The medial preoptic area (MPOA) is a central hub for maternal behavior, integrating hormonal and sensory signals to coordinate adaptive postpartum responses. Although melanin-concentrating hormone (MCH) neurons are well characterized in the lateral hypothalamus, their identity and functional engagement within MPOA circuits remain poorly defined. Here, through integrative reanalysis of a publicly available single-cell RNA sequencing dataset of the mouse MPOA (GSE295610), we identify two transcriptionally distinct Pmch -expressing neuronal populations. Both populations are GABAergic and emerge prominently during mid to late lactation. Lactation is characterized by significant upregulation of Pmch and coordinated enrichment of neuropeptidergic and hormone-responsive genes, including islet amyloid polypeptide ( Iapp ), prodynorphin ( Pdyn ), and prolactin receptor ( Prlr ). Independent single-cell gene expression profiling of FACS-isolated GAD67-GFP neurons from the MPOA further corroborated these findings, confirming the selective emergence of Pmch expression during lactation and its co-expression with neuropeptidergic and hormone-responsive genes. NeuroEstimator-based activity inference demonstrates increased predicted neuronal activity in lactating females, while pseudotime reconstruction reveals a lactation-associated transcriptional shift toward later trajectory states. hdWGCNA analysis identified gene co-expression modules significantly enriched during lactation. Regulatory network inference using SCENIC further revealed activation of activity-dependent transcriptional regulons, including cyclic AMP-responsive element-binding protein 3-like 1 ( Creb3l1 ), early growth response 1 ( Egr1 ), and FBJ osteosarcoma oncogene ( Fos ). These transcriptional programs converge on gene networks associated with synaptic plasticity, regulation of neurogenesis, and broader mechanisms of neuronal plasticity. Notably, these Pmch populations were not annotated in the original study, underscoring the power of systems-level reanalysis to uncover previously unrecognized components of maternal circuitry. Together, our findings provide single-cell evidence that MCH-expressing neurons in the MPOA undergo state-dependent transcriptional reorganization during lactation, suggesting a dynamic role for MCH signaling in postpartum neuroendocrine plasticity.

The lateral hypothalamic area (LHA) is well-known for its conserved role in modulating and driving many forms of innate behaviors, yet the cellular diversity underlying these functions remains incompletely understood. Although neurons expressing the neuropeptide thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus have been well-characterized for their hypophysiotropic functions and role in metabolism and feeding behavior, other hypothalamic TRH-expressing neurons, such as those located in the LHA, have received comparatively little attention. Here, using a viral-targeting approach in a Trh-ires-Cre mouse, we probe the anatomical and electrophysiological properties of TRH-expressing neurons in the LHA (LHA(TRH)). We find that LHA(TRH) neurons make both ascending and descending axonal projections throughout the brain, with particularly dense projections to the regions of the dorsal lateral septum, lateral preoptic, basal forebrain, and dorsal premammillary nuclei. We further define the active and passive membrane properties of LHA(TRH) neurons in slices, as well as the morphology of filled neurons. We find that the distinguishing features of LHA(TRH) neurons, in contrast to intermingled hypocretin/orexin-expressing neurons, are capable of anode-break bursting in a majority of neurons and exhibit rhythmic spontaneous bursting in a smaller subset of neurons. We further compare the electrophysiological features of both anode-break bursting and nonbursting LHA(TRH) neurons, as well as determining that anode-break bursting is mediated by T-type calcium channels. Our work defines both the intrinsic membrane properties, morphology and axonal projections of LHA(TRH) neurons, providing a foundation for understanding their roles in physiology and behavior. NEW & NOTEWORTHY This work provides the first characterization of the axonal projections and distinctive electrophysiological properties that define thyrotropin-releasing hormone (TRH)-expressing neurons in the mouse lateral hypothalamic area (LHA) in acute brain slices. We find that a large majority of LHA TRH neurons exhibit robust rebound burst firing, with a smaller subpopulation exhibiting spontaneous rhythmic burst firing. Together, these findings advance our understanding of how LHA TRH neurons may contribute to regulating physiology and behavior.

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity, many of which are implicated in neuropsychiatric disorders. Many members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell-type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and postsynaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a valuable tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.

KCNQ2 potassium channel variants are linked to developmental and epileptic encephalopathy (DEE). However, the mechanisms by which pathogenic variants, especially those outside known hotspots, such as the S4-S5 linker, lead to disease remain unknown. Here, we examined the H228R variant, a pathogenic mutation in the S4-S5 linker associated with DEE. We tested whether H228R induces KCNQ2 channel mistargeting in addition to its biophysical effects, given recent evidence of impaired trafficking in KCNQ2 DEE variants. We confirmed the H228R variant as a loss-of-function (LOF) when expressed as a homomer and as a dominant-negative when coexpressed with wild-type (WT) KCNQ3. Surprisingly, it exhibited some gain-of-function effects when coexpressed with WT KCNQ2. To determine its cellular localization in vivo, we used male and female heterozygous Kcnq2H228R knock-in mice, some of which die prematurely despite lack of increases in hippocampal excitatory neuron intrinsic excitability. We validated two different KCNQ2 antibodies in hippocampus via immunohistochemistry. These antibodies detected KCNQ2 in axons, with signal loss observed in Kcnq2 knock-out mice. Using these antibodies, we found that the H228R variant caused KCNQ2 channels to concentrate in the soma, strongly reducing their presence in axons. Further, analysis of heterozygous mice expressing both a FLAG-tagged WT KCNQ2 and H228R revealed that the FLAG-WT KCNQ2 could still traffic to axons, indicating that some KCNQ2 channels are correctly targeted within neurons. In summary, our results demonstrate that the LOF H228R variant disrupts the localization of variant KCNQ2 channels, suggesting mislocalization as a general endophenotype of KCNQ2 encephalopathy.

ABSTRACT Lateral hypothalamic GABAergic (LHA GABA ) neurons regulate arousal, feeding, and reward-related behaviors, but how their downstream projections coordinate motivated behaviors across domains remains incompletely defined. Our histological analyses revealed that LHA GABA fibers were distributed across the dorsal pons (DP) subregions, including the peri-locus coeruleus, laterodorsal tegmentum, and Barrington’s nucleus, and extend throughout the lateral preoptic area (LPO), thereby refining existing anatomical descriptions. We then used optogenetics to systematically compare the effects of activating LHA GABA somata and their projections to the DP and LPO across assays of feeding, non-food-directed gnawing, predatory behavior, real-time place preference, and operant self-stimulation. In sated mice, optogenetic activation of LHA GABA somata or their terminals in the DP or LPO increased caloric food intake, whereas non-caloric cellulose intake was minimally affected during terminal stimulation. Across conditions, activation increased gnawing and shredding of non-food objects while reducing inactivity. In cricket hunting, stimulation increased cricket killing and consumption relative to controls. Similarly, all stimulation conditions supported positive-valence and reinforcement-related responding, as indicated by real-time place preference and operant self-stimulation. Together, these results provide new functional evidence that activation of LHA GABA somata and projections to both the DP and LPO recruit largely overlapping behavioral responses across feeding, non-food behavior, predatory hunting, and reinforcement-related assays. These findings support a distributed hypothalamic output architecture in which major ascending and descending LHA GABA pathways contribute to a shared motivational repertoire rather than wholly discrete behavioral functions.

Disclosure: L.J. DiLeone: None. M.S. Antony: None. A.C. Jackson: None. Melanin-concentrating hormone (MCH; encoded for by the Pmch gene) is a neuropeptide expressed by a unique population of neuromodulatory neurons, localized in the mammalian lateral hypothalamic area (LHA). MCH neurons play a pivotal role in a diversity of both sleep- and wake-related innate behaviors such as the regulation of rapid-eye movement (REM) sleep, feeding, memory consolidation, and exploratory behavior. Despite its functional heterogeneity, neurochemical and molecular diversity among MCH neurons is poorly understood. Building upon previous anatomical and developmental studies, our single-cell RNA sequencing of the LHA confirmed that MCH neurons exhibit molecular heterogeneity in the form of two major subpopulations, defined by multiple markers. Notably, one subset of MCH neurons exhibits an enrichment in transcripts expressing cocaine- and amphetamine-regulated transcript (Cartpt) and tachykinin receptor-3 (Tacr3). We hypothesize that MCH neurons are molecularly diverse, with distinct subpopulations differentially contributing to their specialized roles in regulating opposing behaviors through unique anatomical projections. Here, we describe an intersectional genetic strategy for gaining genetic access to a molecularly distinct MCH subpopulation in mice. We crossed a Pmch-Cre; Tacr3-Flpo dual-recombinase mouse to an intersectional reporter line, RC::FLTG to generate a Pmch-Cre; Tacr3-Flpo; RC::FLTG mouse which exhibits fluorescent labelling of MCH(Tacr3+) cell bodies and axons. We first characterized the specificity of the intersectional targeting within the LHA and the differential spatial distribution of labeled vs. unlabeled MCH+ neurons within the LHA. We then identified unique anatomical projection targets of labeled MCH(Tacr3+) neurons within the forebrain and midbrain. Ultimately, this approach will enable us to identify molecularly distinct MCH subpopulations and their anatomical projections, providing important groundwork for future studies aimed at understanding the roles of these neuronal subpopulations in distinct behaviors. Presentation: Saturday, July 12, 2025

It remains unexplored in the field of fear memory whether functional neuronal connectivity between two brain areas is necessary for one sex but not the other. Here, we show that chemogenetic silencing of centromedial (CeM)– Tac2 fibers in the lateral posterior BNST (BNSTpl) decreased fear memory consolidation in male mice but not females. Optogenetic excitation of CeM- Tac2 fibers in the BNSTpl exhibited enhanced inhibitory postsynaptic currents in males compared to females. In vivo calcium imaging analysis revealed a sex-dimorphic fear memory engram in the BNSTpl. Furthermore, in humans, the single-nucleotide polymorphism (SNP) in the Tac2 receptor (rs2765) ( TAC3R ) decreased CeM-BNST connectivity in a fear task, impaired fear memory consolidation, and increased the expression of the TAC3R mRNA in AA-carrier men but not in women. These sex differences in critical neuronal circuits underlying fear memory formation may be relevant to human neuropsychiatric disorders with fear memory alterations such as posttraumatic stress disorder.

While gene regulatory networks underlying hypothalamic development are being characterized, minor intron splicing remains unexplored. Here, we used Nkx2 . 1-Cre to ablate Rnu11 , encoding the minor spliceosome-specific U11 snRNA, in the progenitors of the ventral diencephalon (VD), to study minor intron splicing in hypothalamic development and control of energy balance in mice. Loss of U11 resulted in aberrant minor intron splicing, mitotic stalling, apoptosis, and altered neurogenesis. Mutant mice exhibited gross dysgenesis of hypothalamic architecture, while single-cell RNA sequencing (scRNAseq) revealed aberrant composition of neuronal subtypes implicated in feeding and energy balance. Mutant weanlings failed to thrive, followed by rapid weight gain, resulting in obesity. Assessment of energy imbalance and pair-feeding demonstrated that hyperphagia in adult mutants initiates weight gain, and is compounded by metabolic dysfunction, ultimately resulting in obesity. Our findings suggest a key role of minor intron splicing in the developmental patterning of hypothalamic neuronal subtypes underlying energy balance.

An explosion of recent work has harnessed the power of single-cell biology to reveal the transcriptomic diversity of cell types in the mouse hypothalamus. Steuernagel, Lam and co-authors present HypoMap, a comprehensive single-cell transcriptomic atlas of the mouse hypothalamus.

The lateral hypothalamic area (LHA) is a highly conserved brain region critical for maintaining physiological homeostasis and goal-directed behavior. LHA neurons that express melanin-concentrating hormone (MCH) are key regulators of arousal, energy balance, and motivated behavior. However, cellular and functional diversity among LHAMCH neurons is not well understood. Previous anatomic and molecular data suggest that LHAMCH neurons may be parsed into at least two distinct subpopulations, one of which is enriched in neurokinin-3 receptor (NK3R), the receptor for neurokinin B (NKB), encoded by the Tac2 gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory, and stress in other brain regions, but NKB interactions with LHAMCH neurons are poorly understood. We first identified how LHAMCH subpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHAMCH neurons, we used Cre-dependent retrograde and anterograde viral tracing in male Tac2-Cre mice and identified Tac2/EYFP+ neurons in the bed nucleus of the stria terminalis and central nucleus of the amygdala, the central extended amygdala, as major sources of NKB input onto LHAMCH neurons. In addition to innervating the LHA, these limbic forebrain NKB neurons also project to midbrain and brainstem targets. Finally, using a dual-virus approach, we found that optogenetic activation of these inputs in slices evokes GABA release onto a subset of LHAMCH neurons but lacked specificity for the NK3R+ subpopulation. Overall, these data define parallel tachykininergic/GABAergic limbic forebrain projections that are positioned to modulate multiple nodes of homeostatic and behavioral control.SIGNIFICANCE STATEMENT The LHA orchestrates fundamental behavioral states in the mammalian hypothalamus, including arousal, energy balance, memory, stress, and motivated behavior. The neuropeptide MCH defines one prominent population of LHA neurons, with multiple roles in the regulation of homeostatic behavior. Outstanding questions remain concerning the upstream inputs that control MCH neurons. We sought to define neurochemically distinct pathways in the mouse brain that may communicate with specific MCH neuron subpopulations using viral-based retrograde and anterograde neural pathway tracing and optogenetics in brain slices. Here, we identify a specific neuropeptide-defined forebrain circuit that makes functional synaptic connections with MCH neuron subpopulations. This work lays the foundation for further manipulating molecularly distinct neural circuits that modulate innate behavioral states.

The ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance, and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single-cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell populations in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in selected neuronal populations through fluorescence in situ hybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit neuroanatomical parcellation within MB subdivisions and topographic projections to the thalamus. This single-cell transcriptomic atlas of VPH cell types provides a resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits.

ABSTRACTThe ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell types in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in a selection of neuronal populations through fluorescencein situhybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit a surprising degree of segregation within neuroanatomical subdivisions of the MB, while genetically-defined MB cell types project topographically to the anterior thalamus. This single cell transcriptomic atlas of cell types in the VPH provides a detailed resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits in health and disease.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance, and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single-cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell populations in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in selected neuronal populations through fluorescence in situ hybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit neuroanatomical parcellation within MB subdivisions and topographic projections to the thalamus. This single-cell transcriptomic atlas of VPH cell types provides a resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits. Introduction The ventral posterior hypothalamus (VPH) is a functionally and cytoarchitecturally complex region of the hypothalamus, dominated by the mammillary bodies (MB), a discrete diencephalic structure on the basal surface of the VPH. Surrounding VPH subregions include the premammillary (PM), supramammillary (SUM), and tuberomammillary (TMN) nuclei as well as the caudal arcuate nucleus (Arc) and caudal lateral hypothalamic area (LHA). These subregions are embedded within diverse neural systems, with widespread afferents and efferents, known to regulate distinct physiological and behavioral functions (Saper and Lowell, 2014; Simerly, 2015; Card and Swanson, 2013). The MB are best known as the diencephalic branch of the classic limbic Circuit of Papez (Papez, 1937), which links the hippocampal formation with the anterior thalamus and midbrain/pons, and is critical for spatial memory in both rodent and primate models, as well as episodic memory in humans (Vann and Aggleton, 2004; Vann, 2010; Aggleton et al., 2010; Dillingham et al., 2015; Vann and Nelson, 2015). VPH subregions surrounding the MB are also robust modulators of behavioral state. The ventral and dorsal PM nuclei are implicated in reproductive and defensive behaviors, respectively (Canteras et al., 2008; Donato and Elias, 2011; Leshan and Pfaff, 2014). The SUM is associated with arousal and modulation of theta rhythms (Luppi et al., 2017; Pan and McNaughton, 2004; Vertes, 2015). The Arc is a crucial node in the regulation of hunger, satiety, and reproduction (Graebner et al., 2015; Andermann and Lowell, 2017; Lehman et al., 2010). Finally, the TMN, populated by histamine (HA)-synthesizing neurons, is an important modulator of wakefulness (Brown et al., 2001; Haas et al., 2008; Panula and Nuutinen, 2013). The functional diversity of the VPH is likely explained by cellular heterogeneity among these neuronal populations, the neural circuits they give rise to, and the complex brain-wide networks in which they are embedded. A significant obstacle in understanding the circuit-level mechanisms underlying its function is that VPH neuronal cell type diversity is poorly understood. Here we employ single-cell RNA sequencing (scRNA-seq) to develop a comprehensive molecular census of transcriptionally distinct cell populations in the VPH of both male and female juvenile mice. In our unsupervised analysis of over 16,000 isolated single cells, we identify 18 non-neuronal clusters and 20 distinct neuronal clusters, the majority of which are glutamatergic. Transcriptionally distinct neuronal populations are defined by the unique expression of discriminatory markers that include neuropeptides, transcription factors, calcium-binding proteins, and signaling molecules. We went on to validate differentially expressed genes in a selection of identified neuron populations through multiplexed fluorescence in situ hybridization (FISH), ISH data from the Allen Brain Institute (ABA) (Lein et al., 2007), as well as anterograde tract-tracing in genetically-distinct populations in the MB. Taken together, our identification of the population structure and cellular diversity of VPH cell populations provides a resource for detailed genetic dissection of VPH circuits and interrogation of their specific roles in behavior, in both health and disease states. Results Isolation of single cells from the mouse VPH for transcriptomic analysis To isolate single cells from the mouse VPH for scRNA-seq analysis, we microdissected the region of the VPH from fresh brain slices obtained from a total of five male and five female C57BL/6 mice (P30-34), as described previously (Mickelsen et al., 2017; Mickelsen et al., 2019), in two separate harvests (see Materials and methods). Single-cell suspensions were loaded onto a Chromium Controller (Figure 1a) and processed using the 10x Genomics platform (Zheng et al., 2017). All VPH microdissections were mapped for consistency using anatomical landmarks across the rostrocaudal axis (Figure 1b; Paxinos, 2012). The two harvests used 10x V2 and V3 chemistry, respectively, and both consisted of separate male and female pools for a total of four separate pools. We found little sex-dependent differences within each harvest (Figure 1c) and samples from the two harvests were pooled and batch corrected to account for chemistry-dependent differences (Figure 1—figure supplement 1a; see Materials and methods). In our pooled data set, the median transcripts (UMIs)/cell was 8,336 and the median genes/cell was 3,415 (Figure 1d). We used the 1,500 genes with the highest normalized dispersion for dimensionality reduction using principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) followed by cluster identification using Leiden community detection, identifying a total of 20 clusters in the first iteration of clustering (Figure 1e,f). Neuronal and non-neuronal clusters were segregated using a two-component Gaussian mixture model trained on the per-cluster average expression of four pan-neuronal markers Snap25, Syp, Tubb3, and Elavl2 (Figure 1—figure supplement 1b,c) leading to a binary classification of neuronal and non-neuronal cells (Figure 1e,f). Subsequent clustering of only neuronal cells (20 clusters; Figure 1—figure supplement 2a,c) and only non-neuronal cells (18 clusters; Figure 1—figure supplement 2b,d) showed comparable proportions from each sex and batch. Figure 1 with 5 supplements see all Download asset Open asset Overview of VPH microdissection, single-cell isolation, batch correction, and clustering. (a) Workflow schematic representing the VPH microdissection from coronal mouse brain slices, single-cell dissociation, sequencing library preparation, and bioinformatic analysis (Mickelsen et al., 2019). (b) Location of VPH microdissections mapped onto the coronal mouse brain atlas at distances from bregma of −2.54, –2.70, −2.92, and –3.16 mm. Atlas images were modified from Paxinos, 2012. (c) Two-dimensional UMAP plots representing 16,991 single cells from four sequencing libraries color-coded by mouse sex (left) and the 10x Genomics chemistry version (right) following batch correction. (d) Histograms of unique transcripts (left) and genes (right) were detected in 16,991 single cells after quality control. Dashed vertical lines represent the median transcripts and genes per cell, respectively. (e) Heatmap and (f) UMAP plot showing the first iteration of unsupervised clustering revealing 20 unique clusters. Neuronal populations are disjoint from non-neuronal populations. Identification of marker genes and testing gene specificity with a classification model Throughout this study, we present multiple links between the transcriptional signatures of groups of single cells and their anatomical mapping. A key way in which we represent the shared transcriptional signature of groups of cells is through the presentation of marker genes, the expression of which is over-represented in a given population, as violin plots, which we use to show the distribution of transcripts per cell in each cluster for a given gene. While an individual marker gene may not be sufficient to uniquely describe an individual population, we provide an exhaustive set of markers that, when used in aggregate, specifically and uniquely identify each population described herein. Given that the median number of transcripts per cell is a small multiple of the number of genes per cell (Figure 1d), consistent with similar studies (e.g. Mickelsen et al., 2019; Moffitt et al., 2018; Chen et al., 2017), it is important to verify whether the marker gene expression presented is specific and robust. We first examined the distribution of transcripts captured per gene (Figure 1—figure supplement 2e and f) with the genes that we present as markers typically being the most highly expressed genes in the data set. Moreover, typical marker genes are expressed with at least 10 counts in a cell, and often with substantially more counts (Figure 1—figure supplement 2g). We also verified that the markers presented are sufficient to uniquely describe each population. For this, we built a model that predicts the cluster identity of individual cells using only the expression of a small number of marker genes (Figure 1—figure supplement 3a). We then computed the number of incorrect cluster label predictions (as compared to each cells’ original label as shown in Figure 2b). This computational experiment demonstrates that we can predict the neuronal cluster identity with over 90% accuracy with fewer than only 80 total genes (Figure 1—figure supplement 3b–d). Notably, this is less than 10% of the 1500 genes used to guide the neuronal dimensionality reduction and clustering analysis. Moreover, many of the misclassifications are between related clusters that share markers or within the clusters with uncertain cluster identity (ex. GLUT10, GLUT11, GABA12, GABA13; Figure 1—figure supplement 3e). Most clusters ascribed to discrete anatomical regions can be correctly labeled with almost 100% accuracy with fewer than five marker genes. This model suggests that the marker genes for the populations described in Figures 3, 4, 5, 6 and figure supplements are more than adequate to identify individual cells from these populations. Figure 2 Download asset Open asset Classification of VPH neuronal populations. (a) UMAP plots showing normalized expression of Slc17a6, Slc32a1, Gad1, and Hdc after the second iteration of unsupervised clustering on just neuronal cells. Using these four genes, neurons were classified by a three-class Gaussian mixture model as glutamatergic (GLUT, blue), GABAergic (GABA, yellow), or histaminergic (HA, green). (b) Unsupervised clustering of 20 VPH neuronal cell types shown in a UMAP embedding. (c) Heatmap showing scaled expression of discriminatory genes across all 20 neuronal clusters. (d) Violin plot showing the distribution of normalized expression in each cluster of neurotransmitters (Slc17a6, Slc32a1, Gad1, Gad2) (upper) and discriminatory marker genes (lower). (e) Violin plots showing the distribution of the number of unique transcripts (upper) and the number of genes (lower) in each neuronal cluster. Figure 3 with 1 supplement see all Download asset Open asset Identification of a population of putative PMv neurons and a catecholaminergic PMv subpopulation. (a) UMAP plots showing normalized expression of Tac1, Nos1, Calb2, and Foxp2 enriched in VPHGLUT cluster 7 following Slc32a1 and Slc17a6 (top). (b) Violin plot showing discriminatory marker genes enriched in cluster 7. (c) Mouse brain atlas schematic, modified from Paxinos, 2012, showing the PMv in a coronal section at distance from bregma of −2.46 mm (top). ISH images for Tac1, Nos1, Calb2, and Foxp2 from the ABA (Lein et al., 2007; bottom). (d) Confocal micrographs (40×) of FISH in coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA for Tac1 and Slc17a6 (n = 678 cells, three mice; upper) and Tac1 and Calb2 (n = 963 cells, three mice; lower). Scale bar (applicable to all micrographs) 50 μm. (e) UMAP plots showing normalized expression of markers in VPHGLUT cluster 7 only, including cell type markers for reference (Tac1, Adcyap1, and Slc17a6) and markers that define a subpopulation of putative catecholaminergic neurons (Slc6a3, Slc18a2 and Ddc, but with very low Th). ISH image from the ABA (Lein et al., 2007) showing Slc6a3 expression in the PMv (inset). Figure 4 with 3 supplements see all Download asset Open asset Identification of a population of putative PMd neurons (VPHGLUT cluster 6). (a) UMAP plots showing normalized expression of Cck, Foxb1 (shared with VPHGLUT clusters 1–5), Synpr, Dlk1, Ebf3, and Stxbp6 (enriched in VPHGLUT cluster 6). (b) Violin plot showing discriminatory marker genes enriched in VPHGLUT cluster 6 following Slc32a1 and Slc17a6 (top). (c) Mouse brain atlas schematic (Paxinos, 2012) showing the PMd in a coronal section at distance from bregma of −2.70 mm (top). (d) ISH images for three anterior to posterior coronal sections (approximate distance from bregma −2.46,–2.80, and −2.92 mm) for Synpr (left), Dlk1 (middle), and Stxbp6 (right) from the ABA (Lein et al., 2007; bottom). In each case, expression appears to be enriched in the PMd in anterior sections and largely absent in the MB in the posterior section. Figure 5 with 1 supplement see all Download asset Open asset Identification of histaminergic (HA) neurons in the TMN. (a) UMAP plots showing normalized expression of Hdc, Slc18a2, Wif1, and Maob enriched in VPHHA cluster 17 (circled). (b) Violin plot showing discriminatory marker genes enriched in VPHHA cluster 17 following Slc32a1 and Slc17a6 (top). (c) Mouse brain atlas schematic, modified from Paxinos, 2012, showing the dorsal TMN (TMNd) and ventral TMN (TMNv) in a coronal section at distance from bregma of −2.54 mm (top). ISH images for Hdc, Slc18a2, Wif1, and Maob from the ABA (Lein et al., 2007; bottom). (d) Confocal micrographs (40×) of FISH in coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA in the TMNv (top) for Hdc and Wif1 (n = 415 cells, three mice; upper) and Hdc and Maob (n = 570 cells, three mice; lower) and in the TMNd (bottom) for Hdc and Wif1 (n = 109 cells, three mice; upper) and Hdc and Maob (n = 192 cells, three mice; lower). Scale bar (applicable to all micrographs) 50 μm. Figure 6 with 1 supplement see all Download asset Open asset Identification of lateral mammillary (LM) neurons. (a) UMAP plots showing normalized expression of Tac2, Tcf4, Cplx1, and Pvalb enriched in VPHGLUT cluster 20 (circled). (b) Violin plot showing discriminatory marker genes enriched in VPHGLUT cluster 20 following Slc32a1 and Slc17a6 (top). (c) Mouse brain atlas schematic, modified from Paxinos, 2012, showing the LM in a coronal section at distance from bregma of −2.92 mm (top). ISH images for Tac2, Tcf4, Cplx1, and Pvalb from the ABA (Lein et al., 2007; bottom). (d) Confocal micrographs (40×) of FISH in coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA for Slc17a6 and Tac2 (n = 1210 cells, three mice; upper), Slc17a6 and Pvalb (n = 1057 cells, three mice, upper middle), Slc17a6 and Cplx1 (n = 1269 cells, three mice; lower middle), and Slc17a6 and Tcf4 (n = 1642 cells, three mice; lower), Scale bar (applicable to all micrographs) 50 μm. Major non-neuronal cell types in the VPH Among non-neuronal cell populations in the VPH, we identified 18 distinct clusters distinguished from one another by suites of cell type-specific discriminatory markers (Figure 1—figure supplement 4a–d). We resolved six distinct populations of oligodendrocyte lineage cells arranged in a contiguous strand in a UMAP plot (Figure 1—figure supplement 4a), likely reflecting a developmental gradient of gene expression. These include oligodendrocyte precursor cells (OPC or NG2+ cells; cluster 1), immature oligodendrocytes (cluster 2), and four oligodendrocyte populations (clusters 3–6), all of which exhibit a wave of differentially-expressed genes (Cspg4, Fyn, Ctps, Tspans2, Apod, Klk6, etc.; Figure 1—figure supplement 5a and b) that aligns well with the diversity of functional markers of the oligodendrocyte lineage in the mouse brain identified through previous scRNA-seq analyses (Marques et al., 2016; Zeisel et al., 2018; Saunders et al., 2018). We also resolved three distinct clusters of astrocytes (clusters 7, 8, and 9), all of which are Aqp4+ and Agt+. Clusters 7 and 8 are distinguishable by high expression of Slc7a10 and Htra1 and low expression of Gfap, while cluster 9 exhibits high expression of both Gfap and C4b (Figure 1—figure supplement 5c and d). In nearby clusters, we identified tanycytes (cluster 10: Rax+) and ependymal cells (cluster 11: Ccdc153+, S100a6+; Figure 1—figure supplement 5c and d). In addition, we found other distinct clusters readily identifiable as macrophages (cluster 12: Mrc1+), microglia (cluster 13: Tmem119+), pericytes (cluster 14: Rgs5+), vascular smooth muscle cells (VSMCs; cluster 15: Rgs5+, Acta2+), two populations of putative vascular leptomeningeal cells or VLMCs (cluster 16: Fxyd5+, Slc47a1+; cluster 17: Dcn+) and vascular endothelial cells (VECs; cluster 18: Pecam1+, Slc38a5+; Figure 1—figure supplement 3b). Our identification of major non-neuronal VPH cell types is based on, and closely aligns with, previous scRNA-seq analyses from mouse brain (Marques et al., 2016; Zeisel et al., 2018; Saunders et al., 2018). Diverse populations of excitatory and inhibitory neuronal cell clusters in the VPH Among the neuronal clusters, which contained significantly more genes and UMIs per cell (4334 and 12,065, respectively) than the overall data set, we first examined broad patterns in the expression of fast amino acid and monoamine neurotransmitter markers (Figure 2a). Expression of genes necessary for the synthesis and packaging of the excitatory transmitter glutamate (Slc17a6, encoding the vesicular glutamate transporter 2, VGLUT2) and the inhibitory transmitter GABA (Slc32a1, encoding the vesicular GABA transporter, VGAT) provided a binary classification of Slc17a6+ clusters as glutamatergic (VPHGLUT) and Slc32a1+ clusters as GABAergic (VPHGABA) neurons. This was further supported by the expression of the gene encoding a synthetic enzyme for GABA (Gad1/GAD67) which largely aligns with Slc32a1+ clusters. We found that of the 20 neuronal clusters we identified, thirteen are glutamatergic, six are GABAergic and one (cluster 17) best matched the profile of a unique population of histaminergic (HA) neurons based on the unique expression of histidine decarboxylase (Hdc/HDC; Figure 2a,b). Overall, within these neuronal populations, clusters are distinguished by suites of differentially expressed transcripts (Figure 2c,d) with comparable UMIs/cell and genes/cell (Figure 2e). In the following analyses, we validated the co-expression of key markers and their spatial organization in selected VPH neuronal populations. In all cases, we first attempted to map transcriptionally distinct cell clusters onto specific anatomical subregions within the VPH, by comparing differentially-expressed transcripts with the online database of in situ hybridization (ISH) data from the Allen Brain Atlas (ABA; Lein et al., 2007), followed by co-expression analysis using multiplexed FISH. Transcriptional signatures differentiate ventral and dorsal premammillary (PM) nuclei Among neuronal clusters that correspond to discrete anatomical subregions of the VPH, we identified at least one VPHGLUT population with a suite of differentially-expressed transcripts that appears to closely match the ventral premammillary nucleus (PMv; Figure 3). VPHGLUT cluster 7 is enriched in the following key transcripts: Tac1 (encoding substance P or SP), Nos1 (encoding neuronal nitric oxide synthase or NOS), Calb2 (encoding calretinin), and Foxp2 (encoding the transcription factor forkhead box P2; Figure 3a) along with a host of other differentially expressed markers (Figure 3b). The spatial pattern of expression of each of the markers in Figure 3a were found in the ABA (Lein et al., 2007), and correspond closely to the PMv (Figure 3c). Through FISH co-expression analysis, we found that Tac1, Calb2, and Slc17a6 are extensively co-expressed in a large cluster of neurons in the PMv (Figure 3d). These data broadly align with several known markers for the PMv (Donato and Elias, 2011). For example, SP (Shimada et al., 1988; Larsen, 1992) and NOS (Vincent and Kimura, 1992) are both enriched in the PMv. Another marker that is enriched in VPHGLUT cluster 7 is Adcyap1 (encoding the neuropeptide pituitary adenylate cyclase-activating polypeptide, or PACAP; Figure 3b). Consistent with the role of the PMv in reproductive function (Donato and Elias, 2011; Leshan and Pfaff, 2014), PACAP+ PMv neurons were recently shown to critically regulate female reproductive physiology and fertility (Ross et al., 2018). Interestingly, we also found that a distinct subpopulation of VPHGLUT cluster 7 neurons robustly expresses markers of catecholaminergic neurotransmission including Slc6a3 (encoding the dopamine transporter, DAT), Ddc (encoding DOPA decarboxylase), and Slc18a2 (encoding the vesicular monoamine transporter 2, VMAT2) but low expression of Th (encoding tyrosine hydroxylase; Figure 3e). This cluster corresponds well to a previously identified catcholaminergic (Zoli et al., 1993), Slc6a3+ PMv population (Meister and Elde, 1993) that was more recently found, through circuit and behavioral analyses, to regulate male social behavior (Soden et al., 2016) and aggression (Stagkourakis et al., 2018) in a glutamate-dependent, but dopamine-independent, manner (Soden et al., 2016), consistent with the profile we identified (Slc17a6+, Slc18a2+, Slc6a3+, Ddc+, Th-). To probe further PMv subpopulations, we subjected VPHGLUT cluster 7 to another iteration of clustering and found that it could be parsed into eight subclusters (Figure 3—figure supplement 1a). Each of the eight subclusters expressed Slc17a6 but exhibit differential expression of major markers such as Tac1 and Foxp2 (Figure 3—figure supplement 1b–d). Notably, subclustering revealed two distinct populations of putative catecholaminergic Tac1+/Foxp2+ neurons (clusters 7–1 and −2), both of which express Slc18a2 but only one of which expresses Slc6a3. Taken together, these transcriptomic data provide further biological insight into the repertoire of receptors and signaling molecules expressed by this key behavioral node. Another distinct VPHGLUT population (cluster 6) share a number of common markers with clusters 1–5, for example Cck (encoding the neuropeptide cholecystokinin) and Foxb1 (encoding the transcription factor Forkhead Box B1), which are largely undetectable in clusters 7–10 (Figure 4a,b). Curiously, VPHGLUT cluster 6 also expresses a suite of markers that are enriched in clusters 7–10 (ex. Ebf3, Dlk1, Synpr, Nxph1) but largely undetectable in clusters 1–5 (Figure 4a,b). In particular, Synpr (encoding the presynaptic protein synaptoporin), Dlk1 (encoding delta like non-canonical Notch ligand 1), Ebf3 (encoding early B cell factor 3) and Stxbp6 (encoding the synaptic protein syntaxin binding protein 6) are enriched in VPHGLUT cluster 6 (Figure 4a,b). Examining the expression patterns of Synpr, Dlk1, and Stxbp6 in the ABA (Lein et al., 2007), we found that all three are enriched in a discrete region that appears to correspond well to the dorsal PM (PMd; Figure 4c,d) and are largely undetectable in the more caudal mammillary bodies (MB; Figure 4d). This suggests that VPHGLUT cluster 6 likely represents a PMd population that expresses a number of unique signatures (ex. Synpr, Stxbp6) but shares some commonalities with VPHGLUT clusters 1–5 (ex. Foxb1, Cck, Adcyap1) and VPHGLUT cluster 7, a putative PMv population (ex. Nxph1, Ar). These data suggest that the spatially segregated PMd and PMv may be defined by distinct transcriptional signatures. A neuronal population in the supramammillary (SUM) nuclei Another notable neuronal cluster is VPHGLUT cluster 8. Subjecting this cluster to another iteration of unsupervised clustering revealed six subclusters (Figure 4—figure supplement 1a) which exhibit suites of differentially expressed genes (Figure 4—figure supplement 1b,c). Several of the markers that define distinct subclusters within VPHGLUT cluster 8 are found within anatomically identified neuronal populations in the SUM (Figure 4—figure supplement 1d), only the most ventral portion of which would be included in our microdissection (Figure 1b). These markers, previously identified in rodents include Th (encoding tyrosine hydroxylase, enriched in cluster 8–1; Swanson, 1982) and Nos1 (enriched in cluster 8–5; Yamada et al., 1996; Pedersen et al., 2017). Although all cells in cluster 8 express Slc17a6, and are nominally classified as glutamatergic, at least one subpopulation appears to co-express Slc32a1 and Gad2 but not Gad1 (Figure 4—figure supplement 1e). Subcluster 8–3 closely corresponds to this Slc17a6+/Slc32a1+/Gad2+ subcluster and co-expresses a suite of discriminatory makers including Sema3c, Inhba, Rxfp1 and others (Figure 4—figure supplement 1f). Cross-referencing with the ABA (Lein et al., 2007) shows selective expression of Sem3c, Inhba, and Rxfp1 in the SUM (Figure 4—figure supplement 1f). Another subcluster, cluster 8–4 also expresses a moderate level of Slc32a1 (Figure 4—figure supplement 1c). Interestingly, a unique population of VGLUT2/VGAT co-expressing axons originating in the SUM has been described among projections to the hippocampal dentate gyrus (Boulland et al., 2009; Soussi et al., 2010) and these dual phenotype SUM neurons were found to indeed co-release GABA and glutamate onto neurons of the dentate gyrus (Pedersen et al., 2017; Hashimotodani et al., 2018). Our identification of this VPHGLUT cluster 8 subpopulation is consistent with previous anatomical and functional data defining a unique dentate-projecting, dual phenotype SUM population but would require additional functional evidence to validate. However, the markers identified in our transcriptomic analysis may provide possible strategies for their precise genetic targeting. Furthermore, a microdissection that includes the entirety of the SUM would provide a more comprehensive picture of neuronal cell type diversity within this region. Neuronal populations in the caudal arcuate (Arc) and lateral hypothalamic/tuberal (LHA/Tub) nuclei A number of neuronal clusters are defined by markers known to be enriched in the hypothalamic Arc nucleus, despite our microdissection likely capturing only the most caudal portion of the Arc. Clusters that express Arc-enriched markers (Romanov et al., 2017; Campbell et al., 2017), including cluster 14 (Unc13c, Trh, etc.), cluster 16 (Tac2, Pdyn, etc.), and cluster 18 (Agrp, Sst, etc.) were combined in a second iteration of clustering to reveal eight subclusters defined by differentially expressed genes (Figure 4—figure supplement 2a–c). Cluster 14 parsed into five subclusters with markers that include (Unc13c, Thrb and Trh; Figure 4—figure supplement 2e), that appear to correspond well to a population of cells in the caudal Arc. In particular, using ISH data from the ABA (Lein et al., 2007), both Dlk1 and Thrb (encoding the thyroid hormone receptor beta) are enriched in a cluster surrounding the 3rd ventricle/mammillary recess corresponding to the medial posterior portion of the Arc (Paxinos, 2012) or posterior portion of the periventricular nucleus (Allen Institute Mouse Brain Reference Atlas). Cluster 18 parsed into two subclusters. While both express Otp, one subcluster likely corresponds to well-known Arc AGRP/NPY neurons (Agrp and Npy), and another likely corresponds to a previously identified population of Arc Sst+ neurons such as Sst/Unc13c (Campbell et al., 2017). A highly distinct neuronal cluster, VPHGLUT cluster 16, which did not subcluster further, expresses a suite of markers that identify it as a well-described neuronal cell type in the Arc - kisspeptin-neurokinin B-dynorphin (KNDy) neurons (Figure 4—figure supplement 2f). KNDy neurons are considered to be the gonadotropin-releasing hormone pulse generator controlling release of luteinizing hormone from the anterior pituitary, and therefore essential for fertility and reproduction (Lehman et al., 2010; Moore et al., 2018; Harter et al., 2018). We confirm that this cluster co-expresses the defining neuropeptides Tac2 (encoding neurokinin B) and Pdyn (encoding dynorphin; Figure 4—figure supplement 2c,f). The key markers that define KNDy neurons in our data set are consistent with those identified in previous mouse scRNA-seq data sets, both from broader hypothalamic samples (Chen et al., 2017; Romanov et al., 2017) and Arc-specific samples (Campbell et al., 2017). Consistent with the important role of Arc KNDy neurons in reproduction and fertility, we found high expression of the following hormone receptors: Prlr (encoding the prolactin receptor), Esr1 (encoding the estrogen receptor 1), Ar (encoding the androgen receptor), and Pgr (encoding the progesterone receptor). Other markers that exhibit relatively robust and unique expression in KNDy neurons include Nhlh2, Inhbb, Nr5a2, Rxfp1 and C

The lateral hypothalamic area (LHA) coordinates an array of fundamental behaviors, including sleeping, waking, feeding, stress and motivated behavior. The wide spectrum of functions ascribed to the LHA may be explained by a heterogeneous population of neurons, the full diversity of which is poorly understood. We employed a droplet-based single-cell RNA-sequencing approach to develop a comprehensive census of molecularly distinct cell types in the mouse LHA. Neuronal populations were classified based on fast neurotransmitter phenotype and expression of neuropeptides, transcription factors and synaptic proteins, among other gene categories. We define 15 distinct populations of glutamatergic neurons and 15 of GABAergic neurons, including known and novel cell types. We further characterize a novel population of somatostatin-expressing neurons through anatomical and behavioral approaches, identifying a role for these neurons in specific forms of innate locomotor behavior. This study lays the groundwork for better understanding the circuit-level underpinnings of LHA function.

Histamine was first identified in the brain about 50 years ago, but only in the last few years have researchers gained an understanding of how it regulates sleep/wake behavior. We provide a translational overview of the histamine system, from basic research to new clinical trials demonstrating the usefulness of drugs that enhance histamine signaling. The tuberomammillary nucleus is the sole neuronal source of histamine in the brain, and like many of the arousal systems, histamine neurons diffusely innervate the cortex, thalamus, and other wake-promoting brain regions. Histamine has generally excitatory effects on target neurons, but paradoxically, histamine neurons may also release the inhibitory neurotransmitter GABA. New research demonstrates that activity in histamine neurons is essential for normal wakefulness, especially at specific circadian phases, and reducing activity in these neurons can produce sedation. The number of histamine neurons is increased in narcolepsy, but whether this affects brain levels of histamine is controversial. Of clinical importance, new compounds are becoming available that enhance histamine signaling, and clinical trials show that these medications reduce sleepiness and cataplexy in narcolepsy.

The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding, and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their coexpression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single-cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single-cell quantitative polymerase chain reaction (qPCR) to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components, and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.

Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons ( Hdc -Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep–wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo . We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep. SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain function, but has been difficult to selectively manipulate owing to cellular heterogeneity in this region. Here we use a transgenic mouse to interrogate both the characteristic firing properties of HA neurons and their specific role in maintaining wakefulness. Our results demonstrate that the acute, cell type-specific silencing of HA neurons during wakefulness is sufficient to not only impair arousal but to rapidly and selectively induce slow-wave sleep. This work furthers our understanding of HA-mediated mechanisms that regulate behavioral arousal.

The hypothalamus is among the most phylogenetically conserved regions in the vertebrate brain, reflecting its critical role in maintaining physiological and behavioural homeostasis. By integrating signals arising from both the brain and periphery, it governs a litany of behaviourally important functions essential for survival. In particular, the lateral hypothalamic area (LHA) is central to the orchestration of sleep-wake states, feeding, energy balance and motivated behaviour. Underlying these diverse functions is a heterogeneous assembly of cell populations typically defined by neurochemical markers, such as the well-described neuropeptides hypocretin/orexin and melanin-concentrating hormone. However, anatomical and functional evidence suggests a rich diversity of other cell populations with complex neurochemical profiles that include neuropeptides, receptors and components of fast neurotransmission. Collectively, the LHA acts as a hub for the integration of diverse central and peripheral signals and, through complex local and long-range output circuits, coordinates adaptive behavioural responses to the environment. Despite tremendous progress in our understanding of the LHA, defining the identity of functionally discrete LHA cell types, and their roles in driving complex behaviour, remain significant challenges in the field. In this review, we discuss advances in our understanding of the neurochemical and cellular heterogeneity of LHA neurons and the recent application of powerful new techniques, such as opto- and chemogenetics, in defining the role of LHA circuits in feeding, reward, arousal and stress. From pioneering work to recent developments, we review how the interrogation of LHA cells and circuits is contributing to a mechanistic understanding of how the LHA coordinates complex behaviour.