Distribution of hypocretin-(orexin) immunoreactivity in the central nervous system of Syrian hamsters (Mesocricetus auratus)

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Abstract

The hypocretins are peptides synthesized in neurons of the hypothalamus. Recent studies have suggested a role for these peptides in the regulation of sleep, feeding, and endocrine regulation. The distribution of hypocretin-immunoreactive cell bodies and fibers has been extensively described in rats, but not in other species. This study was designed to examine the distribution of hypocretin immunoreactivity in Syrian hamsters, as important differences in neuropeptide distribution between rats and hamsters have previously been demonstrated. Immunoreactive cell bodies were found primarily in the lateral hypothalamic area and the perifornical area, although a few hypocretin-positive cells were also located in the dorsomedial hypothalamus and the retrochiasmatic area. Fibers were distributed throughout the brain in a pattern similar to that seen in rats. The densest projections were found in the paraventricular nucleus of the thalamus, locus coeruleus, dorsal raphe, and lateroanterior hypothalamus. The innervation of the anterior hypothalamus may be of particular interest as similar cluster of immunoreactivity does not appear to be present in rats. Moderate levels of immunoreactivity could be seen throughout the hypothalamus, the lateral septum, bed nucleus of the stria terminalis, A5 noradrenergic area, and the midline thalamic nuclei. Hypocretin-immunoreactive fibers are present in all lamina of the spinal cord, with the greatest axon densities in lamina 1 and 10. The widespread distribution of hypocretin suggests its involvement in a wide variety of physiological and behavioral processes. Our results in hamsters indicate that the organization of the hypocretin system is strongly conserved across species, suggesting an important role for the peptide and its projections.

Introduction

The hypothalamus contains numerous groups of relatively small, discrete populations of neurons which produce a wide variety of neuropeptides. Some of these neurons, such as those containing oxytocin, send projections to all levels of the central nervous system from the olfactory bulbs to the spinal cord (Zimmerman et al., 1984, Cechetto and Saper, 1988). Because of the wide distribution of the projections of these neurons, they have the potential to influence a large number of diverse and anatomically distinct brain areas. Recently, a novel neuropeptide family, hypocretin [Hcrt] (also called orexin) was reported in the hypothalamus of rats (de Lecea et al., 1998, Sakurai et al., 1998). The hypocretins consist of a single precursor peptide, preprohypocretin, which is then cleaved into two smaller neuroactive peptides, hypocretin-1 (Hcrt-1) and hypocretin-2 (Hcrt-2). The distribution of the two peptides show 100% overlap in the brains of rats (Peyron et al., 1998).

The physiological functions of the hypocretins are not well understood. However, there is now significant data which suggest a major role for Hcrt in arousal and sleep regulation. Hypocretin knockout mice show a behavioral phenotype which is very similar to human narcolepsy (Chemelli et al., 1999), and canine narcolepsy is caused by a mutation of the hypocretin receptor 2 gene (Lin et al., 1999). Although hypocretin is normally detectable in human cerebrospinal fluid, humans with narcolepsy generally have no detectable hypocretin (Nishino et al., 2000). Recent work suggests that narcoleptic humans have few or no hypocretin neurons in their hypothalamus with no change in the distribution of melanin concentrating hormone neurons in the same area of the hypothalamus (Peyron et al., 2000, Thannickal et al., 2000), perhaps due to selective degeneration of the hypocretin system (van den Pol, 2000). Application of Hcrt-1 and Hcrt-2 to the locus coeruleus (LC), a brain region which receives heavy Hcrt innervation in rats and is important in the determination of arousal state, stimulates the firing of noradrenergic neurons (Hagan et al., 1999, Horvath et al., 1999). Increased LC activity is associated with arousal and the inhibition of rapid eye-movement sleep (REM) (Aston-Jones and Bloom, 1981). In rats, some of the most dense Hcrt projections are to areas that are very important in sleep regulation, such as the LC, the dorsal raphe nucleus (DR), and the paraventricular nucleus of the thalamus (PVT) (Peyron et al., 1998). In addition, lesions of the lateral hypothalamus (LH), where many Hcrt-immunoreactive cell bodies are located in rats, alters sleep (Jurkowlaniec et al., 1994, Jurkowlaniec et al., 1996). These data are all consistent for a role for Hcrt in sleep regulation, REM sleep in particular.

Hypocretin is also thought to play a role in the regulation of energy balance. Hcrt-ir neurons are found in the lateral hypothalamus, an area which has a strong influence on feeding behavior (Bernardis and Bellinger, 1996). Hcrt mRNA in the LH is increased by insulin-induced hypoglycemia in rats (Griffond et al., 1999). Furthermore, the amount of Hcrt in the brain decreases in response to fasting in rats. In the LH there is a trend toward an increase in Hcrt content in response to fasting (Mondal et al., 1999), but a trend for a decrease in the rest of the brain. However, it should be noted that in a separate study no change in Hcrt content was found in fasted rats (Taheri et al., 1999). In addition, Hcrt gene expression is downregulated in genetically obese mice (Yamamoto et al., 1999). The effects of Hcrt administration on feeding behavior have varied considerably from study to study. Sakurai et al. (1998) found that intracerebroventricular (ICV) injection of Hcrt stimulated feeding in rats. A later study found that injections Hcrt-1 into the paraventricular nucleus of the hypothalamus (PVN) stimulated food intake during a 2 h interval after the injection (Edwards et al., 1999), but that the induction of feeding by Hcrt-1 was considerably less than that caused by neuropeptide Y (NPY). More recently, it was demonstrated that injection of Hcrt-1 into the LH significantly elevated c-fos immunoreactivity in a large number of energy regulatory sites in the brain (Mullett et al., 2000). The stimulatory effects of hypocretin on feeding may be mediated at least in part by NPY (Jain et al., 2000, Yamanaka et al., 2000). However, another study found that Hcrt-1 stimulated feeding when injected into the LH or perifornical nucleus, but not when injected into the PVN (Sweet et al., 1999). Other studies have found that Hcrt is capable of stimulating gastric acid secretion in rats (Takahashi et al., 1999), increasing metabolic rate in mice (Lubkin and Stricker-Krongrad, 1998), and altering luteinizing hormone release in rats (Pu et al., 1998). Thus, the potential exists for Hcrt to play a role in energy homeostasis as well as in a wide variety of other physiological processes.

The majority of research on the hypocretins to date has been conducted on rats, and to a much lesser extent, mice and primates. A systematic analysis of the distribution of the hypocretin system has been reported only in one species, the rat. It is important, therefore, to examine this peptide in other species in order to determine which of its properties are species-specific versus which are conserved across different mammalian genera. An important example of species-specific peptide distribution is vasopressin. There are significance differences in the distribution of vasopressin in hamsters compared to rats, indicating that the neurochemical organization found in rats may not reflect that found in other species (Ferris et al., 1995). Hamsters are used extensively in research on neuropeptides, sleep-related phenomenon, and bioenergetics. Therefore, we sought to identify the distribution of Hcrt-ir neurons and fibers in Syrian hamster brains.

Section snippets

Animals

Adult male (n=6) and female (n=6) Syrian hamsters (130–170 g, Charles River Labs, Natick, MA) were maintained on a 14:10 light–dark cycle in group housing for at least 2 weeks after arrival. Food and water were available ad libitum. Hamsters were deeply anesthetized with a lethal dose of Nembutal (90 mg/kg) and perfused transcardially with 100 ml 0.9% saline followed by 200 ml 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed, post-fixed overnight at 4°C, immersed in 4%

Results

Representative micrographs for coronal sections through the brain are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 All brain regions containing significant hypocretin immunoreactivity are shown. In addition, Table 1 indicates the relative density of hypocretin-immunoreactive fibers throughout the brain. There were no qualitative differences in cell body or fiber distribution between male and female hamsters, and labeled regions of the brain were very consistent from animal to

Discussion

The distribution of hypocretin immunoreactive cell bodies and fibers in the central nervous system of hamsters is very similar to that found in rats (Peyron et al., 1998). This close inter-species consistency suggests that Hcrt may serve an important, evolutionarily conserved function. The widespread distribution of hypocretin fibers suggests that it may be involved in a wide variety of physiological and behavioral processes. One interesting feature of hypocretin distribution specific to Syrian

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