Localization of orexins and their receptors in the rat olfactory system: possible modulation of olfactory perception by a neuropeptide synthetized centrally or locally

Abstract

Orexin-A and -B, also known as hypocretins, are two neuropeptides acting on feeding and sleep. They are specific ligands for two different receptors belonging to the G-protein coupled receptors family. Orexin fibers and orexin receptor neurons have been previously described in the forebrain olfactory system. Using immunocytochemistry, we showed that both orexin-A and -B as well as their receptors were present at different levels of the olfactory system, from the nasal mucosa to nuclei of the amygdala. A punctuated staining for orexins and their receptors was detected at the apical part of the olfactory epithelium; in the lamina propria of the mucosa, the staining was localized around olfactory nerves. At the ultrastructural level, olfactory neurons and supporting cells were found immunoreactive for orexins and their receptors. The labeling was localized in dendritic knobs and cilia of neurons, in the apical part and microvilli of supporting cells. The finding of immunolabeled cisternae of reticulum strongly suggests a local synthesis of both peptides and receptors, confirmed by RT-PCR experiments. In forebrain and amygdala regions, we detected numerous orexin fibers. Orexin receptors were present in mitral-tufted cells of the bulb and in many neuronal perikarya in the anterior olfactory nuclei, piriform cortex and amygdala nuclei. Altogether, these results show that orexins and their receptors are present at all levels of the olfactory system, from cilia where odors bind to their receptors to central regions where integration of olfactory signals occurs. They suggest a possible modulation of olfactory perception by these neuropeptides.

Introduction

Odors are a main attractant to food sources or prey in most mammals. Among the three molecular sensors developed to probe the environment, the main olfactory system (olfactory mucosa lining the nasal cavity, main olfactory bulb and olfactory structures targeted by mitral-tufted cells) detects small volatile molecules. Olfactory perception, which starts in the olfactory receptor neurons located in the epithelium, is modulated by behavioral states, via hormonal or neuronal regulation: substance-P immunoreactive fibers present in the frog olfactory mucosa (lamina propria and epithelium) modulate the activity of olfactory receptor neurons [5], [6]; in vitro, adrenaline renders newt olfactory neurons more sensitive to the presence or absence of an odorant [26]. Gonadotropin releasing hormone (GnRH) is present in mudpuppies terminal nerve running below the olfactory epithelium and increases the excitability of olfactory receptor neurons in vitro [18]. Dopamine modulates the sensitivity of rat olfactory neurons to odorants via D2 receptors [52].

The recently discovered orexins (OXs), also called hypocretins [15], [44], consist of OXA and OXB which derived from the same 130 amino acid residue prepro-OX precursor. To date, two receptors (OX_SR: OX₁R and OX₂R) belonging to the G protein-coupled receptor family have been described [44]. In the central nervous system, OXs are produced only in neurons of the lateral hypothalamic perifornical area. These neurons project widely in hypothalamic, thalamic and brain stem nuclei, in forebrain regions as well as in the spinal cord. These neuropeptides appear to be involved in several functions including food intake [15], [44], sleep [8], [31], [22], and autonomic and neuroendocrine functions [14], [42], [51].

In the olfactory bulb, a number of neuropeptides and their receptors, possibly involved in the control of food intake, are present: orexin fibers [50], mRNA coding for neuropeptide Y (NPY) [43], NPY-immunoreactive (-ir) fibers and cell bodies [21], [46], leptin receptor [48], and insulin and its receptor modulated by fasting and external sensory experience [2], [19]. Leptin-ir neurons are present in the piriform cortex [35] and messenger RNAs coding for NPY-Y5 receptor subtype are detected in the lateral olfactory tubercle and piriform cortex [17]. Taken together, these data suggest that peptides involved in the control of food intake and body weight homeostasis might exert a modulatory role on smell perception. However, no data are currently available concerning the presence of such neuropeptides in the olfactory epithelium.

Because of the possible role of OXs and their receptors in the regulation of food intake, and because of the high concentration of OX-ir fibers in the forebrain olfactory system, we wondered whether OXs and their receptors might also be present at the first level of odor detection, the nasal mucosa, as well as at higher processing levels of the olfactory message (olfactory nuclei, piriform cortex and amygdala). OXA and B and their receptors were looked for by immunocytochemistry (ICC). The presence of mRNA for these proteins was checked by RT-PCR.