Probes for INS

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.

Viral vectors enable foreign proteins to be expressed in brains of non-genetic species, including non-human primates. However, viruses targeting specific neuron classes have proved elusive. Here we describe viral promoters and strategies for accessing GABAergic interneurons and their molecularly defined subsets in the rodent and primate. Using a set intersection approach, which relies on two co-active promoters, we can restrict heterologous protein expression to cortical and hippocampal somatostatin-positive and parvalbumin-positive interneurons. With an orthogonal set difference method, we can enrich for subclasses of neuropeptide-Y-positive GABAergic interneurons by effectively subtracting the expression pattern of one promoter from that of another. These methods harness the complexity of gene expression patterns in the brain and significantly expand the number of genetically tractable neuron classes across mammals.

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.

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.

Primary afferents transduce environmental stimuli into electrical activity that is transmitted centrally to be decoded into corresponding sensations. However, it remains unknown how afferent populations encode different somatosensory inputs. To address this, we performed two-photon Ca2+ imaging from thousands of dorsal root ganglion (DRG) neurons in anesthetized mice while applying mechanical and thermal stimuli to hind paws. We found that approximately half of all neurons are polymodal and that heat and cold are encoded very differently. As temperature increases, more heating-sensitive neurons are activated, and most individual neurons respond more strongly, consistent with graded coding at population and single-neuron levels, respectively. In contrast, most cooling-sensitive neurons respond in an ungraded fashion, inconsistent with graded coding and suggesting combinatorial coding, based on which neurons are co-activated. Although individual neurons may respond to multiple stimuli, our results show that different stimuli activate distinct combinations of diversely tuned neurons, enabling rich population-level coding.

Cracking the cytoarchitectural organization, activity patterns, and neurotransmitter nature of genetically-distinct cell types in the lateral hypothalamus (LH) is fundamental to develop a mechanistic understanding of how activity dynamics within this brain region are generated and operate together through synaptic connections to regulate circuit function. However, the precise mechanisms through which LH circuits orchestrate such dynamics have remained elusive due to the heterogeneity of the intermingled and functionally distinct cell types in this brain region. Here we reveal that a cell type in the mouse LH identified by the expression of the calcium-binding protein parvalbumin (PVALB; LHPV) is fast-spiking, releases the excitatory neurotransmitter glutamate, and sends long range projections throughout the brain. Thus, our findings challenge long-standing concepts that define neurons with a fast-spiking phenotype as exclusively GABAergic. Furthermore, we provide for the first time a detailed characterization of the electrophysiological properties of these neurons. Our work identifies LHPV neurons as a novel functional component within the LH glutamatergic circuitry.

Neuropathic pain resulting from nerve injury can become persistent and difficult to treat but the molecular signaling responsible for its development remains poorly described. Here, we identify the neuronal stress sensor dual leucine zipper kinase (DLK; Map3k12) as a key molecule controlling the maladaptive pathways that lead to pain following injury. Genetic or pharmacological inhibition of DLK reduces mechanical hypersensitivity in a mouse model of neuropathic pain. Furthermore, DLK inhibition also prevents the spinal cord microgliosis that results from nerve injury and arises distant from the injury site. These striking phenotypes result from the control by DLK of a transcriptional program in somatosensory neurons regulating the expression of numerous genes implicated in pain pathogenesis, including the immune gene Csf1. Thus, activation of DLK is an early event, or even the master regulator, controlling a wide variety of pathways downstream of nerve injury that ultimately lead to chronic pain.