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.

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.

Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat.

Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.

Effects of prolonged wakefulness on c-fos and AP1 activity in young and old rats.

Recent studies have demonstrated that the immediate-early gene c-fos is induced in neuronal populations responsible for specific sleep-wake states. The induction of this gene may be functionally relevant to sleep homeostasis since without the gene mice (c-fos null) take longer to fall asleep and have a selective reduction in slow-wave sleep. This suggests that a build-up of c-fos during wakefulness increases the drive to sleep and lack of c-fos is associated with reduced sleep. Sleep also has an effect on c-Fos serving to eliminate the protein rapidly. Waxing and waning of transcription factors such as c-Fos may influence slow, oscillating events such as sleep and wakefulness. To further examine what role c-Fos may play in regulating sleep, the present study examined the effects of prolonged wakefulness on c-Fos and AP-1 activity in young (3.5 months old) and old (21.5 months old) Sprague--Dawley rats. Previously we found that old rats slept less even after prolonged wakefulness, and other investigators have found that aging is also associated with a decline in c-Fos. In the present study, we reasoned that prolonged wakefulness would also fail to increase c-Fos in old versus young rats. The baseline levels of c-Fos and AP-1 activity were not different between young and old rats. However, in response to 6 or 12 h of prolonged wakefulness, old rats demonstrated significantly less c-Fos and AP-1 activity compared to young rats. These findings suggest that in old rats the mechanism responsible for c-Fos induction in response to wakefulness is deficient. Such a decline at the molecular level could contribute to the decline in sleep that typically occurs with age.

Choline acetyltransferase expression during periods of behavioral activity and across natural sleep-wake states in the basal forebrain.

The present study examined whether the expression of the messenger RNA encoding the protein responsible for acetylcholine synthesis is associated with sleep-wakefulness. Choline acetyltransferase messenger RNA levels were analysed using a semi-quantitative assay in which reverse transcription was coupled to complementary DNA amplification using the polymerase chain reaction. To examine the relationship between steady-state messenger RNA and behavioral activity, rats were killed during the day (4.00 p.m.) or night (4.00 a.m.), and tissue from the vertical and horizontal limbs of the diagonal bands of Broca was analysed. Choline acetyltransferase messenger RNA levels were higher during the day than during the night. The second study examined more closely the association between choline acetyltransferase messenger RNA levels and individual bouts of wakefulness, slow-wave sleep or rapid eye movement sleep. Choline acetyltransferase messenger RNA levels were low during wakefulness, intermediate in slow-wave sleep and high during rapid eye movement sleep. In contrast, protein activity, measured at a projection site of cholinergic neurons of the basal forebrain, was higher during wakefulness than during sleep. These findings suggest that choline acetyltransferase protein and messenger RNA levels exhibit an inverse relationship during sleep and wakefulness. The increased messenger RNA expression during sleep is consistent with a restorative function of sleep.

Expression of c-fos protein by immunohistochemically identified oxytocin neurons in the rat hypothalamus upon osmotic stimulation.

Double immunostaining for c-fos and oxytocin (OXY) was used to study the topography and time course of the metabolic activation of the hypothalamic oxytocinergic system upon osmotic stress in the male rat. Animals injected i.p. with hypertonic saline expressed c-fos-like immunoreactivity (FLI) in the paraventricular (PVN), periventricular (PEV) and supraoptic (SON) hypothalamic nuclei, and in the preoptic and retrochiasmatic regions, as early as 30 min after stimulation and up to 6 h, while these areas were mostly devoid of staining in isotonic saline-injected animals. The activation of the oxytocinergic system peaked at 30 min and declined at different rates in the PVN and in the SON after 90 min. The maximal percentage of OXY neurons expressing FLI upon osmotic stress was about 80% in the SON, PEV and LSN, 60% in the PVN and 50% in the medial preoptic area. Activated OXY neurons were found in both the magnocellular and parvocellular divisions of the system. These data show that OXY nuclei in the rat hypothalamus are differentially activated by osmotic stress. They also suggest a role of OXY in the central as well as in the humoral response to changes in plasma osmolarity.

Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress.

In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.