Gene expression in the rat brain during sleep deprivation and recovery sleep: an Affymetrix GeneChip study.

Previous studies have demonstrated that macromolecular synthesis in the brain is modulated in association with the occurrence of sleep and wakefulness. Similarly, the spectral composition of electroencephalographic activity that occurs during sleep is dependent on the duration of prior wakefulness. Since this homeostatic relationship between wake and sleep is highly conserved across mammalian species, genes that are truly involved in the electroencephalographic response to sleep deprivation might be expected to be conserved across mammalian species. Therefore, in the rat cerebral cortex, we have studied the effects of sleep deprivation on the expression of immediate early gene and heat shock protein mRNAs previously shown to be upregulated in the mouse brain in sleep deprivation and in recovery sleep after sleep deprivation. We find that the molecular response to sleep deprivation and recovery sleep in the brain is highly conserved between these two mammalian species, at least in terms of expression of immediate early gene and heat shock protein family members. Using Affymetrix Neurobiology U34 GeneChips , we also screened the rat cerebral cortex, basal forebrain, and hypothalamus for other genes whose expression may be modulated by sleep deprivation or recovery sleep. We find that the response of the basal forebrain to sleep deprivation is more similar to that of the cerebral cortex than to the hypothalamus. Together, these results suggest that sleep-dependent changes in gene expression in the cerebral cortex are similar across rodent species and therefore may underlie sleep history-dependent changes in sleep electroencephalographic activity.

[Hypocretins/orexins and narcolepsy: from molecules to disease]. / Hypocrétines/orexines et narcolepsie: de la molécule à la pathologie.

In order to foresee a curative treatment for narcolepsy, it is crucial to determine whether or not human narcolepsy is a neurodegenerative disorder, as we suggested, and to understand the mechanisms involved. The current hypothesis regarding the etiology of human narcolepsy is that it is an autoimmune disorder, because of its strong association with the human leukocyte antigen (HLA DQB1*0602), with the Hcrt neurons as the target. This hypothesis is supported by our results (Peyron et al., 2000) and others (Thannickal et al., 2000) showing that Hcrt messengers RNA and mature peptides are absent or greatly reduced in the brain of HLA DQB1*0602 positive narcoleptic patients examined to date. It is of great importance to determine whether the absence of Hcrt is due to a neurodegenerative process or to a default in the transcription process. After a brief review on hypocretins and narcolepsy, we discuss on how to tackle the issue.

Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice.

The hypocretin/orexin ligand-receptor system has recently been implicated in the sleep disorder narcolepsy. During the dark (active) period, null mutants of the prepro-orexin (prepro-hypocretin) gene have cataplectic attacks and increased levels of both rapid eye movement (REM) and non-REM (NREM) sleep. Intracerebroventricular injection of one of the encoded neuropeptides, orexin-A, early in the light period increases wakefulness and reduces REM sleep in the rat, suggesting that this system may be involved in the normal regulation of sleep and wakefulness. To further test this hypothesis, we measured hypocretin (hcrt) mRNA levels by both Northern hybridization and Taqman analysis in mouse and rat hypothalamus after short-term (6 h) sleep deprivation (SD) and 2-4 hours after recovery from SD. Although our SD procedures effectively induced a sleep debt and increased c-fos mRNA expression in the cortex and hypothalamus as described by other investigators, we found that hcrt mRNA levels were not significantly changed in either species either after SD or after recovery from SD. If the hcrt system is involved in normal regulation of sleep and wakefulness, longer periods of SD may be necessary to affect hcrt mRNA levels or changes may occur at the protein rather than mRNA level. Alternatively, this system may also be involved in another function that counterbalances any SD-induced changes in hcrt mRNA levels.

A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains.

We explored the role of hypocretins in human narcolepsy through histopathology of six narcolepsy brains and mutation screening of Hcrt, Hcrtr1 and Hcrtr2 in 74 patients of various human leukocyte antigen and family history status. One Hcrt mutation, impairing peptide trafficking and processing, was found in a single case with early onset narcolepsy. In situ hybridization of the perifornical area and peptide radioimmunoassays indicated global loss of hypocretins, without gliosis or signs of inflammation in all human cases examined. Although hypocretin loci do not contribute significantly to genetic predisposition, most cases of human narcolepsy are associated with a deficient hypocretin system.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Forebrain projections of the rostral nucleus raphe magnus shown by iontophoretic application of choleratoxin b in rats.

The nucleus raphe magnus belongs to the thermoafferent system. Following iontophoretic choleratoxin b injections in its rostral part, a substantial to large number of anterogradely labeled varicose fibres were observed in the medial and lateral preoptic areas, the bed nucleus, the substantia innominata, the ventral pallidum, the median preoptic nucleus, the paraventricular hypothalamic nucleus, the central amygdaloid nucleus and the lateral and dorsal hypothalamic areas. A small to moderate number were seen in the septal nuclei, the diagonal band, the magnocellular preoptic nucleus, the anterior hypothalamic area and the paraventricular and intralaminar thalamic nuclei. After choleratoxin b injections in the preoptic, dorsal and lateral hypothalamic areas, a substantial number of retrogradely labeled serotonin immunonegative neurones were specifically found in the rostral nucleus raphe magnus. Thus, non-serotonergic rostral nucleus raphe magnus cells might directly modulate hypothalamic thermointegrative neurones.

Origin of the dopaminergic innervation of the rat dorsal raphe nucleus.

The aim of the present study was to describe the distribution of dopamine (DA) fibres in the dorsal raphe nucleus (DRN) and to determine their neurones of origin. Using an anti-DA antibody, we observed a moderate density of DA varicose fibres over the DRN and a dense plexus of DA fibres in the ventrolateral central grey. With a sensitive retrograde tracing technique combining the use of cholera toxin subunit b with tyrosine hydroxylase immunohistochemistry, after tracer injections in the DRN, a few double-labelled cells were observed in the ventral tegmental area and the A10 dorsocaudal DA cell group, as already described. In addition, a moderate number of double-labelled cells was seen in the A11 hypothalamic DA cell group.