Probes for INS

The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasisand after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.

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.

Intermale aggression is used to establish social rank. Several neuronal populations have been implicated in aggression, but the circuit mechanisms that shape this innate behavior and coordinate its different components (including attack execution and reward) remain elusive. We show that dopamine transporter-expressing neurons in the hypothalamic ventral premammillary nucleus (PMvDAT neurons) organize goal-oriented aggression in male mice. Activation of PMvDATneurons triggers attack behavior; silencing these neurons interrupts attacks. Regenerative PMvDAT membrane conductances interacting with recurrent and reciprocal excitation explain how a brief trigger can elicit a long-lasting response (hysteresis). PMvDAT projections to the ventrolateral part of the ventromedial hypothalamic and the supramammillary nuclei control attack execution and aggression reward, respectively. Brief manipulation of PMvDAT activity switched the dominance relationship between males, an effect persisting for weeks. These results identify a network structure anchored in PMvDAT neurons that organizes aggressive behavior and, as a consequence, determines intermale hierarchy.

Abstract

Neurotensin (Nts) is a neuropeptide implicated in the regulation of many facets of physiology, including cardiovascular tone, pain processing, ingestive behaviors, locomotor drive, sleep, addiction and social behaviors. Yet, there is incomplete understanding about how the various populations of Nts neurons distributed throughout the brain mediate such physiology. This knowledge gap largely stemmed from the inability to simultaneously identify Nts cell bodies and manipulate them in vivo. One means of overcoming this obstacle is to study NtsCremice crossed onto a Cre-inducible green fluorescent reporter line (NtsCre;GFP mice), as these mice permit both visualization and in vivo modulation of specific populations of Nts neurons (using Cre-inducible viral and genetic tools) to reveal their function. Here we provide a comprehensive characterization of the distribution and relative densities of the Nts-GFP populations observed throughout the male NtsCre;GFP mouse brain, which will pave the way for future work to define their physiologic roles. We also compared the distribution of Nts-GFP neurons with Nts-In situ Hybridization (Nts-ISH) data from the adult mouse brain. By comparing these data sets we can distinguish Nts-GFP populations that may only transiently express Nts during development but not in the mature brain, and hence which populations may not be amenable to Cre-mediated manipulation in adult NtsCre;GFPmice. This atlas of Nts-GFP neurons will facilitate future studies using the NtsCre;GFP line to describe the physiological functions of individual Nts populations and how modulating them may be useful to treat disease.

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.

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.