Research reportOrexin A-like immunoreactivity in the hypothalamus and thalamus of the Syrian hamster (Mesocricetus auratus) and Siberian hamster (Phodopus sungorus), with special reference to circadian structures
Introduction
Orexin A (OXA) and orexin B are putative neuropeptide transmitters derived from the proteolysis of the peptide precursor prepro-orexin [49]. In the central nervous system (CNS) of various mammalian species, including rat, mouse and human, orexin synthesising cells have been localised exclusively to the lateral hypothalamic area (LHA) [8], [9], [36]. Although the orexin-synthesising cells have a restricted distribution, orexin-containing fibres are found throughout the rat brain and spinal cord, occurring in high densities in the thalamus, hypothalamus, locus coeruleus and septal nuclei [8], [9], [36]. The widespread distribution of OXA containing fibres implies that this peptide could play a role in many physiological processes. Indeed, OXA has been implicated in stress [23], gastric acid secretion [55] and feeding.
The presence of orexin cells in the LHA, an area instrumental in body weight regulation [4], [11], is suggestive of a role for the orexins in energy homeostasis; a hypothesis that has subsequently been supported by functional studies [13], [14], [17], [20], [22]. It has been found, in the rat, that continuous infusion of orexin A into the lateral ventricle alters feeding behaviour in a diurnal pattern such that feeding activity is increased during the day phase, but decreased during the night phase [20]. A further investigation has shown that there is a diurnal variation in orexin A-like immunoreactivity (OXA-lir) in the hypothalamus, with levels maximal at early morning (09:00 h) and lowest at early evening (21:00 h) [15], [54]. The expression of prepro-orexin mRNA in the LHA also shows similar diurnal variation, suggesting that orexin synthesis varies over the day–night cycle. These findings suggest that diurnal variation in OXA affects ingestive behaviour by promoting feeding activity only at a specific time of day (e.g. early morning).
Two receptors, orexin receptor 1 and orexin receptor 2, mediate the actions of the orexins in the mammalian CNS [49]. The mRNA for these receptors is widely but differentially distributed throughout the rat brain, i.e. high levels of orexin receptor 1 mRNA are found in the ventromedial hypothalamus, while lower levels of expression are found in the tenia tecta, hippocampus, dorsal raphe and locus coeruleus. High levels of mRNA for orexin receptor 2 are found in the hypothalamic and thalamic paraventricular nuclei, the cerebral cortex, nucleus accumbens and anterior pretectal nucleus [56]. The paraventricular thalamic nucleus is implicated in sleep/wake behaviour [18], [41] and the locus coeruleus is implicated in arousal [19], [21]. The presence of orexin receptors at these loci implies that a relationship exists between orexins and the physiology of the sleep/wake cycle and arousal. Indeed, it has been shown that intracerebroventricular administration of OXA modulates the sleep/wake cycle of rats in a dose dependent fashion [44]. Further evidence for this relationship has been found in a mutation in the orexin receptor 2 gene, which has been shown to be instrumental in canine narcolepsy [29], a disease characterised by disruption of the sleep/wake cycle. Similarly, mice lacking the prepro-orexin gene also exhibit a narcoleptic-like state [7]. Since the timing of the sleep/wake cycle is modulated by the circadian system, it is possible that the orexins interact with the circadian system to regulate the sleep/wake cycle.
The suprachiasmatic nucleus (SCN) contains the main endogenous mammalian circadian pacemaker [24], [28], [46] and, as such, determines the temporal architecture for many behavioural and physiological processes including the sleep/wake cycle, feeding and drinking. The SCN circadian pacemaker is synchronised (entrained) to recurring environmental signals, the most prevalent of which is the daily variation in light levels. Photic information is conveyed directly to the SCN via the monosynaptic retinohypothalamic tract, and indirectly through retinally innervated neurones of the thalamic intergeniculate leaflet (IGL) that in turn project to the SCN via the geniculohypothalamic tract [32]. The IGL is intercalated between the dorsal and ventral nuclei of the thalamic geniculate complex and plays an important role in circadian rhythm regulation by relaying both photic and non-photic phase information to the circadian clock [25], [33], [48]. The SCN is innervated by serotonergic fibres originating from the median raphe, and this projection is thought to modulate the effects of light on the SCN circadian clock [26], [34]. The IGL also receives a serotonergic input from the dorsal raphe nucleus, a region that is involved in sleep activity and when lesioned induces insomnia [34]. In addition, the IGL is innervated by noradrenergic fibres arising from neurons in the locus coeruleus [33], a region that possesses a bidirectional polysynaptic connection to the SCN [27].
The neuroanatomical basis for the possible interaction of the orexins and the circadian system is unknown. In this study we used a polyclonal antiserum to examine the distribution of OXA-lir in the hypothalamus and thalamus of one of the most widely used animal models in circadian neurobiology, the Syrian hamster. We also examined the distribution of OXA-lir in three brainstem regions important in the regulation of the sleep/wake cycle: the dorsal and median raphe nuclei, and the locus coeruleus. To determine whether this pattern of immunostaining was conserved across related species, we also investigated the distribution of OXA-lir in these structures of the Siberian hamster.
Section snippets
Perfusion
Adult male Syrian (n=10; Charles River, Margate, Kent, UK) and Siberian hamsters (n=9; University of Manchester breeding colony), all held on a 16:8-h light/dark regime for at least 3 weeks, were anaesthetised with sodium pentobarbitone (200 mg/ml; Pentoject, Animal Care Ltd., York, UK) during the lights-on phase and intracardially perfused with 0.9% saline followed by 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The brains were removed,
Results
The pattern of immunostaining for OXA-lir cells and fibres was similar in Syrian and Siberian hamsters and the following description of the results therefore applies to both species unless otherwise mentioned. In control sections from both Syrian and Siberian hamsters incubated with preadsorbed primary antisera, OXA-lir was absent from all levels of the brain examined (data not shown). Brain sections from both species incubated in the absence of primary antibody did not show any OXA-lir (data
Discussion
The OXA polyclonal antiserum used in this study revealed a pattern of immunolabelling in the hypothalamus, thalamus, dorsal and median raphe nuclei and locus coeruleus that was similar in Syrian and Siberian hamsters. In the rat CNS, OXA is synthesised only by cells of the LHA [8], [9], [36], [43], a finding that has also been extended to the Siberian hamster [47]. Our study shows a similar distribution of OXA-lir cells in the LHA of Syrian and Siberian hamsters and reveals that in these
Acknowledgements
This work was supported by a BBSRC studentship to P. McGranaghan and BBSRC project grants to Dr H. Piggins. Thanks to Dr D.J. Cutler, R.E. Samuels, Dr A. Coogan and Dr H.E. Reed for their editorial help.
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