {2011}, month = {2011}, pages = {147}, abstract = {

The basal ganglia play a critical role in the regulation of voluntary action in vertebrates. Our understanding of the function of the basal ganglia relies heavily upon anatomical information, but continued progress will require an understanding of the specific functional roles played by diverse cell types and their connectivity. An increasing number of mouse lines allow extensive identification, characterization, and manipulation of specified cell types in the basal ganglia. Despite the promise of genetically modified mice for elucidating the functional roles of diverse cell types, there is relatively little anatomical data obtained directly in the mouse. Here we have characterized the retrograde labeling obtained from a series of tracer injections throughout the dorsal striatum of adult mice. We found systematic variations in input along both the medial-lateral and anterior-posterior neuraxes in close agreement with canonical features of basal ganglia anatomy in the rat. In addition to the canonical features we have provided experimental support for the importance of non-canonical inputs to the striatum from the raphe nuclei and the amygdala. To look for organization at a finer scale we have analyzed the correlation structure of labeling intensity across our entire dataset. Using this analysis we found substantial local heterogeneity within the large-scale order. From this analysis we conclude that individual striatal sites receive varied combinations of cortical and thalamic input from multiple functional areas, consistent with some earlier studies in the rat that have suggested the presence of a combinatorial map.

}, issn = {1662-5129}, doi = {10.3389/fnana.2010.00147}, author = {Pan, Weixing X and Mao, Tianyi and Dudman, Joshua T} } @article {46608, title = {Insulin attenuates the acquisition and expression of ethanol-induced locomotor sensitization in DBA/2J mice.}, journal = {Life Sciences}, volume = {89}, year = {2011}, month = {2011 Dec 19}, pages = {968-74}, abstract = {

AIM: Ethanol-induced locomotor sensitization is a behavioral manifestation of physiological responses to repeated ethanol exposures. While ethanol exerts direct effects on multiple neurotransmitter systems in the brain, ethanol-induced changes in metabolic state, including acute hyperglycemia and inhibition of insulin signaling, also have plausible roles in the expression of ethanol-related behaviors through direct and indirect effects on brain function. The current experiments examined whether insulin administration or the resultant hypoglycemia might attenuate the development of sensitization to the locomotor stimulant effect of ethanol.

MAIN METHODS: Male and female DBA/2J mice received daily injections of 5 or 10 IU/kg insulin before or after a stimulating dose of ethanol and subsequent testing in an automated activity monitor. Blood glucose levels were determined upon the completion of the experiments.

KEY FINDINGS: Insulin injected prior to ethanol blunted the acute stimulant response as well as the acquisition and expression of locomotor sensitization, while insulin given after ethanol did not affect the development of the sensitized response. In a separate experiment, mice given glucose concurrently with insulin developed ethanol-induced locomotor sensitization normally.

SIGNIFICANCE: These experiments suggest that insulin attenuates the development of ethanol-induced locomotor sensitization, and that blood glucose levels can largely account for this effect. Further studies of the role of ethanol-induced metabolic states should provide novel information on the expression of ethanol-related behaviors.

}, keywords = {Animals, Behavior, Animal, Blood Glucose, Central Nervous System Depressants, Dose-Response Relationship, Drug, Ethanol, Female, Glucose, Hypoglycemia, Hypoglycemic Agents, Insulin, Male, Mice, Mice, Inbred DBA, Motor Activity}, issn = {1879-0631}, doi = {10.1016/j.lfs.2011.10.011}, author = {Kliethermes, Christopher L and Heberlein, Ulrike} } @article {2694, title = {Intracellular determinants of hippocampal CA1 place and silent cell activity in a novel environment.}, journal = {Neuron}, volume = {70}, year = {2011}, month = {2011 Apr 14}, pages = {109-20}, abstract = {

For each environment a rodent has explored, its hippocampus contains a map consisting of a unique subset of neurons, called place cells, that have spatially tuned spiking there, with the remaining neurons being essentially silent. Using whole-cell recording in freely moving rats exploring a novel maze, we observed differences in intrinsic cellular properties and input-based subthreshold membrane potential levels underlying this division into place and silent cells. Compared to silent cells, place cells had lower spike thresholds and peaked versus flat subthreshold membrane potentials as a function of animal location. Both differences were evident from the beginning of exploration. Additionally, future place cells exhibited higher burst propensity before exploration. Thus, internal settings appear to predetermine which cells will represent the next novel environment encountered. Furthermore, place cells fired spatially tuned bursts with large, putatively calcium-mediated depolarizations that could trigger plasticity and stabilize the new map for long-term storage. Our results provide new insight into hippocampal memory formation.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2011.03.006}, author = {Epsztein, J{\'e}r{\^o}me and Brecht, Michael and Lee, Albert K} } @article {46300, title = {Intracellular dynamics of virtual place cells.}, journal = {Neural Computation}, volume = {23}, year = {2011}, month = {2011 Mar}, pages = {651-5}, abstract = {

The pattern of spikes recorded from place cells in the rodent hippocampus is strongly modulated by both the spatial location in the environment and the theta rhythm. The phases of the spikes in the theta cycle advance during movement through the place field. Recently intracellular recordings from hippocampal neurons (Harvey, Collman, Dombeck, \& Tank, 2009 ) showed an increase in the amplitude of membrane potential oscillations inside the place field, which was interpreted as evidence that an intracellular mechanism caused phase precession. Here we show that an existing network model of the hippocampus (Tsodyks, Skaggs, Sejnowski, \& McNaughton, 1996 ) can equally reproduce this and other aspects of the intracellular recordings, which suggests that new experiments are needed to distinguish the contributions of intracellular and network mechanisms to phase precession.

}, issn = {1530-888X}, doi = {10.1162/NECO_a_00087}, author = {Romani, Sandro and Sejnowski, Terrence J and Tsodyks, Misha} } @article {2751, title = {Identification of genes controlled by LMX1B in E13.5 mouse limbs.}, journal = {Developmental Dynamics: An Official Publication of the American Association of Anatomists}, volume = {239}, year = {2010}, month = {2010 Aug}, pages = {2246-55}, abstract = {

During limb development, the dorsal limb mesenchyme expression of the transcription factor LMX1B is required for dorsoventral limb patterning. In mice, Lmx1b mutations result in the mirror-image duplication of ventral limb structures and loss of dorsal limb structures. Heterozygous LMX1B mutations in humans cause the Nail-Patella Syndrome characterized by limb, kidney, and eye developmental defects. We used DNA microarrays to compare the mRNAs in E13.5 mouse Lmx1b mutant and wild-type limbs. We report 14 genes that require Lmx1b for their normal expression in the dorsal limb or the restriction of their expression to the ventral limb.

}, keywords = {Animals, Body Patterning, Extremities, Gene Expression, Genes, Homeodomain Proteins, Humans, Messenger, Mice, Oligonucleotide Array Sequence Analysis, RNA, Transcription Factors}, issn = {1097-0177}, doi = {10.1002/dvdy.22357}, author = {Gu, Wendy X W and Kania, Artur} } @article {5689, title = {Identifying neuronal lineages of Drosophila by sequence analysis of axon tracts.}, journal = {The Journal of Neuroscience}, volume = {30}, year = {2010}, month = {2010 Jun 2}, pages = {7538-53}, abstract = {

The Drosophila brain is formed by an invariant set of lineages, each of which is derived from a unique neural stem cell (neuroblast) and forms a genetic and structural unit of the brain. The task of reconstructing brain circuitry at the level of individual neurons can be made significantly easier by assigning neurons to their respective lineages. In this article we address the automation of neuron and lineage identification. We focused on the Drosophila brain lineages at the larval stage when they form easily recognizable secondary axon tracts (SATs) that were previously partially characterized. We now generated an annotated digital database containing all lineage tracts reconstructed from five registered wild-type brains, at higher resolution and including some that were previously not characterized. We developed a method for SAT structural comparisons based on a dynamic programming approach akin to nucleotide sequence alignment and a machine learning classifier trained on the annotated database of reference SATs. We quantified the stereotypy of SATs by measuring the residual variability of aligned wild-type SATs. Next, we used our method for the identification of SATs within wild-type larval brains, and found it highly accurate (93-99\%). The method proved highly robust for the identification of lineages in mutant brains and in brains that differed in developmental time or labeling. We describe for the first time an algorithm that quantifies neuronal projection stereotypy in the Drosophila brain and use the algorithm for automatic neuron and lineage recognition.

}, keywords = {Algorithms, Animals, Automatic Data Processing, Axons, Brain, Brain Mapping, Caspase 3, Cell Lineage, Computer-Assisted, Confocal, Dendrites, Drosophila, Drosophila Proteins, Embryo, Genetically Modified, Green Fluorescent Proteins, Image Processing, Microscopy, Models, Neurological, Neurons, Nonmammalian, Reproducibility of Results, Software}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0186-10.2010}, author = {Cardona, Albert and Saalfeld, Stephan and Arganda, Ignacio and Pereanu, Wayne and Schindelin, Johannes and Hartenstein, Volker.} } @article {47676, title = {Imaging: visualizing the possibilities.}, journal = {Journal of cell science}, volume = {123}, year = {2010}, month = {2010 Nov 1}, pages = {3619-20}, keywords = {Diagnostic Imaging, Fluorescence Recovery After Photobleaching, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Humans, Image Enhancement, Molecular Probes, Photoaffinity Labels}, issn = {1477-9137}, doi = {10.1242/jcs081539}, author = {Lippincott-Schwartz, Jennifer} } @article {1344, title = {Impact of spikelets on hippocampal CA1 pyramidal cell activity during spatial exploration.}, journal = {Science}, volume = {327}, year = {2010}, month = {2010 Jan 22}, pages = {474-7}, abstract = {

In vivo intracellular recordings of hippocampal neurons reveal the occurrence of fast events of small amplitude called spikelets or fast prepotentials. Because intracellular recordings have been restricted to anesthetized or head-fixed animals, it is not known how spikelet activity contributes to hippocampal spatial representations. We addressed this question in CA1 pyramidal cells by using in vivo whole-cell recording in freely moving rats. We observed a high incidence of spikelets that occurred either in isolation or in bursts and could drive spiking as fast prepotentials of action potentials. Spikelets strongly contributed to spiking activity, driving approximately 30\% of all action potentials. CA1 pyramidal cell firing and spikelet activity were comodulated as a function of the animal{\textquoteright}s location in the environment. We conclude that spikelets have a major impact on hippocampal activity during spatial exploration.

}, keywords = {Action Potentials, Animals, CA1 Region, Exploratory Behavior, Hippocampal, Male, Maze Learning, Patch-Clamp Techniques, Pyramidal Cells, Rats, Space Perception, Wistar}, issn = {1095-9203}, doi = {10.1126/science.1182773}, author = {Epsztein, J{\'e}r{\^o}me and Lee, Albert K and Chorev, Edith and Brecht, Michael} } @article {48167, title = {Improving deformable surface meshes through omni-directional displacements and MRFs.}, journal = {Medical image computing and computer-assisted intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention}, volume = {13}, year = {2010}, month = {2010}, pages = {227-34}, abstract = {

Deformable surface models are often represented as triangular meshes in image segmentation applications. For a fast and easily regularized deformation onto the target object boundary, the vertices of the mesh are commonly moved along line segments (typically surface normals). However, in case of high mesh curvature, these lines may intersect with the target boundary at "non-corresponding" positions, or even not at all. Consequently, certain deformations cannot be achieved. We propose an approach that allows each vertex to move not only along a line segment, but within a surrounding sphere. We achieve globally regularized deformations via Markov Random Field optimization. We demonstrate the potential of our approach with experiments on synthetic data, as well as an evaluation on 2 x 106 coronoid processes of the mandible in Cone-Beam CTs, and 56 coccyxes (tailbones) in low-resolution CTs.

}, keywords = {Algorithms, Computer Simulation, Cone-Beam Computed Tomography, Finite Element Analysis, Humans, Imaging, Three-Dimensional, Models, Biological, Radiographic Image Enhancement, Radiographic Image Interpretation, Computer-Assisted, Reproducibility of Results, Sensitivity and Specificity}, author = {Kainmueller, D and Lamecker, H and Seim, H and Zachow, S and Hege, H C} } @article {1245, title = {Increasing depth resolution of electron microscopy of neural circuits using sparse tomographic reconstruction.}, journal = {Computer Vision and Pattern Recognition (CVPR)}, year = {2010}, month = {2010}, pages = {1767-74}, issn = {1063-6919}, doi = {10.1109/CVPR.2010.5539846}, author = {Veeraraghavan, Ashok and Genkin, Alex V and Vitaladevuni, Shiv and Scheffer, Lou and Xu, CShan and Hess, Harald and Fetter, Richard and Cantoni, Marco and Knott, Graham and Chklovskii, Dmitri B} } @article {5687, title = {An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.}, journal = {PLoS Biology}, volume = {8}, year = {2010}, month = {2010}, abstract = {

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

}, keywords = {Animals, Brain, Computer-Assisted, Drosophila melanogaster, Electron, Humans, Image Processing, Imaging, Microscopy, Neurons, Neuropil, Software, Synapses, Three-Dimensional, Transmission}, issn = {1545-7885}, doi = {10.1371/journal.pbio.1000502}, author = {Cardona, Albert and Saalfeld, Stephan and Preibisch, Stephan and Schmid, Benjamin and Cheng, Anchi and Pulokas, Jim and Tomancak, Pavel and Hartenstein, Volker.} } @article {3635, title = {Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system.}, journal = {mBio}, volume = {1}, year = {2010}, month = {2010}, abstract = {

The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.

}, keywords = {Bacterial Proteins, Membrane Transport Proteins, Models, Molecular, Protein Binding, Protein Multimerization, Salmonella typhimurium}, issn = {2150-7511}, doi = {10.1128/mBio.00158-10}, author = {Sanowar, Sarah and Singh, Pragya and Pfuetzner, Richard A and Andr{\'e}, Ingemar and Zheng, Hongjin and Spreter, Thomas and Strynadka, Natalie C J and Gonen, Tamir and Baker, David and Goodlett, David R and Miller, Samuel I} } @proceedings {46792, title = {Into ImgLib{\textemdash}Generic image processing in Java}, year = {2010}, month = {2012 Oct 27}, address = {Mondorf-les-Bains, Luxembourg}, abstract = {

The purpose of ImgLib, a Generic Java Image Processing Library, is to provide an abstract framework enabling Java developers to design and implement data processing algorithms without having to consider dimensionality, type of data (e. g. byte, float, complex float), or strategies for data access (e. g. linear arrays, cells, paged cells). This kind of programming has significant advantages over the classical way. An algorithm written once for a certain class of Type will potentially run on any compatible Type, even if it does not exist yet. Same applies for data access strategies and the number of dimensions.
We achieve this abstraction by accessing data through Iterators and Type interfaces. Iterators guarantee efficient traversal through pixels depending on whether random coordinate access is required or just all pixels have to be visited once, whether real or integer coordinates are accessed, whether coordinates outside of image boundaries are accessed or not. Type interfaces define the supported operators on pixel values (like basic algebra) and hide the underlying basic type from algorithm implementation.

}, author = {Preibisch, Stephan and Tomancak, Pavel and Saalfeld, Stephan} } @article {2785, title = {Ion trap mass analysis at high pressure: an experimental characterization.}, journal = {Journal of Mass Spectrometry}, volume = {45}, year = {2010}, month = {2010 Jan}, pages = {26-34}, abstract = {

In recent years, it has become increasingly interesting to understand the performance of mass spectrometers at pressures much higher than those employed with conventional operating conditions. This interest has been driven by several influences, including demand for the development of reduced-power miniature mass spectrometers, desire for improved ion transfer into and through mass spectrometers, enhanced-yield preparative mass separations, and mass filtering at the atmospheric pressure interface. In this study, an instrument was configured to allow for the performance characterization of a rectilinear ion trap (RIT) at pressures up to 50 mtorr with air used as the buffer gas. The mass analysis efficiency, mass resolution, isolation efficiency, and collision-induced dissociation (CID) efficiency were evaluated at pressures ranging from 1 to 50 mtorr. The extent of degradation of mass resolution, isolation efficiency and ion stability as functions of pressure were characterized. Also, the optimal resonance ejection conditions were obtained at various pressures. Operations at 50 mtorr demonstrated improved CID efficiency in addition to peak widths of 2 and 5 m/z units (full width at half-maximum, FWHM) for protonated caffeine (m/z 195) and Ultramark (m/z 1521) respectively.

}, keywords = {Equipment Design, Ions, Mass Spectrometry, Pressure, Proteins}, issn = {1096-9888}, doi = {10.1364/AO.50.001792}, author = {Song, Qingyu and Xu, Wei and Smith, Scott A and Gao, Liang and Chappell, William J and Cooks, R Graham and Ouyang, Zheng} } @article {6346, title = {IP3 receptor sensitization during in vivo amphetamine experience enhances NMDA receptor plasticity in dopamine neurons of the ventral tegmental area.}, journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {30}, year = {2010}, month = {2010 May 12}, pages = {6689-99}, abstract = {

Synaptic plasticity in the mesolimbic dopamine (DA) system is critically involved in reward-based conditioning and the development of drug addiction. Ca2+ signals triggered by postsynaptic action potentials (APs) drive the induction of synaptic plasticity in the CNS. However, it is not clear how AP-evoked Ca2+ signals and the resulting synaptic plasticity are altered during in vivo exposure to drugs of abuse. We have recently described long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated transmission onto DA neurons that is induced in a manner dependent on bursts of APs. LTP induction requires amplification of burst-evoked Ca2+ signals by preceding activation of metabotropic glutamate receptors (mGluRs) generating inositol 1,4,5-trisphosphate (IP3). In this study, using brain slices prepared from male rats, we show that repeated in vivo exposure to the psychostimulant amphetamine (5 mg/kg, i.p., 3-7 d) upregulates mGluR-dependent facilitation of burst-evoked Ca2+ signals in DA neurons of the ventral tegmental area (VTA). Protein kinase A (PKA)-induced sensitization of IP3 receptors mediates this upregulation of mGluR action. As a consequence, NMDAR-mediated transmission becomes more susceptible to LTP induction after repeated amphetamine exposure. We have also found that the magnitude of amphetamine-conditioned place preference (CPP) in behaving rats correlates with the magnitude of mGluR-dependent Ca2+ signal facilitation measured in VTA slices prepared from these rats. Furthermore, the development of amphetamine CPP is significantly attenuated by intra-VTA infusion of the PKA inhibitor H89. We propose that enhancement of mGluR-dependent NMDAR plasticity in the VTA may promote the learning of environmental stimuli repeatedly associated with amphetamine experience.

}, keywords = {4, 5-Trisphosphate Receptors, Amphetamine, Animals, Calcium, Central Nervous System Stimulants, Classical, Conditioning, Cyclic AMP-Dependent Protein Kinases, Dopamine, Inositol 1, Isoquinolines, Long-Term Potentiation, Male, N-Methyl-D-Aspartate, Neurons, Protein Kinase Inhibitors, Rats, Receptors, Space Perception, Sprague-Dawley, Sulfonamides, Synaptic Transmission, Ventral Tegmental Area}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.4453-09.2010}, author = {Ahn, Kee-Chan and Bernier, Brian E and Harnett, Mark T and Morikawa, Hitoshi} } @article {2148, title = {Imaging informatics for personalised medicine: applications and challenges.}, journal = {International Journal of Functional Informatics and Personalised Medicine}, volume = {2}, year = {2009}, month = {2009}, pages = {125-35}, abstract = {

Imaging informatics has emerged as a major research theme in biomedicine in the last few decades. Currently, personalised, predictive and preventive patient care is believed to be one of the top priorities in biomedical research and practice. Imaging informatics plays a major role in biomedicine studies. This paper reviews main applications and challenges of imaging informatics in biomedicine.

}, issn = {1756-2112}, doi = {10.1007/s12021-010-9090-x}, author = {Liu, Tianming and Peng, Hanchuan and Zhou, Xiaobo} } @article {1646, title = {Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.}, journal = {Nature Methods}, volume = {6}, year = {2009}, month = {2009 Dec}, pages = {875-81}, abstract = {

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

}, keywords = {Animals, Brain, Caenorhabditis elegans, Calcium, Cell Line, Drosophila melanogaster, Fluorescence Resonance Energy Transfer, Humans, Mice, Neurons}, issn = {1548-7105}, doi = {10.1038/nmeth.1398}, author = {Tian, Lin and Hires, S Andrew and Mao, Tianyi and Huber, Daniel and Chiappe, M Eugenia and Chalasani, Sreekanth H and Petreanu, Leopoldo and Akerboom, Jasper and McKinney, Sean A and Schreiter, Eric R and Bargmann, Cornelia I and Jayaraman, Vivek and Svoboda, Karel and Looger, Loren L} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} } @article {2796, title = {In situ SIMS analysis and reactions of surfaces prepared by soft landing of mass-selected cations and anions using an ion trap mass spectrometer.}, journal = {Journal of the American Society for Mass Spectrometry}, volume = {20}, year = {2009}, month = {2009 Jun}, pages = {949-56}, abstract = {

Mass-selected polyatomic cations and anions, produced by electrosonic spray ionization (ESSI), were deposited onto polycrystalline Au or fluorinated self-assembled monolayer (FSAM) surfaces by soft landing (SL), using a rectilinear ion trap (RIT) mass spectrometer. Protonated and deprotonated molecules, as well as intact cations and anions generated from such molecules as peptides, inorganic catalysts, and fluorescent dyes, were soft-landed onto the surfaces. Analysis of the modified surfaces was performed in situ by Cs(+) secondary ion mass spectrometry (SIMS) using the same RIT mass analyzer to characterize the sputtered ions as that used to mass select the primary ions for SL. Soft-landing times as short as 30 s provided surfaces that yielded good quality SIMS spectra. Chemical reactions of the surfaces modified by SL were generated in an attached reaction chamber into which the surface was transferred under vacuum. For example, a surface on which protonated triethanolamine had been soft landed was silylated using vapor-phase chlorotrimethylsilane before being returned still under vacuum to the preparation chamber where SIMS analysis revealed the silyloxy functionalization. SL and vapor-phase reactions are complementary methods of surface modification and in situ surface analysis by SIMS is a simple way to characterize the products produced by either technique.

}, issn = {1879-1123}, doi = {10.1364/AO.50.001792}, author = {Nie, Zongxiu and Li, Guangtao and Goodwin, Michael P and Gao, Liang and Cyriac, Jobin and Cooks, R Graham} } @article {4277, title = {Infernal 1.0: inference of RNA alignments.}, journal = {Bioinformatics}, volume = {25}, year = {2009}, month = {2009 May 15}, pages = {1335-7}, abstract = {

SUMMARY: INFERNAL builds consensus RNA secondary structure profiles called covariance models (CMs), and uses them to search nucleic acid sequence databases for homologous RNAs, or to create new sequence- and structure-based multiple sequence alignments. AVAILABILITY: Source code, documentation and benchmark downloadable from http://infernal.janelia.org. INFERNAL is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X.

}, keywords = {Databases, Nucleic Acid, Nucleic Acid Conformation, RNA, Sequence Alignment, Sequence Analysis, Software}, issn = {1367-4811}, doi = {10.1093/bioinformatics/btp157}, author = {Nawrocki, Eric P and Kolbe, Diana L and Eddy, Sean R.} } @article {927, title = {Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {106}, year = {2009}, month = {2009 Mar 3}, pages = {3125-30}, abstract = {

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

}, keywords = {Animals, Cell Line, Cercopithecus aethiops, Fluorescence, Humans, Imaging, Interference, Microscopy, Microtubules, Three-Dimensional}, issn = {1091-6490}, doi = {10.1073/pnas.0813131106}, author = {Shtengel, Gleb and Galbraith, James A and Galbraith, Catherine G and Lippincott-Schwartz, Jennifer and Gillette, Jennifer M and Manley, Suliana and Sougrat, Rachid and Waterman, Clare M and Kanchanawong, Pakorn and Davidson, Michael W and Fetter, Richard D and Hess, Harald F} } @article {2959, title = {I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions.}, journal = {Biophysical Journal}, volume = {94}, year = {2008}, month = {2008 Jun}, pages = {4971-83}, abstract = {

A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.

}, keywords = {Equipment Design, Equipment Failure Analysis, Image Enhancement, Imaging, Lenses, Microscopy, Nanotechnology, Reproducibility of Results, Sensitivity and Specificity, Three-Dimensional}, issn = {1542-0086}, doi = {10.1529/biophysj.107.120352}, author = {Shao, Lin and Isaac, Berith and Uzawa, Satoru and Agard, David A and Sedat, John W and Gustafsson, Mats G L} } @article {2347, title = {Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance.}, journal = {American Journal of Physiology. Endocrinology and Metabolism}, volume = {295}, year = {2008}, month = {2008 Aug}, pages = {E356-67}, abstract = {

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

}, keywords = {Aminoimidazole Carboxamide, AMP-Activated Protein Kinases, Animals, Basal Metabolism, Body Temperature, Body Weight, Calorimetry, Dietary Fats, Fatty Acids, Inbred C57BL, Indirect, Male, Mice, Multienzyme Complexes, Muscle, Newborn, Nonesterified, Obesity, Oxygen Consumption, Protein-Serine-Threonine Kinases, Random Allocation, Ribonucleotides, Skeletal, Thermogenesis, Triglycerides}, issn = {0193-1849}, doi = {10.1523/JNEUROSCI.3773-10.2011}, author = {Kus, Vladimir and Prazak, Tomas and Brauner, Petr and Hensler, Michal and Kuda, Ondrej and Flachs, Pavel and Janovska, Petra and Medrikova, Dasa and Rossmeisl, Martin and Jilkova, Zuzana and Stefl, Bohumir and Pastalkova, Eva and Drahota, Zdenek and Houstek, Josef and Kopecky, Jan} } @proceedings {5678, title = {Inferring elapsed time from stochastic neural processes.}, volume = {20}, year = {2008}, month = {2008}, publisher = {MIT press}, abstract = {

Many perceptual processes and neural computations, such as speech recognition, motor control and learning, depend on the ability to measure and mark the passage of time. However, the processes that make such temporal judgements possible are unknown. A number of different hypothetical mechanisms have been advanced, all of which depend on the known, temporally predictable evolution of a neural or psychological state, possibly through oscillations or the gradual decay of a memory trace. Alternatively, judgements of elapsed time might be based on observations of temporally structured, but stochastic processes. Such processes need not be specific to the sense of time; typical neural and sensory processes contain at least some statistical structure across a range of time scales. Here, we investigate the statistical properties of an estimator of elapsed time which is based on a simple family of stochastic process.

}, author = {Ahrens MB and Sahani M.} } @article {5673, title = {Inferring input nonlinearities in neural encoding models.}, journal = {Network}, volume = {19}, year = {2008}, month = {2008}, pages = {35-67}, abstract = {

We describe a class of models that predict how the instantaneous firing rate of a neuron depends on a dynamic stimulus. The models utilize a learnt pointwise nonlinear transform of the stimulus, followed by a linear filter that acts on the sequence of transformed inputs. In one case, the nonlinear transform is the same at all filter lag-times. Thus, this "input nonlinearity" converts the initial numerical representation of stimulus value to a new representation that provides optimal input to the subsequent linear model. We describe algorithms that estimate both the input nonlinearity and the linear weights simultaneously; and present techniques to regularise and quantify uncertainty in the estimates. In a second approach, the model is generalized to allow a different nonlinear transform of the stimulus value at each lag-time. Although more general, this model is algorithmically more straightforward to fit. However, it has many more degrees of freedom than the first approach, thus requiring more data for accurate estimation. We test the feasibility of these methods on synthetic data, and on responses from a neuron in rodent barrel cortex. The models are shown to predict responses to novel data accurately, and to recover several important neuronal response properties.

}, keywords = {Models, Neural Networks (Computer), Neurological, Neurons, Nonlinear Dynamics}, issn = {1361-6536}, doi = {10.1080/09548980701813936}, author = {Ahrens, Misha B and Paninski, Liam and Sahani, Maneesh} } @article {2503, title = {Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone.}, journal = {Developmental Biology}, volume = {324}, year = {2008}, month = {2008 Dec 15}, pages = {258-65}, abstract = {

At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.

}, keywords = {Animals, Cells, Cloning, Cultured, Developmental, Ecdysterone, Gene Expression Regulation, Insect Proteins, Insulin, Juvenile Hormones, Larva, Manduca, Messenger, Molecular, Receptor, RNA, RNA Interference, Signal Transduction, Somatomedins, Sucrose, Wing}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2008.09.017}, author = {Koyama, Takashi and Syropyatova, Maria O and Riddiford, Lynn M} } @article {45960, title = {Interactions of lipids with aquaporin-0 and other membrane proteins.}, journal = {Pfl{\"u}gers Archiv - European Journal of Physiology}, volume = {456}, year = {2008}, month = {2008 Jul}, pages = {651-61}, abstract = {

The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.

}, keywords = {Animals, Aquaporins, Crystallization, Eye Proteins, Humans, Lipid Metabolism, Membrane Proteins, Models, Molecular, X-Ray Diffraction}, issn = {0031-6768}, doi = {10.1007/s00424-007-0353-9}, author = {Hite, Richard K and Gonen, Tamir and Harrison, Stephen C and Walz, Thomas} } @article {1731, title = {Interfacial structures of acidic and basic aqueous solutions.}, journal = {Journal of the American Chemical Society}, volume = {130}, year = {2008}, month = {2008 Oct 1}, pages = {13033-9}, abstract = {

Phase-sensitive sum-frequency vibrational spectroscopy was used to study water/vapor interfaces of HCl, HI, and NaOH solutions. The measured imaginary part of the surface spectral responses provided direct characterization of OH stretch vibrations and information about net polar orientations of water species contributing to different regions of the spectrum. We found clear evidence that hydronium ions prefer to emerge at interfaces. Their OH stretches contribute to the "ice-like" band in the spectrum. Their charges create a positive surface field that tends to reorient water molecules more loosely bonded to the topmost water layer with oxygen toward the interface, and thus enhances significantly the "liquid-like" band in the spectrum. Iodine ions in solution also like to appear at the interface and alter the positive surface field by forming a narrow double-charge layer with hydronium ions. In NaOH solution, the observed weak change of the "liquid-like" band and disappearance of the "ice-like" band in the spectrum indicates that OH(-) ions must also have excess at the interface. How they are incorporated in the interfacial water structure is, however, not clear.

}, issn = {1520-5126}, doi = {10.1021/ja8021297}, author = {Tian, Chuanshan and Ji, Na and Waychunas, Glenn A and Shen, Y Ron} } @article {2345, title = {Internally generated cell assembly sequences in the rat hippocampus.}, journal = {Science}, volume = {321}, year = {2008}, month = {2008 Sep 5}, pages = {1322-7}, abstract = {

A long-standing conjecture in neuroscience is that aspects of cognition depend on the brain{\textquoteright}s ability to self-generate sequential neuronal activity. We found that reliably and continually changing cell assemblies in the rat hippocampus appeared not only during spatial navigation but also in the absence of changing environmental or body-derived inputs. During the delay period of a memory task, each moment in time was characterized by the activity of a particular assembly of neurons. Identical initial conditions triggered a similar assembly sequence, whereas different conditions gave rise to different sequences, thereby predicting behavioral choices, including errors. Such sequences were not formed in control (nonmemory) tasks. We hypothesize that neuronal representations, evolved for encoding distance in spatial navigation, also support episodic recall and the planning of action sequences.

}, keywords = {Action Potentials, Animal, Animals, Behavior, Choice Behavior, Cues, Hippocampus, Interneurons, Long-Evans, Male, Maze Learning, Memory, Mental Recall, Models, Motor Activity, Neurological, Pyramidal Cells, Rats}, issn = {1095-9203}, doi = {10.1523/JNEUROSCI.3773-10.2011}, author = {Pastalkova, Eva and Itskov, Vladimir and Amarasingham, Asohan and Buzs{\'a}ki, Gy{\"o}rgy} } @article {2850, title = {Isolation of highly purified yeast nuclei for nuclease mapping of chromatin structure.}, journal = {Methods in Molecular Biology}, volume = {463}, year = {2008}, month = {2008}, pages = {43-53}, abstract = {

Probing chromatin structure with nucleases is a well-established method for determining the accessibility of DNA to gene regulatory proteins and measuring competency for transcription. A hallmark of many silent genes is the presence of translationally positioned nucleosomes over their promoter regions, which can be inferred by the sensitivity of the underlying DNA to nucleases, particularly micrococcal nuclease. The quality of this data is highly dependent upon the nuclear preparation, especially if the digestion products are analyzed by high-resolution detection methods such as reiterative primer extension. Here we describe a method to isolate highly purified nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the positions of nucleosomes at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the commonly used chromatin mapping and detection procedures.

}, keywords = {Cell Nucleus, Chromatin, DNA, Fungal, Genetic, Micrococcal Nuclease, Mycology, Promoter Regions, Ribonucleoside Diphosphate Reductase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1940-6029}, doi = {10.1007/978-1-59745-406-3_3}, author = {Reese, Joseph C and Zhang, Hesheng and Zhang, Zhengjian} } @article {6497, title = {Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics.}, journal = {Molecular Microbiology}, volume = {66}, year = {2007}, month = {2007 Dec}, pages = {1080-91}, abstract = {

Bacterial small non-coding RNAs (sRNAs) are being recognized as novel widespread regulators of gene expression in response to environmental signals. Here, we present the first search for sRNA-encoding genes in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, performed by a genome-wide computational analysis of its intergenic regions. Comparative sequence data from eight related alpha-proteobacteria were obtained, and the interspecies pairwise alignments were scored with the programs eQRNA and RNAz as complementary predictive tools to identify conserved and stable secondary structures corresponding to putative non-coding RNAs. Northern experiments confirmed that eight of the predicted loci, selected among the original 32 candidates as most probable sRNA genes, expressed small transcripts. This result supports the combined use of eQRNA and RNAz as a robust strategy to identify novel sRNAs in bacteria. Furthermore, seven of the transcripts accumulated differentially in free-living and symbiotic conditions. Experimental mapping of the 5{\textquoteright}-ends of the detected transcripts revealed that their encoding genes are organized in autonomous transcription units with recognizable promoter and, in most cases, termination signatures. These findings suggest novel regulatory functions for sRNAs related to the interactions of alpha-proteobacteria with their eukaryotic hosts.

}, keywords = {Bacterial, Blotting, Computational Biology, DNA, Double-Stranded, Fabaceae, Genetic, Genome, Intergenic, Northern, Nucleic Acid Conformation, Promoter Regions, RNA, Sinorhizobium meliloti, Transcription, Untranslated}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2007.05978.x}, author = {del Val, Coral and Rivas, Elena and Torres-Quesada, Omar and Toro, Nicol{\'a}s and Jim{\'e}nez-Zurdo, Jos{\'e} I} } @article {46405, title = {IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system.}, journal = {The Journal of Clinical Investigation}, volume = {117}, year = {2007}, month = {2007 Jun}, pages = {1538-49}, abstract = {

ST2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II- and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-kappaB, it inhibited angiotensin II- and phenylephrine-induced phosphorylation of inhibitor of NF-kappa B alpha (I kappa B alpha) and NF-kappaB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myocardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2(-/-) mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening, more fibrosis, and impaired survival compared with WT littermates. Furthermore, recombinant IL-33 treatment reduced hypertrophy and fibrosis and improved survival after TAC in WT mice, but not in ST2(-/-) littermates. Thus, IL-33/ST2 signaling is a mechanically activated, cardioprotective fibroblast-cardiomyocyte paracrine system, which we believe to be novel. IL-33 may have therapeutic potential for beneficially regulating the myocardial response to overload.

}, keywords = {Animals, Biomechanical Phenomena, Cardiotonic Agents, Cells, Cultured, Fibroblasts, Humans, Interleukins, Membrane Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Myocytes, Cardiac, NF-kappa B, Rats, Receptors, Cell Surface, Receptors, Interleukin, Recombinant Proteins, Signal Transduction}, issn = {0021-9738}, doi = {10.1172/JCI30634}, author = {Sanada, Shoji and Hakuno, Daihiko and Higgins, Luke J and Schreiter, Eric R and McKenzie, Andrew N J and Lee, Richard T} } @article {46415, title = {An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans.}, journal = {Cell}, volume = {129}, year = {2007}, month = {2007 May 18}, pages = {787-99}, abstract = {

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Body Patterning, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Calcium Signaling, Cell Communication, Cell Differentiation, Connexins, Functional Laterality, Gap Junctions, Membrane Proteins, Molecular Sequence Data, Nerve Net, Nervous System, Neurons, Olfactory Pathways}, issn = {0092-8674}, doi = {10.1016/j.cell.2007.02.052}, author = {Chuang, Chiou-Fen and Vanhoven, Miri K and Fetter, Richard D and Verselis, Vytas K and Bargmann, Cornelia I} } @article {45738, title = {Internal and external constraints in the evolution of morphological allometries in a butterfly.}, journal = {Evolution}, volume = {61}, year = {2007}, month = {2007 Dec}, pages = {2958-70}, abstract = {

Much diversity in animal morphology results from variation in the relative size of morphological traits. The scaling relationships, or allometries, that describe relative trait size can vary greatly in both intercept and slope among species or other animal groups. Yet within such groups, individuals typically exhibit low variation in relative trait size. This pattern of high intra- and low intergroup variation may result from natural selection for particular allometries, from developmental constraints restricting differential growth among traits, or both. Here we explore the relative roles of short-term developmental constraints and natural selection in the evolution of the intercept of the allometry between the forewing and hindwing of a butterfly. First, despite a strong genetic correlation between these two traits, we show that artificial selection perpendicular to the forewing-hindwing scaling relationship results in rapid evolution of the allometry intercept. This demonstrates an absence of developmental constraints limiting intercept evolution for this scaling relationship. Mating experiments in a natural environment revealed strong stabilizing selection favoring males with the wild-type allometry intercept over those with derived intercepts. Our results demonstrate that evolution of this component of the forewing-hindwing allometry is not limited by developmental constraints in the short term and that natural selection on allometry intercepts can be powerful.

}, keywords = {Animals, Biological Evolution, Body Size, Butterflies, Genetic Variation, Phenotype, Selection, Genetic, Wing}, issn = {0014-3820}, doi = {10.1111/j.1558-5646.2007.00249.x}, author = {Frankino, W Anthony and Zwaan, Bas J and Stern, David L and Brakefield, Paul M} } @article {1102, title = {Intraspecies regulation of ribonucleolytic activity.}, journal = {Biochemistry}, volume = {46}, year = {2007}, month = {2007 Nov 13}, pages = {13131-40}, abstract = {

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of coevolution higher than that for nonobligate protein-protein interactions. The coevolution of the proteins involved in certain nonobligate interactions is, however, essential to cell survival. To gain insight into the coevolution of one such nonobligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.

}, keywords = {Amino Acid Sequence, Animals, Biological Evolution, Cattle, Enzyme Inhibitors, Humans, Hydrogen Peroxide, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Pancreatic, Placental Hormones, Protein Conformation, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Ribonuclease, Sequence Alignment}, issn = {0006-2960}, doi = {10.1021/bi701521q}, author = {Johnson, R Jeremy and Lavis, Luke D and Raines, Ronald T} } @article {2404, title = {IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback.}, journal = {Genes \& Development}, volume = {21}, year = {2007}, month = {2007 Jan 15}, pages = {175-83}, abstract = {

It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Here, we report evidence for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. Our studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions.

}, keywords = {5{\textquoteright} Untranslated Regions, Animals, Biological, Drosophila melanogaster, Drosophila Proteins, Eukaryotic Initiation Factor-4E, Feedback, Forkhead Transcription Factors, Gene Expression Regulation, Genetic, Insulin, Models, Phosphorylation, Physiological, Protein Biosynthesis, Receptor, Ribosomes, Signal Transduction, Transcription, Transfection}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Marr, Michael T and D{\textquoteright}Alessio, Joseph A and Puig, Oscar and Tjian, Robert} } @article {1586, title = {Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.}, journal = {FEMS Microbiology Letters}, volume = {260}, year = {2006}, month = {2006 Jul}, pages = {69-76}, abstract = {

The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, some of which catalyze unique conversions. Moreover, the enzymes appear not to be regulated allosterically, but rather at transcriptional level. To elucidate details of the gene expression control, the transcription initiation sites of the glycolytic genes in Pyrococcus furiosus have been mapped by primer extension analysis and the obtained promoter sequences have been compared with upstream regions of non-glycolytic genes. Apart from consensus sequences for the general transcription factors (TATA-box and BRE) this analysis revealed the presence of a potential transcription factor binding site (TATCAC-N(5)-GTGATA) in glycolytic and starch utilizing promoters of P. furiosus and several thermococcal species. The absence of this inverted repeat in Pyrococcus abyssi and Pyrococcus horikoshii probably reflects that their reduced catabolic capacity does not require this regulatory system. Moreover, this phyletic pattern revealed a TrmB-like regulator (PF0124 and TK1769) which may be involved in recognizing the repeat. This Thermococcales glycolytic regulon, with more than 20 genes, is the largest regulon that has yet been described for Archaea.

}, keywords = {Base Sequence, Binding Sites, Genetic, Glycolysis, Molecular Sequence Data, Promoter Regions, Pyrococcus, Regulon, Thermococcus, Transcription Initiation Site}, issn = {0378-1097}, doi = {10.1111/j.1574-6968.2006.00292.x}, author = {van de Werken, Harmen J G and Verhees, Corn{\'e} H and Akerboom, Jasper and de Vos, Willem M and van der Oost, John} } @article {1489, title = {Image diffusion using saliency bilateral filter.}, journal = {Medical Image Computing and Computer-Assisted Intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention}, volume = {9}, year = {2006}, month = {2006}, pages = {67-75}, abstract = {

Image diffusion can smooth away noise and small-scale structures while retaining important features, thereby enhancing the performances of many image processing algorithms such as image compression, segmentation and recognition. In this paper, we present a novel diffusion algorithm for which the filtering kernels vary according to the perceptual saliency of boundaries in the input images. The boundary saliency is estimated through a saliency measure which is generally determined by curvature changes, intensity gradient and the interaction of neighboring vectors. The connection between filtering kernels and perceptual saliency makes it possible to remove small-scale structures and preserves significant boundaries adaptively. The effectiveness of the proposed approach is validated by experiments on various medical images including the color Chinese Visible Human data set and gray MRI brain images.

}, keywords = {Algorithms, Artificial Intelligence, Automated, Brain, Cluster Analysis, Computer-Assisted, Diffusion Magnetic Resonance Imaging, Humans, Image Enhancement, Image Interpretation, Magnetic Resonance Imaging, Pattern Recognition, Reproducibility of Results, Sensitivity and Specificity}, author = {Xie, Jun and Heng, Pheng-Ann and Ho, Simon S M and Shah, Mubarak} } @article {2083, title = {Imaging intracellular fluorescent proteins at nanometer resolution. (With commentary)}, journal = {Science}, volume = {313}, year = {2006}, month = {2006 Sep 15}, pages = {1642-5}, abstract = {

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method{\textendash}termed photoactivated localization microscopy{\textendash}to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Commentary: The original PALM paper by myself and my friend and co-inventor Harald Hess, spanning the before- and after-HHMI eras. Submitted and publicly presented months before other publications in the same year, the lessons of the paper remain widely misunderstood: 1) localization precision is not resolution; 2) the ability to resolve a few molecules by the Rayleigh criterion in a diffraction limited region (DLR) does not imply the ability to resolve structures of arbitrary complexity at the same scale; 3) true resolution well beyond the Abbe limit requires the ability to isolate and localize hundreds or thousands of molecules in one DLR; and 4) certain photoactivatable fluorescent proteins (PA-FPs) and caged dyes can be isolated and precisely localized at such densities; yielding true resolution down to \ 20 nm. The molecular densities we demonstrate (105 molecules/m2) are more than two orders of magnitude greater than in later papers that year (implying ten-fold better true resolution) {\textendash} indeed, these papers demonstrate densities only comparable to earlier spectral or photobleaching based isolation methods. We validate our claims by correlative electron microscopy, and demonstrate the outstanding advantages of PA-FPs for superresolution microscopy: minimally perturbative sample preparation; high labeling densities; close binding to molecular targets; and zero non-specific background.

}, keywords = {Actins, Algorithms, Animals, Cell Line, Cell Membrane, Cercopithecus aethiops, COS Cells, Fluorescence, Focal Adhesions, gag, Gene Products, HIV-1, Light, Luminescent Proteins, Lysosomes, Microscopy, Mitochondria, Nanotechnology, Organelles, Photobleaching, Proteins, Pseudopodia, Recombinant Fusion Proteins, Vinculin}, issn = {1095-9203}, doi = {10.1126/science.1127344}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16902090}, author = {Betzig, Eric and Patterson, George H and Sougrat, Rachid and Lindwasser, O Wolf and Olenych, Scott and Bonifacino, Juan S and Davidson, Michael W and Lippincott-Schwartz, Jennifer and Hess, Harald F} } @article {3928, title = {In situ background estimation in quantitative fluorescence imaging.}, journal = {Biophysical Journal}, volume = {90}, year = {2006}, month = {2006 Apr 1}, pages = {2534-47}, abstract = {

Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of our method and give example applications in ratiometric calcium imaging of bulk-stained olfactory bulb slices. Converting the fluorescence signals into [Ca2+] gives resting values consistent with earlier single-cell staining results, and odorant-induced [Ca2+] transients can be quantitatively compared in different cells. Using these examples we show that inaccurate background subtraction introduces large errors (easily in the range of 100\%) in the assessment of both resting [Ca2+] and [Ca2+] dynamics. The proposed method allows us to avoid such errors.

}, keywords = {Algorithms, Animals, Biophysics, Brain, Calcium, Computer Simulation, Computer-Assisted, Confocal, Fluorescence, Image Processing, Likelihood Functions, Microscopy, Models, Normal Distribution, Olfactory Bulb, Olfactory Nerve, Regression Analysis, Reproducibility of Results, Software, Statistical, Time Factors, Xenopus laevis}, issn = {0006-3495}, doi = {10.1529/biophysj.105.070854}, author = {Chen, Tsai-Wen and Lin, Bei-Jung and Brunner, Edgar and Schild, Detlev} } @article {1543, title = {An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly.}, journal = {Nature Biotechnology}, volume = {24}, year = {2006}, month = {2006 Mar}, pages = {358-62}, abstract = {

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.

}, keywords = {Antiviral Agents, Biological Assay, Boron Compounds, Capsid, Capsid Proteins, Dimerization, Fluorescence, Fluorescent Dyes, Hepatitis B virus, Kinetics, Light, Models, Molecular, Radiation, Scattering, Virus Assembly}, issn = {1087-0156}, doi = {10.1038/nbt1187}, author = {Stray, Stephen J and Johnson, Jennifer M and Kopek, Benjamin G and Zlotnick, Adam} } @article {46629, title = {Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics.}, journal = {Cell}, volume = {127}, year = {2006}, month = {2006 Oct 6}, pages = {213-26}, abstract = {

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

}, keywords = {Actins, Adaptor Proteins, Signal Transducing, Alcohol Drinking, Animals, Behavior, Animal, Cells, Cultured, Central Nervous System Depressants, Cerebellum, Cytoskeletal Proteins, Cytoskeleton, Ethanol, Female, Humans, Male, Mice, Mice, Knockout, Neurons, Receptors, N-Methyl-D-Aspartate, Recombinant Proteins, Signal Transduction}, issn = {0092-8674}, doi = {10.1016/j.cell.2006.09.011}, author = {Offenh{\"a}user, Nina and Castelletti, Daniela and Mapelli, Lisa and Soppo, Blanche Ekalle and Regondi, Maria Cristina and Rossi, Paola and D{\textquoteright}Angelo, Egidio and Frassoni, Carolina and Amadeo, Alida and Tocchetti, Arianna and Pozzi, Benedetta and Disanza, Andrea and Guarnieri, Douglas and Betsholtz, Christer and Scita, Giorgio and Heberlein, Ulrike and Di Fiore, Pier Paolo} } @article {3918, title = {Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons.}, journal = {Neuron}, volume = {50}, year = {2006}, month = {2006 Apr 20}, pages = {291-307}, abstract = {

Although radial oblique dendrites are a major synaptic input site in CA1 pyramidal neurons, little is known about their integrative properties. We have used multisite two-photon glutamate uncaging to deliver different spatiotemporal input patterns to single branches while simultaneously recording the uncaging-evoked excitatory postsynaptic potentials and local Ca2+ signals. Asynchronous input patterns sum linearly in spite of the spatial clustering and produce Ca2+ signals that are mediated by NMDA receptors (NMDARs). Appropriately timed and sized input patterns ( approximately 20 inputs within approximately 6 ms) produce a supralinear summation due to the initiation of a dendritic spike. The Ca2+ signals associated with synchronous input were larger and mediated by influx through both NMDARs and voltage-gated Ca2+ channels (VGCCs). The oblique spike is a fast Na+ spike whose duration is shaped by the coincident activation of NMDAR, VGCCs, and transient K+ currents. Our results suggest that individual branches can function as single integrative compartments.

}, keywords = {Animals, Calcium Channels, Dendrites, Excitatory Postsynaptic Potentials, Glutamic Acid, N-Methyl-D-Aspartate, Patch-Clamp Techniques, Pyramidal Cells, Rats, Receptors, Sprague-Dawley, Synapses, Synaptic Transmission}, issn = {0896-6273}, doi = {10.1016/j.neuron.2006.03.016}, author = {Losonczy, Attila and Magee, Jeffrey C} } @article {2855, title = {Isolation of yeast nuclei and micrococcal nuclease mapping of nucleosome positioning.}, journal = {Methods in Molecular Biology }, volume = {313}, year = {2006}, month = {2006}, pages = {245-55}, abstract = {

Chromatin structure and nucleosome positioning play a crucial role in gene expression regulation. Nucleosome positioning is often inferred by the protection of underlying DNA to nucleases. Because nucleases are excluded by plasma membranes, chromatin mapping requires isolating nuclei from cells and digesting the chromatin in situ with nucleases. The quality of this data is highly dependent on the nuclei preparation. Here we describe a method to isolate nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the chromatin structure at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the common chromatin mapping and detection procedures.

}, keywords = {Cell Fractionation, Cell Nucleus, Chromatin, DNA, Fungal, Gene Expression Regulation, Genes, Micrococcal Nuclease, Mycology, Nucleosomes, Saccharomyces cerevisiae}, issn = {1064-3745}, author = {Zhang, Zhengjian and Reese, Joseph C} } @article {5821, title = {An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression.}, journal = {Development Genes \& Evolution}, volume = {215}, year = {2005}, month = {2005 Sep}, pages = {482-88}, abstract = {

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed.

}, keywords = {Animals, Developmental, Dose-Response Relationship, Drug, Embryo, Epidermis, Gene Expression Regulation, In Situ Hybridization, Myosin Heavy Chains, Nonmammalian, Planarians, Time Factors, Tissue Preservation}, issn = {0949-944X}, doi = {10.1007/s00427-005-0003-1}, author = {Cardona, Albert and Fern{\'a}ndez, Juani and Solana, Jordi and Romero, Rafael} } @article {2412, title = {In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.}, journal = {Cell}, volume = {123}, year = {2005}, month = {2005 Dec 29}, pages = {1241-53}, abstract = {

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington{\textquoteright}s disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. Here, we report the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. We demonstrate that both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. Our results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus.

}, keywords = {Animals, Biological Assay, Cell-Free System, Cells, Cercopithecus aethiops, COS Cells, Cultured, Dopamine D2, Down-Regulation, Genetic, Mice, Mutation, Nerve Tissue Proteins, Neurons, Nuclear Proteins, Protein Binding, Protein Subunits, Rats, Receptors, Sp1 Transcription Factor, TFII, Transcription, Transcription Factor TFIID, Transcription Factors}, issn = {0092-8674}, doi = {10.1073/pnas.1100640108}, author = {Zhai, Weiguo and Jeong, Hyunkyung and Cui, Libin and Krainc, Dimitri and Tjian, Robert} } @article {5490, title = {Insulin signaling in the nervous system regulates ethanol intoxication in Drosophila melanogaster.}, journal = {Nature Neuroscience}, volume = {8}, year = {2005}, month = {2005 Jan}, pages = {18-9}, abstract = {

The insulin signaling pathway regulates multiple physiological processes, including energy metabolism, organismal growth, aging and reproduction. Here we show that genetic manipulations in Drosophila melanogaster that impair the function of insulin-producing cells or of the insulin-receptor signaling pathway in the nervous system lead to increased sensitivity to the intoxicating effects of ethanol. These findings suggest a previously unknown role for this highly conserved pathway in regulating the behavioral responses to an addictive drug.

}, keywords = {Alcoholic Intoxication, Animals, Brain, Drosophila melanogaster, Ethanol, Insulin, Neurons, Receptor, Signal Transduction}, issn = {1097-6256}, doi = {10.1038/nn1363}, author = {Corl, Ammon B and Rodan, Aylin R and Heberlein, Ulrike} } @article {1985, title = {Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit.}, journal = {Nature Neuroscience}, volume = {8}, year = {2005}, month = {2005 Mar}, pages = {354-64}, abstract = {

Microcircuits composed of principal neuron and interneuron dendrites have an important role in shaping the representation of sensory information in the olfactory bulb. Here we establish the physiological features governing synaptic signaling in dendrodendritic microcircuits of olfactory bulb glomeruli. We show that dendritic gamma-aminobutyric acid (GABA) release from periglomerular neurons mediates inhibition of principal tufted cells, retrograde inhibition of sensory input and lateral signaling onto neighboring periglomerular cells. We find that L-type dendritic Ca(2+) spikes in periglomerular cells underlie dendrodendritic transmission by depolarizing periglomerular dendrites and activating P/Q type channels that trigger GABA release. Ca(2+) spikes in periglomerular cells are evoked by powerful excitatory inputs from a single principal cell, and glutamate release from the dendrites of single principal neurons activates a large ensemble of periglomerular cells.

}, keywords = {1, 3-Pyridinecarboxylic acid, 4-dihydro-2, 6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Animals, Bicuculline, Cadmium, Calcium, Calcium Channel Agonists, Calcium Channel Blockers, Calcium Signaling, Chelating Agents, Dendrites, Diagnostic Imaging, Dihydropyridines, Dose-Response Relationship, Drug, Drug Interactions, Egtazic Acid, Electric Stimulation, GABA Antagonists, gamma-Aminobutyric Acid, Membrane Potentials, Methyl ester, Neural Conduction, Neural Inhibition, Neural Networks (Computer), Neurons, Newborn, Nickel, Nimodipine, Olfactory Bulb, Patch-Clamp Techniques, Phosphinic Acids, Potassium, Propanolamines, Pyrimidines, Rats, Signal Transduction, Sprague-Dawley, Synapses, Synaptic Transmission, Tetrodotoxin, Time Factors, Valine}, issn = {1097-6256}, doi = {10.1038/nn1403}, author = {Murphy, Gabe J and Darcy, Daniel P and Isaacson, Jeffry S} } @article {2319, title = {Individual differences in trait anxiety predict the response of the basolateral amygdala to unconsciously processed fearful faces.}, journal = {Neuron}, volume = {44}, year = {2004}, month = {2004 Dec 16}, pages = {1043-55}, abstract = {

Responses to threat-related stimuli are influenced by conscious and unconscious processes, but the neural systems underlying these processes and their relationship to anxiety have not been clearly delineated. Using fMRI, we investigated the neural responses associated with the conscious and unconscious (backwardly masked) perception of fearful faces in healthy volunteers who varied in threat sensitivity (Spielberger trait anxiety scale). Unconscious processing modulated activity only in the basolateral subregion of the amygdala, while conscious processing modulated activity only in the dorsal amygdala (containing the central nucleus). Whereas activation of the dorsal amygdala by conscious stimuli was consistent across subjects and independent of trait anxiety, activity in the basolateral amygdala to unconscious stimuli, and subjects{\textquoteright} reaction times, were predicted by individual differences in trait anxiety. These findings provide a biological basis for the unconscious emotional vigilance characteristic of anxiety and a means for investigating the mechanisms and efficacy of treatments for anxiety.

}, keywords = {Adult, Amygdala, Anxiety, Brain Mapping, Emotions, Facial Expression, Fear, Female, Humans, Individuality, Male, Photic Stimulation, Predictive Value of Tests, Reaction Time, Unconscious (Psychology)}, issn = {0896-6273}, doi = {10.3389/fnana.2010.00147}, author = {Etkin, Amit and Klemenhagen, Kristen C and Dudman, Joshua T and Rogan, Michael T and Hen, Ren{\'e} and Kandel, Eric R and Hirsch, Joy} } @article {2133, title = {On the initiation and propagation of dendritic spikes in CA1 pyramidal neurons.}, journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {24}, year = {2004}, month = {2004 Dec 8}, pages = {11046-56}, abstract = {

Under certain conditions, regenerative voltage spikes can be initiated locally in the dendrites of CA1 pyramidal neurons. These are interesting events that could potentially provide neurons with additional computational abilities. Using whole-cell dendritic recordings from the distal apical trunk and proximal tuft regions and realistic computer modeling, we have determined that highly synchronized and moderately clustered inputs are required for dendritic spike initiation: approximately 50 synaptic inputs spread over 100 mum of the apical trunk/tuft need to be activated within 3 msec. Dendritic spikes are characterized by a more depolarized voltage threshold than at the soma [-48 +/- 1 mV (n = 30) vs -56 +/- 1 mV (n = 7), respectively] and are mainly generated and shaped by dendritic Na+ and K+ currents. The relative contribution of AMPA and NMDA currents is also important in determining the actual spatiotemporal requirements for dendritic spike initiation. Once initiated, dendritic spikes can easily reach the soma, but their propagation is only moderately strong, so that it can be modulated by physiologically relevant factors such as changes in the V(m) and the ionic composition of the extracellular solution. With effective spike propagation, an extremely short-latency neuronal output is produced for greatly reduced input levels. Therefore, dendritic spikes function as efficient detectors of specific input patterns, ensuring that the neuronal response to high levels of input synchrony is a precisely timed action potential output.

}, keywords = {Action Potentials, AMPA, Animals, Dendrites, Electrodes, Excitatory Postsynaptic Potentials, Ion Channels, Models, N-Methyl-D-Aspartate, Neurological, Pyramidal Cells, Rats, Receptors, Sprague-Dawley}, issn = {1529-2401}, doi = {10.1002/cbic.201000254}, author = {Gasparini, Sonia and Migliore, Michele and Magee, Jeffrey C} } @article {2137, title = {Impaired regulation of synaptic strength in hippocampal neurons from GluR1-deficient mice.}, journal = {The Journal of Physiology}, volume = {552}, year = {2003}, month = {2003 Oct 1}, pages = {35-45}, abstract = {

Neurons of the central nervous system (CNS) exhibit a variety of forms of synaptic plasticity, including associative long-term potentiation and depression (LTP/D), homeostatic activity-dependent scaling and distance-dependent scaling. Regulation of synaptic neurotransmitter receptors is currently thought to be a common mechanism amongst many of these forms of plasticity. In fact, glutamate receptor 1 (GluR1 or GluRA) subunits containing L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors have been shown to be required for several forms of hippocampal LTP and a particular hippocampal-dependent learning task. Because of this importance in associative plasticity, we sought to examine the role of these receptors in other forms of synaptic plasticity in the hippocampus. To do so, we recorded from the apical dendrites of hippocampal CA1 pyramidal neurons in mice lacking the GluR1 subunit (GluR1 -/-). Here we report data from outside-out patches that indicate GluR1-containing receptors are essential to the extrasynaptic population of AMPA receptors, as this pool was nearly empty in the GluR1 -/- mice. Additionally, these receptors appear to be a significant component of the synaptic glutamate receptor pool because the amplitude of spontaneous synaptic currents recorded at the site of input and synaptic AMPA receptor currents evoked by focal glutamate uncaging were both substantially reduced in these mice. Interestingly, the impact on synaptic weight was greatest at distant synapses such that the normal distance-dependent synaptic scaling used by these cells to counter dendritic attenuation was lacking in GluR1 -/- mice. Together the data suggest that the highly regulated movement of GluR1-containing AMPA receptors between extrasynaptic and synaptic receptor pools is critically involved in establishing two functionally diverse forms of synaptic plasticity: LTP and distance-dependent scaling.

}, keywords = {AMPA, Animals, Hippocampus, Inbred C57BL, Kinetics, Long-Term Potentiation, Mice, Mutant Strains, Neuronal Plasticity, Patch-Clamp Techniques, Pyramidal Cells, Receptors, Synapses}, issn = {0022-3751}, doi = {10.1002/cbic.201000254}, author = {Andrasfalvy, Bertalan K and Smith, Mark A and Borchardt, Thilo and Sprengel, Rolf and Magee, Jeffrey C} } @article {2014, title = {In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation.}, journal = {Neuron}, volume = {39}, year = {2003}, month = {2003 Sep 11}, pages = {1005-17}, abstract = {

In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.

}, keywords = {Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Calcium Channels, Cells, Cellular, Cultured, L-Type, Mechanoreceptors, Mechanotransduction, Membrane Potentials, Membrane Proteins, Neurons, Touch}, issn = {0896-6273}, author = {Suzuki, Hiroshi and Kerr, Rex and Bianchi, Laura and Fr{\o}kjaer-Jensen, Christian and Slone, Dan and Xue, Jian and Gerstbrein, Beate and Driscoll, Monica and Schafer, William R} } @article {2292, title = {Inducible nitric oxide synthase (NOS2) promoter CCTTT repeat polymorphism: relationship to in vivo nitric oxide production/NOS activity in an asymptomatic malaria-endemic population.}, journal = {The American Journal of Tropical Medicine and Hygiene}, volume = {69}, year = {2003}, month = {2003 Dec}, pages = {569-73}, abstract = {

Polymorphisms in the inducible nitric oxide synthase gene (NOS2) promoter have been associated with clinical outcome from malaria. These include a CCTTT repeat (CCTTTn) 2.5 kilobases upstream from the NOS2 transcription start site, and two single nucleotide substitutions: G{\textendash}\>C at position -954 (G-954C), and C{\textendash}\>T at position -1173 (C-1173T). Although hypothesized to influence NO production in vivo, the functional relevance of (CCTTT)n and G-954C is uncertain because disease association studies have yielded inconsistent results. This study found no association between CCTTT repeat number and levels of plasma NO metabolites or peripheral blood mononuclear cell NOS activity in a cohort of asymptomatic malaria-exposed coastal Papua New Guineans 1-60 years old. This suggests that (CCTTT)n does not independently influence NOS2 transcription in vivo. Neither the G-954C nor the C-1173T polymorphisms were identified in this population, indicating the variability and complexity of selection for NOS2 promoter polymorphisms in different malaria-endemic populations.

}, keywords = {Endemic Diseases, Female, Genetic, Genetic Predisposition to Disease, Humans, Malaria, Male, Nitric Oxide, Nitric Oxide Synthase, Nitric Oxide Synthase Type II, Nucleic Acid, Oceanic Ancestry Group, Papua New Guinea, Polymorphism, Promoter Regions, Repetitive Sequences}, issn = {0002-9637}, author = {Boutlis, Craig S and Hobbs, Maurine R and Marsh, Robyn L and Misukonis, Mary A and Tkachuk, Ariana N and Lagog, Moses and Booth, Jennifer and Granger, Donald L and Bockarie, Moses J and Mgone, Charles S and Levesque, Marc C and Weinberg, J Brice and Anstey, Nicholas M} } @article {45954, title = {Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier.}, journal = {Experimental Eye Research}, volume = {77}, year = {2003}, month = {2003 Nov}, pages = {567-74}, abstract = {

It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space.

}, keywords = {Animals, Aquaporins, Cell Differentiation, Cell Membrane, Cytoplasm, Extracellular Space, Eye Proteins, Lens, Crystalline, Membrane Glycoproteins, Rats, Sheep}, issn = {0014-4835}, author = {Grey, Angus C and Jacobs, Marc D and Gonen, Tamir and Kistler, Joerg and Donaldson, Paul J} } @article {1649, title = {Intensity versus identity coding in an olfactory system.}, journal = {Neuron}, volume = {39}, year = {2003}, month = {2003 Sep 11}, pages = {991-1004}, abstract = {

We examined the encoding and decoding of odor identity and intensity by neurons in the antennal lobe and the mushroom body, first and second relays, respectively, of the locust olfactory system. Increased odor concentration led to changes in the firing patterns of individual antennal lobe projection neurons (PNs), similar to those caused by changes in odor identity, thus potentially confounding representations for identity and concentration. However, when these time-varying responses were examined across many PNs, concentration-specific patterns clustered by identity, resolving the apparent confound. This is because PN ensemble representations changed relatively continuously over a range of concentrations of each odorant. The PNs{\textquoteright} targets in the mushroom body-Kenyon cells (KCs)-had sparse identity-specific responses with diverse degrees of concentration invariance. The tuning of KCs to identity and concentration and the patterning of their responses are consistent with piecewise decoding of their PN inputs over oscillation-cycle length epochs.

}, keywords = {Action Potentials, Animals, Female, Grasshoppers, Male, Mushroom Bodies, Neurons, Odors, Reaction Time, Smell}, issn = {0896-6273}, author = {Stopfer, Mark and Jayaraman, Vivek and Laurent, Gilles} } @article {45894, title = {Intracellular correlate of EPSP-spike potentiation in CA1 pyramidal neurons is controlled by GABAergic modulation.}, journal = {Hippocampus}, volume = {13}, year = {2003}, month = {2003}, pages = {801-5}, abstract = {

The hippocampus has been used extensively as a model to study plastic changes in the brain{\textquoteright}s neural circuitry. Immediately after high-frequency stimulation to hippocampal Schaffer collateral axons, a dramatic change occurs in the relationship between the presynaptic CA3 and the postsynaptic CA1 pyramidal neurons. For a fixed excitatory postsynaptic potential (EPSP), there arises an increased likelihood of action potential generation in the CA1 pyramidal neuron. This phenomenon is called EPSP-spike (E-S) potentiation. We explored E-S potentiation, using patch-clamp techniques in the hippocampal slice preparation. A specific protocol was developed to measure the action potential probability for a given synaptic strength, which allowed us to quantify the amount of E-S potentiation for a single neuron. E-S potentiation was greatest when gamma-aminobutyric acid (GABA)ergic inhibition was intact, suggesting that modulation of inhibition is a major aspect of E-S potentiation. Expression of E-S potentiation also correlated with a reduced action-potential threshold, which was greatest when GABAergic inhibition was intact. Conditioning stimuli produced a smaller threshold reduction when inhibition was blocked, but some reduction also occurred in the absence of a conditioning stimulus. Together, these results suggest that E-S potentiation is caused primarily through a reduction of GABAergic inhibition, leading to larger EPSPs and reduced action potential threshold. Our findings do not rule out, however, the possibility that modulation of voltage-gated conductances also contributes to E-S potentiation.

}, keywords = {Action Potentials, Animals, Dendrites, Excitatory Postsynaptic Potentials, GABA Antagonists, GABA-A Receptor Antagonists, GABA-B Receptor Antagonists, gamma-Aminobutyric Acid, Hippocampus, In Vitro Techniques, Intracellular Fluid, Neural Inhibition, Neuronal Plasticity, Patch-Clamp Techniques, Pyramidal Cells, Rats, Rats, Wistar, Receptors, GABA-A, Receptors, GABA-B, Synaptic Transmission}, issn = {1050-9631}, doi = {10.1002/hipo.10129}, author = {Staff, Nathan P and Spruston, Nelson} } @article {46639, title = {Invertebrate models of drug abuse.}, journal = {Journal of Neurobiology}, volume = {54}, year = {2003}, month = {2003 Jan}, pages = {161-78}, abstract = {

Susceptibility to drug addiction depends on genetic and environmental factors and their complex interactions. Studies with mammalian models have identified molecular targets, neurochemical systems, and brain regions that mediate some of the addictive properties of abused drugs. Yet, our understanding of how the primary effects of drugs lead to addiction remains incomplete. Recently, researchers have turned to the invertebrate model systems Drosophila melanogaster and Caenorhabditis elegans to dissect the mechanisms by which abused drugs modulate behavior. Due to their sophisticated genetics, relatively simple anatomy, and their remarkable molecular similarity to mammals, these invertebrate models should provide useful insights into the mechanisms of drug action. Here we review recent behavioral and genetic studies in flies and worms on the effects of ethanol, cocaine, and nicotine, three of the most widely abused drugs in the world.

}, keywords = {Alcoholic Intoxication, Animals, Behavior, Animal, Caenorhabditis elegans, Cocaine, Disease Models, Animal, Dopamine, Drosophila melanogaster, Drug Tolerance, Ethanol, Learning, Memory, Nicotine, Substance-Related Disorders}, issn = {0022-3034}, doi = {10.1002/neu.10166}, author = {Wolf, Fred W and Heberlein, Ulrike} } @article {2681, title = {Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration.}, journal = {Neuron}, volume = {39}, year = {2003}, month = {2003 Jul 17}, pages = {217-25}, abstract = {

Local axon degeneration is a common pathological feature of many neurodegenerative diseases and peripheral neuropathies. While it is believed to operate with an apoptosis-independent molecular program, the underlying molecular mechanisms are largely unknown. In this study, we used the degeneration of transected axons, termed "Wallerian degeneration," as a model to examine the possible involvement of the ubiquitin proteasome system (UPS). Inhibiting UPS activity by both pharmacological and genetic means profoundly delays axon degeneration both in vitro and in vivo. In addition, we found that the fragmentation of microtubules is the earliest detectable change in axons undergoing Wallerian degeneration, which among other degenerative events, can be delayed by proteasome inhibitors. Interestingly, similar to transected axons, degeneration of axons from nerve growth factor (NGF)-deprived sympathetic neurons could also be suppressed by proteasome inhibitors. Our findings suggest a possibility that inhibiting UPS activity may serve to retard axon degeneration in pathological conditions.

}, keywords = {Amino Acids, Animal, Animals, Axons, Benzimidazoles, Blotting, Calpain, Cells, Chelating Agents, Cultured, Cysteine Endopeptidases, Cysteine Proteinase Inhibitors, Cytoskeleton, Disease Models, Drug Interactions, Egtazic Acid, Endopeptidases, Ganglia, Immunohistochemistry, Leupeptins, Microtubules, Multienzyme Complexes, Nerve Growth Factor, Newborn, Optic Nerve, Optic Nerve Injuries, Peptide Fragments, Proteasome Endopeptidase Complex, Rats, Sympathetic, Time Factors, Tubulin, Ubiquitin, Wallerian Degeneration, Western}, issn = {0896-6273}, author = {Zhai, Qiwei and Wang, Jing and Kim, Anna and Liu, Qing and Watts, Ryan and Hoopfer, Eric and Mitchison, Timothy and Luo, Liqun and He, Zhigang} } @article {1782, title = {Independent anchoring and assembly mechanisms of INAD signaling complexes in Drosophila photoreceptors.}, journal = {The Journal of Neuroscience: The Official Journal of the Society for Neuroscience}, volume = {21}, year = {2001}, month = {2001 Jan 1}, pages = {150-8}, abstract = {

In Drosophila photoreceptors the multivalent PDZ protein INAD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. Previously, we showed that the subcellular localization of INAD signaling complexes is critical for signaling. Now we have examined how INAD complexes are anchored and assembled in photoreceptor cells. We find that trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between INAD and TRP, cause the mislocalization of the entire transduction complex. The INAD-TRP interaction is not required for targeting but rather for anchoring of complexes, because INAD and TRP can be targeted independently of each other. We also show that, in addition to its scaffold role, INAD functions to preassemble transduction complexes. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location.

}, keywords = {Animals, Calcium Channels, Cell Membrane, CHO Cells, Cricetinae, Drosophila, Drosophila Proteins, Eye Proteins, Invertebrate, Macromolecular Substances, Mutagenesis, Mutation, Ocular, Photoreceptor Cells, Precipitin Tests, Protein Kinase C, Protein Structure, Protein Transport, Site-Directed, Tertiary, TRPC Cation Channels, Type C Phospholipases, Vision}, issn = {1529-2401}, author = {Tsunoda, S and Sun, Y and Suzuki, E and Zuker, C} } @article {46432, title = {Ionic liquids based on FeCl(3) and FeCl(2). Raman scattering and ab initio calculations.}, journal = {Inorganic Chemistry}, volume = {40}, year = {2001}, month = {2001 May 7}, pages = {2298-304}, abstract = {

We have prepared ionic liquids by mixing either iron(II) chloride or iron(III) chloride with 1-butyl-3-methylimidazolium chloride (BMIC). Iron(II) chloride forms ionic liquids from a mole ratio of 1 FeCl(2)/3 BMIC to almost 1 FeCl(2)/1 BMIC. Both Raman scattering and ab initio calculations indicate that FeCl(4)(2-) is the predominant iron-containing species in these liquids. Iron(III) chloride forms ionic liquids from a mole ratio of 1 FeCl(3)/1.9 BMIC to 1.7 FeCl(3)/1 BMIC. When BMIC is in excess, Raman scattering indicates the presence of FeCl(4-). When FeCl(3) is in excess, Fe(2)Cl(7-) begins to appear and the amount of Fe(2)Cl(7-) increases with increasing amounts of FeCl(3). Ionic liquids were also prepared from a mixture of FeCl(2) and FeCl(3) and are discussed. Finally, we have used both Hartree-Fock and density functional theory methods to compute the optimized structures and vibrational spectra for these species. An analysis of the results using an all-electron basis set, 6-31G, as well as two different effective core potential basis sets, LANL2DZ and CEP-31G is presented.

}, issn = {0020-1669}, author = {Sitze, M S and Schreiter, E R and Patterson, E V and Freeman, R G} } @article {2854, title = {Identification of a yeast transcription factor IID subunit, TSG2/TAF48.}, journal = {The Journal of Biological Chemistry}, volume = {275}, year = {2000}, month = {2000 Jun 9}, pages = {17391-8}, abstract = {

The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAF(II)68/61p to analyze its function in vivo. We provide biochemical and genetic evidence that the C-terminal alpha-helix of TAF(II)68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAF(II) other than TAF(II)145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAF(II) mutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAF(II)68/61p in vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAF(II)48p is the histone 2A-like dimerization partner for the histone 2B-like TAF(II)68/61p in the yeast TFIID complex.

}, keywords = {Mutagenesis, Phenotype, Protein Structure, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Secondary, TATA-Binding Protein Associated Factors, Temperature, TFII, Transcription Factor TFIID, Transcription Factors}, issn = {0021-9258}, doi = {10.1074/jbc.M001635200}, author = {Reese, J C and Zhang, Z and Kurpad, H} } @article {1167, title = {Initiation of mitochondrial DNA replication by transcription and R-loop processing.}, journal = {The Journal of Biological Chemistry}, volume = {273}, year = {1998}, month = {1998 Nov 13}, pages = {30614-21}, abstract = {

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.

}, keywords = {Base Sequence, Catalytic Domain, Cell Line, Circular, DNA, DNA Replication, Endoribonucleases, Genetic, Humans, Mitochondrial, Molecular Sequence Data, Nucleic Acid Conformation, Ribonucleoproteins, RNA, Transcription}, issn = {0021-9258}, doi = {10.1101/gad.1352105}, author = {Lee, D Y and Clayton, D A} } @article {1567, title = {Introduction of fluorescent allosteric signal transduction elements into glucose-binding protein: construction of a novel glucose sensor.}, journal = {Journal of the American Chemical Society}, year = {1998}, month = {1998}, pages = {7-11}, author = {Marvin, Jonathan S and Hellinga, H W} } @article {45903, title = {Interneurons in the stratum lucidum of the rat hippocampus: an anatomical and electrophysiological characterization.}, journal = {J Comp Neurol}, volume = {385}, year = {1997}, month = {1997 Sep 1}, pages = {427-40}, abstract = {

The anatomical and electrophysiological properties of neurons in the stratum lucidum of the CA3 subfield of the hippocampus were examined by using patch-pipette recordings combined with biocytin staining. This method facilitated the analysis of the morphological features and passive and active properties of a recently described class of spiny neurons in the stratum lucidum, as well as aspiny neurons in this region. Some, but not all, synaptic inputs of both types of neurons were found to arise from the mossy fiber system. The axons of spiny neurons in the stratum lucidum were heavily collateralized, terminating primarily in the stratum lucidum and stratum radiatum of CA3, and to a lesser extent in the stratum pyramidale and stratum oriens. Only a few axonal projections were found that extended beyond the CA3 region into CA1 and the hilus. Aspiny neurons fell into two classes: those projecting axons to the stratum lucidum and stratum radiatum of CA3 and those with axon terminations mainly in the stratum pyramidale and stratum oriens. The electrophysiological properties of spiny and aspiny neurons in the stratum lucidum were similar, but on average, the aspiny neurons had significantly higher maximal firing rates and narrower action potential half-widths. The results demonstrate that a diverse population of neurons exists in the region of mossy fiber termination in area CA3. These neurons may be involved in local-circuit feedback, or feed-forward systems controlling the flow of information through the hippocampus.

}, keywords = {Animals, Axons, Dendrites, Hippocampus, Interneurons, Neurons, Patch-Clamp Techniques, Rats, Rats, Wistar, Synapses, Synaptic Transmission}, issn = {0021-9967}, author = {Spruston, N and L{\"u}bke, J and Frotscher, M} } @article {1214, title = {In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.}, journal = {The Journal of Cell Biology}, volume = {135}, year = {1996}, month = {1996 Dec}, pages = {1685-700}, abstract = {

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

}, keywords = {Anaphase, Animals, Base Sequence, CHO Cells, Chromatids, Chromatin, Chromosomes, Cricetinae, DNA, Electron, Gene Amplification, Gene Dosage, Green Fluorescent Proteins, In Situ Hybridization, Lac Operon, Luminescent Proteins, Microscopy, Mitosis, Molecular Sequence Data, Nucleic Acid, Recombinant Proteins, Repetitive Sequences, Staining and Labeling, Time Factors, Yeasts}, issn = {0021-9525}, author = {Robinett, C C and Straight, A and Li, G and Willhelm, C and Sudlow, G and Murray, A and Belmont, A S} } @article {1173, title = {Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies.}, journal = {Nature}, volume = {351}, year = {1991}, month = {1991 May 16}, pages = {236-9}, abstract = {

Defects in mitochondrial DNA (mtDNA) are associated with several different human diseases, including the mitochondrial encephalomyopathies. The mutations include deletions but also duplications and point mutations. Individuals with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) carry a common A-to-G substitution in a highly conserved portion of the gene for transfer RNA(Leu(UUR)). Although the MELAS mutation may be comparable to the defect in the tRNA(Lys) gene associated with MERRF (myoclonus epilepsy associated with ragged-red fibres), it is also embedded in the middle of a tridecamer sequence necessary for the formation of the 3{\textquoteright} ends of 16S ribosomal RNA in vitro. We found that the MELAS mutation results in severe impairment of 16S rRNA transcription termination, which correlates with a reduced affinity of the partially purified termination protein for the MELAS template. This suggests that the molecular defect in MELAS is the inability to produce the correct type and quantity of rRNA relative to other mitochondrial gene products.

}, keywords = {Base Sequence, Blotting, Brain Diseases, Chromosome Mapping, DNA, DNA Mutational Analysis, Genetic, Humans, Mitochondria, Mitochondrial, Molecular Sequence Data, Muscle, Mutation, Peptide Termination Factors, Ribosomal, RNA, Transcription, Transfer, Western}, issn = {0028-0836}, doi = {10.1101/gad.1352105}, author = {Hess, J F and Parisi, M A and Bennett, J L and Clayton, D A} } @article {1195, title = {Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex. An intron-containing gene with a conserved sequence occurring in the intron of COX4.}, journal = {The Journal of Biological Chemistry}, volume = {265}, year = {1990}, month = {1990 Dec 5}, pages = {20813-21}, abstract = {

A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9. QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471. The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2. The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron. The intron is 213 base pairs long and contains previously reported 5{\textquoteright} donor, 3{\textquoteright} acceptor, and TACTAAC sequences necessary for splicing. The splice junctions, as well as the 5{\textquoteright} end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9. In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4. The deduced amino acid sequence of the yeast subunit 9 is 39\% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex. If conservative substitutions are allowed for, the two proteins are 56\% similar. The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex. Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted. Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5\% of wild-type ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b. When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra. These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)

}, keywords = {Amino Acid Sequence, Base Sequence, Cell Nucleus, Chromosome Deletion, Cloning, Electron Transport Complex III, Electron Transport Complex IV, Fungal, Genes, Genomic Library, Introns, Macromolecular Substances, Models, Molecular, Molecular Sequence Data, Protein Conformation, Restriction Mapping, Saccharomyces cerevisiae}, issn = {0021-9258}, author = {Phillips, J D and Schmitt, M E and Brown, T A and Beckmann, J D and Trumpower, B L} } @article {1180, title = {In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.}, journal = {Cell}, volume = {42}, year = {1985}, month = {1985 Oct}, pages = {951-8}, abstract = {

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

}, keywords = {Animals, Base Sequence, Cattle, Cloning, DNA, DNA Replication, DNA Restriction Enzymes, Genetic, Humans, KB Cells, Macromolecular Substances, Mitochondrial, Molecular, Nucleic Acid Conformation, Templates}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Wong, T W and Clayton, D A} } @article {977, title = {Isolation and structure of a rhodopsin gene from D. melanogaster.}, journal = {Cell}, volume = {40}, year = {1985}, month = {1985 Apr}, pages = {851-8}, abstract = {

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Cattle, Chromosome Mapping, DNA, Drosophila melanogaster, Genes, Messenger, Nucleic Acid Hybridization, Poly A, Retinal Pigments, Rhodopsin, RNA, Tissue Distribution}, issn = {0092-8674}, doi = {10.1186/gb-2007-8-7-r145}, author = {Zuker, C S and Cowman, A F and Rubin, G M} }