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17 Janelia Publications

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    01/18/23 | Mesolimbic dopamine adapts the rate of learning from action.
    Coddington LT, Lindo SE, Dudman JT
    Nature. 2023 Jan 18:. doi: 10.1038/s41586-022-05614-z

    Recent success in training artificial agents and robots derives from a combination of direct learning of behavioural policies and indirect learning through value functions. Policy learning and value learning use distinct algorithms that optimize behavioural performance and reward prediction, respectively. In animals, behavioural learning and the role of mesolimbic dopamine signalling have been extensively evaluated with respect to reward prediction; however, so far there has been little consideration of how direct policy learning might inform our understanding. Here we used a comprehensive dataset of orofacial and body movements to understand how behavioural policies evolved as naive, head-restrained mice learned a trace conditioning paradigm. Individual differences in initial dopaminergic reward responses correlated with the emergence of learned behavioural policy, but not the emergence of putative value encoding for a predictive cue. Likewise, physiologically calibrated manipulations of mesolimbic dopamine produced several effects inconsistent with value learning but predicted by a neural-network-based model that used dopamine signals to set an adaptive rate, not an error signal, for behavioural policy learning. This work provides strong evidence that phasic dopamine activity can regulate direct learning of behavioural policies, expanding the explanatory power of reinforcement learning models for animal learning.

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    11/18/22 | TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
    Espinosa-Medina I, Feliciano D, Belmonte-Mateos C, Linda Miyares R, Garcia-Marques J, Foster B, Lindo S, Pujades C, Koyama M, Lee T
    Neuron. 2022 Nov 18:. doi: 10.1016/j.neuron.2022.10.035

    During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

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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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    09/01/22 | A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility
    Shu-Hsien Sheu , Srigokul Upadhyayula , Vincent Dupuy , Song Pang , Andrew L. Lemire , Deepika Walpita , H. Amalia Pasolli , Fei Deng , Jinxia Wan , Lihua Wang , Justin Houser , Silvia Sanchez-Martinez , Sebastian E. Brauchi , Sambashiva Banala , Melanie Freeman , C. Shan Xu , Tom Kirchhausen , Harald F. Hess , Luke Lavis , Yu-Long Li , Séverine Chaumont-Dubel , David E. Clapham
    Cell. 2022 Sep 01;185(18):3390-3407. doi: 10.1016/j.cell.2022.07.026

    Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

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    07/27/20 | A general method to optimize and functionalize red-shifted rhodamine dyes.
    Grimm JB, Tkachuk AN, Xie L, Choi H, Mohar B, Falco N, Schaefer K, Patel R, Zheng Q, Liu Z, Lippincott-Schwartz J, Brown TA, Lavis LD
    Nature Methods. 2020 Jul 27:. doi: 10.1038/s41592-020-0909-6

    Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

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    03/04/20 | Recurrent interactions in local cortical circuits.
    Peron S, Pancholi R, Voelcker B, Wittenbach JD, Ólafsdóttir HF, Freeman J, Svoboda K
    Nature. 2020 Mar 04;579(7798):256-59. doi: 10.1038/s41586-020-2062-x

    Most cortical synapses are local and excitatory. Local recurrent circuits could implement amplification, allowing pattern completion and other computations. Cortical circuits contain subnetworks that consist of neurons with similar receptive fields and increased connectivity relative to the network average. Cortical neurons that encode different types of information are spatially intermingled and distributed over large brain volumes, and this complexity has hindered attempts to probe the function of these subnetworks by perturbing them individually. Here we use computational modelling, optical recordings and manipulations to probe the function of recurrent coupling in layer 2/3 of the mouse vibrissal somatosensory cortex during active tactile discrimination. A neural circuit model of layer 2/3 revealed that recurrent excitation enhances sensory signals by amplification, but only for subnetworks with increased connectivity. Model networks with high amplification were sensitive to damage: loss of a few members of the subnetwork degraded stimulus encoding. We tested this prediction by mapping neuronal selectivity and photoablating neurons with specific selectivity. Ablation of a small proportion of layer 2/3 neurons (10-20, less than 5% of the total) representing touch markedly reduced responses in the spared touch representation, but not in other representations. Ablations most strongly affected neurons with stimulus responses that were similar to those of the ablated population, which is also consistent with network models. Recurrence among cortical neurons with similar selectivity therefore drives input-specific amplification during behaviour.

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    01/16/20 | Cortical pattern generation during dexterous movement is input-driven.
    Sauerbrei BA, Guo J, Cohen JD, Mischiati M, Guo W, Kabra M, Verma N, Mensh B, Branson K, Hantman AW
    Nature. 2020 Jan 16;577(7790):386-91. doi: 10.1038/s41586-019-1869-9

    The motor cortex controls skilled arm movement by sending temporal patterns of activity to lower motor centres. Local cortical dynamics are thought to shape these patterns throughout movement execution. External inputs have been implicated in setting the initial state of the motor cortex, but they may also have a pattern-generating role. Here we dissect the contribution of local dynamics and inputs to cortical pattern generation during a prehension task in mice. Perturbing cortex to an aberrant state prevented movement initiation, but after the perturbation was released, cortex either bypassed the normal initial state and immediately generated the pattern that controls reaching or failed to generate this pattern. The difference in these two outcomes was probably a result of external inputs. We directly investigated the role of inputs by inactivating the thalamus; this perturbed cortical activity and disrupted limb kinematics at any stage of the movement. Activation of thalamocortical axon terminals at different frequencies disrupted cortical activity and arm movement in a graded manner. Simultaneous recordings revealed that both thalamic activity and the current state of cortex predicted changes in cortical activity. Thus, the pattern generator for dexterous arm movement is distributed across multiple, strongly interacting brain regions.

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    10/30/18 | The subiculum is a patchwork of discrete subregions.
    Cembrowski MS, Wang L, Lemire AL, Copeland M, DiLisio SF, Clements J, Spruston N
    eLife. 2018 Oct 30;7:. doi: 10.7554/eLife.37701

    In the hippocampus, the classical pyramidal cell type of the subiculum acts as a primary output, conveying hippocampal signals to a diverse suite of downstream regions. Accumulating evidence suggests that the subiculum pyramidal cell population may actually be comprised of discrete subclasses. Here, we investigated the extent and organizational principles governing pyramidal cell heterogeneity throughout the mouse subiculum. Using single-cell RNA-seq, we find that the subiculum pyramidal cell population can be deconstructed into eight separable subclasses. These subclasses were mapped onto abutting spatial domains, ultimately producing a complex laminar and columnar organization with heterogeneity across classical dorsal-ventral, proximal-distal, and superficial-deep axes. We further show that these transcriptomically defined subclasses correspond to differential protein products and can be associated with specific projection targets. This work deconstructs the complex landscape of subiculum pyramidal cells into spatially segregated subclasses that may be observed, controlled, and interpreted in future experiments.

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    10/11/18 | Thy1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a for neuronal population imaging in vivo.
    Dana H, Novak O, Guardado-Montesino M, Fransen JW, Hu A, Borghuis BG, Guo C, Kim DS, Svoboda K
    PloS One. 2018;13(10):e0205444. doi: 10.1371/journal.pone.0205444

    Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

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    04/10/18 | Dissociable structural and functional hippocampal outputs via distinct subiculum cell classes.
    Cembrowski MS, Phillips MG, DiLisio SF, Shields BC, Winnubst J, Chandrashekar J, Bas E, Spruston N
    Cell. 2018 Apr 10;173(5):1280-92. doi: 10.1016/j.cell.2018.03.031

    The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.

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