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3605 Publications

Showing 1-10 of 3605 results
12/16/22 | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics.
Lu Z, Xu CS, Hayworth KJ, Pang S, Shinomiya K, Plaza SM, Scheffer LK, Rubin GM, Hess HF, Rivlin PK, Meinertzhagen IA
Frontiers in Neural Circuits. 2022 Dec 16;16:917251. doi: 10.3389/fncir.2022.917251

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly , in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the brain. These requirements include: good preservation of ultrastructural detail, high level of staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.

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03/18/23 | Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch
Annie Handler , Qiyu Zhang , Song Pang , Tri M. Nguyen , Michael Iskols , Michael Nolan-Tamariz , Stuart Cattel , Rebecca Plumb , Brianna Sanchez , Karyl Ashjian , Aria Shotland , Bartianna Brown , Madiha Kabeer , Josef Turecek , Genelle Rankin , Wangchu Xiang , Elisa C. Pavarino , Nusrat Africawala , Celine Santiago , Wei-Chung Allen Lee , C. Shan Xu , David D. Ginty
bioRxiv. 2023 Mar 18:. doi: 10.1101/2023.03.17.533188

Specialized mechanosensory end organs within mammalian skin—hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles—enable our perception of light, dynamic touch1. In each of these end organs, fast-conducting mechanically sensitive neurons, called Aβ low-threshold mechanoreceptors (Aβ LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aβ LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli16. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo2715 and Aβ RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aβ RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aβ RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aβ RA-LTMR activation in which axon protrusions anchor Aβ RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

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03/16/23 | Social association predicts immunological similarity in rewilded mice
A. E. Downie , O. Oyesola , R. S. Barre , Q. Caudron , Y.-H. Chen , E. J. Dennis , R. Garnier , K. Kiwanuka , A. Menezes , D. J. Navarrete , O. Mondragón-Palomino , J. B. Saunders , C. K. Tokita , K. Zaldana , K. Cadwell , P. Loke , A. L. Graham
bioRxiv. 2023 Mar 16:. doi: 10.1101/2023.03.15.532825

Environmental influences on immune phenotypes are well-documented, but our understanding of which elements of the environment affect immune systems, and how, remains vague. Behaviors, including socializing with others, are central to an individual’s interaction with its environment. We tracked behavior of rewilded laboratory mice of three inbred strains in outdoor enclosures and examined contributions of behavior, including social associations, to immune phenotypes. We found that the more associated two individuals were, the more similar their immune phenotypes were. Social association was particularly predictive of similar memory T and B cell profiles and was more influential than sibling relationships or worm infection status. These results highlight the importance of social networks for immune phenotype and reveal important immunological correlates of social life.

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03/15/23 | Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome
Kailash Venkatraman , Christopher T Lee , Guadalupe C. Garcia , Arijit Mahapatra , Guy Perkins , Keun-Young Kim , Hilda Amalia Pasolli , Sebastien Phan , Jennifer Lippincott-Schwartz , Mark Ellisman , Padmini Rangamani , Itay Budin
bioRxiv. 2023 Mar 15:. doi: 10.1101/2023.03.13.532310

The inner mitochondrial membrane (IMM) is the site of bulk ATP generation in cells and has a broadly conserved lipid composition enriched in unsaturated phospholipids and cardiolipin (CL). While proteins that shape the IMM and its characteristic cristae membranes (CM) have been defined, specific mechanisms by which mitochondrial lipids dictate its structure and function have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions shape CM morphology and ATP generation. When modulating fatty acid unsaturation in engineered yeast strains, we observed that loss of di-unsaturated phospholipids (PLs) led to a breakpoint in IMM topology and respiratory capacity. We found that PL unsaturation modulates the organization of ATP synthases that shape cristae ridges. Based on molecular modeling of mitochondrial-specific membrane adaptations, we hypothesized that conical lipids like CL buffer against the effects of saturation on the IMM. In cells, we discovered that loss of CL collapses the IMM at intermediate levels of PL saturation, an effect that is independent of ATP synthase oligomerization. To explain this interaction, we employed a continuum modeling approach, finding that lipid and protein-mediated curvatures are predicted to act in concert to form curved membranes in the IMM. The model highlighted a snapthrough instability in cristae tubule formation, which could drive IMM collapse upon small changes in composition. The interaction between CL and di-unsaturated PLs suggests that growth conditions that alter the fatty acid pool, such as oxygen availability, could define CL function. While loss of CL only has a minimal phenotype under standard laboratory conditions, we show that its synthesis is essential under microaerobic conditions that better mimic natural yeast fermentation. Lipid and protein-mediated mechanisms of curvature generation can thus act together to support mitochondrial architecture under changing environments.

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03/15/23 | Fast and sensitive GCaMP calcium indicators for imaging neural populations.
Zhang Y, Rozsa M, Liang Y, Bushey D, Wei Z, Zheng J, Reep D, Broussard GJ, Tsang A, Tsegaye G, Narayan S, Obara CJ, Lim J, Patel R, Zhang R, Ahrens MB, Turner GC, Wang SS, Korff WL, Schreiter ER, Svoboda K, Hasseman JP, Kolb I, Looger LL
Nature. 2023 Mar 15:. doi: 10.1038/s41586-023-05828-9

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

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03/13/23 | Connectome-constrained deep mechanistic networks predict neural responses across the fly visual system at single-neuron resolution
Janne K. Lappalainen , Fabian D. Tschopp , Sridhama Prakhya , Mason McGill , Aljoscha Nern , Kazunori Shinomiya , Shin-ya Takemura , Eyal Gruntman , Jakob H. Macke , Srinivas C. Turaga
bioRxiv. 2023 Mar 13:. doi: 10.1101/2023.03.11.532232

We can now measure the connectivity of every neuron in a neural circuit, but we are still blind to other biological details, including the dynamical characteristics of each neuron. The degree to which connectivity measurements alone can inform understanding of neural computation is an open question. Here we show that with only measurements of the connectivity of a biological neural network, we can predict the neural activity underlying neural computation. We constructed a model neural network with the experimentally determined connectivity for 64 cell types in the motion pathways of the fruit fly optic lobe but with unknown parameters for the single neuron and single synapse properties. We then optimized the values of these unknown parameters using techniques from deep learning, to allow the model network to detect visual motion. Our mechanistic model makes detailed experimentally testable predictions for each neuron in the connectome. We found that model predictions agreed with experimental measurements of neural activity across 24 studies. Our work demonstrates a strategy for generating detailed hypotheses about the mechanisms of neural circuit function from connectivity measurements. We show that this strategy is more likely to be successful when neurons are sparsely connected—a universally observed feature of biological neural networks across species and brain regions.

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03/10/23 | Drosophila antennae are dispensable for gravity orientation
Nikolay Kladt , Michael B. Reiser
bioRxiv. 2023 Mar 10:. doi: 10.1101/2023.03.08.531317

The nearly constant downward force of gravity has powerfully shaped the behaviors of many organisms [1]. Walking flies readily orient against gravity in a behavior termed negative gravitaxis. In Drosophila this behavior is studied by observing the position of flies in vials [24] or simple mazes [59]. These assays have been used to conduct forward-genetic screens [568] and as simple tests of locomotion deficits [1012]. Despite this long history of investigation, the sensory basis of gravitaxis is largely unknown [1]. Recent studies have implicated the antennae as a major mechanosensory input [34], but many details remain unclear. Fly orientation behavior is expected to depend on the direction and amplitude of the gravitational pull, but little is known about the sensitivity of flies to these features of the environment. Here we directly measure the gravity-dependent orientation behavior of flies walking on an adjustable tilted platform, that is inspired by previous insect studies [1316]. In this arena, flies can freely orient with respect to gravity. Our findings indicate that flies are exquisitely sensitive to the direction of gravity’s pull. Surprisingly, this orientation behavior does not require antennal mechanosensory input, suggesting that other sensory structures must be involved.

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03/10/23 | The connectome of an insect brain.
Winding M, Pedigo BD, Barnes CL, Patsolic HG, Park Y, Kazimiers T, Fushiki A, Andrade IV, Khandelwal A, Valdes-Aleman J, Li F, Randel N, Barsotti E, Correia A, Fetter RD, Hartenstein V, Priebe CE, Vogelstein JT, Cardona A, Zlatic M
Science. 2023 Mar 10;379(6636):eadd9330. doi: 10.1126/science.add9330

Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an entire insect brain ( larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses. We characterized neuron types, hubs, feedforward and feedback pathways, as well as cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.

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03/09/23 | Smart Lattice Light Sheet Microscopy for imaging rare and complex cellular events
Yu Shi , Jimmy S. Tabet , Daniel E. Milkie , Timothy A. Daugird , Chelsea Q. Yang , Andrea Giovannucci , Wesley R. Legant
bioRxiv. 2023 Mar 09:. doi: 10.1101/2023.03.07.531517

Light sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.

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Looger LabGENIE
03/08/23 | Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network
Vincent Magloire , Leonid P. Savtchenko , Sergyi Sylantyev , Thomas P. Jensen , Nicholas Cole , Jonathan S. Marvin , Loren L. Looger , Dimitri M. Kullmann , Matthew C. Walker , Ivan Pavlov , Dmitri A. Rusakov
Current Biology. 2023 Mar 08:. doi: 10.1016/j.cub.2023.02.051

Mechanisms that entrain and drive rhythmic epileptiform discharges remain debated. Traditionally, this quest has been focusing on interneuronal networks driven by GABAergic connections that activate synaptic or extrasynaptic receptors. However, synchronised interneuronal discharges could also trigger a transient elevation of extracellular GABA across the tissue volume, thus raising tonic GABAA receptor conductance (Gtonic) in multiple cells. Here, we use patch-clamp GABA ‘sniffer’ and optical GABA sensor to show that periodic epileptiform discharges are preceded by region-wide, rising waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to mechanistic principles underpinning this relationship. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells, selective optogenetic stimulation of fast-spiking interneurons. Critically, we manipulate GABA uptake to suppress extracellular GABA waves but not synaptic GABAergic transmission, which shows a clear effect on rhythm generation. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA actions in pacing regenerative rhythmic activity in brain networks.

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