The development of hypocretin (orexin) deficiency in hypocretin/ataxin-3 transgenic rats.

Narcolepsy is linked to a widespread loss of neurons containing the neuropeptide hypocretin (HCRT), also named orexin. A transgenic (TG) rat model has been developed to mimic the neuronal loss found in narcoleptic humans. In these rats, HCRT neurons gradually die as a result of the expression of a poly-glutamine repeat under the control of the HCRT promoter. To better characterize the changes in HCRT-1 levels in response to the gradual HCRT neuronal loss cerebrospinal fluid (CSF) HCRT-1 levels were measured in various age groups (2-82 weeks) of wild-type (WT) and TG Sprague-Dawley rats. TG rats showed a sharp decline in CSF HCRT-1 level at week 4 with levels remaining consistently low (26%+/-9%, mean+/-S.D.) thereafter compared with WT rats. In TG rats, HCRT-1 levels were dramatically lower in target regions such as the cortex and brainstem (100-fold), indicating decreased HCRT-1 levels at terminals. In TG rats, CSF HCRT-1 levels significantly increased in response to 6 h of prolonged waking, indicating that the remaining HCRT neurons can be stimulated to release more neuropeptide. Rapid eye movement (REM) sleep in TG rats (n=5) was consistent with a HCRT deficiency. In TG rats HCRT immunoreactive (HCRT-ir) neurons were present in the lateral hypothalamus (LH), even in old rats (24 months) but some HCRT-ir somata were in various stages of disintegration. The low output of these neurons is consistent with a widespread dysfunction of these neurons, and establishes this model as a tool to investigate the consequences of partial hypocretin deficiency.

Beneficial effects of regular exercise on sleep in old F344 rats.

With aging there is a significant decline in the normal architecture of sleep and a reduction in the diurnal amplitude of core body temperature. Regular moderate exercise has been shown to have a positive impact in the elderly and here we investigate whether sleep-wake patterning can also be improved. Young (3 months) and old (22 months) F344 rats were exercised once a day for 50min at night onset over an 8-week period. Thereafter, polysomnographic recordings were obtained immediately after exercise. To determine the lasting consequences of exercise, sleep was also recorded 2 days and 2 weeks after exercise had ended. Old rats that were exercised had a significant weight loss, were awake more during the last third of their active period, had less sleep fragmentation and the amplitude of the diurnal rhythm of core body temperature was significantly increased. Old exercised rats also had an overall increase in the amplitude of EEG power (0.5-16Hz) during wake and theta EEG power during REM sleep. In young rats regular exercise increased EEG delta power (0.5-4Hz) during NREM sleep. Our data indicate regular exercise in old rats improves sleep architecture, EEG power and diurnal rhythm of temperature.

Insomnia following hypocretin2-saporin lesions of the substantia nigra.

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.

The diurnal rhythm of adenosine levels in the basal forebrain of young and old rats.

There are significant decrements in sleep with age. These include fragmentation of sleep, increased wake time, decrease in the length of sleep bouts, decrease in the amplitude of the diurnal rhythm of sleep, decrease in rapid eye movement sleep and a profound decrease in electroencephalogram Delta power (0.3-4 Hz). Old rats also have less sleep in response to 12 h-prolonged wakefulness (W) indicating a reduction in sleep drive with age. The mechanism contributing to the decline in sleep with aging is not known but cannot be attributed to loss of neurons implicated in sleep since the numbers of neurons in the ventral lateral preoptic area, a region implicated in generating sleep, is similar between young (3.5 months) and old (21.5 months) rats. One possibility for the reduced sleep drive with age is that sleep-wake active neurons may be stimulated less as a result of a decline in endogenous sleep factors. Here, we test this hypothesis by focusing on the purine, adenosine (AD), one such sleep factor that increases after prolonged W. In experiment 1, microdialysis measurements of AD in the basal forebrain at 1 h intervals reveal that old (21.5 months) rats have more extracellular levels of AD compared with young rats across the 24 h diurnal cycle. In experiment 2, old rats kept awake for 6 h (first half of lights-on period) accumulated more AD compared with young rats. If old rats have more AD then why do they sleep less? To investigate whether changes in sensitivity of the AD receptor contribute to the decline in sleep, experiments 3 and 4 determined that for the same concentration of AD or the AD receptor 1 agonist, cyclohexyladenosine, old rats have less sleep compared with young rats. We conclude that even though old rats have more AD, a reduction in the sensitivity of the AD receptor to the ligand does not transduce the AD signal at the same strength as in young rats and may be a contributing factor to the decline in sleep drive in the elderly.

Effects of lateral hypothalamic lesion with the neurotoxin hypocretin-2-saporin on sleep in Long-Evans rats.

Narcolepsy, a disabling neurological disorder characterized by excessive daytime sleepiness, sleep attacks, sleep fragmentation, cataplexy, sleep-onset rapid eye movement sleep periods and hypnagogic hallucinations was recently linked to a loss of neurons containing the neuropeptide hypocretin. There is considerable variability in the severity of symptoms between narcoleptic patients, which could be related to the extent of neuronal loss in the lateral hypothalamus. To investigate this possibility, we administered two concentrations (90 ng or 490 ng in a volume of 0.5 microl) of the neurotoxin hypocretin-2-saporin, unconjugated saporin or saline directly to the lateral hypothalamus and monitored sleep, the entrained and free-running rhythm of core body temperature and activity. Neurons stained for hypocretin or for the neuronal specific marker were counted in the perifornical area, dorsomedial and ventromedial nucleus of the hypothalamus. More neuronal nuclei (NeuN) cells were destroyed by the higher concentration of hypocretin-2-saporin (-55%) compared with the lower concentration (-34%) in the perifornical area, although both concentrations lesioned the hypocretin neurons almost equally well (high concentration=91%; low concentration=88%). The high concentration of hypocretin-2-saporin also lesioned neurons in the dorsomedial nucleus of the hypothalamus and ventromedial nucleus of the hypothalamus. Narcoleptic-like sleep behavior was produced by both concentrations of the hypocretin-2-saporin. The high concentration produced a larger increase in non-rapid eye movement sleep amounts during the normally active night cycle than low concentration. Neither concentration of hypocretin-2-saporin disrupted the phase or period of the core temperature or activity rhythms. The low concentration of unconjugated saporin did not significantly lesion hypocretin or neurons and did not alter sleep. The high concentration of unconjugated saporin produced some loss of neuronal nuclei-immunoreactive (NeuN-ir) neurons and hypocretin immunoreactive neurons, but only a transient increase in non-rapid eye movement sleep. These results led us to conclude that the extent of hypocretin neuronal loss together with an accompanying loss of cells in the lateral hypothalamus may explain the differences in severity of symptoms seen in human narcolepsy.

Extracellular serotonin levels in the medullary reticular formation during normal sleep and after REM sleep deprivation.

Rapid eye movement (REM) sleep is hypothesized to result from the activity of REM sleep-generating and REM sleep-inhibiting neurons. The serotoninergic (5-HT) neurons of the dorsal raphe nucleus (DRN) represents one such population of REM-sleep inhibiting neurons since they are silent during REM sleep. Consistent with the decrease in activity of 5-HT neurons, the brain extracellular levels of 5-HT are lower during REM sleep compared to wakefulness. It is not known whether serotonin release is also reduced as a consequence of REM sleep rebound. Using microdialysis sampling coupled to HPLC-ECD, we measured the extracellular levels of 5-HT and its metabolite (5-HIAA) in the medial medullary reticular formation (mMRF) of freely behaving rats during normal sleep, REM sleep deprivation as well as during REM sleep rebound. We found that the levels 5-HT and 5-HIAA were significantly decreased by REM sleep deprivation. The reduction of 5-HT release was maintained during REM sleep rebound but the extracellular level of its main metabolite was increased. In addition, even during REM sleep rebound, 5-HT release during sleep was low compared to wakefulness. Taken together these data support the permissive role of 5-HT neurotransmission for REM sleep expression.

Hypothalamic regulation of sleep.

The recent discovery linking narcolepsy, a sleep disorder characterized by very short REM sleep latency, with a neuropeptide that regulates feeding and energy metabolism, provides a way to understand how several behaviors may be disrupted as a result of a defect in this peptide. In this chapter we review the evidence linking hypocretin and sleep, including our own studies, and propose that a defect in the lateral hypothalamus that also involves the hypocretin neurons is likely to produce a disturbance in sleep, mood, appetite, and rhythms.

Changes in sleep after acute and repeated administration of nicotine in the rat.

RATIONALE: Acetylcholine clearly plays a role in regulating sleep. This influence may involve nicotinic systems because several studies have demonstrated that nicotine treatment alters sleep. However, the literature that suggests an effect of nicotine treatment on sleep is contradictory, perhaps because different doses and routes of administration were used. OBJECTIVE: The studies reported here evaluated the effects of several doses of nicotine on REM sleep in the rat. METHODS: Male Wistar rats were prepared with a set of sleep recording electrodes and, following habituation to the test chamber, were used in one of three studies: a) a dose-response analysis of an acute dose of nicotine on REM sleep measured during the first 4 h after injection; b) a chronic treatment experiment; or c) a mecamylamine blockade experiment. RESULTS: Acute nicotine administration decreased REM sleep in a dose-dependent fashion; significant effects were observed following injection with the 0.5 and 1.0 mg/kg doses. A decrease in slow wave sleep and an increase in wakefulness were also observed. Mecamylamine by itself did not affect REM sleep, but it blocked the effects on sleep produced by nicotine when given 30 min before a 1 mg/kg dose of nicotine. Rats that had been injected once daily with a 0.1 mg/kg dose of nicotine showed an increase in REM sleep after the third injection, whereas rats that had been chronically treated with a higher dose (0.5 mg/kg) displayed a reduction in REM and total sleep time. CONCLUSION: These findings argue that the effects of both acute and chronic nicotine treatment on sleep are influenced by the dose of nicotine used.

Decrease in muscarinic M2 receptors from synaptosomes in the pons and hippocampus after REM sleep deprivation in rats.

The effects of both REM sleep deprivation and its recovery on pontine and hippocampus muscarinic M2 receptors were investigated in synaptosomes using [3H]-AF-DX 384 as a ligand. Animals were divided into three groups: REM sleep deprivation group (small platforms 6.5 cm of diameter); stress group (large platforms 14 cm of diameter) and cage control group. In a second experiment REM sleep-deprived animals were allowed 48 h of recovery. REM sleep-deprived rats showed a reduction in M2 receptors compared with both intact and stress groups. Changes in M2 receptors were also observed after 48 h of recovery from REM sleep deprivation only in hippocampus. The enhancement of acetylcholine release during both REM sleep deprivation and recovery could explain the present findings.

Repeated REM sleep deprivation after chronic haloperidol administration in the rat.

Repeated haloperidol administration produces up-regulation of dopamine (DA) receptors. REM sleep deprivation (REMSD) does also, but in addition, has been shown to produce REM sleep rebound. Should DA receptor up-regulation play a role in REM sleep rebound, haloperidol could conceivably have effects similar to those observed following REMSD. This is the central question investigated in this study. Male Wistar rats were prepared for sleep recordings. They were randomly assigned to the following groups: group 1, REMSD by small platforms (40 h REMSD + 8 h recording); group 2, was the large platform control group (40 h in large platforms + 8 h of recording); group 3, received 2-week daily administration of haloperidol (3 mg/kg, i.p.) plus REMSD (40 h REMSD + 8 h of recording); group 4, 2-week administration of haloperidol (3 mg/kg) without sleep manipulation and at the end 40 h were allowed to elapse, following which 8 h of sleep recordings was carried out. In each group the sleep manipulation and/or sleep recordings were repeated five consecutive times. Repeated REMSD produced increases of REM sleep time after each recovery in group 1. Large platforms did not produce increases of REM sleep during the recovery trials. The 2-week administration of haloperidol plus REMSD prevented REM sleep rebound (group 3). The 2-week administration of haloperidol without sleep manipulation (group 4) produced a REM sleep reduction. Dopamine modulation seems not to be important for REM sleep rebound. Hypersensitivity of DA receptors developed after REMSD may be an epiphenomenon associated with this sleep manipulation, but seems not to participate in REM sleep enhancement after REMSD.