Chronic Suppression of Hypothalamic Cell Proliferation and Neurogenesis Induces Aging-Like Changes in Sleep-Wake Organization in Young Mice.

Aging is associated with sleep-wake disruption, dampening of circadian amplitudes, and a reduced homeostatic sleep response. Aging is also associated with a decline in hypothalamic cell proliferation. We hypothesized that the aging-related decline in cell-proliferation contributes to the dysfunction of preoptic-hypothalamic sleep-wake and circadian systems and consequent sleep-wake disruption. We determined if cytosine-ß-D-arabinofuranoside (AraC), an antimitotic agent known to suppress hypothalamic cell proliferation and neurogenesis, causes sleep-wake instability in young mice. The sleep-wake profiles were compared during baseline, during 4 weeks of artificial cerebrospinal fluid (aCSF) + 5-bromo-2'-deoxyuridine (BrdU) or AraC+BrdU infusion into the lateral ventricle, and 8 weeks after treatments. The sleep-wake architecture after AraC treatment was further compared with sleep-wake profiles in aged mice. Compared to aCSF+BrdU, 4 weeks of AraC+BrdU infusion significantly decreased (-96%) the number of BrdU+ cells around the third ventricular wall and adjacent preoptic-hypothalamic area and produced a) sleep disruption during the light phase with decreases in non-rapid eye movement (nonREM) (-9%) and REM sleep (-21%) amounts, and increased numbers of shorter (<2 min; 142 versus 98 episodes/12 h) and decreased numbers of longer (>5 min; 19 versus 26 episodes/12 h) nonREM sleep episodes; and b) wake disruption during the dark phase, with increased numbers of shorter (138 versus 91 episodes/12 h) and decreased numbers of longer active waking (17 versus 24 episodes/12 h) episodes. AraC-treated mice also exhibited lower delta activity within nonREM recovery sleep. The sleep-wake architecture of AraC-treated mice was similar to that observed in aged mice. These findings are consistent with a hypothesis that a decrease in hypothalamic cell proliferation/neurogenesis is detrimental to sleep-wake and circadian systems and may underlie sleep-wake disturbance in aging.

Adenosine A(2A) receptors regulate the activity of sleep regulatory GABAergic neurons in the preoptic hypothalamus.

The median preoptic nucleus (MnPN) and the ventrolateral preoptic area (VLPO) are two hypothalamic regions that have been implicated in sleep regulation, and both nuclei contain sleep-active GABAergic neurons. Adenosine is an endogenous sleep regulatory substance, which promotes sleep via A1 and A2A receptors (A2AR). Infusion of A2AR agonist into the lateral ventricle or into the subarachnoid space underlying the rostral basal forebrain (SS-rBF), has been previously shown to increase sleep. We examined the effects of an A2AR agonist, CGS-21680, administered into the lateral ventricle and the SS-rBF on sleep and c-Fos protein immunoreactivity (Fos-IR) in GABAergic neurons in the MnPN and VLPO. Intracerebroventricular administration of CGS-21680 during the second half of lights-on phase increased sleep and increased the number of MnPN and VLPO GABAergic neurons expressing Fos-IR. Similar effects were found with CGS-21680 microinjection into the SS-rBF. The induction of Fos-IR in preoptic GABAergic neurons was not secondary to drug-induced sleep, since CGS-21680 delivered to the SS-rBF significantly increased Fos-IR in MnPN and VLPO neurons in animals that were not permitted to sleep. Intracerebroventricular infusion of ZM-241385, an A2AR antagonist, during the last 2 h of a 3-h period of sleep deprivation caused suppression of subsequent recovery sleep and reduced Fos-IR in MnPN and VLPO GABAergic neurons. Our findings support a hypothesis that A2AR-mediated activation of MnPN and VLPO GABAergic neurons contributes to adenosinergic regulation of sleep.

Hypothalamic versus neocortical control of sleep.

PURPOSE OF REVIEW: Regions of the neocortex most strongly activated during waking exhibit increased sleep intensity during subsequent sleep. The novel concept that aspects of sleep homeostasis are determined locally in the cortex contrasts with the established views that global changes in neocortical activity during sleep are achieved through inhibition of ascending arousal systems that originate in the brainstem and hypothalamus. RECENT FINDINGS: Experiments in animals and humans document asymmetries in neocortical electroencephalogram (EEG) slow-wave activity (SWA), a marker of homeostatic sleep need, as a result of functional activity during waking. In addition to local, use-dependent augmentation of EEG SWA and evoked potentials, expression of plasticity-related genes and of sleep-regulatory cytokines and neuromodulators have been shown to be elevated in a use-dependent manner in neocortex. The functional consequences of local sleep are hypothesized to involve regulation of synaptic plasticity, synaptic homeostasis and energy balance. SUMMARY: The evidence for use-dependent modulation of neocortical activity during sleep is compelling and provides novel insights into sleep function. However, local changes in neocortex are generally expressed on a background of global sleep. It remains to be determined if events initiated in the cortex have global sleep-promoting effects and how neocortical and hypothalamic mechanisms of sleep control interact.

Growth hormone-releasing hormone activates sleep regulatory neurons of the rat preoptic hypothalamus.

We examined whether growth hormone-releasing hormone (GHRH) may promote non-rapid eye movement (NREM) sleep via activation of GABAergic neurons in the preoptic area. Male Sprague-Dawley rats were implanted with EEG, EMG electrodes and a unilateral intracerebroventricular cannula. Groups of rats received injections (3 microl icv) with gonadotropin-releasing hormone (GHRH) (0.1 nmol/100 g body wt) or equal volume of physiological saline at the onset of the dark period and were permitted spontaneous sleep for 90 min. Separate groups of rats were sleep deprived by gentle handling for 90 min, beginning at the time of GHRH or saline injection, at the onset of the dark period. Other groups of rats received intracerebroventricular octreotide (somatostatin analog OCT) injections, intracerebroventricular injection of one of two doses of competitive GHRH antagonist, or intracerebroventricular saline injection at light onset and were then permitted 90 min spontaneous sleep-waking. Rats were killed immediately after the 90-min sleep/wake monitoring period. Brain tissue was processed for immunohistochemistry for c-Fos protein and glutamic acid decarboxylase (GAD). Single c-Fos and dual Fos-GAD cell counts were determined in the median preoptic nucleus (MnPN), and in the core and the extended parts of the ventrolateral preoptic nucleus (cVLPO and exVLPO). Intracerebroventricular GHRH elicited a significant increase in NREM sleep amount. Double-labeled Fos+GAD cell counts were significantly elevated after GHRH injection in the MnPN and VLPO in both undisturbed and sleep-deprived groups. OCT and GHRH antagonist significantly decreased NREM sleep amount compared with control rats. OCT injection increased single c-Fos-labeled cell counts in the MnPN, but not in the VLPO. Double-labeled cell counts were significantly reduced after OCT and the high dose of GHRH antagonist injection in all areas examined. These findings identify GABAergic neurons in the MnPN and VLPO as potential targets of the sleep-regulatory actions of GHRH.

Functional correlates of activity in neurons projecting from the lamina terminalis to the ventrolateral periaqueductal gray.

The lamina terminalis (LT) consists of the organum vasculosum of the LT (OVLT), the median preoptic nucleus (MnPO) and the subfornical organ (SFO). All subdivisions of the LT project to the ventrolateral periaqueductal gray (vlPAG). The LT and the vlPAG are implicated in several homeostatic and behavioral functions, including body fluid homeostasis, thermoregulation and the regulation of sleep and waking. By combining visualization of c-Fos protein and retrograde neuroanatomical tracer we have examined the functional correlates of LT-vlPAG projection neurons. Rats were injected with retrograde tracer into the vlPAG and, following a 1-week recovery period, they were subjected to either hypertonic saline administration (0.5 M NaCl, 1 mL/100 g i.p.), 24-h water deprivation, isoproterenol administration (increases circulating angiotensin II; 50 microg/kg s.c.), heat exposure (39 degrees C for 60 min) or permitted 180 min spontaneous sleep. Retrogradely labeled neurons from the vlPAG and double-labelled neurons were then identified and quantified throughout the LT. OVLT-vlPAG projection neurons were most responsive to hypertonic saline and water deprivation. SFO-vlPAG projection neurons were most active following isoproterenol administration, and MnPO-vlPAG projection neurons displayed significantly more Fos immunostaining following water deprivation, heat exposure and sleep. These results support the existence of functional subdivisions of LT-vlPAG-projecting neurons, and indicate three patterns of activity that correspond to thermal and sleep wake regulation, osmotic or hormonal stimuli.

Hypothalamic regulation of sleep and arousal.

Normal waking is associated with neuronal activity in several chemically defined ascending arousal systems. These include monoaminergic neurons in the brainstem and posterior hypothalamus, cholinergic neurons in the brainstem and basal forebrain, and hypocretin (orexin) neurons in the lateral hypothalamus. Collectively, these systems impart tonic activation to their neuronal targets in the diencephalon and neocortex that is reflected in the low-voltage fast-frequency electroencephalogram patterns of wakefulness. Neuronal discharge in these arousal systems declines rapidly at sleep onset. Transitions from waking to sleep, therefore, involve coordinated inhibition of multiple arousal systems. An important source of sleep-related inhibition of arousal arises from neurons located in the preoptic hypothalamus. These preoptic neurons are strongly activated during sleep, exhibiting sleep/waking state-dependent discharge patterns that are the reciprocal of that observed in the arousal systems. The majority of preoptic sleep regulatory neurons synthesize the inhibitory neurotransmitter GABA. Anatomical and functional evidence supports the hypothesis that GABAergic neurons in the median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) exert inhibitory control over the monoaminergic systems and the hypocretin system during sleep. Recent findings indicate that MnPN and VLPO neurons integrate homeostatic aspects of sleep regulation and are important targets for endogenous sleep factors, such as adenosine and growth hormone releasing hormone.

Hypothalamic control of sleep.

A sleep-promoting function for the rostral hypothalamus was initially inferred from the presence of chronic insomnia following damage to this brain region. Subsequently, it was determined that a unique feature of the preoptic hypothalamus and adjacent basal forebrain is the presence of neurons that are activated during sleep compared to waking. Preoptic area "sleep-active" neurons have been identified by single and multiple-unit recordings and by the presence of the protein product of the c-Fos gene in the neurons of sleeping animals. Sleep-active neurons are located in several subregions of the preoptic area, occurring with high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Neurons in the vlPOA contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, GABA. A majority of MnPN neurons activated during sleep contain GABA. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Cumulative evidence indicates that preoptic area neurons function to promote sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent studies suggest a role for preoptic area neurons in the homeostatic aspects of the regulation of both rapid eye movement (REM) and non-REM (NREM) sleep and as a potential target for endogenous somnongens, such as cytokines and adenosine.

Hypothalamic regulation of sleep and arousal.

The hypnogenic function of the rostral hypothalamic region, particularly the preoptic area (POA) was established previously on the basis of lesion, neuronal unit recording, and neurochemical and thermal stimulation studies. Recent studies have mapped the locations of putative sleep-promoting neurons in the POA using c-Fos immunostaining techniques and confirmed these findings with electrophysiological methods. Segregated groups of sleep-active neurons have been localized in the ventrolateral POA (vlPOA) and median preoptic nucleus (MnPN). MnPN and vlPOA sleep-active neurons express the inhibitory transmitter, GABA. In vlPOA neurons, GABA is co-localized with a second inhibitory transmitter, galanin. Descending projections from these sites terminate in putative arousal-promoting cell groups, including histaminergic, serotonergic, orexinergic, noradrenergic, and cholinergic neurons. These findings suggest the hypothesis that non-REM sleep occurs as a consequence of GABAergic and galaninergic inhibition of arousal-promoting neurons resulting from activation of vlPOA and MnPN sleep-promoting neurons. In support of this hypothesis, it was shown that putative sleep-promoting and arousal-promoting neurons exhibit reciprocal changes in discharge across the sleep-wake cycle and that GABA release in wake-promoting sites increases during nonREM sleep. In addition, some POA sleep-active neurons are warm-sensitive. Local POA warming inhibits discharge of multiple arousal-promoting neuronal groups. POA warming, unit recording, and lesion studies also show that POA regulates the amount of delta EEG activity within nonREM sleep, and index of the depth of sleep. Finally, there is evidence that arousal systems inhibit vlPOA and MnPN neurons and the POA hypnogenic mechanism. Mutually-inhibitory interactions between sleep-promoting and arousal-promoting systems are hypothesized to form a functional sleep-wake switch.