Neurokinin B-Expressing Neurons of the Central Extended Amygdala Mediate Inhibitory Synaptic Input onto Melanin-Concentrating Hormone Neuron Subpopulations.

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.

Cellular taxonomy and spatial organization of the murine ventral posterior hypothalamus.

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.

Single-cell transcriptomic analysis of the lateral hypothalamic area reveals molecularly distinct populations of inhibitory and excitatory neurons.

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: neural circuits and new medications.

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.

Neurochemical Heterogeneity Among Lateral Hypothalamic Hypocretin/Orexin and Melanin-Concentrating Hormone Neurons Identified Through Single-Cell Gene Expression Analysis.

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.

Hypothalamic Tuberomammillary Nucleus Neurons: Electrophysiological Diversity and Essential Role in Arousal Stability.

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.

Hubs and spokes of the lateral hypothalamus: cell types, circuits and behaviour.

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.

Antagonistic interplay between hypocretin and leptin in the lateral hypothalamus regulates stress responses.

The hypothalamic-pituitary-adrenal (HPA) axis functions to coordinate behavioural and physiological responses to stress in a manner that depends on the behavioural state of the organism. However, the mechanisms through which arousal and metabolic states influence the HPA axis are poorly understood. Here using optogenetic approaches in mice, we show that neurons that produce hypocretin (Hcrt)/orexin in the lateral hypothalamic area (LHA) regulate corticosterone release and a variety of behaviours and physiological hallmarks of the stress response. Interestingly, we found that Hcrt neuronal activity and Hcrt-mediated stress responses were inhibited by the satiety hormone leptin, which acts, in part, through a network of leptin-sensitive neurons in the LHA. These data demonstrate how peripheral metabolic signals interact with hypothalamic neurons to coordinate stress and arousal and suggest one mechanism through which hyperarousal or altered metabolic states may be linked with abnormal stress responses.

Relative contribution of TARPs γ-2 and γ-7 to cerebellar excitatory synaptic transmission and motor behavior.

Transmembrane AMPA receptor regulatory proteins (TARPs) play an essential role in excitatory synaptic transmission throughout the central nervous system (CNS) and exhibit subtype-specific effects on AMPA receptor (AMPAR) trafficking, gating, and pharmacology. The function of TARPs has largely been determined through work on canonical type I TARPs such as stargazin (TARP γ-2), absent in the ataxic stargazer mouse. Little is known about the function of atypical type II TARPs, such as TARP γ-7, which exhibits variable effects on AMPAR function. Because γ-2 and γ-7 are both strongly expressed in multiple cell types in the cerebellum, we examined the relative contribution of γ-2 and γ-7 to both synaptic transmission in the cerebellum and motor behavior by using both the stargazer mouse and a γ-7 knockout (KO) mouse. We found that the loss of γ-7 alone had little effect on climbing fiber (cf) responses in Purkinje neurons (PCs), yet the additional loss of γ-2 all but abolished cf responses. In contrast, γ-7 failed to make a significant contribution to excitatory transmission in stellate cells and granule cells. In addition, we generated a PC-specific deletion of γ-2, with and without γ-7 KO background, to examine the relative contribution of γ-2 and γ-7 to PC-dependent motor behavior. Selective deletion of γ-2 in PCs had little effect on motor behavior, yet the additional loss of γ-7 resulted in a severe disruption in motor behavior. Thus, γ-7 is capable of supporting a component of excitatory transmission in PCs, sufficient to maintain essentially normal motor behavior, in the absence of γ-2.

Probing TARP modulation of AMPA receptor conductance with polyamine toxins.

The properties of synaptic AMPA receptors (AMPARs) depend on their subunit composition and association with transmembrane AMPAR regulatory proteins (TARPs). Although both GluA2 incorporation and TARP association have been shown to influence AMPAR channel conductance, the manner in which different TARPs modulate the mean channel conductance of GluA2-containing AMPARs is unknown. Using ultrafast agonist application and nonstationary fluctuation analysis, we found that TARP subtypes differentially increase the mean channel conductance, but not the peak open probability, of recombinant GluA2-containing AMPARs. TARP γ-8, in particular, enhances mean channel conductance to a greater degree than γ-2, γ-3, or γ-4. We then examined the action of a use-dependent antagonist of GluA2-containing AMPARs, philanthotoxin-74 (PhTx-74), on recombinant AMPARs and on GluA2-containing AMPARs in cerebellar granule neurons from stargazer mice transfected with TARPs. We found that the rate and extent of channel block varies with TARP subtype, in a manner that correlates linearly with mean channel conductance. Furthermore, block of GluA2-containing AMPARs by polyamine toxins varied depending on whether channels were activated by the full agonist glutamate or the partial agonist kainate, consistent with conductance state-dependent block. Block of GluA2-lacking AMPARs by PhTx-433 is also modulated by TARP association and is a function of agonist efficacy. Our data indicate that channel block by polyamine toxins is sensitive to the mean channel conductance of AMPARs, which varies with TARP subtype and agonist efficacy. Furthermore, our results illustrate the utility of polyamine toxins as sensitive probes of AMPAR channel conductance and suggest the possibility that TARPs may influence their channel properties by selectively stabilizing specific channel conformations, rather than altering the pore structure.

The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits.

Ionotropic glutamate receptors (iGluRs) underlie rapid, excitatory synaptic signaling throughout the CNS. After years of intense research, our picture of iGluRs has evolved from them being companionless in the postsynaptic membrane to them being the hub of dynamic supramolecular signaling complexes, interacting with an ever-expanding litany of other proteins that regulate their trafficking, scaffolding, stability, signaling, and turnover. In particular, the discovery that transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary subunits that are critical determinants of their trafficking, gating, and pharmacology has changed the way we think about iGluR function. Recently, a number of novel transmembrane proteins have been uncovered that may also serve as iGluR auxiliary proteins. Here we review pivotal developments in our understanding of the role of TARPs in AMPA receptor trafficking and gating, and provide an overview of how newly discovered transmembrane proteins expand our view of iGluR function in the CNS.

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells.

The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.

Stargazin (TARP gamma-2) is required for compartment-specific AMPA receptor trafficking and synaptic plasticity in cerebellar stellate cells.

In the cerebellar cortex, parallel fiber-to-stellate cell (PF-SC) synapses exhibit a form of synaptic plasticity manifested as a switch in the subunit composition of synaptic AMPA receptors (AMPARs) from calcium-permeable, GluA2-lacking to calcium-impermeable, GluA2-containing receptors. Here, we examine the role of stargazin (γ-2), canonical member of the transmembrane AMPAR regulatory protein (TARP) family, in the regulation of GluA2-lacking AMPARs and synaptic plasticity in SCs from epileptic and ataxic stargazer mutant mice. We found that AMPAR-mediated synaptic transmission is severely diminished in stargazer SCs, and that the rectification index (RI) of AMPAR current is reduced. Activity-dependent plasticity in the rectification of synaptic AMPARs is also impaired in stargazer SCs. Despite the dramatic loss in synaptic AMPARs, extrasynaptic AMPARs are preserved. We then examined the role of stargazin in regulating the rectification of extrasynaptic AMPARs in nucleated patches and found, in contrast to previous reports, that wild-type extrasynaptic AMPARs have moderate RI values (average RI = 0.38), while those in stargazer SCs are low (average RI = 0.24). The GluA2-lacking AMPAR blocker, philanthotoxin-433 (PhTx-433), was used as an alternative measure of GluA2 content in wild-type and stargazer SCs. Despite the difference in RI, PhTx-433 sensitivity of both synaptic and extrasynaptic AMPARs remains unchanged, suggesting that the dramatic changes in RI and the impairment in synaptic plasticity observed in the stargazer mouse are not the result of a specific impairment in GluA2 trafficking. Together, these data suggest that stargazin regulates compartment-specific AMPAR trafficking, as well as activity-dependent plasticity in synaptic AMPAR rectification at cerebellar PF-SC synapses.

Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach.

The precise subunit composition of synaptic ionotropic receptors in the brain is poorly understood. This information is of particular importance with regard to AMPA-type glutamate receptors, the multimeric complexes assembled from GluA1-A4 subunits, as the trafficking of these receptors into and out of synapses is proposed to depend upon the subunit composition of the receptor. We report a molecular quantification of synaptic AMPA receptors (AMPARs) by employing a single-cell genetic approach coupled with electrophysiology in hippocampal CA1 pyramidal neurons. In contrast to prevailing views, we find that GluA1A2 heteromers are the dominant AMPARs at CA1 cell synapses (approximately 80%). In cells lacking GluA1, -A2, and -A3, synapses are devoid of AMPARs, yet synaptic NMDA receptors (NMDARs) and dendritic morphology remain unchanged. These data demonstrate a functional dissociation of AMPARs from trafficking of NMDARs and neuronal morphogenesis. This study provides a functional quantification of the subunit composition of AMPARs in the CNS and suggests novel roles for AMPAR subunits in receptor trafficking.

State-dependent enhancement of subthreshold A-type potassium current by 4-aminopyridine in tuberomammillary nucleus neurons.

A-type potassium current (I(A)) both activates and inactivates at subthreshold voltages. We asked whether there is steady-state I(A) at subthreshold voltages, using dissociated mouse tuberomammillary nucleus neurons, pacemaking neurons with large I(A) currents in which subthreshold I(A) might regulate firing frequency. With slow depolarizing voltage ramps (20 mV/s), there was no discernible component of steady-state outward current in the range of -70 to -40 mV. However, faster ramps of 50-100 mV/s, similar to the rate of spontaneous depolarization during pacemaking, did evoke subthreshold outward currents. Ramp-evoked current at subthreshold voltages was unaffected by 10 mM tetraethylammonium and likely represents I(A), because its voltage dependence overlaps that of I(A) activation (midpoint near -44 mV) and inactivation (midpoint near -85 mV). However, although 4-aminopyridine (4-AP) inhibited peak I(A) activated by step depolarizations as expected (IC50, approximately 1 mM), ramp-evoked current was instead dramatically enhanced (current at -40 mV evoked by 50 mV/s ramp enhanced >15-fold by 10 mM 4-AP). In cell-attached recordings of spontaneous pacemaking, 10 mM 4-AP slowed rather than speeded firing, consistent with enhancement of subthreshold I(A). Also consistent with such enhancement, 4-AP also greatly increased the latency to first spike after long hyperpolarizations. The striking enhancement of I(A) during depolarizing ramps can be explained by a model in which 4-AP binds tightly to closed channels but must unbind before channels can inactivate. Thus, the state dependence of 4-AP binding to the channels underlying I(A) can result in effects on firing patterns opposite to those expected from simple block of I(A).

Mechanism of spontaneous firing in dorsomedial suprachiasmatic nucleus neurons.

We studied acutely dissociated neurons from the dorsomedial (shell) region of the rat suprachiasmatic nucleus (SCN) with the aim of determining the ionic conductances that underlie spontaneous firing. Most isolated neurons were spontaneously active, firing rhythmically at an average frequency of 8 +/- 4 Hz. After application of TTX, oscillatory activity generally continued, but more slowly and at more depolarized voltages; these oscillations were usually blocked by 2 microm nimodipine. To quantify the ionic currents underlying normal spontaneous activity, we voltage clamped cells using a segment of the spontaneous activity of each cell as voltage command and then used ionic substitution and selective blockers to isolate individual currents. TTX-sensitive sodium current flowed throughout the interspike interval, averaging -3 pA at -60 mV and -11 pA at -55 mV. Calcium current during the interspike interval was, on average, fourfold smaller. Except immediately before spikes, calcium current was outweighed by calcium-activated potassium current, and in current clamp, nimodipine usually depolarized cells and slowed firing only slightly (average, approximately 8%). Thus, calcium current plays only a minor role in pacemaking of dissociated SCN neurons, although it can drive oscillatory activity with TTX present. During normal pacemaking, the early phase of spontaneous depolarization (-85 to -60 mV) is attributable mainly to background conductance; cells have relatively depolarized resting potentials (with firing stopped by TTX and nimodipine) of -55 to -50 mV, although input resistance is high (9.5 +/- 4.1 GOmega). During the later phase of pacemaking (positive to -60 mV), TTX-sensitive sodium current is dominant.