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2194 Publications

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    12/22/22 | Neuronal cell types, projections, and spatial organization of the central amygdala
    O’Leary TP, Kendrick RM, Bristow BN, Sullivan KE, Wang L, Clements J, Lemire AL, Cembrowski MS
    iScience. 2022 Dec 22;25(12):105497. doi: 10.1016/j.isci.2022.105497

    The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a “bottom-up” understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

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    11/15/22 | Channel-independent function of UNC-9/Innexin in spatial arrangement of GABAergic synapses in .
    Hendi A, Niu L, Snow AW, Ikegami R, Wang Z, Mizumoto K
    eLife. 2022 Nov 15;11:. doi: 10.7554/eLife.80555

    Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in , in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

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    11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
    Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
    bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

    Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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    11/10/22 | Robotic Multi-Probe-Single-Actuator Inchworm Neural Microdrive
    Smith R, Kolb I, Tanaka S, Lee A, Harris T, Barbic M
    eLife. 2022 Nov 10:. doi: 10.7554/eLife.71876

    Electrophysiology is one of the major experimental techniques used in neuroscience. The favorable spatial and temporal resolution as well as the increasingly larger site counts of brain recording electrodes contribute to the popularity and importance of electrophysiology in neuroscience. Such electrodes are typically mechanically placed in the brain to perform acute or chronic freely moving animal measurements. The micro positioners currently used for such tasks employ a single translator per independent probe being placed into the targeted brain region, leading to significant size and weight restrictions. To overcome this limitation, we have developed a miniature robotic multi-probe neural microdrive that utilizes novel phase-change-material-filled resistive heater micro-grippers. The microscopic dimensions, gentle gripping action, independent electronic actuation control, and high packing density of the grippers allow for micrometer-precision independent positioning of multiple arbitrarily shaped parallel neural electrodes with only a single piezo actuator in an inchworm motor configuration. This multi-probe-single-actuator design allows for significant size and weight reduction, as well as remote control and potential automation of the microdrive. We demonstrate accurate placement of multiple independent recording electrodes into the CA1 region of the rat hippocampus in vivo in acute and chronic settings. Thus, our robotic neural microdrive technology is applicable towards basic neuroscience and clinical studies, as well as other multi-probe or multi-sensor micro-positioning applications.

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    11/07/22 | Cellpose 2.0: how to train your own model.
    Pachitariu M, Stringer C
    Nature Methods. 2022 Nov 07:. doi: 10.1038/s41592-022-01663-4

    Pretrained neural network models for biological segmentation can provide good out-of-the-box results for many image types. However, such models do not allow users to adapt the segmentation style to their specific needs and can perform suboptimally for test images that are very different from the training images. Here we introduce Cellpose 2.0, a new package that includes an ensemble of diverse pretrained models as well as a human-in-the-loop pipeline for rapid prototyping of new custom models. We show that models pretrained on the Cellpose dataset can be fine-tuned with only 500-1,000 user-annotated regions of interest (ROI) to perform nearly as well as models trained on entire datasets with up to 200,000 ROI. A human-in-the-loop approach further reduced the required user annotation to 100-200 ROI, while maintaining high-quality segmentations. We provide software tools such as an annotation graphical user interface, a model zoo and a human-in-the-loop pipeline to facilitate the adoption of Cellpose 2.0.

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    Looger Lab
    11/07/22 | Chemically stable fluorescent proteins for advanced microscopy.
    Campbell BC, Paez-Segala MG, Looger LL, Petsko GA, Liu CF
    Nature Methods. 2022 Nov 07:. doi: 10.1038/s41592-022-01660-7

    We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

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    11/04/22 | Facemap: a framework for modeling neural activity based on orofacial tracking
    Atika Syeda , Lin Zhong , Renee Tung , Will Long , Marius Pachitariu , Carsen Stringer
    bioRxiv. 2022 Nov 04:. doi: 10.1101/2022.11.03.515121

    Recent studies in mice have shown that orofacial behaviors drive a large fraction of neural activity across the brain. To understand the nature and function of these signals, we need better computational models to characterize the behaviors and relate them to neural activity. Here we developed Facemap, a framework consisting of a keypoint tracking algorithm and a deep neural network encoder for predicting neural activity. We used the Facemap keypoints as input for the deep neural network to predict the activity of ∼50,000 simultaneously-recorded neurons and in visual cortex we doubled the amount of explained variance compared to previous methods. Our keypoint tracking algorithm was more accurate than existing pose estimation tools, while the inference speed was several times faster, making it a powerful tool for closed-loop behavioral experiments. The Facemap tracker was easy to adapt to data from new labs, requiring as few as 10 annotated frames for near-optimal performance. We used Facemap to find that the neuronal activity clusters which were highly driven by behaviors were more spatially spread-out across cortex. We also found that the deep keypoint features inferred by the model had time-asymmetrical state dynamics that were not apparent in the raw keypoint data. In summary, Facemap provides a stepping stone towards understanding the function of the brainwide neural signals and their relation to behavior.

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    11/02/22 | Cap-dependent translation initiation monitored in living cells.
    Gandin V, English BP, Freeman M, Leroux L, Preibisch S, Walpita D, Jaramillo M, Singer RH
    Nature Communications. 2022 Nov 02;13(1):6558. doi: 10.1038/s41467-022-34052-8

    mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5'cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.

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    Sternson Lab
    11/02/22 | Characterization of Ultrapotent Chemogenetic Ligands for Research Applications in Nonhuman Primates.
    Raper J, Eldridge MA, Sternson SM, Shim JY, Fomani GP, Richmond BJ, Wichmann T, Galvan A
    ACS Chemical Neuroscience. 2022 Nov 02;13(21):3118-3125. doi: 10.1021/acschemneuro.2c00525

    Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of "Pharmacologically Selective Actuator Modules" (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent "Pharmacologically Selective Effector Molecules" (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates, it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus monkeys received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817 (0.064 mg/kg) or uPSEM792 (0.87 mg/kg), and plasma and cerebrospinal fluid samples were collected over 48 h. Both compounds exhibited good brain penetrance, relatively slow washout, and negligible conversion to potential metabolites─varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered the heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

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    10/28/22 | High-throughput automated methods for classical and operant conditioning of larvae.
    Croteau-Chonka EC, Clayton MS, Venkatasubramanian L, Harris SN, Jones BM, Narayan L, Winding M, Masson J, Zlatic M, Klein KT
    eLife. 2022 Oct 28;11:. doi: 10.7554/eLife.70015

    Learning which stimuli (classical conditioning) or which actions (operant conditioning) predict rewards or punishments can improve chances of survival. However, the circuit mechanisms that underlie distinct types of associative learning are still not fully understood. Automated, high-throughput paradigms for studying different types of associative learning, combined with manipulation of specific neurons in freely behaving animals, can help advance this field. The Drosophila melanogaster larva is a tractable model system for studying the circuit basis of behaviour, but many forms of associative learning have not yet been demonstrated in this animal. Here, we developed a high-throughput (i. e. multi-larva) training system that combines real-time behaviour detection of freely moving larvae with targeted opto- and thermogenetic stimulation of tracked animals. Both stimuli are controlled in either open- or closed-loop, and delivered with high temporal and spatial precision. Using this tracker, we show for the first time that Drosophila larvae can perform classical conditioning with no overlap between sensory stimuli (i. e. trace conditioning). We also demonstrate that larvae are capable of operant conditioning by inducing a bend direction preference through optogenetic activation of reward-encoding serotonergic neurons. Our results extend the known associative learning capacities of Drosophila larvae. Our automated training rig will facilitate the study of many different forms of associative learning and the identification of the neural circuits that underpin them.

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