VLPO Neurons Research Articles

Tenuigenin promotes non-rapid eye movement sleep via the GABAA receptor and exerts somnogenic effect in a MPTP mouse model of Parkinson's disease

Sleep disturbances are commonly non-motor symptoms in Parkinson's diseases (PD). However, standard dopamine replacement therapies for the treatment of motor symptoms often prove inadequate in combating sleep disturbances. Previous studies conducted by our research group have reported the neuroprotective effects of tenuigenin, a natural extract from Polygala tenuifolia root, which has been traditionally employed in treating insomnia. The objective of this study was to investigate the potential of tenuigenin in modulating sleep-wake behaviors and elucidate the underlying mechanisms. We employed EEG/EMG recordings to evaluate the impact of tenuigenin on sleep-wake profiles. Furthermore, we utilized c-Fos immunostaining, whole-cell patch clamping and local field potentials (LFP) recording to explore the mechanisms involved in sleep-promoting effects of tenuigenin. Additionally, we examined the effects of tenuigenin on sleep-promoting in MPTP PD mice. Here, we found tenuigenin demonstrated a significant increase in NREM sleep and a reduction in sleep latency in mice, without altering the EEG power density. Moreover, tenuigenin increased c-Fos expression in the ventrolateral preoptic area (VLPO) and stimulated sleep-promoting neurons in VLPO. The sleep-promoting effects of tenuigenin were abolished when mice were pretreated with flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. Furthermore, tenuigenin was found to ameliorate sleep disturbances in MPTP-induced mice. The results suggesting that tenuigenin facilitated a type of NREM sleep comparable to physiological NREM sleep through interaction with the GABAA receptor. Additionally, tenuigenin demonstrated improvements in sleep disturbances in MPTP-induced PD mice, suggesting its potential as a sleep-promoting substance, particularly for PD patients experiencing sleep disturbances.

Calcium Dynamics of the Ventrolateral Preoptic GABAergic Neurons during Spontaneous Sleep-Waking and in Response to Homeostatic Sleep Demands.

The ventrolateral preoptic area (VLPO) contains GABAergic sleep-active neurons. However, the extent to which these neurons are involved in expressing spontaneous sleep and homeostatic sleep regulatory demands is not fully understood. We used calcium (Ca2+) imaging to characterize the activity dynamics of VLPO neurons, especially those expressing the vesicular GABA transporter (VGAT) across spontaneous sleep-waking and in response to homeostatic sleep demands. The VLPOs of wild-type and VGAT-Cre mice were transfected with GCaMP6, and the Ca2+ fluorescence of unidentified (UNID) and VGAT cells was recorded during spontaneous sleep-waking and 3 h of sleep deprivation (SD) followed by 1 h of recovery sleep. Although both VGAT and UNID neurons exhibited heterogeneous Ca2+ fluorescence across sleep-waking, the majority of VLPO neurons displayed increased activity during nonREM/REM (VGAT, 120/303; UNID, 39/106) and REM sleep (VGAT, 32/303; UNID, 19/106). Compared to the baseline waking, VLPO sleep-active neurons (n = 91) exhibited higher activity with increasing SD that remained elevated during the recovery period. These neurons also exhibited increased Ca2+ fluorescence during nonREM sleep, marked by increased slow-wave activity and REM sleep during recovery after SD. These findings support the notion that VLPO sleep-active neurons, including GABAergic neurons, are components of neuronal circuitry that mediate spontaneous sleep and homeostatic responses to sustained wakefulness.

Divergent Neural Activity in the VLPO During Anesthesia and Sleep.

The invention of general anesthesia (GA) represents a significant advance in modern clinical practices. However, the exact mechanisms of GA are not entirely understood. Because of the multitude of similarities between GA and sleep, one intriguing hypothesis is that anesthesia may engage the sleep-wake regulation circuits. Here, using fiber photometry and micro-endoscopic imaging of Ca2+ signals at both population and single-cell levels, it investigates how various anesthetics modulate the neural activity in the ventrolateral preoptic nucleus (vLPO), a brain region essential for the initiation of sleep. It is found that different anesthetics primarily induced suppression of neural activity and tended to recruit a similar group of vLPO neurons; however, each anesthetic caused comparable modulations of both wake-active and sleep-active neurons. These results demonstrate that anesthesia creates a different state of neural activity in the vLPO than during natural sleep, suggesting that anesthesia may not engage the same vLPO circuits for sleep generation.

Activation of the Ventrolateral Preoptic Neurons Projecting to the Perifornical-Hypothalamic Area Promotes Sleep: DREADD Activation in Wild-Type Rats.

The ventrolateral preoptic area (VLPO) predominantly contains sleep-active neurons and is involved in sleep regulation. The perifornical-hypothalamic area (PF-HA) is a wake-regulatory region and predominantly contains wake-active neurons. VLPO GABAergic/galaninergic neurons project to the PF-HA. Previously, the specific contribution of VLPO neurons projecting to the PF-HA (VLPO > PF-HAPRJ) in sleep regulation in rats could not be investigated due to the lack of tools that could selectively target these neurons. We determined the contribution of VLPO > PF-HAPRJ neurons in sleep regulation by selectively activating them using designer receptors exclusively activated by designer drugs (DREADDs) in wild-type Fischer-344 rats. We used a combination of two viral vectors to retrogradely deliver the Cre-recombinase gene, specifically, in VLPO > PF-HA neurons, and further express hM3Dq in those neurons to selectively activate them for delineating their specific contributions to sleep–wake functions. Compared to the control, in DREADD rats, clozapine-N-oxide (CNO) significantly increased fos-expression, a marker of neuronal activation, in VLPO > PF-HAPRJ neurons (2% vs. 20%, p < 0.01) during the dark phase. CNO treatment also increased nonREM sleep (27% vs. 40%, p < 0.01) during the first 3 h of the dark phase, when rats are typically awake, and after exposure to the novel environment (55% vs. 65%; p < 0.01), which induces acute arousal during the light phase. These results support a hypothesis that VLPO > PF-HAPRJ neurons constitute a critical component of the hypothalamic sleep–wake regulatory circuitry and promote sleep by suppressing wake-active PF-HA neurons.

Astrocyte-derived adenosine excites sleep-promoting neurons in the ventrolateral preoptic nucleus: Astrocyte-neuron interactions in the regulation of sleep.

Although ATP and/or adenosine derived from astrocytes are known to regulate sleep, the precise mechanisms underlying the somnogenic effects of ATP and adenosine remain unclear. We selectively expressed channelrhodopsin-2 (ChR2), a light-sensitive ion channel, in astrocytes within the ventrolateral preoptic nucleus (VLPO), which is an essential brain nucleus involved in sleep promotion. We then examined the effects of photostimulation of astrocytic ChR2 on neuronal excitability using whole-cell patch-clamp recordings in two functionally distinct types of VLPO neurons: sleep-promoting GABAergic projection neurons and non-sleep-promoting local GABAergic neurons. Optogenetic stimulation of VLPO astrocytes demonstrated opposite outcomes in the two types of VLPO neurons. It led to the inhibition of non-sleep-promoting neurons and excitation of sleep-promoting neurons. These responses were attenuated by blocking of either adenosine A1 receptors or tissue-nonspecific alkaline phosphatase (TNAP). In contrast, exogenous adenosine decreased the excitability of both VLPO neuron populations. Moreover, TNAP was expressed in galanin-negative VLPO neurons, but not in galanin-positive sleep-promoting projection neurons. Taken together, these results suggest that astrocyte-derived ATP is converted into adenosine by TNAP in non-sleep-promoting neurons. In turn, adenosine decreases the excitability of local GABAergic neurons, thereby increasing the excitability of sleep-promoting GABAergic projection neurons. We propose a novel mechanism involving astrocyte-neuron interactions in sleep regulation, wherein endogenous adenosine derived from astrocytes excites sleep-promoting VLPO neurons, and thus decreases neuronal excitability in arousal-related areas of the brain.

The NAergic locus coeruleus-ventrolateral preoptic area neural circuit mediates rapid arousal from sleep

The locus coeruleus (LC), which is located in the brain stem, plays an important role in promoting arousal. However, the neural circuitry underlying this function remains unclear. Using cortical electroencephalography combined with optrode recording, we found that LC noradrenergic (LCNA) neurons exhibit high activity during wakefulness, while suppressing the activity of these neurons causes a reduction in wakefulness. Viral tracing showed that LCNA neurons directly project to the ventrolateral preoptic area (VLPO) and that optogenetic activation of the noradrenergic (NAergic) LC-VLPO (NAergicLC-VLPO) neural circuit promotes arousal. Optrode recordings in the VLPO revealed two functionally distinct neuronal populations that were stimulated in response to the optogenetic activation of LCNA neurons. Consistently, we identified two types of VLPO neurons that exhibited different responses to NAergic projections from the LC mediated by discrete adrenergic receptors. Together, our results demonstrate that the NAergicLC-VLPO neural circuit is a critical pathway for controlling wakefulness and that a synergistic effect is produced by inhibition of sleep-active neurons in the VLPO through α2 receptors and activation of wake-active neurons in the VLPO through α1 and β receptors.

The Interaction Between the Ventrolateral Preoptic Nucleus and the Tuberomammillary Nucleus in Regulating the Sleep-Wakefulness Cycle.

The ventrolateral preoptic nucleus (VLPO) in the anterior hypothalamus and the tuberomammillary nucleus (TMN) in the posterior hypothalamus are critical regions which involve the regulation of sleep-wakefulness flip-flop in the central nervous system. Most of the VLPO neurons are sleep-promoting neurons, which co-express γ-aminobutyric acid (GABA) and galanin, while TMN neurons express histamine (HA), a key wake-promoting neurotransmitter. Previous studies have shown that the two regions are innervated between each other, but how to regulate the sleep-wake cycle are not yet clear. Here, bicuculline (Bic), a GABAA-receptor antagonist, L-glutamate (L-Glu), an excitatory neurotransmitter, and triprolidine (Trip), a HA1 receptor (HRH1) inhibitor, were bilaterally microinjected into TMN or VLPO after surgically implanting the electroencephalogram (EEG) and electromyography (EMG) electrode recording system. Microinjecting L-Glu into VLPO during the night significantly increased the NREM sleep time, and this phenomenon was weakened after selectively blocking GABAA receptors with Bic microinjected into TMN. Those results reveal that VLPO neurons activated, which may inhibit TMN neurons inducing sleep via GABAA receptors. On the contrary, exciting TMN neurons by L-Glu during the day, the wakefulness time was significantly increased. These phenomena were reversed by blocking HRH1 with Trip microinjected into VLPO. Those results reveal that TMN neuron activating may manipulate VLPO neurons via HRH1, and induce wakefulness. In conclusion, VLPO GABAergic neurons and TMN histaminergic neurons may interact with each other in regulating the sleep-wake cycle.

Activation of Preoptic GABAergic or Glutamatergic Neurons Modulates Sleep-Wake Architecture, but Not Anesthetic State Transitions.

The precise mechanism of general anesthesia remains unclear. In the last two decades, there has been considerable focus on the hypothesis that anesthetics co-opt the neural mechanisms regulating sleep. This hypothesis is supported by ample correlative evidence at the level of sleep-promoting nuclei, but causal investigations of potent inhaled anesthetics have not been conducted. Here, we tested the hypothesis that chemogenetic activation of discrete neuronal subpopulations within the median preoptic nucleus (MnPO) and ventrolateral preoptic nucleus (VLPO) of the hypothalamus would modulate sleep/wake states and alter the time to loss and resumption of consciousness associated with isoflurane, a potent halogenated ether in common clinical use. We show that activating MnPO/VLPO GABAergic or glutamatergic neurons does not alter anesthetic induction or recovery time. However, activation of these neuronal subpopulations did alter sleep-wake architecture. Notably, we report the novel finding that stimulation of VLPO glutamatergic neurons causes a strong increase in wakefulness. We conclude that activation of preoptic GABAergic or glutamatergic neurons that increase sleep or wakefulness does not substantively influence anesthetic state transitions. These data indicate that the correlative evidence for a mechanistic overlap of sleep and anesthesia at the level of an individual nucleus might not necessarily have strong causal significance.

Critical Dynamics and Coupling in Bursts of Cortical Rhythms Indicate Non-Homeostatic Mechanism for Sleep-Stage Transitions and Dual Role of VLPO Neurons in Both Sleep and Wake.

Origin and functions of intermittent transitions among sleep stages, including brief awakenings and arousals, constitute a challenge to the current homeostatic framework for sleep regulation, focusing on factors modulating sleep over large time scales. Here we propose that the complex micro-architecture characterizing sleep on scales of seconds and minutes results from intrinsic non-equilibrium critical dynamics. We investigate θ- and δ-wave dynamics in control rats and in rats where the sleep-promoting ventrolateral preoptic nucleus (VLPO) is lesioned (male Sprague-Dawley rats). We demonstrate that bursts in θ and δ cortical rhythms exhibit complex temporal organization, with long-range correlations and robust duality of power-law (θ-bursts, active phase) and exponential-like (δ-bursts, quiescent phase) duration distributions, features typical of non-equilibrium systems self-organizing at criticality. We show that such non-equilibrium behavior relates to anti-correlated coupling between θ- and δ-bursts, persists across a range of time scales, and is independent of the dominant physiologic state; indications of a basic principle in sleep regulation. Further, we find that VLPO lesions lead to a modulation of cortical dynamics resulting in altered dynamical parameters of θ- and δ-bursts and significant reduction in θ-δ coupling. Our empirical findings and model simulations demonstrate that θ-δ coupling is essential for the emerging non-equilibrium critical dynamics observed across the sleep-wake cycle, and indicate that VLPO neurons may have dual role for both sleep and arousal/brief wake activation. The uncovered critical behavior in sleep- and wake-related cortical rhythms indicates a mechanism essential for the micro-architecture of spontaneous sleep-stage and arousal transitions within a novel, non-homeostatic paradigm of sleep regulation.SIGNIFICANCE STATEMENT We show that the complex micro-architecture of sleep-stage/arousal transitions arises from intrinsic non-equilibrium critical dynamics, connecting the temporal organization of dominant cortical rhythms with empirical observations across scales. We link such behavior to sleep-promoting neuronal population, and demonstrate that VLPO lesion (model of insomnia) alters dynamical features of θ and δ rhythms, and leads to significant reduction in θ-δ coupling. This indicates that VLPO neurons may have dual role for both sleep and arousal/brief wake control. The reported empirical findings and modeling simulations constitute first evidences of a neurophysiological fingerprint of self-organization and criticality in sleep- and wake-related cortical rhythms; a mechanism essential for spontaneous sleep-stage and arousal transitions that lays the bases for a novel, non-homeostatic paradigm of sleep regulation.

Neurons in the rat ventral lateral preoptic area are essential for the warm-evoked inhibition of brown adipose tissue and shivering thermogenesis.

To determine the role of neurons in the ventral part of the lateral preoptic area (vLPO) in CNS thermoregulation. In vivo electrophysiological and neuropharmacological were used toevaluate the contribution of neurons in the vLPO to the regulation of brown adipose tissue (BAT) thermogenesis and muscle shivering in urethane/chloralose-anaesthetized rats. Nanoinjections of NMDA targeting the medial preoptic area (MPA) and the vLPO suppressed the cold-evoked BAT sympathetic activity (SNA), reduced the BAT temperature (TBAT ), expired CO2 , mean arterial pressure (MAP), and heart rate. Inhibition of vLPO neurons with muscimol or AP5/CNQX elicited increases in BAT SNA, TBAT , tachycardia, and small elevations in MAP. The BAT thermogenesis evoked by AP5/CNQX in vLPO was inhibited by the activation of MPA neurons. The inhibition of BAT SNA by vLPO neurons does not require a GABAergic input to dorsomedial hypothalamus (DMH), but MPA provides a GABAergic input to DMH. The activation of vLPO neurons inhibits the BAT thermogenesis evoked by NMDA in the rostral raphe pallidus (rRPa), but not that after bicuculline in rRPa. The BAT thermogenesis elicited by vLPO inhibition is dependent on glutamatergic inputs to DMH and rRPa, but these excitatory inputs do not arise from MnPO neurons. The activation of neurons in the vLPO also inhibits cold- and prostaglandin-evoked muscle shivering, and vLPO inhibition is sufficient to evoke shivering. The vLPO contains neurons that are required for the warm ambient-evoked inhibition of muscle shivering and of BAT thermogenesis, mediated through a direct or indirect GABAergic input to rRPa from vLPO.

Neurons in the Ventral Lateral Preoptic (vLPO) Area Inhibit Brown Adipose Tissue (BAT) Thermogenesis

Neurons in the preoptic area of the hypothalamus play important roles in thermoregulation. However, the subregions of the preoptic hypothalamus are heterogeneous, such that neuronal activation at some sites elicits hyperthermia, while others evoke hypothermia. Here we show that in urethane/chloralose‐anesthetized, artificially‐ventilated rats, neurons in the vLPO (0.0 to −1.44 mm caudal to bregma, 0.8 to 1.5 mm lateral to the midline, and −8.0 to 9.8 mm below the dura surface, incisor bar in flat skull position) have a hypothermic effect mediated by inhibition of sympathetic nerve activity (SNA) and BAT thermogenesis. The activation of cvLPO neurons with nanoinjections of NMDA (0.2 mM, 60 nl) evoked a complete inhibition of cold‐evoked BAT thermogenesis: BAT SNA (−97 %, p = 0.01), BAT temperature (TBAT, −0.4 °C, p = 0.01), expired CO2, (Exp CO2, −0.4 %, p = 0.0004), mean arterial pressure (MAP, −9 mmHg, p = 0.01) and heart rate (HR, −53 bpm, p = 0.0005). Similar BAT sympathoinhibitory responses were obtained with nanoinjections of bicuculline (BIC, 1.0 mM, 60 nl) in the vLPO. Under thermoneutral conditions, the inhibition of cvLPO neurons by activation of local GABAA receptors (muscimol, 1.2 mM, 60 nl) increased BAT SNA (+99 % p = 0.0001), TBAT (+2.2 °C, p = 0.0001), Exp CO2 (+1.0 %, p = 0.00003), and HR (+58 bpm, p = 0.007), with no change in MAP (p = 0.5). Nanoinjections of muscimol, or the glutamate receptor antagonists, AP5/CNQX, in the dorsomedial hypothalamus (DMH), or in the rostral raphe pallidus (rRPa), reversed the activation of BAT thermogenesis evoked by inhibition of vLPO neurons. Activation of neurons in vLPO inhibited the BAT thermogenesis evoked by BIC nanoinjections in the DMH, but not the increase in BAT SNA evoked by BIC nanoinjections in the rRPa. We conclude that vLPO contains BAT sympathoinhibitory neurons that mediate the warm ambient‐evoked inhibition of BAT thermogenesis through a direct or indirect GABAergic input to rRPa. Similarly to the cold‐evoked activation of BAT thermogenesis, that following inhibition of vLPO neurons requires the glutamatergic, BAT sympathoexcitatory pathway from DMH to BAT sympathetic premotor neurons in rRPa. These novel findings a key population of BAT sympathoinhibitory neurons in the central pathway mediating the skin warming‐evoked inhibition of BAT thermogenesis, requiring the addition of a BAT sympathoinhibitory pathway between vLPO and rRPa to the current model of central autonomic thermoregulation.Support or Funding InformationThis work was supported by NIH R01‐NS091066 (S.F.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ventral Lateral Preoptic (vLPO) Neurons Inhibit Brown Adipose Tissue Thermogenesis during Warming of the Preoptic Area

Brown adipose tissue (BAT) and shivering thermogenesis and cutaneous vasoconstriction are inhibited in warm climatic conditions to reduce heat production and increase heat dissipation, thereby maintaining core body temperature homeostasis. We tested the hypothesis that the hypothalamic preoptic area (POA) contains warm sensitive neurons (WSNs) that are activated by local warming to produce inhibition of BAT activity. In urethane/α‐chloralose‐anesthetized, artificially‐ventilated rats, warming the POA bilaterally with a thermode (two silver rods connected to a peltier device) increased POA temperature from 35 to 40°C and promptly inhibited skin cooling (35–36°C)‐evoked BAT sympathetic nerve activity (SNA) (from 1061±2.7 to 102±0.1 that corresponds to 100% reduction, p&lt;0.001) and reduced BAT temperature (1.7±0.2, p&lt;0.001), core temperature (0.3±0.1, p&lt;0.05), expired CO2 (0.7±0.1%, p&lt;0.001), heart rate (61±10, p&lt;0.001) and mean arterial pressure (14±6, p&lt;0.05). Bilateral nanoinjections of the GABAA‐agonist, muscimol (500 mM, 100 nl), into the ventral lateral preoptic area (vLPO) prevented (p&lt;0.001) the local POA warming‐induced inhibition of BAT SNA and the reductions in BAT and core temperatures. Blockade of ionotropic glutamate receptors in vLPO with nanoinjections of AP5/CNQX also prevented (p&lt;0.001) the local POA warming‐induced inhibitions of BAT thermogenesis. POA warming failed (p&gt;0.05) to inhibit the elevated BAT SNA following blockade of local GABAA receptors with nanoinjections of bicuculline (BIC) in the rostral raphe pallidus (rRPa), but still effectively inhibited the elevated BAT SNA following BIC nanoinjections in the dorsomedial hypothalamus (DMH). Our results demonstrate that the POA warming‐evoked inhibition of BAT thermogenesis requires activation of GABAA receptors in rRPa, but not those in DMH. In addition, the finding that glutamate receptor blockade in vLPO prevents the POA warming‐induced inhibition of BAT activity, is consistent with WSNs glutamatergically exciting BAT sympathoinhibitory neurons in vLPO to produce the POA warming‐induced inhibition of BAT thermogenesis. These novel findings elucidate the central pathway mediating POA warming‐evoked inhibition of BAT thermogenesis and require the addition of a BAT sympathoinhibitory pathway between vLPO and rRPa to the current model of central autonomic thermoregulation.Support or Funding InformationSupported by NIH R01091066 (SFM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

0133 OREXIN MEDIATES FEED-FORWARD INHIBITION OF VLPO SLEEP-ACTIVE NEURONS - A MECHANISM FOR CONTROLLING AROUSAL

The ventrolateral preoptic area (VLPO) plays an essential role in the initiation and maintenance of sleep. It contains a cluster of sleep-active neurons that are GABAergic and co-expresses galanin (VLPOGABA/Gal). VLPO is innervated by wake-promoting neurons and the VLPOGABA/Gal neurons are strongly inhibited by noradrenaline, carbachol and serotonin. VLPO also receives input from orexin neurons and administration of orexin in VLPO arouses mice from sleep suggesting that orexin might be inhibiting the sleep-active VLPOGABA/Gal neurons. However, orexin is an excitatory peptide, thus orexin’s effect in the VLPO remains unclear. In this study we investigate the effect of orexin on VLPO neurons in brain slices. We recorded VLPO neurons in brain slices from WT and Vgat-IRES-cre mice. We filled the recorded cells with biocytin for anatomical localization. We recorded from VLPO GFP-labelled GABAergic neurons in Vgat-IRES-cre mice that were injected in VLPO with an AAV-flex-GFP. We identify VLPO-sleep active neurons based on the inhibitory responses to noradrenaline or carbachol and/or by the presence of VGAT and galanine mRNAs using single cell RT-PCR. We found a dual response to orexin in VLPO. About 60% of VLPO neurons were excited by orexin but 40% were inhibited through release of GABA. The VLPO neurons that were inhibited by orexin were also inhibited by carbachol or noradrenaline suggesting that they could be the VLPOGABA/Gal sleep-active neurons. We tested this hypothesis by recording VLPO GABAergic neurons (GFP-labelled). Orexin increased the frequency of spontaneous IPSCs in 50% of the VLPO GABAergic neurons and these neurons expressed galanin mRNA. Orexin inhibits VLPOGABA/Gal sleep-active neuron by increasing GABAergic afferent input. This GABAergic input could originate from the local neurons that are directly activated by orexin. We propose that during wakefulness VLPOGABA/Gal sleep-active neurons are strongly and directly inhibited by wake-promoting signals, such as noradrenaline, carbachol and serotonin. Whereas, orexin could act by activating local GABAergic neurons that in turn produce feed-forward inhibition of VLPOGABA/Gal sleep-promoting neurons. R01NS091126 and P01HL095491.

Sedative Effect of Sophora flavescens and Matrine.

The present study investigated the sedative effects of Sophora flavescens (SF) and its bioactive compound, matrine through performing locomotor activity test and the electroencephalography (EEG) analysis in the rat. The underlying neural mechanism of their beneficial effects was determined by assessing c-Fos immunoreactivity and serotonin (5-HT) in the brain utilizing immunohistochemical method and enzyme-linked immunosorbent assay. The results showed that SF and matrine administration had an effect on normalization of caffeine-induced hyperactivity and promoting a shift toward non-rapid eye movement (NREM) sleep. c-Fos-immunoreactivity and 5-HT level in the ventrolateral preoptic nucleus (VLPO), a sleep promoting region, were increased in the both SF and matrine-injected groups. In conclusion, SF and its bioactive compound, matrine alleviated caffeine-induced hyperactivity and promoted NREM sleep by activating VLPO neurons and modulating serotonergic transmission. It is suggested that SF might be a useful natural alternatives for hypnotic medicine.

Multiparametric characterization of neuronal subpopulations in the ventrolateral preoptic nucleus

The characterization of neuronal properties is a necessary first step toward understanding how the ventrolateral preoptic nucleus (VLPO) neuronal network regulates slow-wave sleep (SWS). Indeed, the electrophysiological heterogeneity of VLPO neurons suggests the existence of subtypes that could differently contribute in SWS induction and maintenance. The aim of the present study was to define cell classes in the VLPO using an unsupervised clustering classification method. Electrophysiological features extracted from 289 neurons recorded in whole-cell patch-clamp allowed the identification of three main classes of VLPO neurons subdivided into five distinct subpopulations (cluster 1, 2a, 2b, 3a and 3b). The high occurrence of a low-threshold calcium spike (LTS) was one of the most distinctive features of cluster 1 and 3. Since sleep-promoting neurons are generally identified by their ability to generate an LTS and by their inhibitory response to noradrenaline (NA), 189 neurons from our dataset were also tested for this neurotransmitter. Neurons from cluster 3 were the most frequently inhibited by NA. Biocytin labeling and Neurolucida reconstructions of 112 neurons furthermore revealed a small dendritic arbor of cluster 3b neurons compared, in particular, to cluster 2b neurons. Altogether, we performed an exhaustive characterization of VLPO neuronal subtypes that is a crucial step toward a better understanding of the neuronal network within the VLPO and thereby sleep physiology.

Serotonin differentially modulates excitatory and inhibitory synaptic inputs to putative sleep-promoting neurons of the ventrolateral preoptic nucleus

The role of serotonin (5-HT) in sleep-wake regulation has been a subject of intense debate and remains incompletely understood. In the ventrolateral preoptic nucleus (VLPO), the main structure that triggers non-rapid eye movement (NREM) sleep, putative sleep-promoting (PSP) neurons were shown ex vivo to be either inhibited (Type-1) or excited (Type-2) by 5-HT application. To determine the complex action of this neurotransmitter on PSP neurons, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, sIPSCs, mEPSCs and mIPSCs) in response to bath application of 5-HT. We established in mouse acute VLPO slices that 5-HT reduces spontaneous and miniature EPSC and IPSC frequencies to Type-1 neurons, whereas 5-HT selectively increases sIPSC and mIPSC frequencies to Type-2 VLPO neurons. We further determined that Type-1 neurons display a lower action potential threshold and a smaller soma size than Type-2 neurons. Finally, single-cell RT-PCR designed to identify the 13 serotonergic receptor subtypes revealed the specific mRNA expression of the 5-HT1A,B,D,F receptors by Type-1 neurons. Furthermore, the 5-HT2A-C,4,7 receptors were found to be equivalently expressed by both neuronal types. Altogether, our results establish that the excitatory and inhibitory inputs to Type-1 and Type-2 VLPO PSP neurons are differentially regulated by 5-HT. Electrophysiological, morphological and molecular differences were also identified between these two neuronal types. Our results provide new insights regarding the orchestration of sleep regulation by 5-HT release, and strongly suggest that Type-2 neurons could play a permissive role, whereas Type-1 neurons could have an executive role in sleep induction and maintenance.

Disrupted Sleep in Narcolepsy: Exploring the Integrity of Galanin Neurons in the Ventrolateral Preoptic Area.

To examine the integrity of sleep-promoting neurons of the ventrolateral preoptic nucleus (VLPO) in postmortem brains of narcolepsy type 1 patients. Postmortem examination of five narcolepsy and eight control brains. VLPO galanin neuron count did not differ between narcolepsy patients (11,151 ± 3,656) and controls (13,526 ± 9,544). A normal number of galanin-immunoreactive VLPO neurons in narcolepsy type 1 brains at autopsy suggests that VLPO cell loss is an unlikely explanation for the sleep fragmentation that often accompanies the disease.

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose.

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity.

Promotion of Wakefulness and Energy Expenditure by Orexin-A in the Ventrolateral Preoptic Area.

The ventrolateral preoptic area (VLPO) and the orexin/hypocretin neuronal system are key regulators of sleep onset, transitions between vigilance states, and energy homeostasis. Reciprocal projections exist between the VLPO and orexin/hypocretin neurons. Although the importance of the VLPO to sleep regulation is clear, it is unknown whether VLPO neurons are involved in energy balance. The purpose of these studies was to determine if the VLPO is a site of action for orexin-A, and which orexin receptor subtype(s) would mediate these effects of orexin-A. We hypothesized that orexin-A in the VLPO modulates behaviors (sleep and wakefulness, feeding, spontaneous physical activity [SPA]) to increase energy expenditure. Sleep, wakefulness, SPA, feeding, and energy expenditure were determined after orexin-A microinjection in the VLPO of male Sprague-Dawley rats with unilateral cannulae targeting the VLPO. We also tested whether pretreatment with a dual orexin receptor antagonist (DORA, TCS-1102) or an OX2R antagonist (JNJ-10397049) blocked the effects of orexin-A on the sleep/wake cycle or SPA, respectively. Orexin-A injected into the VLPO significantly increased wakefulness, SPA, and energy expenditure (SPA-induced and total) and reduced NREM sleep and REM sleep with no effect on food intake. Pretreatment with DORA blocked the increase in wakefulness and the reduction in NREM sleep elicited by orexin-A, and the OX2R antagonist reduced SPA stimulated by orexin-A. These data show the ventrolateral preoptic area is a site of action for orexin-A, which may promote negative energy balance by modulating sleep/wakefulness and stimulating spontaneous physical activity and energy expenditure.

Glucose Induces Slow-Wave Sleep by Exciting the Sleep-Promoting Neurons in the Ventrolateral Preoptic Nucleus: A New Link between Sleep and Metabolism.

Although the brain circuitry underlying vigilance states is well described, the molecular mechanisms responsible for sleep onset remain largely unknown. Combining in vitro and in vivo experiments, we demonstrate that glucose likely contributes to sleep onset facilitation by increasing the excitability of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO). We find here that these neurons integrate energetic signals such as ambient glucose directly to regulate vigilance states accordingly. Glucose-induced excitation of sleep-promoting VLPO neurons should therefore be involved in the drowsiness that one feels after a high-sugar meal. This novel mechanism regulating the activity of VLPO neurons reinforces the fundamental and intimate link between sleep and metabolism.