VLPO Neurons Research Articles

Suprachiasmatic Modulation of Noradrenaline Release in the Ventrolateral Preoptic Nucleus

As the major brain circadian pacemaker, the suprachiasmatic nucleus (SCN) is known to influence the timing of sleep and waking. We thus investigated here the effect of SCN stimulation on neurons of the ventrolateral preoptic nucleus (VLPO) thought to be involved in promoting sleep. Using an acute in vitro preparation of the rat anterior hypothalamus/preoptic area, we found that whereas single-pulse stimulations of the SCN evoked standard fast ionotropic IPSPs and EPSPs, train stimulations unexpectedly evoked a long-lasting inhibition (LLI). Such LLIs could also be evoked in VLPO neurons by pressure application of NMDA within the SCN, indicating the specific activation of SCN neurons. This LLI was shown to result from the presynaptic facilitation of noradrenaline release, because it was suppressed in presence of yohimbine, a selective antagonist of alpha2-adrenoreceptors. The LLI depended on the opening of a potassium conductance, because it was annulled at E(K) and could be reversed below E(K). These results show that the SCN can provide an LLI of the sleep-promoting VLPO neurons that could play a role in the circadian organization of the sleep-waking cycle.

Nicotinic enhancement of the noradrenergic inhibition of sleep-promoting neurons in the ventrolateral preoptic area.

According to multiple lines of evidence, neurons in the ventrolateral preoptic area (VLPO) that contain GABA promote sleep by inhibiting neurons of the arousal systems. Reciprocally, transmitters used by these systems, including acetylcholine (ACh) and noradrenaline (NA), exert an inhibitory action on the VLPO neurons. Because nicotine, an agonist of ACh, acts as a potent stimulant, we queried whether it might participate in the cholinergic inhibition of these sleep-promoting cells. Indeed, we found that ACh inhibits the VLPO neurons through a nicotinic, as well as a muscarinic, action. As evident in the presence of atropine, the non-muscarinic component was mimicked by epibatidine, a nonselective nicotinic ACh receptor (nAChR) agonist and was blocked by dihydro-beta-erythroidine, a nonselective nAChR antagonist. It was not, however, blocked by methyllycaconitine, a selective antagonist of the alpha7 subtype, indicating that the action was mediated by non-alpha7 nAChRs. The nicotinic inhibition was attributed to a presynaptic facilitation of NA release because it persisted in the presence of tetrodotoxin and was blocked by yohimbine and RS 79948, which are both selective antagonists of alpha2 adrenergic receptors. Sleep-promoting VLPO neurons are thus dually inhibited by ACh through a muscarinic postsynaptic action and a nicotinic presynaptic action on noradrenergic terminals. Such dual complementary actions allow ACh and nicotine to enhance wakefulness by inhibiting sleep-promoting systems while facilitating other wake-promoting systems.

Effect of the Wake-Promoting Agent Modafinil on Sleep-Promoting Neurons from the Ventrolateral Preoptic Nucleus: an In Vitro Pharmacologic Study

The pharmacologic profile of modafinil, an increasingly popular wake-promoting drug for narcolepsy treatment, differs from those of classic psychostimulants such as amphetamine. However, its brain targets are still a matter of debate. We hypothesized that modafinil could increase waking by inhibiting the sleep-promoting neurons from the ventrolateral preoptic nucleus (VLPO). Such action could be direct or indirect via the potentiation of inhibition mediated by waking neurotransmitters. We thus studied the effect of modafinil on the membrane potential and firing rate of VLPO neurons recorded in rat-brain slices. We further determined whether pretreatment with modafinil modifies the effect of noradrenaline, carbachol, serotonin, histamine, dopamine, or clonidine. Pretreatment with modafinil specifically increased the inhibition of VLPO neurons induced by noradrenaline but had no effect when applied alone or in combination with other substances. Pretreatment with nisoxetine, a selective noradrenaline reuptake blocker, similarly increased the noradrenaline-induced inhibition of VLPO cells. Further, the potentiation by modafinil was minimized when modafinil and nisoxetine were applied together. These results suggest that modafinil blocks the reuptake of noradrenaline by the noradrenergic terminals on sleep-promoting neurons from the VLPO. Such a mechanism could be at least partially responsible for the wake-promoting effect of modafinil.

Effects of adenosine on gabaergic synaptic inputs to identified ventrolateral preoptic neurons

The ventrolateral preoptic nucleus (VLPO) is a key regulator of behavioral state that promotes sleep by directly inhibiting brain regions that maintain wakefulness. Subarachnoid administration of adenosine (AD) or AD agonists promotes sleep and induces expression of Fos protein in VLPO neurons. Therefore, activation of VLPO neurons may contribute to the somnogenic actions of AD. To define the mechanism through which AD activates VLPO neurons, we prepared hypothalamic slices from 9 to 12-day-old rat pups and recorded from 43 neurons in the galaninergic VLPO cluster; nine neurons contained galanin mRNA by post hoc in situ hybridization. Bath application of AD (20 μM) to seven of these neurons had no direct effect but caused a significant decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents in the presence of tetrodotoxin, indicating a presynaptic site of action. We conclude that AD-mediated disinhibition increases the excitability of VLPO neurons thus contributing to the somnogenic properties of AD.

Alpha 2-Adrenoceptor-mediated presynaptic modulation of GABAergic transmission in mechanically dissociated rat ventrolateral preoptic neurons.

The ventrolateral preoptic nucleus (VLPO) is a key nucleus involved in the homeostatic regulation of sleep-wakefulness. Little is known, however, about the cellular mechanisms underlying its role in sleep regulation and how the neurotransmitters, such as GABA and noradrenaline (NA), are involved. In the present study we investigated GABAergic transmission to acutely dissociated VLPO neurons using an enzyme-free, mechanical dissociation procedure in which functional terminals remained adherent and we investigated how this GABAergic transmission was modulated by NA. As previously reported in slices, NA hyperpolarized multipolar VLPO neurons and depolarized bipolar VLPO neurons. NA also inhibited the release of GABA onto multipolar VLPO neurons but had no effect on GABAergic transmission to bipolar neurons. The inhibition of release was mediated by presynaptic alpha(2) adrenoceptors coupled to N-ethylmaleimide (NEM)-sensitive G-proteins which appeared to act via inhibition of adenylate cyclase and subsequent decreases in protein kinase A activity. The inhibition of GABA release did not, however, involve an inhibition of external Ca(2+) influx. The results indicate that all VLPO neurons contain GABAergic inputs and that the different morphological subgroups of VLPO neurons are correlated not only to different postsynaptic responses to NA but also to different presynaptic NA responses. Furthermore our results demonstrate an additional mechanism by which NA can modulate the excitability of multipolar VLPO neurons which may have important implications for its role in regulating sleep/wakefulness.

Estradiol differentially regulates lipocalin-type prostaglandin D synthase transcript levels in the rodent brain: Evidence from high-density oligonucleotide arrays and in situ hybridization.

Microarrays comprise an efficient approach to discovering large numbers of differentially expressed mRNA transcripts in the CNS resulting from changes in hormonal milieu. We used high-density oligonucleotide microarrays to examine the short- and long-term actions of estradiol (E(2)) on the transcriptomes from the medial basal hypothalamus and other brain regions of E(2)-treated (10 microg) adult female mice. Our results have revealed several unanticipated gene regulations. Most striking is lipocalin prostaglandin D(2) synthase (L-PGDS), which catalyzes the conversion of prostaglandin (PG) H(2) to PGD(2), a neuromodulator involved in a variety of functions, including sleep, pain, and odor responses. In situ hybridization revealed significant increases in L-PGDS expression in the arcuate and ventromedial nucleus of the medial basal hypothalamus compared with vehicle controls. The magnitude of these changes is approximately 2-fold and suggests a modulatory role for PGD(2) in E(2)-controlled neuroendocrine secretions and behaviors. Surprisingly, L-PGDS gene expression is reduced 2-fold after E(2) treatment in the ventrolateral preoptic area (VLPO), the suspected site of action for the sleep-promoting effects of PGD(2). Finally, whereas L-PGDS has been reported to be expressed primarily in oligodendrocytes of the adult rodent brain, we demonstrate, immunocytochemically, that L-PGDS is also expressed in a population of VLPO neurons. Thus, our data suggest the intriguing possibility that E(2) modulation of L-PGDS plays a role in the regulation of sleep-wake states through hitherto unknown mechanisms in VLPO neurons and through hormone-dependent neuronal-glial cooperation.

Ventrolateral preoptic nucleus contains sleep-active, galaninergic neurons in multiple mammalian species

The ventrolateral preoptic nucleus (VLPO) is a group of sleep-active neurons that has been identified in the hypothalamus of rats and is thought to inhibit the major ascending monoaminergic arousal systems during sleep; lesions of the VLPO cause insomnia. Identification of the VLPO in other species has been complicated by the lack of a marker for this cell population, other than the expression of Fos during sleep. We now report that a high percentage of the sleep-active (Fos-expressing) VLPO neurons express mRNA for the inhibitory neuropeptide, galanin, in nocturnal rodents (mice and rats), diurnal rodents (degus), and cats. A homologous (i.e. galanin mRNA-containing cell group) is clearly distinguishable in the ventrolateral region of the preoptic area in diurnal and nocturnal monkeys, as well as in humans. Galanin expression may serve to identify sleep-active neurons in the ventrolateral preoptic area of the mammalian brain. The VLPO appears to be a critical component of sleep circuitry across multiple species, and we hypothesize that shrinkage of the VLPO with advancing age may explain sleep deficits in elderly humans.

Electrophysiological analysis of suprachiasmatic nucleus projections to the ventrolateral preoptic area in the rat.

The circadian pacemaker housed in the suprachiasmatic nucleus (SCN) synchronizes daily sleep-wake cycles, presumably by modulating the sleep-wake regulatory system, including ventrolateral preoptic area (VLPO) neurons. We used whole-cell patch-clamp recording to study the projections from the SCN to the VLPO in horizontal slices of rat hypothalamus. Single-pulse stimulation of the SCN region elicited postsynaptic currents (PSCs) in 20 of 66 neurons (30%) recorded within the VLPO region as verified by intracellular biocytin labelling. At a holding potential of -60 mV, the evoked PSCs had an amplitude of 17.6 +/- 3.2 pA (SEM) and a latency of 6.3 +/- 0.5 ms (n = 10). There was a trend for simple excitatory postsynaptic currents (EPSCs) to be evoked in the VLPO cluster, simple inhibitory postsynaptic currents (IPSCs) in the extended VLPO, and a combination of EPSCs and IPSCs in both regions. IPSCs were blocked reversibly by bicuculline (10 microm, n = 11). In both the presence and absence of bicuculline, EPSCs had fast and slow components that were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microm; n = 7), and (+/-)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 microm, n = 6), respectively. Reversal potentials for the evoked IPSCs and EPSCs were consistent with mediation via GABAA and ionotropic glutamate receptors, respectively. These results suggest that the SCN region provides both inhibitory and excitatory inputs to single VLPO neurons, which are mediated, respectively, by GABAA receptors and by both non-NMDA and NMDA glutamate receptors. These projections may play important roles in conveying circadian input to systems in the preoptic area that regulate sleep and waking.

Perchance to sleep…

Why and how do we sleep? The history of sleep research is full of stories of dedicated human researchers living in caves, and experiments on animal brainstems, but we still do not really know the answers to the fundamental questions of how and why (and therefore also why not). The latest research, however, is beginning to confirm the answer to how.From the results of in vivo animal experiments, neurons that induce the onset of sleep are thought to reside in the ventrolateral preoptic nucleus (VLPO) at the base of the forebrain1xActivation of ventrolateral preoptic neurons during sleep. Sherin, J.E. et al. Science. 1996; 271: 216–219Crossref | PubMedSee all

Effect of Lesions of the Ventrolateral Preoptic Nucleus on NREM and REM Sleep

Neurons in the ventrolateral preoptic nucleus (VLPO) in rats show c-fos activation after sleep and provide GABAergic innervation of the major monoamine arousal systems, suggesting that they may be a necessary part of the brain circuitry that produces sleep. We examined the effects on sleep behavior in rats of cell-specific damage to the VLPO by microinjection of ibotenic acid. Severe lesions of the central cell cluster of the VLPO ( approximately 80-90% cell loss bilaterally) caused a 60-70% decrease in delta power and a 50-60% decrease in nonrapid-eye-movement (NREM) sleep time (p < 0.001). The number of remaining Fos-immunoreactive neurons in the VLPO cell cluster was linearly related to NREM sleep time (r = 0.77; p < 0.001) and total electroencephalogram delta power (r = 0. 79; p < 0.001) but not to rapid-eye-movement (REM) sleep (r = 0.35; p > 0.10). Lesions in the region containing scattered VLPO neurons medial or dorsal to the cell cluster caused smaller changes in NREM sleep time (24.5 or 15%, respectively) but were more closely associated with loss of REM sleep (r = 0.74; p < 0.01). The insomnia caused by bilateral VLPO lesions persisted for at least 3 weeks. Lesions of the VLPO caused no change in mean body temperature or its circadian variation; after small lesions of the ventromedial preoptic nucleus, body temperature showed normal circadian variation but a wider temperature range, and sleep behavior was not affected. These experiments delineate distinct preoptic sites with primary effects on the regulation of NREM sleep, REM sleep, and body temperature.

Identification of sleep-promoting neurons in vitro.

The neurons responsible for the onset of sleep are thought to be located in the preoptic area and more specifically, in the ventrolateral preoptic nucleus (VLPO). Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain gamma-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project is a key factor for promoting sleep.

Molecular mechanisms of sleep-wake regulation: a role of prostaglandin D2.

Prostaglandin (PG) D2 is a major prostanoid in the brains of rats and other mammals, including humans. When PGD synthase (PGDS), the enzyme that produces PGD2 in the brain, was inhibited by the intracerebroventricular infusion of its selective inhibitors, i.e. tetravalent selenium compounds, the amount of sleep decreased both time and dose dependently. The amount of sleep of transgenic mice, in which the human PGDS gene had been incorporated, increased several fold under appropriate conditions. These data indicate that PGDS is a key enzyme in sleep regulation. In situ hybridization, immunoperoxidase staining and direct enzyme activity determination of tissue samples revealed that PGDS is hardly detectable in the brain parenchyma but is localized in the membrane systems surrounding the brain, namely, the arachnoid membrane and choroid plexus, from which it is secreted into the cerebrospinal fluid (CSF) to become beta-trace, a major protein component of the CSF. PGD2 exerts its somnogenic activity by binding to PGD2 receptors exclusively localized at the ventrorostral surface of the basal forebrain. When PGD2 was infused into the subarachnoid space below the rostral basal forebrain, striking expression of proto-oncogene Fos immunoreactivity (FosIR) was observed in the ventrolateral preoptic area (VLPO), a putative sleep centre, concurrent with sleep induction. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus (TMN), a putative wake centre. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO neurons and downregulation of the wake neurons in the TMN area. Adenosine may be involved in the signal transduction associated with PGD2.

Innervation of Histaminergic Tuberomammillary Neurons by GABAergic and Galaninergic Neurons in the Ventrolateral Preoptic Nucleus of the Rat

The tuberomammillary nucleus (TMN) is the major source of histaminergic innervation of the mammalian brain and is thought to play a major role in regulating wake-sleep states. We recently found that sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) provide a major input to the TMN, but the specificity of this projection and the neurotransmitters involved remain unknown. In this study, we examined the relationship of VLPO efferents to the TMN using both retrograde and anterograde tracing, combined with immunocytochemistry. We found that the descending projection from the VLPO selectively targets the cell bodies and proximal dendrites of the histaminergic TMN. In addition, VLPO axons could be traced into the brainstem, where they provided terminals in the the serotoninergic dorsal and median raphe nuclei, and the core of the noradrenergic locus coeruleus. Approximately 80% of the VLPO neurons that were retrogradely labeled by tracer injections including the TMN were immunoreactive either for galanin or for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA. Virtually all of the galaninergic neurons in the VLPO were also GAD positive. Our results indicate that the VLPO may provide inhibitory GABAergic and galaninergic inputs to the cell bodies and proximal dendrites of the TMN and other components of the ascending monoaminergic arousal system. Because these cell groups are simultaneously inhibited during sleep, the VLPO sleep-active neurons may play a key role in silencing the ascending monoaminergic arousal system during sleep.

Studies on the sleep-promoting mechanisms mediated by adenosine A 2A receptors

Daily cycles of wakefulness and sleep are regulated by coordinated interactions between wakefulness- and sleep-regulating neural circuitry. Wakefulness is associated with neuronal activity in cholinergic neurons in the brainstem and basal forebrain, monoaminergic neurons in the brainstem and posterior hypothalamus, and hypocretin (orexin) neurons in the lateral hypothalamus that act in a coordinated manner to stimulate cortical activation on the one hand and behavioral arousal on the other hand. Each of these neuronal groups subserves distinct aspects of wakefulness-related functions of the brain. Normal transitions from wakefulness to sleep involve sleep-related inhibition and/or disfacilitation of the multiple arousal systems. The cell groups that shut off the network of arousal systems, at sleep onset, occur with high density in the ventral lateral preoptic area (VLPO) and the median preoptic nucleus (MnPN) of the hypothalamus. Preoptic neurons are activated during sleep and exhibit sleep--wake state-dependent discharge patterns that are reciprocal of that observed in several arousal systems. Neurons in the VLPO contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). The majority of MnPN sleep-active neurons synthesize GABA. VLPO and MnPN neurons are sources of projections to arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Mechanisms of sleep induction by these nuclei are hypothesized to involve GABA-mediated inhibition of multiple arousal systems. Normal cycling between discrete behavioral states is mediated by the combined influence of a sleep need that increases with continued wakefulness and an intrinsic circadian oscillation. This chapter will review anatomical and functional properties of populations of sleep- /wake-regulating neurons, focusing on recent findings supporting functional significance of the VLPO and MnPN in the regulation of sleep--wake homeostasis. Evidence indicating that MnPN and VLPO neurons have different, but complementary sleep regulatory functions will be summarized. Potential mechanisms that function to couple activity in these two sleep-regulatory neurons will be discussed.

Brucellosis in children.

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. <b>SIGNIFICANCE STATEMENT</b> 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.