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.
Abstract
Cannabidiol (CBD), a major non-psychoactive phytocannabinoid in cannabis, is an effective treatment for some forms of epilepsy and pain. At high concentrations, CBD interacts with a huge ...variety of proteins, but which targets are most relevant for clinical actions is still unclear. Here we show that CBD interacts with Na
v
1.7 channels at sub-micromolar concentrations in a state-dependent manner. Electrophysiological experiments show that CBD binds to the inactivated state of Na
v
1.7 channels with a dissociation constant of about 50 nM. The cryo-EM structure of CBD bound to Na
v
1.7 channels reveals two distinct binding sites. One is in the IV-I fenestration near the upper pore. The other binding site is directly next to the inactivated “wedged” position of the Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV, which mediates fast inactivation. Consistent with producing a direct stabilization of the inactivated state, mutating residues in this binding site greatly reduced state-dependent binding of CBD. The identification of this binding site may enable design of compounds with improved properties compared to CBD itself.
Cannabidiol (CBD), a chemical found in the
plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a ...large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.
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 male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons (
-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
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.
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 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.
Background:To investigate the relationship between age and both short- and long-term outcomes of off-pump coronary bypass grafting (OPCAB).Methods and Results:The 780 patients undergoing OPCAB were ...divided into 3 groups: 262 aged <65 years (young), 329 aged 65–74 years (early elderly), and 189 aged >75 years (late elderly), and retrospectively analyzed. Thefollow-up rate was 94.9%, and mean follow-up period was 5.6±3.4 years. In-hospital mortality rates were similar among the groups (0.8% in young, 1.2% in early elderly, and 1.1% in late elderly; P=0.862). In logistic regression analysis, the risk factor for predicting major complications was the New York Heart Association (NYHA) classification (odds ratio: 1.555, P=0.001), and not age. The 10-year estimated rates free from cardiac death (89.6±3.2, 95.0±2.1, and 96.5±2.1%, log rank, P=0.16) and cardiac events (71.8±3.8, 66.8±4.3, and 59.9±7.7%, P=0.61) were not significantly different among the groups. In multivariate Cox models, independent risk factors predicting cardiac events were the NYHA classification (hazard ratio (HR): 1.265, P=0.009), and ejection fraction (HR: 0.986, P=0.016), but not age (young HR: 1.0, early elderly HR: 1.276; P=0.210, late elderly HR: 0.910; P=0.707).Conclusions:Both short- and long-term cardiac outcomes of OPCAB are not influenced by age at operation. (Circ J 2015; 79: 2177–2185)
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.
LHA cell types, circuits and behaviour. Schematic representation of a parasagittal section of a rodent brain depicting the boundary of the hypothalamus (pink) and the location of the LHA (red). Through both short‐ and long‐range projections throughout the CNS, LHA neurons coordinate diverse aspects of physiology and behaviour including reward, feeding, sleep/wake, stress/anxiety and neuroendocrine and autonomic function among others. The inset in the foreground depicts a portion of a coronal brain section, highlighting the approximate boundaries of the LHA. The LHA is composed of a heterogeneous population of neurons defined by a variety of neurochemical markers such as neuropeptides (e.g. Hcrt/Ox, MCH), receptors (e.g. LepRb), and markers of fast inhibitory (GABA) and excitatory (glutamate) neurotransmission.
Sleep and wakefulness are evolutionary conserved across species and proper maintenance of vigilance states, and the transitions between them, is essential for optimal health and survival. In ...particular, during wakefulness, arousal levels are continuously fluctuating owing to the need to adapt to the ever-shifting internal states and external environment. Therefore, tight regulation of arousal is crucial and dysfunction can lead to hypoarousal or hyperarousal that are characteristic of neuropsychiatric disorders, including stress and anxiety-related disorders.The lateroposterior hypothalamus (LPH) is a heterogenous subregion of the hypothalamus critical for modulating behavioral homeostasis, including arousal. The LPH is home to three major and unique arousal-related cell types, which includes the monoaminergic histamine (HA) neurons and neuropeptidergic melanin-concentrating hormone (MCH) neurons and hypocretin/orexin (Hcrt/OX) neurons. The LPH is a component of the ascending arousal network and sends extensive but diffuse projections throughout the central nervous system to regulate diverse physiological functions, including arousal, wakefulness (cortical activation), stress, feeding, energy balance and motivated behaviors. Whether subpopulations exist that mediate distinct aspects of physiological and behavioral functions remains unclear and requires further investigation on a multifaceted level, including the electrophysiological properties, neurochemical profile, projection patterns and circuitry.Classical work has considered HA neurons and MCH neurons to be homogenous. However, in both cell types, recent studies have shed light on their heterogeneity and alluded to potential subpopulations. In Chapter 2, we characterize a novel transgenic line to characterize the electrophysiological properties of HA neurons and neighboring non-HA neurons in vitro. We identified electrophysiological diversity between HA and non-HA neurons, but also among HA neurons. In addition, we optogenetically silenced HA neurons in vivo and found that acute silencing not only impairs wakefulness but promotes slow-wave sleep. We provide the first direct evidence on the effect of acutely silenced HA neurons. In Chapter 3, we investigate the anatomical and functional diversity of MCH neurons, in which a subset have been shown to uniquely express the neurokinin 3 receptor (NK3R), a G protein-coupled receptor. Using anatomical tracing techniques and slice electrophysiology, we discovered a novel limbic forebrain hypothalamic circuit that provides neuropeptidergic and GABAergic modulation of MCH neurons. Taken together, this thesis lays the groundwork advancing our understanding of how neuronal diversity in the LPH may underlie functional diversity.
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 LHA
neurons is not well understood. Previous anatomic and molecular data suggest that LHA
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
gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory, and stress in other brain regions, but NKB interactions with LHA
neurons are poorly understood. We first identified how LHA
subpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHA
neurons, we used Cre-dependent retrograde and anterograde viral tracing in male
Cre mice and identified
/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 LHA
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 LHA
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.
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.
Cannabidiol (CBD), a major non-psychoactive phytocannabinoid in cannabis, is an effective treatment for some forms of epilepsy and pain. At high concentrations, CBD interacts with a huge variety of ...proteins, but which targets are most relevant for clinical actions is still unclear. Here we show that CBD interacts with Na
1.7 channels at sub-micromolar concentrations in a state-dependent manner. Electrophysiological experiments show that CBD binds to the inactivated state of Na
1.7 channels with a dissociation constant of about 50 nM. The cryo-EM structure of CBD bound to Na
1.7 channels reveals two distinct binding sites. One is in the IV-I fenestration near the upper pore. The other binding site is directly next to the inactivated "wedged" position of the Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV, which mediates fast inactivation. Consistent with producing a direct stabilization of the inactivated state, mutating residues in this binding site greatly reduced state-dependent binding of CBD. The identification of this binding site may enable design of compounds with improved properties compared to CBD itself.
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