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SummaryDisinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory and excitatory neurons suggests that each circuit motif is controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a newly-developed genetically-encoded GRP sensor and trans-synaptic tracing we reveal that GRP regulates VIP cells via extrasynaptic diffusion from several putative local and long-range sources. In vivo photometry and CRISPR/Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and that GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Brain function depends on forming and maintaining connections between neurons of specific types, ensuring neural function while allowing the plasticity necessary for cellular and behavioral dynamics. However, systematic descriptions of how brain cell types organize into synaptic networks and which molecules instruct these relationships are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its “starter” postsynaptic cell to that cell’s presynaptic partners1. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNA profiles – and thus cell identities and molecular states – of each host cell. We used SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, leveraging a system similar to those used to identify synaptogenic molecules. We found that the molecular identity (cell type/subtype) of the starter cell predicted the number and types of cells that had synapsed onto it. Rabies transmission tended to occur into cells with RNA-expression signatures related to developmental maturation and synaptic transmission. The estimated size of a cell’s presynaptic network, relative to that of other cells of the same type, associated with increased expression of Arpp21 and Cdh13. By tracking individual virions and their clonal progeny as they travel among host cells, single-cell, single-virion genomic technologies offer new opportunities to map the synaptic organization of neural circuits in health and disease.

The release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the pattern of urine release is modulated by external and internal factors and transmitted to the spinal cord via the pontine micturition center (PMC). Here, we exploited a behavioral paradigm in which mice, depending on strain, social experience, and sensory context, either vigorously cover an arena with small urine spots or deposit urine in a few isolated large spots. We refer to these micturition modes as, respectively, high and low territory-covering micturition (TCM) and find that the presence of a urine stimulus robustly induces high TCM in socially isolated mice. Comparison of the brain networks activated by social isolation and by urine stimuli to those upstream of the PMC identified the lateral hypothalamic area as a potential modulator of micturition modes. Indeed, chemogenetic manipulations of the lateral hypothalamus can switch micturition behavior between high and low TCM, overriding the influence of social experience and sensory context. Our results suggest that both inhibitory and excitatory signals arising from a network upstream of the PMC are integrated to determine context- and social-experience-dependent micturition patterns.

Viral vectors are essential tools for the study of neural circuits, with glycoprotein-deleted rabies viruses being widely used for monosynaptic retrograde tracing to map connectivity between specific cell types in the nervous system. However, the use of rabies virus is limited by the cytotoxicity and the inflammatory responses these viruses trigger. While components of the rabies virus genome contribute to its cytotoxic effects, the function of other neuronal and non-neuronal cells within the vicinity of the infected host neurons in either effecting or mitigating virally-induced tissue damage are still being elucidated. Here, we analyzed 60,212 single-cell RNA profiles to assess both global and cell-type-specific transcriptional responses in the mouse dorsal raphe nucleus (DRN) following intracranial injection of glycoprotein-deleted rabies viruses and axonal infection of dorsal raphe serotonergic neurons. Gene pathway analyses revealed a down-regulation of genes involved in metabolic processes and neurotransmission following infection. We also identified several transcriptionally diverse leukocyte populations that infiltrate the brain and are distinct from resident immune cells. Cell type-specific patterns of cytokine expression showed that antiviral responses were likely orchestrated by Type I and Type II interferon signaling from microglia and infiltrating CD4+ T cells, respectively. Additionally, we uncovered transcriptionally distinct states of microglia along an activation trajectory that may serve different functions, which range from surveillance to antigen presentation and cytokine secretion. Intercellular interactions inferred from transcriptional data suggest that CD4+ T cells facilitate microglial state transitions during the inflammatory response. Our study uncovers the heterogeneity of immune cells mediating neuroinflammatory responses and provides a critical evaluation of the compatibility between rabies-mediated connectivity mapping and single-cell transcriptional profiling. These findings provide additional insights into the distinct contributions of various cell types in mediating different facets of antiviral responses in the brain and will facilitate the design of strategies to circumvent immune responses to improve the efficacy of viral gene delivery.

The lateral habenula (LHb) is an epithalamic brain structure critical for processing and adapting to negative action outcomes. However, despite the importance of LHb to behavior and the clear anatomical and molecular diversity of LHb neurons, the neuron types of the habenula remain unknown. Here, we use high-throughput single-cell transcriptional profiling, monosynaptic retrograde tracing, and multiplexed FISH to characterize the cells of the mouse habenula. We find five subtypes of neurons in the medial habenula (MHb) that are organized into anatomical subregions. In the LHb, we describe four neuronal subtypes and show that they differentially target dopaminergic and GABAergic cells in the ventral tegmental area (VTA). These data provide a valuable resource for future study of habenular function and dysfunction and demonstrate neuronal subtype specificity in the LHb-VTA circuit.

The controlled and volitional release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the decision to release urine is modulated by external and internal factors such as environmental stimuli and social history and is transmitted to the spinal cord via the pontine micturition center (PMC). The neural pathways by which social experience and sensory stimuli interact to control PMC activity and regulate micturition are unclear. Here we establish a behavioral paradigm in which mice, depending on their strain, social experience, and immediate sensory context, display either high or low territory-covering micturition (TCM). We demonstrate that social context is represented by coordinated global activity changes in the urination network upstream of the PMC, whereas sensory context is represented by the activation of discrete nodes in the network. Furthermore, we show that the lateral hypothalamic area (LHA), which is directly upstream of PMC, is a key node that can switch micturition behavior between high and low TCM modes.

The lateral habenula (LHb) is an epithalamic brain structure critical for processing and adapting to negative action outcomes. However, despite the importance of LHb to behavior and the clear anatomical and molecular diversity of LHb neurons, the neuron types of the habenula remain unknown. Here we use high-throughput single-cell transcriptional profiling, monosynaptic retrograde tracing, and multiplexed FISH to characterize the cells of the mouse habenula. We find 5 subtypes of neurons in the medial habenula (MHb) that are organized into anatomical subregions. In the LHb we describe 4 neuronal subtypes and show that they differentially target dopaminergic and GABAergic cells in the ventral tegmental area (VTA). These data provide a valuable resource for future study of habenular function and dysfunction and demonstrate neuronal subtype specificity in the LHb-VTA circuit.

SUMMARYThe thalamic parafascicular nucleus (PF), an excitatory input to the basal ganglia, is targeted with deep-brain-stimulation to alleviate a range of neuropsychiatric symptoms. Furthermore, PF lesions disrupt the execution of correct motor actions in uncertain environments. Nevertheless, the circuitry of the PF and its contribution to action selection are poorly understood. We find that, in mice, PF forms the densest subcortical projection to the striatum. This projection arises from transcriptionally- and physiologically-distinct classes of PF neurons that are also reciprocally connected with functionally-distinct cortical regions, differentially innervate striatum neurons, and are not synaptically connected in PF. Thus, mouse PF contains heterogeneous neurons that are organized into parallel and independent associative, limbic, and motor circuits. Furthermore, these subcircuits share motifs of cortical-PF-cortical and cortical-PF-striatum organization that allow each PF subregion, via its precise connectivity with cortex, to coordinate diverse inputs to striatum.

The dorsal raphe nucleus (DRN) is an important source of neuromodulators in the brain and has been implicated in a wide variety of behavioral and neurological disorders. Although mostly studied as a source of serotonin, the DRN is comprised of multiple cell types that are subdivided into distinct anatomical subregions. However, the complex and incompletely characterized cellular organization of the DRN has impeded efforts to investigate the distinct circuit and behavioral functions of its subdomains. Here we used high-throughput single-cell RNA sequencing within situhybridization and viral tracing to develop a map of transcriptional and spatial profiles of cells in and around the mouse DRN. Our studies reveal the molecular and spatial organization of multiple neuron subtypes that are the cellular bases of functionally and anatomically distinct serotonergic subsystems, and provide a resource for the design of strategies for further dissection of these subsystems and their diverse functions.

The striatum, the entry nucleus of the basal ganglia, lacks laminar or columnar organization of its principal cells; nevertheless, functional data suggest that it is spatially organized. Here we examine whether the connectivity and synaptic organization of striatal GABAergic interneurons contributes to such spatial organization. Focusing on the two main classes of striatal GABAergic interneurons (fast-spiking interneurons [FSIs] and low-threshold-spiking interneurons [LTSIs]), we apply a combination of optogenetics and viral tracing approaches to dissect striatal microcircuits in mice. Our results reveal fundamental differences between the synaptic organizations of both interneuron types. FSIs target exclusively striatal projection neurons (SPNs) within close proximity and form strong synapses on the proximal somatodendritic region. In contrast, LTSIs target both SPNs and cholinergic interneurons, and synaptic connections onto SPNs are made exclusively over long distances and onto distal dendrites. These results suggest fundamentally different functions of FSIs and LTSIs in shaping striatal output.

BACKGROUND: The nucleus accumbens (NAc) is an extensively studied brain region implicated in pathological motivated behaviors such as drug addiction and is comprised predominantly of two discrete populations of neurons, dopamine receptor-1 and dopamine receptor-2 expressing medium spiny neurons (D1-MSNs and D2-MSNs, respectively). It is unclear whether these populations receive inputs from different brain areas and whether input regions to these cell-types undergo distinct structural adaptations in response to administration of addictive drugs such as cocaine. METHODS: Using a modified rabies virus-mediated tracing method, we present a comprehensive brain-wide monosynaptic input map to D1- and D2-MSNs in the NAc. Next, we analyze nearly 2,000 dendrites and over 125,000 spines of neurons across four separate input brain regions (prelimbic cortex (PrL), medial orbitofrontal cortex (MO), basolateral amygdala (BLA), and ventral hippocampus (vHPC)) at four separate time points during cocaine administration and withdrawal to examine changes in spine density in response to cocaine. RESULTS: D1- and D2-MSNs display overall similar input profiles, with the exception that D1-MSNs receive significantly more input from the MO. We find that neurons in distinct brain areas (PrL, MO, BLA, and vHPC) projecting to D1- and D2-MSNs display different adaptations in dendritic spine density changes during different stages of cocaine administration and withdrawal. CONCLUSIONS: While NAc D1- and D2-MSNs receive input from generally similar brain structures, spine density changes in specific input regions in response to cocaine administration are quite distinct and dynamic. While many previous studies have focused on input-specific postsynaptic changes in NAc MSNs in response to cocaine use, these findings emphasize the dramatic changes that also can occur in the afferent input regions as well.

Social behaviours in species as diverse as honey bees and humans promote group survival but often come at some cost to the individual. Although reinforcement of adaptive social interactions is ostensibly required for the evolutionary persistence of these behaviours, the neural mechanisms by which social reward is encoded by the brain are largely unknown. Here we demonstrate that in mice oxytocin acts as a social reinforcement signal within the nucleus accumbens core, where it elicits a presynaptically expressed long-term depression of excitatory synaptic transmission in medium spiny neurons. Although the nucleus accumbens receives oxytocin-receptor-containing inputs from several brain regions, genetic deletion of these receptors specifically from dorsal raphe nucleus, which provides serotonergic (5-hydroxytryptamine; 5-HT) innervation to the nucleus accumbens, abolishes the reinforcing properties of social interaction. Furthermore, oxytocin-induced synaptic plasticity requires activation of nucleus accumbens 5-HT1B receptors, the blockade of which prevents social reward. These results demonstrate that the rewarding properties of social interaction in mice require the coordinated activity of oxytocin and 5-HT in the nucleus accumbens, a mechanistic insight with implications for understanding the pathogenesis of social dysfunction in neuropsychiatric disorders such as autism.

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior.

Chronic stress is a strong diathesis for depression in humans and is used to generate animal models of depression. It commonly leads to several major symptoms of depression, including dysregulated feeding behaviour, anhedonia and behavioural despair. Although hypotheses defining the neural pathophysiology of depression have been proposed, the critical synaptic adaptations in key brain circuits that mediate stress-induced depressive symptoms remain poorly understood. Here we show that chronic stress in mice decreases the strength of excitatory synapses on D1 dopamine receptor-expressing nucleus accumbens medium spiny neurons owing to activation of the melanocortin 4 receptor. Stress-elicited increases in behavioural measurements of anhedonia, but not increases in measurements of behavioural despair, are prevented by blocking these melanocortin 4 receptor-mediated synaptic changes in vivo. These results establish that stress-elicited anhedonia requires a neuropeptide-triggered, cell-type-specific synaptic adaptation in the nucleus accumbens and that distinct circuit adaptations mediate other major symptoms of stress-elicited depression.

Ventral tegmental area (VTA) dopamine neurons have important roles in adaptive and pathological brain functions related to reward and motivation. However, it is unknown whether subpopulations of VTA dopamine neurons participate in distinct circuits that encode different motivational signatures, and whether inputs to the VTA differentially modulate such circuits. Here we show that, because of differences in synaptic connectivity, activation of inputs to the VTA from the laterodorsal tegmentum and the lateral habenula elicit reward and aversion in mice, respectively. Laterodorsal tegmentum neurons preferentially synapse on dopamine neurons projecting to the nucleus accumbens lateral shell, whereas lateral habenula neurons synapse primarily on dopamine neurons projecting to the medial prefrontal cortex as well as on GABAergic (γ-aminobutyric-acid-containing) neurons in the rostromedial tegmental nucleus. These results establish that distinct VTA circuits generate reward and aversion, and thereby provide a new framework for understanding the circuit basis of adaptive and pathological motivated behaviours.