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191 Publications

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    09/14/20 | Novel fluorescent ligands enable single-molecule localization microscopy of the dopamine transporter.
    Guthrie D, Klein Herenbrink C, Lycas M, Ku T, Bonifazi A, DeVree B, Mathiasen S, Javitch J, Grimm JB, Lavis LD, Gether U, Newman AH
    ACS Chemical Neuroscience. 2020 Sep 14:. doi: 10.1021/acschemneuro.0c00397

    The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

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    09/10/20 | Inpainting Networks Learn to Separate Cells in Microscopy Images
    Wolf S, Hamprecht FA, Funke J
    British Machine Vision Conference. 2020 Sep:

    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.

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    09/09/20 | Cell-type specific outcome representation in primary motor cortex.
    Lavzin M, Levy S, Benisty H, Dubin U, Brosh Z, Aeed F, Mensh BD, Schiller Y, Meir R, Barak O, Talmon R, Hantman AW, Schiller J
    Neuron. 2020 Sep 9;107(5):954-71. doi: 10.1016/j.neuron.2020.06.006

    Adaptive movements are critical to animal survival. To guide future actions, the brain monitors different outcomes, including achievement of movement and appetitive goals. The nature of outcome signals and their neuronal and network realization in motor cortex (M1), which commands the performance of skilled movements, is largely unknown. Using a dexterity task, calcium imaging, optogenetic perturbations, and behavioral manipulations, we studied outcome signals in murine M1. We find two populations of layer 2-3 neurons, “success”- and “failure” related neurons that develop with training and report end-result of trials. In these neurons, prolonged responses were recorded after success or failure trials, independent of reward and kinematics. In contrast, the initial state of layer-5 pyramidal tract neurons contains a memory trace of the previous trial’s outcome. Inter-trial cortical activity was needed to learn new task requirements. These M1 reflective layer-specific performance outcome signals, can support reinforcement motor learning of skilled behavior.

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    09/07/20 | A connectome and analysis of the adult central brain.
    Scheffer LK, Xu CS, Januszewski M, Lu Z, Takemura S, Hayworth KJ, Huang GB, Shinomiya K, Maitlin-Shepard J, Berg S, Clements J, Hubbard PM, Katz WT, Umayam L, Zhao T, Ackerman D, Blakely T, Bogovic J, Dolafi T, Kainmueller D, Kawase T, Khairy KA, Leavitt L, Li PH, Lindsey L, Neubarth N, Olbris DJ, Otsuna H, Trautman ET, Ito M, Bates AS, Goldammer J, Wolff T, Svirskas R, Schlegel P, Neace E, Knecht CJ, Alvarado CX, Bailey DA, Ballinger S, Borycz JA, Canino BS, Cheatham N, Cook M, Dreher M, Duclos O, Eubanks B, Fairbanks K, Finley S, Forknall N, Francis A, Hopkins GP, Joyce EM, Kim S, Kirk NA, Kovalyak J, Lauchie SA, Lohff A, Maldonado C, Manley EA, McLin S, Mooney C, Ndama M, Ogundeyi O, Okeoma N, Ordish C, Padilla N, Patrick CM, Paterson T, Phillips EE, Phillips EM, Rampally N, Ribeiro C, Robertson MK, Rymer JT, Ryan SM, Sammons M, Scott AK, Scott AL, Shinomiya A, Smith C, Smith K, Smith NL, Sobeski MA, Suleiman A, Swift J, Takemura S, Talebi I, Tarnogorska D, Tenshaw E, Tokhi T, Walsh JJ, Yang T, Horne JA, Li F, Parekh R, Rivlin PK, Jayaraman V, Costa M, Jefferis GS, Ito K, Saalfeld S, George R, Meinertzhagen IA, Rubin GM, Hess HF, Jain V, Plaza SM
    Elife. 2020 Sep 07;9:. doi: 10.7554/eLife.57443

    The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly . Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

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    09/02/20 | Neurotransmitter Classification from Electron Microscopy Images at Synaptic Sites in Drosophila
    Eckstein N, Bates AS, Du M, Hartenstein V, Jefferis GS, Funke J
    bioRxiv. 2020 Sep 2:. doi: 10.1101/2020.06.12.148775

    High-resolution electron microscopy (EM) of nervous systems enables the reconstruction of neural circuits at the level of individual synaptic connections. However, for invertebrates, such as Drosophila melanogaster, it has so far been unclear whether the phenotype of neurons or synapses alone is sufficient to predict specific functional properties such as neurotransmitter identity. Here, we show that in Drosophila melanogaster artificial convolutional neural networks can confidently predict the type of neurotransmitter released at a synaptic site from EM images alone. The network successfully discriminates between six types of neurotransmitters (GABA, glutamate, acetylcholine, serotonin, dopamine, and octopamine) with an average accuracy of 87% for individual synapses and 94% for entire neurons, assuming each neuron expresses only one neurotransmitter. This result is surprising as there are often no obvious cues in the EM images that human observers can use to predict neurotransmitter identity. We apply the proposed method to quantify whether, similar to the ventral nervous system (VNS), all hemilineages in the Drosophila melanogaster brain express only one fast acting transmitter within their neurons. To test this principle, we predict the neurotransmitter identity of all identified synapses in 89 hemilineages in the Drosophila melanogaster adult brain. While the majority of our predictions show homogeneity of fast-acting neurotransmitter identity within a single hemilineage, we identify a set of hemilineages that express two fast-acting neurotransmitters with high statistical significance. As a result, our predictions are inconsistent with the hypothesis that all neurons within a hemilineage express the same fast-acting neurotransmitter in the brain of Drosophila melanogaster.

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    09/01/20 | Actin chromobody imaging reveals sub-organellar actin dynamics.
    Schiavon CR, Zhang T, Zhao B, Moore AS, Wales P, Andrade LR, Wu M, Sung T, Dayn Y, Feng JW, Quintero OA, Shadel GS, Grosse R, Manor U
    Nature Methods. 2020 Sep 01;17(9):917-21. doi: 10.1038/s41592-020-0926-5

    The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed 'actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

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    09/01/20 | Extensive and spatially variable within-cell-type heterogeneity across the basolateral amygdala.
    O'Leary TP, Sullivan KE, Wang L, Clements J, Lemire AL, Cembrowski MS
    eLife. 2020 Sep 01;9:. doi: 10.7554/eLife.59003

    The basolateral amygdala complex (BLA), extensively connected with both local amygdalar nuclei as well as long-range circuits, is involved in a diverse array of functional roles. Understanding the mechanisms of such functional diversity will be greatly informed by understanding the cell-type-specific landscape of the BLA. Here, beginning with single-cell RNA sequencing, we identified both discrete and graded continuous gene-expression differences within the mouse BLA. Via in situ hybridization, we next mapped this discrete transcriptomic heterogeneity onto a sharp spatial border between the basal and lateral amygdala nuclei, and identified continuous spatial gene-expression gradients within each of these regions. These discrete and continuous spatial transformations of transcriptomic cell-type identity were recapitulated by local morphology as well as long-range connectivity. Thus, BLA excitatory neurons are a highly heterogenous collection of neurons that spatially covary in molecular, cellular, and circuit properties. This heterogeneity likely drives pronounced spatial variation in BLA computation and function.

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    08/30/20 | Parvalbumin+ and Npas1+ Pallidal neurons have distinct circuit topology and function.
    Pamukcu A, Cui Q, Xenias HS, Berceau BL, Augustine EC, Fan I, Hantman AW, Lerner TN, Boca SM, Chan CS
    Journal of Neuroscience. 2020 Aug 30:
    08/27/20 | Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity.
    Clark EA, Rutlin M, Capano L, Aviles S, Saadon JR, Taneja P, Zhang Q, Bullis JB, Lauer T, Myers E, Schulmann A, Forrest D, Nelson SB
    eLife. 2020 Aug 27;9:. doi: 10.7554/eLife.52370

    Retinoic Acid-Related Orphan Receptor Beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ, , is down-regulated without RORβ, and knock-out alone disrupts TCA organization in adult barrels.

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    08/20/20 | Rational design of bioavailable photosensitizers for manipulation and imaging of biological systems.
    Binns TC, Ayala AX, Grimm JB, Tkachuk AN, Castillon GA, Phan S, Zhang L, Brown TA, Liu Z, Adams SR, Ellisman MH, Koyama M, Lavis LD
    Cell Chemical Biology. 2020 Aug 20;27(8):1063-72. doi: 10.1016/j.chembiol.2020.07.001

    Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

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