Melanin-concentrating hormone (MCH) modulates the activity of dorsal raphe neurons.

Hypothalamic neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized in the postero-lateral hypothalamus and incerto-hypothalamic area. These neurons send dense projections to the dorsal raphe nucleus (DRN). Serotonergic neurons of the DRN are involved in the control of sleep and play a critical role in major depression. Previously, we demonstrated that microinjections of MCH into the DRN resulted in an increase in REM sleep and produce a depressive-like effect. In the present study we examined the mechanisms that mediate these effects by employing neuroanatomical and electrophysiological techniques. First, we determined that rhodamine-labeled MCH (R-MCH), when microinjected into the lateral ventricle, is internalized in serotonergic and non-serotonergic DRN neurons in rats and cats. These data strongly suggest that these neurons express MCHergic receptors. Second, in rats, we demonstrated that the microinjection of MCH into the lateral ventricle results in a significant decrease in the firing rate in 59% of the neurons recorded in the DRN; the juxtacellular administration of MCH reduced the discharge in 80% of these neurons. Some of the neurons affected by MCH were likely serotonergic on the basis of their electrophysiological and pharmacological properties. We conclude that MCH reduces the activity of serotonergic neurons of the DRN. These and previous data suggest that the MCHergic modulation of serotonergic activity within the DRN is involved in the regulation of REM sleep as well as in the pathophysiology of depressive disorders.

Hypocretinergic and non-hypocretinergic projections from the hypothalamus to the REM sleep executive area of the pons.

Within the postero-lateral hypothalamus neurons that utilize hypocretin or melanin-concentrating hormone (MCH) as neuromodulators are co-distributed. These neurons have been involved in the control of behavioral states, and a deficit in the hypocretinergic system is the pathogenic basis of narcolepsy with cataplexy. In this report, utilizing immunohistochemistry and retrograde tracing techniques, we examined the hypocretinergic innervation of the nucleus pontis oralis (NPO), which is the executive site that is responsible for the generation of REM sleep in the cat. The retrograde tracer cholera toxin subunit b (CTb) was administered in pontine regions where carbachol microinjections induced REM sleep. Utilizing immunohistochemical techniques, we found that approximately 1% of hypocretinergic neurons in the tuberal area of the hypothalamus project to the NPO. In addition, approximately 6% of all CTb+ neurons in this region were hypocretinergic. The hypocretinergic innervation of the NPO was also compared with the innervation of the same site by MCH-containing neurons. More than three times as many MCHergic neurons were found to project to the NPO compared with hypocretinergic cells; both neuronal types exhibited bilateral projections. We also identified a group of non-hypocretinergic non-MCHergic neuronal group of neurons that were intermingled with both hypocretinergic and MCHergic neurons that also projected to this same brainstem region. These neurons were grater in number that either hypocretin or MCH-containing neurons; their soma size was also smaller and their projections were mainly ipsilateral. The present anatomical data suggest that hypocretinergic, MCHergic and an unidentified companion group of neurons of the postero-lateral hypothalamus participate in the regulation of the neuronal activity of NPO neurons, and therefore, are likely to participate in the control of wakefulness and REM sleep.

The amygdala and the pedunculopontine tegmental nucleus: interactions controlling active (rapid eye movement) sleep.

There is a consensus that active sleep (AS; i.e., REM sleep) is produced by cholinergic projections from the pedunculopontine tegmental nuclei (PPT) that activate AS-on neurons in the nucleus pontis oralis (NPO) that are components of the AS-Generator. However, there is a growing body of evidence indicating that other sites, such as the amygdala, also participate in the control of AS by inducing the discharge of AS-Generator neurons. In this regard, we recently reported that there are direct, excitatory (glutamatergic) projections from the central nucleus of the amygdala (CNA) to presumptive AS-Generator neurons in the NPO. We therefore hypothesized that the CNA and the PPT act alone, as well as in concert, to promote AS. To test this hypothesis, the effects of stimulation of the CNA and the PPT on the activity of NPO neurons, recorded intracellularly, were examined in urethane-anesthetized rats. Stimulation of either the CNA or the PPT evoked short-latency excitatory postsynaptic potentials (EPSPs) in the same neurons within the NPO. The amplitude of PPT-evoked EPSPs that were recorded from NPO neurons increased by 20.1 to 58.6% when stimulation of the PPT was preceded by stimulation of the CNA at an interval of 0 to 12 ms: maximal potentiation occurred at an interval of 4 to 6 ms. Concurrent subthreshold stimulation of the CNA and the PPT resulted in the discharge of NPO neurons. NPO neurons that were activated following CNA and/or PPT stimulation were identified morphologically and found to be multipolar with diameters >20 µm; similar neurons in the same NPO site have been previously identified as AS-Generator neurons. The present data demonstrate the presence of converging excitatory synaptic inputs from the CNA and the PPT that are capable of promoting the discharge of AS-Generator neurons in the NPO. Therefore, we suggest that the occurrence of AS depends upon interactions between cholinergic projections from the PPT and glutamatergic projections from the CNA as well as inputs from other sites that project to AS-Generator neurons.

Apnea produces excitotoxic hippocampal synapses and neuronal apoptosis.

Obstructive sleep apnea (OSA) results in the degeneration of neurons in the hippocampus that eventuates in neurocognitive deficits. We were therefore interested in determining the effects of apnea on monosynaptic excitatory processes in a hippocampal pathway (cornu ammonis 3-cornu ammonis 1, CA3-CA1) that has been shown to mediate the processing of cognitive information. In addition, to substantiate an anatomical basis for the cognitive dysfunction that occurs in OSA patients, we examined the effects of apnea with respect to neurodegenerative changes (apoptosis) in the same hippocampal pathway. In order to determine the effects of apnea, an automated system for the generation and analysis of single and recurrent periods of apnea was developed. Utilizing this system, the field excitatory postsynaptic potential (fEPSP) generated by pyramidal neurons in the CA1 region of the hippocampus was monitored in α-chloralose anesthetized rats following stimulation of glutamatergic afferents in the CA3 region. A stimulus-response (input-output) curve for CA3-CA1 synaptic activity was determined. In addition, a paired-pulse paradigm was employed to evaluate, electrophysiologically, the presynaptic release of glutamate. Changes in the synaptic efficacy were assessed following single episodes of apnea induced by ventilatory arrest (60 to 80 s duration, mean=72 s; mean oxygen desaturation was 53% of normoxia level). Apnea resulted in a significant potentiation of the amplitude (mean=126%) and slope (mean=117%) of the baseline CA1 fEPSP. This increase in the fEPSP was accompanied by a significant decrease in the amplitude (71%) and slope (81%) of normalized paired-pulse facilitation (PPF) ratios. Since the potentiation of the fEPSP is inversely proportional to changes in PPF ratio, the potentiated fEPSP accompanied by the reduced PPF reveals that apnea produces an abnormal increase in the preterminal release of glutamate that results in the over-activation (and calcium overloading) of hippocampal CA1 neurons. Thus, we conclude that individual episodes of apnea result in the development of excitotoxic processes in the hippocampal CA3-CA1 pathway that is critically involved in the processing of cognitive information. Morphologically, the deleterious effect of recurrent apnea was substantiated by the finding of apoptosis in CA1 neurons of apneic (but not normoxic) animals.

Projections from the central nucleus of the amygdala to the nucleus pontis oralis in the rat: an anterograde labeling study.

The present study was designed to elucidate the neuronal projections from the amygdala to the nucleus pontis oralis (NPO). We propose that glutamatergic cells in the central nucleus of the amygdala (CNA) activate neurons in the NPO, which is the critical brainstem site that is responsible for the generation and maintenance of active (REM) sleep. Phaseolus vulgaris-leucoagglutinin (PHA-L), an anterograde transported neuronal tracer, was iontophoresed into the CNA of adult male Sprague-Dawley rats. After a survival time of 7-8 days, the animals were perfused with a fixative and brain tissue was prepared for histological analysis. Sections of the NPO and CNA, which were immunostained with an antibody against PHA-L, were examined with light microscopy. In addition, in order to identify the phenotype of PHA-L-labeled fibers and terminals in the NPO, a double immunohistochemical technique was employed with antibodies against PHA-L and the vesicular glutamate transporter type 2 (VGluT2). Numerous PHA-L-labeled axons and terminals were found in the NPO ipsilateral to the injection site in the CNA. Within the NPO, the majority of labeled fibers were located in the dorsolateral portion of the caudal part of the nucleus. Double-labeling immunostaining studies revealed that PHA-L-labeled axons and terminals in the NPO were glutamatergic. The present demonstration of direct, excitatory (glutamatergic) projections from the CNA to the NPO provide an anatomical basis for the amygdalar control of active sleep.

Hypocretinergic neurons are activated in conjunction with goal-oriented survival-related motor behaviors.

Hypocretinergic neurons are located in the area of the lateral hypothalamus which is responsible for mediating goal-directed, survival-related behaviors. Consequently, we hypothesize that the hypocretinergic system functions to promote these behaviors including those patterns of somatomotor activation upon which they are based. Further, we hypothesize that the hypocretinergic system is not involved with repetitive motor activities unless they occur in conjunction with the goal-oriented behaviors that are governed by the lateral hypothalamus. In order to determine the veracity of these hypotheses, we examined Fos immunoreactivity (as a marker of neuronal activity) in hypocretinergic neurons in the cat during: a) Exploratory Motor Activity; b) Locomotion without Reward; c) Locomotion with Reward; and d) Wakefulness without Motor Activity. Significantly greater numbers of hypocretinergic neurons expressed c-fos when the animals were exploring an unknown environment during Exploratory Motor Activity compared with all other paradigms. In addition, a larger number of Hcrt+Fos+neurons were activated during Locomotion with Reward than during Wakefulness without Motor Activity. Finally, very few hypocretinergic neurons were activated during Locomotion without Reward and Wakefulness without Motor Activity, wherein there was an absence of goal-directed activities. We conclude that the hypocretinergic system does not promote wakefulness per se or motor activity per se but is responsible for mediating specific goal-oriented behaviors that take place during wakefulness. Accordingly, we suggest that the hypocretinergic system is responsible for controlling the somatomotor system and coordinating its activity with other systems in order to produce successful goal-oriented survival-related behaviors that are controlled by the lateral hypothalamus.

The role of mesopontine NGF in sleep and wakefulness.

The microinjection of nerve growth factor (NGF) into the cat pontine tegmentum rapidly induces rapid eye movement (REM) sleep. To determine if NGF is involved in naturally-occurring REM sleep, we examined whether it is present in mesopontine cholinergic structures that promote the initiation of REM sleep, and whether the blockade of NGF production in these structures suppresses REM sleep. We found that cholinergic neurons in the cat dorso-lateral mesopontine tegmentum exhibited NGF-like immunoreactivity. In addition, the microinjection of an oligodeoxyribonucleotide (OD) directed against cat NGF mRNA into this region resulted in a reduction in the time spent in REM sleep in conjunction with an increase in the time spent in wakefulness. Sleep and wakefulness returned to baseline conditions 2 to 5 days after antisense OD administration. The preceding antisense OD-induced effects occurred in conjunction with the suppression of NGF-like immunoreactivity within the site of antisense OD injection. These data support the hypothesis that NGF is involved in the modulation of naturally-occurring sleep and wakefulness.

Projection neurons from the central nucleus of the amygdala to the nucleus pontis oralis.

The present retrograde labeling study was designed to determine the presence and pattern of projections from individual subdivisions of the central nucleus of the amygdala (CNA) to the nucleus pontis oralis (NPO), which is a critical brainstem site involved in the generation and maintenance of active (REM) sleep. Projections from the CNA were labeled with the retrograde tracer cholera toxin B-subunit (CTB), which was injected, unilaterally, via microiontophoresis, into the NPO. Sections of the amygdala were immunostained in order to identify CTB-labeled CNA neurons and CNA neurons that contained CTB plus the vesicular glutamate transporter 2 (VGLUT2), which is a marker for glutamatergic neurons. Histological analyses revealed that retrogradely labeled neurons that project to the NPO were localized, ipsilaterally, within the medial, lateral, and capsular subdivisions of the CNA. In addition, a substantial proportion (24%) of all retrogradely labeled CNA neurons also exhibited VGLUT2 immunoreactivity. The present study demonstrates that glutamatergic neurons, which are present within various subdivisions of the CNA, project directly to the NPO. These data lend credence to the hypothesis that NPO neurons that are involved in the control of active sleep are activated by glutamatergic projections from the amygdala.

Prevention of apnea-induced apoptosis in NREM- and REM-generating nuclei of adult guinea pigs.

The present study was designed to investigate the effects of recurrent periods of apnea/hypoxia on the morphology of neurons in sites that control NREM and REM sleep. In addition, we determined whether the administration of a GABA agonist, eszopiclone, was capable of preventing the degenerative, i.e., apoptotic, sequelae of hypoxia in these sleep-promoting neurons. Adult guinea pigs were divided into control (normoxic) and hypoxic groups; a separate group of hypoxic animals was administered eszopiclone. Recurrent periods of hypoxia and normoxia lasted for a duration of 3h. Subsequently, the brains were sectioned, and areas in the CNS that control NREM sleep as well as REM sleep were examined after staining with an antibody raised against single-stranded DNA, which labels apoptotic neurons. In the group of control (normoxic) animals, apoptotic neurons were not observed in CNS regions that control NREM or REM sleep. In hypoxic animals, a large number of apoptotic neurons were found in the preceding regions. In the hypoxic animals that were administered eszopiclone, there were almost no apoptotic neurons in the brain regions that control NREM or REM sleep. These results demonstrate that recurrent periods of apnea induce extensive apoptosis in CNS nuclei that control NREM and REM sleep and that eszopiclone is capable of preventing neuronal degeneration in these sites. We suggest that the degeneration of neurons in sites that control the states of sleep is responsible for those sleep disturbances that arise as a consequence of hypoxia in individuals with sleep-related breathing disorders.

Apnea produces neuronal degeneration in the pons and medulla of guinea pigs.

Obstructive sleep apnea and other sleep-related breathing disorders result in recurrent periods of oxygen deprivation (hypoxia), hypercapnia and an increase in the cellular production of reactive oxygen species (oxidative stress-related injury). Individuals with these disorders suffer from a variety of cellular abnormalities that result in cardiopulmonary dysfunctions, disturbances in sleep and other pathologies. In the present experiment, using an animal model of sleep apnea, we determined that the degeneration of neurons and glia, due to apoptosis, occurs in specific regions of the pons and medulla. Adult guinea pigs, which were divided into control (normoxic) and experimental (hypoxic) groups, were anesthetized with alpha-chloralose and immobilized with Flaxedil. Apnea (hypoxia) was induced by ventilatory arrest in order to desaturate the oxyhemoglobin to 75% SpO(2). A sequence of apnea, followed by ventilation with recovery to >95% SpO(2), was repeated for a period of 3h. At the end of the period of recurrent apnea, the animals were perfused and brain sections were immunostained with a mouse monoclonal antibody raised against single-stranded DNA (ssDNA). Apoptotic neurons and glia, which were not found in the control group of animals, were present in brainstem regions in hypoxic group of animals; these regions involved in the control of respiration (e.g., the parafacial respiratory group and the ventral respiratory group), cardiovascular functions (e.g., the nucleus ambiguus, the nucleus tractus solitarius and the dorsal motor nucleus of the vagus) as well as REM sleep (the nucleus pontis oralis) and wakefulness (e.g., the dorsal raphe and locus ceruleus). We suggest apoptotic neurons and glia in critical areas of the pons and medulla results in many of the comorbidities experienced by patients with sleep-disordered breathing pathologies.

State-dependent pattern of Fos protein expression in regionally-specific sites within the preoptic area of the cat.

Clinical and experimental data have shown that the preoptic area of the hypothalamus (POA) is involved in the generation and maintenance of NREM sleep. However, the activity of specific populations of POA neurons during REM sleep, NREM sleep and different waking conditions is still not firmly established. Consequently, we performed a quantitative, regionally-specific analysis of the Fos immunoreactivity of neurons in the POA of the cat during NREM sleep and REM sleep induced by microinjections of carbachol into the nucleus pontis oralis (REMc), as well as during quiet and alert wakefulness. We observed that while the total number of Fos immunoreactive neurons in the POA did not change as a function of these behavioral states, state-specific differences in neuronal activity were detected in restricted regions of the POA. An increase in the number of Fos+ neurons was observed in the rostral tip of the suprachiasmatic nucleus (SCN) during NREM (83.4+/-25.6) compared to quiet wakefulness (5.1+/-1.3, p<0.05) but not with the other behavioral states. In the median preoptic nucleus (MnPN), the number of Fos immunoreactive neurons was greater during NREM sleep (39.5+/-6.1) compared with quiet wakefulness (13.5+/-1.4, p<0.05) and REMc (16.2+/-2.0, p<0.05). State-specific Fos immunoreactive neurons were not observed in the ventro-lateral preoptic nucleus (VLPO). Finally, there was no significant increase in the number of Fos+ neurons during REMc in any of the subregions of the POA. In conclusion, within the POA, a selective neuronal activation during NREM sleep was found only in the MnPN. In addition, our data suggest a potential role of the SCN in NREM sleep. Finally, based on the distribution of Fos+ neurons in the entire POA, we conclude that the neuronal network involved in the regulation of NREM sleep is dispersed and intermingled with waking-related neurons.

MCHergic projections to the nucleus pontis oralis participate in the control of active (REM) sleep.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are located in the lateral hypothalamus and incerto-hypothalamic area and project diffusely throughout the central nervous system, including areas that participate in the generation and maintenance of sleep and wakefulness. Recent studies have shown that hypothalamic MCHergic neurons are active during active sleep (AS), and that intraventricular microinjections of MCH induce AS sleep; however, there are no data available regarding the manner in which MCHergic neurons participate in the control of this behavioral state. Utilizing immunohistochemical and retrograde tracing techniques, we examined, in the cat, projections from MCHergic neurons to the nucleus pontis oralis (NPO), which is considered to be the executive area that is responsible for the generation and maintenance of AS. In addition, we explored the effects on sleep and waking states produced by the microinjection of MCH into the NPO. We first determined that MCHergic fibers and terminals are present in the NPO. We also found that when a retrograde tracer (cholera toxin subunit B) was placed in the NPO MCHergic neurons of the hypothalamus were labeled. When MCH was microinjected into the NPO, there was a significant increase in the amount of AS (19.8+/-1.4% versus 11.9+/-0.2%, P<0.05) and a significant decrease in the latency to AS (10.4+/-4.2 versus 26.6+/-2.3 min, P<0.05). The preceding anatomical and functional data support our hypothesis that the MCHergic system participates in the regulation of AS by modulating neuronal activity in the NPO.

Recurrent apnea induces neuronal apoptosis in the guinea pig forebrain.

Obstructive sleep apnea (OSA) and sleep-disordered breathing (SDB) can result in impaired cognition and mental acuity, and the generation of mood disorders, including depression. However, the mechanisms of neuronal damage for these complications have not been elucidated. Accordingly, using immunohistochemical technique with monoclonal antibody against single-stranded DNA, we examined the morphological effects of chronic recurrent apnea on neurons in the hippocampus and related forebrain sites in guinea pigs. Our results show that a large number of neurons labeled by anti-ssDNA antibody were present in the cingulate, insular and frontal cortices, the hippocampus and the amygdala in conjunction with periods of recurrent apnea. However, no labeling was observed in comparable regions of the brain in control guinea pigs. In the cortices of experimental animals, labeled neurons were detected mainly in the superficial layers (II-III) in the frontal, insular and cingulate cortex. In the hippocampus, most labeled neurons were located in the CA1 region, in which most of stained neurons were observed in strata pyramidal, while only a few positive neurons were located in the strata radiatum and the strata oriens. In addition, a large number of labeled neurons were also detected in the central nucleus of amygdala in the guinea pigs underwent recurrent periods of apnea. The present data indicate that recurrent apnea results in cell death in the hippocampus and related forebrain regions via mechanisms of apoptosis, which may represent the basis for the clinical complications of obstructive sleep apnea and sleep-disordered breathing.

Eszopiclone prevents excitotoxicity and neurodegeneration in the hippocampus induced by experimental apnea.

STUDY OBJECTIVE: This study was designed to determine the effects of eszopiclone on apnea-induced excitotoxic synaptic processes and apoptosis in the hippocampus. DESIGN: Recurrent periods of apnea, which consisted of a sequence of apnea (75% SpO2), followed by ventilation with recovery to normoxia (> 95% SpO2), were induced for a period of three hours in anesthetized guinea pigs. The CA3 Schaffer collateral pathway in the hippocampus was stimulated and the field excitatory postsynaptic potential (fEPSP) response was recorded in CA1. Animals in the experimental group received an intravenous injection of eszopiclone (3 mg/kg) 10 min prior to the initiation of the periods of recurrent apnea, and once every 60 min thereafter; control animals received comparable injections of vehicle. At the end of the 3-h period of recurrent apnea, the animals were perfused, and hippocampal sections were immunostained in order to determine the presence of apoptosis, i.e., programmed cell death. ANALYSES AND RESULTS: Apnea resulted in a persistent increase in synaptic responsiveness of CA1 neurons as determined by analyses of the fEPSP. Eszopiclone antagonized the apnea-induced increase in the fEPSP. Morphological analyses revealed significant apoptosis of CA1 neurons in control animals; however, there was no significant apoptosis in eszopiclone-treated animals. CONCLUSIONS: Eszopiclone was determined to suppress the apnea-induced hyperexcitability of hippocampal CA1 neurons, thereby reducing/eliminating neurotoxicity. These data lend credence to our hypothesis that eszopiclone, exclusive of its hypnotic actions, has the capacity to function as a potent neuroprotective agent.

Melanin-concentrating hormone (MCH) immunoreactivity in non-neuronal cells within the raphe nuclei and subventricular region of the brainstem of the cat.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized within the postero-lateral hypothalamus and zona incerta. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes such as energy homeostasis and sleep. In the present report, we examined the distribution of MCH immunoreactivity in the brainstem of the cat. In addition to MCH+ axons, we found MCH-immunoreactive cells that have not been previously described either in the midbrain raphe nuclei or in the periaqueductal and periventricular areas. These MCH+ cells constituted: 1. ependymal cells that lined the fourth ventricle and aqueduct, 2. ependymal cells with long basal processes that projected deeply into the subventricular (subaqueductal) parenchyma, and, 3. cells in subventricular regions and the midbrain raphe nuclei. The MCH+ cells in the midbrain raphe nuclei were closely related to neuronal processes of serotonergic neurons. Utilizing Neu-N and GFAP immunohistochemistry we determined that the preceding MCH+ cells were neither neurons nor astrocytes. However, we found that vimentin, an intermediate-filament protein that is used as a marker for tanycytes, was specifically co-localized with MCH in these cells. We conclude that MCH is present in tanycytes whose processes innervate the midbrain raphe nuclei and adjacent subependymal regions. Because tanycytes are specialized cells that transport substances from the cerebrospinal fluid (CSF) to neural parenchyma, we suggest that MCH is absorbed from the CSF by tanycytes and subsequently liberate to act upon neurons of brainstem nuclei.

Apnea promotes glutamate-induced excitotoxicity in hippocampal neurons.

Patients with obstructive sleep apnea (OSA) exhibit hippocampal damage and cognitive deficits. To determine the effect of apnea on the synaptic transmission in the hippocampus, we performed electrophysiological studies in an in vivo guinea pig model of OSA. Specifically, we determined the cornu ammonis region 1 (CA1) field excitatory postsynaptic potential (fEPSP) response to cornu ammonis region 3 (CA3) stimulation and examined the presynaptic mechanisms underlying the changes in the fEPSP. Single episodes of apnea resulted in a maximal potentiation of the fEPSPs at 1 to 3 min after the termination of each episode of apnea. The mean amplitude and slope of the post-apneic fEPSP was significantly larger compared with the pre-apneic control. These changes were accompanied by a significant decrease in the paired-pulse facilitation ratio during the post-apneic period compared with the pre-apneic control. The N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801, when applied locally to the CA1 recording site by pressure ejection, blocked the apnea-induced potentiation of the fEPSP. In the experimental animals that were subjected to extended periods of recurrent apnea, CA1 neurons exhibited positive immunoreactivity for fragmented DNA strands, which indicates apoptotic cell death. The present results demonstrate that apnea-induced potentiation of the hippocampal CA1 fEPSP is mediated by an NMDA receptor mechanism. We therefore conclude that recurrent apnea produces abnormally high levels of glutamate that results in the apoptosis of CA1 neurons. We hypothesize that this damage is reflected by the cognitive deficits that are commonly observed in patients with breathing disorders such as OSA.

MCH-containing neurons in the hypothalamus of the cat: searching for a role in the control of sleep and wakefulness.

Neurons that utilize melanin-concentrating hormone (MCH) and others that employ hypocretin as neurotransmitter are located in the hypothalamus and project diffusely throughout the CNS, including areas that participate in the generation and maintenance of the states of sleep and wakefulness. In the present report, immunohistochemical methods were employed to examine the distribution of MCHergic and hypocretinergic neurons. In order to test the hypothesis that the MCHergic system is capable of influencing specific behavioral states, we studied Fos immunoreactivity in MCH-containing neurons during (1) quiet wakefulness, (2) active wakefulness with motor activity, (3) active wakefulness without motor activity, (4) quiet sleep and (5) active sleep induced by carbachol (AS-carbachol). We determined that MCHergic neuronal somata in the cat are intermingled with hypocretinergic neurons in the dorsal and lateral hypothalamus, principally in the tuberal and tuberomammillary regions; however, hypocretinergic neurons extended more in the anterior-posterior axis than MCHergic neurons. Axosomatic and axodendritic contacts were common between these neurons. In contrast to hypocretinergic neurons, which are known to be active during motor activity and AS-carbachol, Fos immunoreactivity was not observed in MCH-containing neurons in conjunction with any of the preceding behavioral conditions. Non-MCHergic, non-hypocretinergic neurons that expressed c-fos during active wakefulness with motor activity were intermingled with MCH and hypocretin-containing neurons, suggesting that these neurons are related to some aspect of motor function. Further studies are required to elucidate the functional sequela of the interactions between MCHergic and hypocretinergic neurons and the phenotype of the other neurons that were active during motor activity.

Age-related changes in cholinergic neurons in the laterodorsal and the pedunculo-pontine tegmental nuclei of cats: a combined light and electron microscopic study.

In aged cats, light microscopic studies revealed significant decrease in the soma size of choline acetyltransferase (ChAT)-positive neurons in the laterodorsal and pedunculo-pontine tegmental nuclei (LDT and PPT), compared with adult control animals. In addition, a significant reduction of the total dendritic length and total dendritic segment number of ChAT-positive neurons was detected in both the LDT and PPT of aged cats. However, in contrast to the changes of soma and dendrites, no significant changes in the number of ChAT-positive neurons in aged were found comparing to that in the control cats in both the LDT and PPT; nor were there differences in the staining intensity of the somata of neurons in the adult and aged cats. Electron microscopic analysis highlighted degenerative changes in cholinergic neurons in the LDT and PPT of aged cats which included somata with intracytoplasmic vacuoles, darkened mitochondria, depletion of dendritic microtubules and severe demyelination of axons. These data indicate that profound atrophic changes occur in cholinergic systems of the LDT and PPT as a consequence of the aging process. These alterations likely reflect the cellular bases for the age-related changes in REM sleep that occur in old animals.

Age-related changes of hypocretin in basal forebrain of guinea pig.

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) have been implicated in a wide variety of functions including sleep and wakefulness as well as related behaviors. Many of these functions of the hypocretins involve the activation of cholinergic neurons in the basal forebrain (BF). These neurons have been shown to exhibit age-related changes in a variety of species. In the present experiment, in adult and aged guinea pigs, we compared hypocretin immunoreactivity in regions of the BF that include the medial septal nucleus (MS), the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB) and the magocellular preoptic nucleus (MCPO). In adult guinea pigs (3-5 months of age), all of the preceding BF regions contained dense hypocretin fibers with varicosities. On the contrary, in old guinea pigs (27-28 months), although the MS exhibited a similar intensity of hypocretin immunoreactivity compared with the adult guinea pig, there was a significant decrease in the intensity of immunoreactivity of hypocretinergic fibers in the VDB, HDB and MCPO. These data indicate that the hypocretinergic innervation of specific nuclei of the BF is compromised during the aging process. We suggest that the reduction in hypocretinergic innervation of the BF nuclei may contribute to the age-related changes in the states of sleep and wakefulness as well as deficits in related systems that occur in old age.

Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat.

The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d(+)/CTb(+) or nNOS(+)/ CTb(+) double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 +/- 15 double-labeled neurons on the ipsilateral side and 33 +/- 14 on the contralateral side. CTb injections into the mXII resulted in 40 +/- 17 double-labeled neurons in this region on the ipsilateral side and 16 +/- 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 +/- 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons.