Probes for INS

Background Aging is accompanied by altered thinking (cognition) and feeling (mood), functions that depend on information processing by brain cortical cell microcircuits. We hypothesized that age-associated long-term functional and biological changes are mediated by gene transcriptomic changes within neuronal cell-types forming cortical microcircuits, namely excitatory pyramidal cells (PYC) and inhibitory GABAergic neurons expressing vasoactive intestinal peptide (Vip), somatostatin (Sst) and parvalbumin (Pvalb). Methods To test this hypothesis, we assessed locomotor, anxiety-like and cognitive behavioral changes between young (2 months, n=9) and old (22 months, n=12) male C57BL/6 mice, and performed frontal cortex cell-type specific molecular profiling, using laser-capture microscopy and RNA sequencing. Results were analyzed by neuroinformatics and validated by fluorescent in situ hybridization. Results Old-mice displayed increased anxiety and reduced working memory. The four cell-types displayed distinct age-related transcriptomes and biological pathway profiles, affecting metabolic and cell signaling pathways, and selective markers of neuronal vulnerability (Ryr3), resilience (Oxr1), and mitochondrial dynamics (Opa1), suggesting high age-related vulnerability of PYCs, and variable degree of adaptation in GABAergic neurons. Correlations between gene expression and behaviors suggest that changes in cognition and anxiety associated with age are partly mediated by normal age-related cell changes, and that additional age-independent decreases in synaptic and signaling pathways, notably in PYC and SST-neurons further contribute to behavioral changes. Conclusions Our study demonstrates cell-dependent differential vulnerability and coordinated cell-specific cortical microcircuit molecular changes with age. Collectively, the results suggest intrinsic molecular links between aging, cognition and mood-related behaviors with SST-neurons contributing evenly to both behavioral conditions.

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of socialinformation in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.

The experience of rewarding or aversive stimuli is encoded by distinct afferents to dopamine (DA) neurons of the ventral tegmental area (VTA). Several neuromodulatory systems including oxytocin regulate DA neuron excitability and synaptic transmission that process socially meaningful stimuli. We and others have recently characterized oxytocinergic modulation of activity in mouse VTA DA neurons, but the mechanisms underlying oxytocinergic modulation of synaptic transmission in DA neurons remain poorly understood. Here, we find that oxytocin application or optogenetic release decrease excitatory synaptic transmission, via long lasting, presynaptic, endocannabinoid-dependent mechanisms. Oxytocin modulation of excitatory transmission alters the magnitude of short and long-term depression. We find that only some glutamatergic projections to DA neurons express CB1 receptors. Optogenetic stimulation of three major VTA inputs demonstrates that oxytocin modulation is limited to projections that show evidence of CB1R transcripts. Thus, oxytocin gates information flow into reward circuits in a temporally selective and pathway-specific manner.