VGAT Neurons Research Articles

A hypothalamic circuit for circadian regulation of corticosterone secretion.

The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for the daily active cycle and is impaired in chronic depression. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN) which governs the activity of neurons in the paraventricular nucleus of the hypothalamus that produce the corticotropin-releasing hormone (PVHCRH neurons). The dorsomedial nucleus of the hypothalamus (DMH) serves as a crucial intermediary, being innervated by the SCN both directly and via relays in the subparaventricular zone, and projecting axons to the PVH, thereby exerting influence over the cortisol/corticosterone rhythm. However, the role and synaptic mechanisms by which DMH neurons regulate the daily rhythm of Cort secretion has not been explored. We found that either ablating or acutely inhibiting the DMH glutamatergic (DMHVglut2) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMHVglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVHCRH neurons through glutamate release (the DMHVglut2 → PVHCRH pathway). Similarly, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMHVgat) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of DMHVgat neurons increased Cort, although with a lower magnitude compared to DMHVglut2 neuron stimulation, suggesting a role in disinhibiting PVHCRH neurons. Supporting this hypothesis, we found that rostral DMHVgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVHCRH neurons, and that activating the DMHVgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVHCRH neurons. Finally, ablation of cvPVHVgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMHVgat → cvPVHVgat → PVHCRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVHCRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between SCN, DMH, and PVH.

Activity of GABA neurons in the zona incerta and ventral lateral periaqueductal grey is biased towards sleep.

As in various brain regions the activity of gamma-aminobutyric acid (GABA) neurons is largely unknown, we measured in vivo changes in calcium fluorescence in GABA neurons in the zona incerta (ZI) and the ventral lateral periaqueductal grey (vlPAG), two areas that have been implicated in regulating sleep. vGAT-Cre mice were implanted with sleep electrodes, microinjected with rAAV-DIO-GCaMP6 into the ZI (n = 6) or vlPAG (n = 5) (isoflurane anesthesia) and a GRIN (Gradient-Index) lens inserted atop the injection site. Twenty-one days later, fluorescence in individual vGAT neurons was recorded over multiple REM cycles. Regions of interest corresponding to individual vGAT somata were automatically extracted with PCA-ICA analysis. In the ZI, 372 neurons were identified. Previously, we had recorded the activity of 310 vGAT neurons in the ZI and we combined the published dataset with the new dataset to create a comprehensive dataset of ZI vGAT neurons (total neurons = 682; mice = 11). In the vlPAG, 169 neurons (mice = 5) were identified. In both regions, most neurons were maximally active in REM sleep (R-Max; ZI = 51.0%, vlPAG = 60.9%). The second most abundant group was W-Max (ZI = 23.9%, vlPAG = 25.4%). In the ZI, but not in vlPAG, there were neurons that were NREMS-Max (11.7%). vlPAG had REMS-Off neurons (8.3%). In both areas, there were two minor classes: wake/REMS-Max and state indifferent. In the ZI, the NREMS-Max neurons fluoresced 30 s ahead of sleep onset. These descriptive data show that the activity of GABA neurons is biased in favor of sleep in two brain regions implicated in sleep.

Optogenetic stimulation of pre–Bötzinger complex reveals novel circuit interactions in swallowing–breathing coordination

The coordination of swallowing with breathing, in particular inspiration, is essential for homeostasis in most organisms. While much has been learned about the neuronal network critical for inspiration in mammals, the pre-Bötzinger complex (preBötC), little is known about how this network interacts with swallowing. Here we activate within the preBötC excitatory neurons (defined as Vglut2 and Sst neurons) and inhibitory neurons (defined as Vgat neurons) and inhibit and activate neurons defined by the transcription factor Dbx1 to gain an understanding of the coordination between the preBötC and swallow behavior. We found that stimulating inhibitory preBötC neurons did not mimic the premature shutdown of inspiratory activity caused by water swallows, suggesting that swallow-induced suppression of inspiratory activity is not directly mediated by the inhibitory neurons in the preBötC. By contrast, stimulation of preBötC Dbx1 neurons delayed laryngeal closure of the swallow sequence. Inhibition of Dbx1 neurons increased laryngeal closure duration and stimulation of Sst neurons pushed swallow occurrence to later in the respiratory cycle, suggesting that excitatory neurons from the preBötC connect to the laryngeal motoneurons and contribute to the timing of swallowing. Interestingly, the delayed swallow sequence was also caused by chronic intermittent hypoxia (CIH), a model for sleep apnea, which is 1) known to destabilize inspiratory activity and 2) associated with dysphagia. This delay was not present when inhibiting Dbx1 neurons. We propose that a stable preBötC is essential for normal swallow pattern generation and disruption may contribute to the dysphagia seen in obstructive sleep apnea.

Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity

ObjectiveCav3.2, a T-type low voltage-activated calcium channel widely expressed throughout the central nervous system, plays a vital role in neuronal excitability and various physiological functions. However, the effects of Cav3.2 on energy homeostasis remain unclear. Here, we examined the role of Cav3.2 expressed by hypothalamic GABAergic neurons in the regulation of food intake and body weight in mice and explored the underlying mechanisms. MethodsMale congenital Cana1h (the gene coding for Cav3.2) global knockout (Cav3.2KO) mice and their wild type (WT) littermates were first used for metabolic phenotyping studies. By using the CRISPR-Cas9 technique, Cav3.2 was selectively deleted from GABAergic neurons in the arcuate nucleus of the hypothalamus (ARH) by specifically overexpressing Cas9 protein and Cav3.2-targeting sgRNAs in ARH Vgat (VgatARH) neurons. These male mutants (Cav3.2KO-VgatARH) were used to determine whether Cav3.2 expressed by VgatARH neurons is required for the proper regulation of energy balance. Subsequently, we used an electrophysiological patch-clamp recording in ex vivo brain slices to explore the impact of Cav3.2KO on the cellular excitability of VgatARH neurons. ResultsMale Cav3.2KO mice had significantly lower food intake than their WT littermate controls when fed with either a normal chow diet (NCD) or a high-fat diet (HFD). This hypophagia phenotype was associated with increased energy expenditure and decreased fat mass, lean mass, and total body weight. Selective deletion of Cav3.2 in VgatARH neurons resulted in similar feeding inhibition and lean phenotype without changing energy expenditure. These data provides an intrinsic mechanism to support the previous finding on ARH non-AgRP GABA neurons in regulating diet-induced obesity. Lastly, we found that naringenin extract, a predominant flavanone found in various fruits and herbs and known to act on Cav3.2, decreased the firing activity of VgatARH neurons and reduced food intake and body weight. These naringenin-induced inhibitions were fully blocked in Cav3.2KO-VgatARH mice. ConclusionOur results identified Cav3.2 expressed by VgatARH neurons as an essential intrinsic modulator for food intake and energy homeostasis, which is a potential therapeutic target in the treatment of obesity.

066 Noradrenaline and acetylcholine inhibit sleep-promoting neurons of ventrolateral preoptic area through a local GABAergic circuit

Abstract Introduction The ventrolateral preoptic (VLPO) nucleus is a key area involved in the initiation and maintenance of sleep. During wakefulness, sleep-promoting galanin neurons in the VLPO are directly inhibited by arousal signals including noradrenaline and acetylcholine. We have found that while these neurotransmitters directly inhibit VLPO galanin neurons, they also activate GABAergic neurons in the VLPO that do not express galanin. We propose that when activated by monoaminergic and cholinergic inputs, these local VLPO GABAergic neurons provide an additional inhibition of the VLPO galanin sleep-promoting neurons. We tested this model in brain slices in mice. Methods We studied VLPO galanin neurons in mouse brain slices using patch-clamp recordings. We recorded from fluorescently labeled VLPO galanin neurons following the injection of a cre-dependent AAV encoding for mCherry, into the VLPO of Gal-cre mice. For the optogenetic studies we expressed channelrhodopsin-2 (ChR-2) in VLPO VGAT neurons and mCherry in galanin neurons by injecting a flp-dependent and a cre-dependent AAV encoding respectively for ChR2 and mCherry into the VLPO of VGAT-flp::Gal-cre mice. We photo-stimulated local GABAergic neurons and recorded from labeled VLPO galanin neurons. Noradrenaline, carbachol and receptor antagonists were bath-applied. Results Noradrenaline and carbachol inhibited VLPO galanin neurons by alpha-2 and muscarinic receptors and these effects were maintained in the presence of tetrodotoxin (TTX) indicating, as previously proposed, a direct inhibitory effect of noradrenaline and carbachol on VLPO galanin neurons. In addition, both noradrenaline and carbachol increased the frequency of spontaneous inhibitory post-synaptic currents (sIPSCs) of VLPO galanin neurons, suggesting an additional inhibitory action on VLPO galanin neurons. Finally, optogenetic stimulation of local VLPO GABAergic neurons produced short latency, TTX-resistant, opto-evoked IPSCs in VLPO galanin neurons. Both noradrenaline and carbachol increased the amplitude of these opto-evoked IPSCs by the activation of alpha-1 and muscarinic receptors. Conclusion Our results demonstrate that noradrenaline and acetylcholine inhibit VLPO galanin neurons directly and indirectly. Both noradrenaline and acetylcholine increase GABAergic afferent inputs to VLPO galanin neurons by activating local GABAergic neurons. We propose that during wakefulness this feedforward inhibition provides additional inhibition of VLPO galanin sleep-promoting neurons. Support (if any) NS091126 and HL149630

Activity of a subset of vesicular GABA-transporter neurons in the ventral zona incerta anticipates sleep onset.

Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely behaving mice, we used microendoscopy to monitor calcium (Ca2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons. vGAT-Cre mice (male = 3; female = 2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max, 5.9% wake/REM max, while 9.9% were state-indifferent. In the NREM-max group, Ca2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.

Distinct hypothalamic control of same- and opposite-sex mounting behaviour in mice.

Animal behaviours that are superficially similar can express different intents in different contexts, but how this flexibility is achieved at the level of neural circuits is not understood. For example, males of many species can exhibit mounting behaviour towards same- or opposite-sex conspecifics1, but it is unclear whether the intent and neural encoding of these behaviours are similar or different. Here we show that female- and male-directed mounting in male laboratory mice are distinguishable by the presence or absence of ultrasonic vocalizations (USVs)2-4, respectively. These and additional behavioural data suggest that most male-directed mounting is aggressive, although in rare cases it can be sexual. We investigated whether USV+ and USV- mounting use the same or distinct hypothalamic neural substrates. Micro-endoscopic imaging of neurons positive for oestrogen receptor1 (ESR1) in either the medial preoptic area (MPOA) or the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) revealed distinct patterns of neuronal activity during USV+ and USV- mounting, and the type of mounting could be decoded from population activity in either region. Intersectional optogenetic stimulation of MPOA neurons that express ESR1andvesicular GABA transporter (VGAT) (MPOAESR1∩VGAT neurons) robustly promoted USV+ mounting, and converted male-directed attack to mounting with USVs. By contrast, stimulation of VMHvl neurons that express ESR1 (VMHvlESR1 neurons) promoted USV- mounting, and inhibited the USVs evoked by female urine. Terminal stimulation experiments suggest that these complementary inhibitory effects are mediated by reciprocal projections between the MPOA and VMHvl. Together, these data identify a hypothalamic subpopulation that is genetically enriched for neurons that causally induce a male reproductive behavioural state, and indicate that reproductive and aggressive states are represented by distinct population codes distributed between MPOAESR1 and VMHvlESR1 neurons, respectively. Thus, similar behaviours that express different internal states are encoded by distinct hypothalamic neuronal populations.

Ultra-sparse Connectivity within the Lateral Hypothalamus

SummaryThe lateral hypothalamic area (LH) is a vital controller of arousal, feeding, and metabolism [1, 2], which integrates external and internal sensory information. Whereas sensory and whole-body output properties of LH cell populations have received much interest, their intrinsic synaptic organization has remained largely unstudied. Local inhibitory and excitatory connections could help integrate and filter sensory information and mutually inhibitory connections [3] could allow coordinating activity between LH cell types, some of which have mutually exclusive behavioral effects, such as LH VGLUT2 and VGAT neurons [4, 5, 6, 7] and orexin- (ORX) and melanin-concentrating hormone (MCH) neurons [8, 9, 10]. However, classical Golgi staining studies did not find interneurons with locally ramifying axons in the LH [11, 12], and nearby subthalamic and thalamic areas lack local synaptic connectivity [13, 14]. Studies with optogenetic circuit mapping within the LH have demonstrated only a minority of connections when a large pool of presynaptic neurons was activated [15, 16, 17, 18, 19]. Because multiple patch clamp has not been used to study LH connectivity, aside from a limited dataset of MCH neurons where no connections were discovered [15], we used quadruple whole-cell recordings to screen connectivity within the LH with standard methodology we previously used in the neocortex [20, 21, 22]. Finding a lack of local connectivity, we used optogenetic circuit mapping to study the strength of LH optogenetic responses and network oscillations, which were consistent with ultra-sparse intrinsic connectivity within the LH. These results suggest that input from other brain structures is decisive for selecting active populations in the LH.

Optogenetic stimulation of the pre‐Bötzinger Complex and the control of breathing in a mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder well known for its progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). Much less known, yet clinically very important, are the detrimental effects on breathing associated with this disease. Studies in the 6‐hydroxydopamine (6‐OHDA) rat model of PD revealed reductions of respiratory frequency (fR) and NK1r‐immunoreactivity in the pre‐Bötzinger complex (preBötC), a medullary region critical for generating inspiration. To unravel mechanisms that underlie breathing disturbances in PD, we used a transgenic approach to specifically label or stimulate different excitatory (Dbx1‐ERT2Cre, Vglut2‐Cre) and inhibitory (Vgat‐Cre) neural populations in the preBötC. Homozygous Cre mice were bred with homozygous Ai6 (ZsGreen) or Ai32 (ChR2) mice. Adult offspring then received bilateral injections of vehicle or 6‐OHDA (10 μg/μl) into the striatum (CPu) to induce PD; and Ai32 mice also received preBötC fiber optic cannulation. 10–12 days later, whole body plethysmography was used to measure ventilation at rest and during optogenetic photo‐stimulation of the preBötC in unanaesthetized and unrestrained mice. As expected, fR at rest was reduced by 24.9% in PD mice (154.6 ± 6.0 vs. vehicle: 205.9 ± 3.7 bpm, p<0.05). Dbx1 photo‐stimulation during the inspiratory phase did not affect the rhythm in control animals, but increased fR by 17% in PD animals. Similarly, Vglut2 photo‐stimulation in control animals did not change the rhythm, but increased fR by 30% in PD animals. No changes were elicited by photo‐stimulation during expiration. Photo‐stimulation of preBötC inhibitory Vgat neurons during inspiration increased fR by 21%, but only in vehicle‐injected mice; no changes were observed in PD mice. Histology confirmed that 6‐OHDA injections reduced the number of catecholaminergic neurons in the SNpc by 69% (150.4 ± 4.9 vs. vehicle: 494.7 ± 14.5 cells). Surprisingly, the number of Dbx1 and Vglut2 neurons in the preBötC and the adjacent ventral respiratory column (VRC) were also reduced by 47.6 and 17.3%, respectively (p<0.05), whereas the number of Vgat neurons did not change. In conclusion, our study reveals that the 6‐OHDA model of PD is associated with a specific reduction in the number of excitatory neurons within the preBötC, which includes rhythmogenic Dbx1 neurons. Photo‐stimulating specifically these neurons can restore breathing in this PD mouse model.Support or Funding InformationNIH and FAPESP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thermoregulation through the glutamatergic neurons in vestibular nucleus complex

The vestibular system is one of the sensory systems which contributes to the sense of balance and spatial orientation. This also participates in the autonomic nervous response, which stimulation of the peripheral vestibular organs induces sympathoexcitation. This response is observed in both rodents and human beings, and we have reported that vestibular system contributes to the arterial pressure response during postural change as one of the feedforward control system (Morita, Abe et al., Sci Rep, 2016). In the other autonomic nervous responses, stimulation of the otolith organs in the inner ear induces hypothermia; exposure to the hypergravity environment decreases body temperature by 8 degree Celsius in mice. This response was attenuated by vestibular lesion or genetic deletion of otolith. In order to elucidate the central mechanism of hypergravity‐induced hypothermia, we examined the role of Vglut2, Vgat and ChAT neurons in vestibular nucleus complex (VNC) on thermoregulation in mice. We used Vglut2‐cre, Vgat‐cre, and ChAT‐cre mice to manipulate each neuron in VNC. Based on the previous work (Abe et al., Nat Neurosci, 2017), AAV‐DIO type viral vector was injected in VNC to express photo sensors (channelrhodopsin or archaerhodopsin) for optogenetics or hM3D(Gq) for chemogenetics. Unilateral photostimulation of the Vglut2 neurons in VNC induced body tilt to the ipsilateral side, while photoinhibition induced body tilt to the contralateral side. In Vgat‐cre mice, opposite response was observed compared with Vglut2‐cre mouse. Photostimulation of ChAT neurons did not show any responses. Chemogenetics stimulation of Vglut2 neurons showed hypothermia with increasing in activity, while Vgat stimulation increased body temperature with decreasing in activity. Deletion of Vglut2 neurons in VNC using AAV2–DIO–taCasp3–TEVp attenuated hypothermia induced by hypergravity exposure. On the other hand, hypothermia was still observed by deletion of Vgat neurons in VNC. Interestingly, chemogenetics stimulation of Vglut2 neurons in VNC 2 days before hypergravity exposure, the hypothermia was attenuated. Taken together, hypothermia induced by hypergravity exposure is due to activation of Vglut2 neurons in VNC.Support or Funding InformationGrant‐in‐Aid for Scientific Research on Innovative Areas 15H05940 (HM) and 18H04974 (CA), Grant‐in‐Aid for Scientific Research (C) 18K06850 (HM), The Takeda Science Foundation (CA), The Uehara Memorial Foundation (CA), Kato Memorial Bioscience Foundation (CA), The Nakatomi Foundation (CA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic mapping of downstream network activity following CNO-induced activation of hM3Dq in BNST VGAT neurons

Metabolic mapping of downstream network activity following CNO-induced activation of hM3Dq in BNST VGAT neurons

Somatostatin Neurons in the Basal Forebrain Promote High-Calorie Food Intake

Obesity has become a global issue, and the overconsumption of food is thought to be a major contributor. However, the regulatory neural circuits that regulate palatable food consumption remain unclear. Here, we report that somatostatin (SOM) neurons and GABAergic (VGAT) neurons in the basal forebrain (BF) play specific roles in regulating feeding. Optogenetic stimulation of BF SOM neurons increased fat and sucrose intake within minutes and promoted anxiety-like behaviors. Furthermore, optogenetic stimulation of projections from BF SOM neurons tothe lateral hypothalamic area (LHA) selectively resulted in fat intake. In addition, activation of BF VGAT neurons rapidly induced general food intake and gnawing behaviors. Whole-brain mapping of inputs and outputs showed that BF SOM neurons form bidirectional connections with several brain areas important in feeding and regulation of emotion. Collectively, these results suggest that BF SOM neurons play a selective role in hedonic feeding.

An Inhibitory Septum to Lateral Hypothalamus Circuit That Suppresses Feeding.

Our results demonstrate that top-down projections from the septum to the hypothalamus control food intake negatively. Given the known role for both of these brain regions in the control of feeding and emotion-related behaviors, these findings reveal previously unknown neural circuitry that is likely implicated in emotional aspects of food intake and provide new insights into the development of therapeutic targets for the treatment of eating disorders.

A Novel Population of Wake-Promoting GABAergic Neurons in the Ventral Lateral Hypothalamus

The largest synaptic input to the sleep-promoting ventrolateral preoptic area (VLPO) [1] arises from the lateral hypothalamus [2], a brain area associated with arousal [3-5]. However, the neurochemical identity of the majority of these VLPO-projecting neurons within the lateral hypothalamus (LH), as well as their function in the arousal network, remains unknown. Herein we describe a population of VLPO-projecting neurons in the LH that express the vesicular GABA transporter (VGAT; a marker for GABA-releasing neurons). In addition to the VLPO, these neurons also project to several other established sleep and arousal nodes, including the tuberomammillary nucleus, ventral periaqueductal gray, and locus coeruleus. Selective and acute chemogenetic activation of LH VGAT(+) neurons was profoundly wake promoting, whereas acute inhibition increased sleep. Because of its direct and massive inputs to the VLPO, this population may play a particularly important role in sleep-wake switching.

Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks.

The lateral hypothalamus (LH) controls energy balance. LH melanin-concentrating-hormone (MCH) and orexin/hypocretin (OH) neurons mediate energy accumulation and expenditure, respectively. MCH cells promote memory and appropriate stimulus-reward associations; their inactivation disrupts energy-optimal behaviour and causes weight loss. However, MCH cell dynamics during wakefulness are unknown, leaving it unclear if they differentially participate in brain activity during sensory processing. By fiberoptic recordings from molecularly defined populations of LH neurons in awake freely moving mice, we show that MCH neurons generate conditional population bursts. This MCH cell activity correlates with novelty exploration, is inhibited by stress and is inversely predicted by OH cell activity. Furthermore, we obtain brain-wide maps of monosynaptic inputs to MCH and OH cells, and demonstrate optogenetically that VGAT neurons in the amygdala and bed nucleus of stria terminalis inhibit MCH cells. These data reveal cell-type-specific LH dynamics during sensory integration, and identify direct neural controllers of MCH neurons.