Ventral Tuberomammillary Nucleus Research Articles

Structural Basis for Histaminergic Regulation of Neural Circuits in the Mouse Olfactory Bulb.

Odor information is modulated by centrifugal inputs from other brain regions to the olfactory bulb (OB). Neurons containing monoamines, such as serotonin, acetylcholine, and noradrenaline, are well known as centrifugal inputs; however, the role of histamine, which is also present in the OB, is not well understood. In this study, we examined the histaminergic neurons projecting from the hypothalamus to the OB. We used an antibody against histidine decarboxylase (HDC), a synthesizing enzyme of histamine, to identify histaminergic neurons and assess their localization within the OB and the ultrastructure of their fibers and synapses using multiple immunostaining laser microscopy, ultra-high voltage electron microscopy (EM), and EM to confirm their relationships with other neurons. To further identify the origin nucleus of the histaminergic neurons projecting to the OB, we injected the retrograde tracer FluoroGold and analyzed the pathway to the OB anterogradely. HDC-immunoreactive (-ir) fibers were abundant in the olfactory nerve (ON) layer compared to other monoamines. HDC-ir neurons received asymmetrical synapses from ONs and formed synapses containing pleomorphic vesicles with variable postsynaptic densities to non-ON elements, thus forming serial synapses. We also confirmed that histaminergic neurons project from the rostral ventral tuberomammillary nucleus to the granule cell layer of the OB and, for the first time, successfully visualized their axons from the hypothalamus to the OB. These findings indicate that histamine may regulate odor discrimination in the OB, suggesting a regulatory relationship between hypothalamic function and olfaction. We thus elucidate morphological mechanisms with tuberomammillary nucleus-derived histaminergic neurons involved in olfactory information.

Study on the protective mechanism of neuropeptide S in neonates with hypoxic ischemic encephalopathy

Hypoxic-ischemic brain damage (HIBD) is a severe complication of neonatal asphyxia that contributes significantly to neonatal mortality, cerebral palsy, and delays in intellectual and motor development. Neuropeptide S (NPS) plays a role in the regulation of various physiological processes. This study aimed to determine the morphological localization of NPS in hypothalamic target neurons during HIBD, providing a basis for further investigation of HIBD. Seven-day-old SD neonatal male rats were assigned to a sham group and a model group to establish the HIBD model. Then, the rats in the model group were further averagely divided into the NPS group and the normal saline group. Immunohistochemical staining of Fos immunoreactivity (Fos-IR) found that NPS administration resulted in a significant increase in the count of Fos-IR neurons in the suprachiasmatic nucleus (122%), paraventricular nucleus (108%), dorsal tuberomammillary nucleus (174% and 386%), ventromedial hypothalamic nucleus (116%), arcuate nucleus (167%), perifornical nucleus (320%), ventral tuberomammillary nucleus (441%), and lateral hypothalamic area (278%) (P < 0.0001), compared to the normal saline group. During HIBD, NPS can protect the above neurons and activate the above target neurons in the hypothalamus to participate in the sleep and wake cycle, mood, diet, circadian rhythm, temperature and neuroendocrine regulation.

Involvement of the tuberomammillary nucleus of the hypothalamus in the modulation of nociception and joint edema in a model of monoarthritis

AimsInvestigate the involvement of the histaminergic projections from tuberomammillary nucleus (TMN) to the spinal cord in the modulation of nociception and peripheral edema in a model of monoarthritis. Main methodsSubacute monoarthritis was induced by an intraarticular injection of carrageenan followed by LPS 72 h later. Disability and joint edema were assessed at the 3rd hour after LPS and at every hour up to 6 h. Key findingsIntrathecal administration of histamine potentiated joint incapacitation and edema, while the H1R antagonist cetirizine decreased both. The H3R agonist immepip decreased both incapacitation and edema, while the H3R antagonist thioperamide had the opposite effect. The microinjection of glutamate into the ventral TMN (vTMN) caused an increase of incapacitation and articular edema, whereas the blockade of this nucleus by cobalt chloride inhibited both parameters. Intrathecal administration of cetirizine prevented the increase of incapacitation and joint edema caused by glutamate microinjection into the vTMN. Similarly, an intrathecal injection of the NKCC1 cotransporter inhibitor bumetanide prevented the effects of glutamate microinjection into the vTMN, whereas coadministration of histamine with bumetanide only inhibited the potentiation of joint edema. A microinjection of orexin B into the vTMN potentiated incapacitation and joint edema, while coadministration of the OX1/2 receptor antagonist almorexant with orexin B did not. SignificanceThese data support the notion that TMN participates in the modulation of a peripheral inflammatory process by means of histaminergic projections to the spinal cord, and the hypothalamus may trigger TMN activation by means of glutamate and orexin.

Reduced c-Fos expression in orexin neurons of the lateral hypothalamic area and the locus coeruleus following injection of spinosin into mice.

Spinosin, a major component of Samen Ziziphi spinosae, has been shown to modulate sedation and hypnosis; however, the underlying neuronal mechanisms of its stimulatory effects remain unclear. In the present study, we injected spinosin (15 mg/kg) or saline into mice, which were killed after 90 min. We isolated the brains, which were immunohistochemically stained for c-Fos as a biomarker for neuronal activation and assessed the expression profile of c-Fos in various sleep-arousal brain areas. Our findings revealed that there were no statistically significant differences in the expression of c-Fos in the nucleus accumbens and ventrolateral preoptic area, the vertical limb of the diagonal band nucleus, horizontal limb of the diagonal band nucleus, ventral tuberomammillary nucleus, ventral tegmental area, and dorsal raphe nucleus relative to saline between saline and spinosin-treated mice. Unlike saline, spinosin markedly decreased c-Fos expression in the lateral hypothalamic area (LHA) as well as the locus coeruleus (LC). Compared to the saline injection, the application of spinosin also resulted in a marked decrease in c-Fos expression in the LHA orexin neurons. These findings suggest that spinosin administration results in a restricted pattern of c-Fos expression within the LHA orexin neurons and the LC, suggesting that this particular neuronal inactivation contributes to sedation and hypnosis.

A comparison of the orexin receptor distribution in the brain between diurnal Nile grass rats (Arvicanthis niloticus) and nocturnal mice (Mus musculus)

The neuropeptide orexin/hypocretin regulates a wide range of behaviors and physiology through its receptors OX1R and OX2R, or HCRTR-1 and HCRTR-2. Although the distributions of these receptors have been established in nocturnal rodents, their distributions in the brain of diurnal species have not been studied. In the present study, we examined spatial patterns of OX1R and OX2R mRNA expression in diurnal Nile grass rats (Arvicanthis niloticus) by in situ hybridization and compared them with those in nocturnal mice (Mus musculus). Both receptors showed similar spatial patterns between species in most brain regions. However, species-specific expression was found in several regions that are mainly implicated in regulation of sleep/wakefulness, emotion and cognition. OX1R expression was detected in the caudate putamen and ventral tuberomammillary nucleus only in grass rats, while it was detected in the bed nucleus of the stria terminalis, medial division, posteromedial part only in mice. The distribution of OX2R mRNA was mostly consistent between the two species, although it was more widely expressed in the ventral tuberomammillary nucleus in grass rats compared to mice. These results suggest that neuronal pathways of the orexin system differ between chronotypes, and these differences could underlie the distinct profiles in behaviors and physiology between diurnal and nocturnal species.

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

Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons (Hdc-Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep-wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep.SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain function, but has been difficult to selectively manipulate owing to cellular heterogeneity in this region. Here we use a transgenic mouse to interrogate both the characteristic firing properties of HA neurons and their specific role in maintaining wakefulness. Our results demonstrate that the acute, cell type-specific silencing of HA neurons during wakefulness is sufficient to not only impair arousal but to rapidly and selectively induce slow-wave sleep. This work furthers our understanding of HA-mediated mechanisms that regulate behavioral arousal.

Evidence for a histaminergic input from the ventral tuberomammillary nucleus to the solitary tract nucleus involved in arterial pressure regulation.

The tuberomammillary nucleus (TMN) of the posterior hypothalamus has a high density of histaminergic neurons, the projection fibers of which are present in many areas of the brain, including the nucleus tractus solitarius (NTS), which controls arterial pressure (AP). In this study, we investigated whether the TMN–NTS pathway is involved in central cardiovascular regulation. Bicuculline, a gamma‐aminobutyric acid type A (GABAA) receptor antagonist, was microinjected into the ventral TMN of anesthetized rats and its effects on AP and heart rate (HR) were observed. We also evaluated the effect of cetirizine, an H1 receptor antagonist, microinjected into the NTS on cardiovascular responses induced by electrical stimulation of the TMN. Both AP and HR increased following bicuculline microinjection into the ventral TMN. Similar pressor and tachycardic responses were observed after electrical stimulation of the ventral TMN. Microinjection of cetirizine into the NTS partially inhibited the pressor response but had no effect on HR. Finally, the treadmill test was associated with a high level of c‐Fos expression in both ventral TMN and NTS neurons. These results suggest that the TMN–NTS pathway is involved in regulation of AP, presumably under a high‐arousal phase, such as that during exercise.

Leptin in the Hindbrain Facilitates Phosphorylation of STAT3 in the Hypothalamus

Leptin receptors (ObRb) in the forebrain and hindbrain are each independently recognized as important mediators of leptin‐induced changes in energy balance. We recently used sub‐threshold doses of leptin to show that chronic activation of both hypothalamic and brainstem ObRb is required to reduce body fat. The objective of this study was to identify selectively activated brain nuclei in rats receiving chronic infusions of sub‐threshold leptin in both forebrain and hindbrain. Male Sprague Dawley rats were fitted with 3rd and 4th ventricle cannulas. Alzet pumps were used to infuse either saline (S) or leptin (L) (0.1 µg/24h in 3rd, 0.6 µg/24h in 4th) for 6 days, resulting in four treatment groups SS, SL, LS, LL (3th‐4th). There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Double ventricle infused rats (LL) showed a 50% inhibition of food intake, 30% loss in body fat, and an increase in ObRb activation marker phosphorylated STAT3 (pSTAT3‐ir) in the arcuate nucleus (Arc), ventromedial (VMH), dorsomedial (DMH) and posterior hypothalamus compared to other groups. No differences in pSTAT3‐ir were observed in midbrain or brainstem nuclei despite a 6 fold higher infusion of leptin into the 4th ventricle than the 3rd. Delta Fos B‐ir, a chronic neuronal activation marker, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the Arc, VMH, DMH and ventral tuberomammillary nucleus showed a significant increase in LL rats compared to other groups. These data imply that the simultaneous activation of both forebrain and hindbrain ObRb is required to reduce body fat and that leptin in the hindbrain facilitates phosphorylation of STAT3 in hypothalamic areas involved in the control of energy balance.

Hypothalamic and basal ganglia projections to the posterior thalamus: Possible role in modulation of migraine headache and photophobia

Migraine attacks are typically described as unilateral, throbbing pain that is usually accompanied by nausea, vomiting, and exaggerated sensitivities to light, noise and smell. The headache phase of a migraine attack is mediated by activation of the trigeminovascular pathway; a nociceptive pathway that originates in the meninges and carries pain signals through meningeal nociceptors to the spinal trigeminal nucleus and from there to the cortex through relay neurons in the thalamus. Recent studies in our lab have identified a population of trigeminovascular neurons in the posterior (Po) and lateral posterior (LP) thalamic nuclei that may be involved in the perception of whole-body allodynia (abnormal skin sensitivity) and photophobia (abnormal sensitivity to light) during migraine. The purpose of the current study was to identify sub-cortical areas that are in position to directly regulate the activity of these thalamic trigeminovascular neurons. Such process begins with anatomical mapping of neuronal projections to the posterior thalamus of the rat by performing discrete injections of the retrograde tracer Fluorogold into the Po/LP region. Such injections yielded retrogradely labeled neurons in the nucleus of the diagonal band of Broca, the dopaminergic cells group A11/A13, the ventromedial and ventral tuberomammillary nuclei of the hypothalamus. We also found that some of these neurons contain acetylcholine, dopamine, cholecystokinin and histamine, respectively. Accordingly, we speculate that these forebrain/hypothalamic projections to Po and LP may play a role in those migraine attacks triggered by disrupted sleep, skipping meals and emotional reactions.

Neuropeptide S promotes wakefulness through activation of the posterior hypothalamic histaminergic and orexinergic neurons

In spite of the initial and pivotal findings that the newly identified neuropeptide S (NPS) promotes arousal associated with locomotor and anxiolytic-like effects, the mechanisms through which NPS acts to modulate sleep-waking states remain unclear. The present study was undertaken to investigate in the rat the effects of i.c.v. injection of NPS on the EEG, sleep-wake cycle, and brain c-Fos expression. NPS at 0.1 and 1 nmol increased significantly wakefulness (W) during the first 2 h (54.7±3.2 and 64.9±2.1 min, respectively, vs. 41.4±2.5 min seen with saline injections, P<0.01 and P<0.001), accompanied by an increase in EEG high frequency activities (14.5–60 Hz). In the meanwhile, slow wave sleep (SWS) and paradoxical sleep (PS) decreased significantly. Ex-vivo Fos immunohistochemistry in the posterior hypothalamus revealed that, as compared with saline-treated rats, NPS enhanced c-Fos expression in histaminergic neurons by 76.0% in the ventral tuberomammillary nucleus (TMN) and 57.8% in the dorsal TMN, and in orexinergic neurons by 28.2% in the perifornical nucleus (PeF), 24.3% in the dorsomedial hypothalamic nucleus (DMH), and 13.7% in the lateral hypothalamic area (LH) of the posterior hypothalamus. The NPS-induced c-Fos expression in histaminergic neurons and orexinergic neurons where NPS receptor (NPSR) mRNA is highly expressed, suggests that NPS activates histaminergic and orexinergic neurons to promote W.

Ascending caudal medullary catecholamine pathways drive sickness-induced deficits in exploratory behavior: Brain substrates for fatigue?

Immune challenges can lead to marked behavioral changes, including fatigue, reduced social interest, anorexia, and somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. Part of the neurocircuitry influencing behavior associated with illness likely includes viscerosensory nuclei located in the caudal brainstem, based on findings that inactivation of the dorsal vagal complex (DVC) can prevent social withdrawal. These brainstem nuclei contribute multiple neuronal projections that target different components of autonomic and stress-related neurocircuitry. In particular, catecholaminergic neurons in the ventrolateral medulla (VLM) and DVC target the hypothalamus and drive neuroendocrine responses to immune challenge, but their particular role in sickness behavior is not known. To test whether this catecholamine pathway also mediates sickness behavior, we compared effects of DVC inactivation with targeted lesion of the catecholamine pathway on exploratory behavior, which provides an index of motivation and fatigue, and associated patterns of brain activation assessed by immunohistochemical detection of c-Fos protein. LPS treatment dramatically reduced exploratory behavior, and produced a pattern of increased c-Fos expression in brain regions associated with stress and autonomic adjustments paraventricular hypothalamus (PVN), bed nucleus of the stria terminalis (BST), central amygdala (CEA), whereas activation was reduced in regions involved in exploratory behavior (hippocampus, dorsal striatum, ventral tuberomammillary nucleus, and ventral tegmental area). Both DVC inactivation and catecholamine lesion prevented reductions in exploratory behavior and completely blocked the inhibitory LPS effects on c-Fos expression in the behavior-associated regions. In contrast, LPS-induced activation in the CEA and BST was inhibited by DVC inactivation but not by catecholamine lesion. The findings support the idea that parallel pathways from immune-sensory caudal brainstem sources target distinct populations of forebrain neurons that likely mediate different aspects of sickness. The caudal medullary catecholaminergic projections to the hypothalamus may significantly contribute to brain mechanisms that induce behavioral “fatigue” in the context of physiological stressors.

Sleep-waking discharge of ventral tuberomammillary neurons in wild-type and histidine decarboxylase knock-out mice

Using extracellular single-unit recordings, we have determined the characteristics of neurons in the ventral tuberomammillary nucleus (VTM) of wild-type (WT) and histidine decarboxylase knock-out (HDC-KO) mice during the sleep-waking cycle. The VTM neurons of HDC-KO mice showed no histamine immunoreactivity, but were immunoreactive for the histaminergic (HA) neuron markers adenosine deaminase and glutamic acid decarboxylase 67. In the VTM of WT mice, we found waking (W)-specific, non-W-specific W-active, sleep-active, W and paradoxical sleep (PS)-active, and state-indifferent neuron groups. We previously demonstrated in WT mice that only W-specific neurons are histaminergic and that they are characterized by a triphasic broad action potential. In the VTM of HDC-KO mice, we found all these groups of state-dependent and state-indifferent neurons, including W-specific neurons that were characterized by a triphasic broad action potential and a W-specific slow tonic discharge, as in WT mice. The W-specific neurons ceased firing before the onset of electroencephalogram (EEG) synchronization, the first EEG sign of sleep, and remained silent during both slow-wave sleep (SWS) and PS. At the transition from SWS to W, they discharged after the onset of EEG activation, the first EEG sign of W. They either responded to an arousing stimulus with a long delay or did not respond. They therefore presented exactly the same characteristics as those seen in the VTM of WT mice. Thus VTM neurons deprived of their natural transmitter histamine still exhibit the firing properties of W-specific HA neurons.

The natriuretic effect of oxytocin blocks medial tuberomammillary polydipsia and polyuria in male rats

Lesions of the tuberomammillary complex, a neuroanatomical system closely related to the hypothalamic supraoptic and paraventricular nuclei, induce strong polydipsia in male rats. It was recently demonstrated that this increase in water intake is immediate, persistent, follows circadian rhythms and appears to be related to sodium regulation. The present study found that urine osmolality was significantly lower in tuberomammillary-lesioned animals vs. their respective controls at 8:00 h after surgery. Therefore, the aim of the present study was to examine the natriuretic effect of intraperitoneal oxytocin (OT) administration on medial ventral tuberomammillary nucleus (E3) polydipsia and polyuria of lesioned and control male rats. At 24:00 h post-lesion, OT blocked the hyperdipsic and polyuric responses of E3-lesioned animals but not those of non-lesioned controls, which did however significantly increase their water intake. Moreover, urinary osmolality and sodium excretion increased in E3 -lesioned animals that received OT but not in lesioned controls receiving physiological saline (992 +/- 187.19 vs. 215.83 +/- 23.39 mOsm/kg; 1.68 +/- 0.13 vs. 0.47 +/- 0.1 mEq/L). At 48:00 h post-lesion, OT administration also induced a higher intake of water and of simultaneously offered hypertonic NaCl (1.5%) in E3-lesioned animals. These results are interpreted in terms of the hypothalamic systems involved in sodium and water homeostasis.

Lipopolysaccharide suppresses activation of the tuberomammillary histaminergic system concomitant with behavior: A novel target of immune-sensory pathways

Infection and inflammation strongly inhibit a variety of behaviors, including exploration, social interaction, and food intake. The mechanisms that underlie sickness behavior remain elusive, but appear to involve fatigue and a state of hypo-arousal. Because histaminergic neurons in the ventral tuberomammillary nucleus of the hypothalamus (VTM) play a crucial role in the mediation of alertness and behavioral arousal, we investigated whether the histaminergic system represents a target for immune activation and, if so, whether modulation by ascending medullary immune-sensitive projections represents a possible mechanism. Rats were injected intraperitoneally with either the pro-inflammatory stimulus lipopolysaccharide (LPS) or saline, and exposed to one of various behavioral tests that would induce motivated behavior (exploration, play behavior, social interaction, sweetened milk consumption). Upon kill, brains were processed for c-Fos and histidine decarboxylase immunoreactivity. LPS treatment reduced behavioral activity and blocked behavioral test-associated c-Fos induction in histaminergic neurons of the VTM. These effects of LPS were prevented by prior inactivation of the caudal medullary dorsal vagal complex (DVC) with a local anesthetic. To determine whether LPS-responsive brainstem projection neurons might provide a link from the DVC to the VTM, the tracer Fluorogold was iontophoresed into the VTM a week prior to experiment. Retrogradely labeled neurons that expressed c-Fos in response to LPS treatment included catecholaminergic neurons within the nucleus of the solitary tract and ventrolateral medulla. These findings support the hypothesis that the histaminergic system represents an important component in the neurocircuitry relevant for sickness behavior that is linked to ascending pathways originating in the lower brainstem.

Hyperphagia and increased body weight induced by lesions of the ventral tuberomammillary system

Individual lesions of the caudal (E1) or rostral (E2) ventral tuberomammillary nuclei induced hyperphagia in Wistar rats. The aim of the present study was to examine the additive or united effects of combined lesions of E1 + E2 in food intake. E1 + E2 lesions produced an immediate, potent and permanent hyperphagic response that was greater than that observed in groups with individual lesions (E1 or E2) during the 22-day observation period. Furthermore, at the end of this period (Days 17/22), the combined lesions induced a greater increase in body weight compared with groups with individual lesions. As previously reported, polydipsia is also induced by lesions of the ventral tuberomammillary subnuclei. However, in this case, responses of the individual polydipsic groups did not differ from those found in the E1 + E2 ventral tuberomammillary group. These results are interpreted in terms of the hypothalamic systems involved in food and water intake.

Lesions of tuberomammillary nuclei induce differential polydipsic and hyperphagic effects

This study aimed to examine the function of the tuberomammillary complex in water and food intake of Wistar rats. The results show that lesions restricted to tuberomammillary subnuclei: caudal ventral tuberomammillary nucleus (E1), rostral ventral tuberomammillary nucleus (E2), medial ventral tuberomammillary nucleus (E3) or medial dorsal tuberomammillary nucleus (E4), induce a strong and persistent polydipsia with specific characteristics for each nucleus. Interestingly, the distribution of tuberomammillary hyperdipsia throughout the day was similar to that in non-lesioned animals, in contrast to the lack of rhythmicity observed in rats with anodic lesion to median eminence. This polydipsia appears to be independent of food intake, as food deprivation for 22 h did not significantly reduce the water intake. Finally, lesions in ventral tuberomammillary nuclei E1 and E2 induce hyperphagia, confirming a possible role for the tuberomammillary complex in food intake. This increase in food intake is not observed after lesions in medial subnuclei E3 and E4. These results are interpreted in terms of the hypothalamic systems involved in the consumption of both food and water.

The premammillary hypothalamic area of the ewe: anatomical characterization of a melatonin target area mediating seasonal reproduction.

Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor alpha, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

Single cns neurons link both central motor and cardiosympathetic systems: a double-virus tracing study

Two anatomical experiments were performed to test the hypothesis that single CNS neurons link the central areas that regulate the somatomotor and sympathetic systems. First, the retrograde neuronal tracer cholera toxin β-subunit was injected into the lateral parafascicular thalamic nucleus, a region that projects to both the motor cortex and striatum. Several days later, a second injection of the retrograde transneuronal tracer, pseudorabies virus (PRV), was made in the same rats in the stellate ganglion, which provides the main sympathetic supply to the heart. Using immunohistochemical methods, we demonstrate that the cholinergic neurons of the pedunculopontine tegmental nucleus (PPN) are connected to both systems. The second experiment used two isogenic strains of Bartha PRV as double transneuronal tracers. One virus contained the unique gene for green fluorescent protein (GFP) and the other had the unique gene for β-galactosidase (β-gal). GFP-PRV was injected in the stellate ganglion and β-gal-PRV was injected into the primary motor cortex. Double-labeled neurons were found in the lateral hypothalamic area (50% contained orexin) and PPN (approximately 95% were cholinergic). Other double-labeled neurons were identified in the deep temporal lobe (viz., amygdalohippocampal zone and lateral entorhinal cortex), posterior hypothalamus, ventral tuberomammillary nucleus, locus coeruleus, laterodorsal tegmental nucleus, periaqueductal gray matter, dorsal raphe nucleus, and nucleus tractus solitarius. These results suggest these putative command neurons integrate the somatomotor and cardiosympathetic functions and may affect different behaviors (viz., arousal, sleep, and/or locomotion).

Orexins (hypocretins) directly excite tuberomammillary neurons.

Wakefulness has recently been shown to depend upon the newly identified orexin (or hypocretin) neuropeptides by the findings that alteration in their precursor protein, their receptors or the neurons that produce them leads to the sleep disorder narcolepsy in both animals and humans. The questions of how and where these brain peptides act to maintain wakefulness remain unresolved. The purpose of the present study was to determine whether the orexins could directly affect hypothalamic histaminergic neurons, which are known to contribute to the state of wakefulness by their diffuse projections through the brain. Using brain slices, we recorded in the ventral tuberomammillary nuclei from neurons identified as histaminergic on the basis of their previously described morphological and electrophysiological characteristics and found that they were depolarized and excited by the orexins through a direct postsynaptic action. We then compared the depolarizing effect of orexin A and B and found that they were equally effective upon these cells. This latter finding suggests that the effect of orexins is mediated by orexin type 2 receptors, which are those lacking in narcoleptic dogs. Our results therefore show that the histaminergic neurons of the tuberomammillary nuclei represent an important target for the orexin system in the maintenance of wakefulness.

Rhythms in Fos expression in brain areas related to the sleep-wake cycle in the diurnal Arvicanthis niloticus.

Most mammals show daily rhythms in sleep and wakefulness controlled by the primary circadian pacemaker, the suprachiasmatic nucleus (SCN). Regardless of whether a species is diurnal or nocturnal, neural activity in the SCN and expression of the immediate-early gene product Fos increases during the light phase of the cycle. This study investigated daily patterns of Fos expression in brain areas outside the SCN in the diurnal rodent Arvicanthis niloticus. We specifically focused on regions related to sleep and arousal in animals kept on a 12:12-h light-dark cycle and killed at 1 and 5 h after both lights-on and lights-off. The ventrolateral preoptic area (VLPO), which contained cells immunopositive for galanin, showed a rhythm in Fos expression with a peak at zeitgeber time (ZT) 17 (with lights-on at ZT 0). Fos expression in the paraventricular thalamic nucleus (PVT) increased during the morning (ZT 1) but not the evening activity peak of these animals. No rhythm in Fos expression was found in the centromedial thalamic nucleus (CMT), but Fos expression in the CMT and PVT was positively correlated. A rhythm in Fos expression in the ventral tuberomammillary nucleus (VTM) was 180 degrees out of phase with the rhythm in the VLPO. Furthermore, Fos production in histamine-immunoreactive neurons of the VTM cells increased at the light-dark transitions when A. niloticus show peaks of activity. The difference in the timing of the sleep-wake cycle in diurnal and nocturnal mammals may be due to changes in the daily pattern of activity in brain regions important in sleep and wakefulness such as the VLPO and the VTM.