Interactive reportDistribution of orexin neurons in the adult rat brain1
Introduction
The hypothalamus has long been thought to play an important role in the regulation of food intake and energy homeostasis. It is well known that lesions of the lateral hypothalamic area (LHA) result in hypophagia and weight loss, while lesions of the ventromedial hypothalamic area (VMH) produce the hyperphagic obesity. These are known as the `LHA syndrome' and `VMH syndrome', respectively 1, 4, 6, 16, 30.
However, the effect of lesions of the LHA is controversial. A fundamental technical problem with electrolytic lesions is that both cell bodies and axons are destroyed with this procedure. Thus, it is impossible to distinguish whether the behavioral effects manifested after such lesioning are attributable to loss of nerve somata or fibers of passage. It has been suggested that aphagia following LHA lesions is due to damage to fibers of passage, rather than destruction of key populations of neurons residing in the LHA [26]. Ungerstedt showed that most of the LHA lesion syndrome could be demonstrated in rats with hindbrain lesions that damaged the ascending nigrostriatal pathway [40]. Morgane reported that the LHA lesion syndrome was caused by far lateral lesions [27]. The far lateral area was shown by Marshall et al. [26]and Ungerstedt [40]to contain dopamine fibers from the nigrostriatal pathway. These observations suggest that the `LHA syndrome' is due to the destruction of dopaminergic fibers, rather than LHA neurons. However, cell-specific lesioning of the LHA induced by ibotenic acid, which destroys neuronal somata without damaging fibers of passage, also produced aphagia, although this was less severe compared with the outcome of electrolytic lesioning [43]. This finding suggests that LHA neurons have important roles in regulating feeding behavior, although all of the symptoms observed in the LHA syndrome cannot be attributed to a lesion of the LHA neurons.
Similarly, the effects of lesions of the VMH are also controversial. It has been demonstrated that tracts lateral to the VMH were responsible for the hyperphagia caused by electrolytic VMH lesions [10]. It was also shown that lesions restricted to the VMH did not produce the so-called VMH hyperphagic syndrome. Bray et al. demonstrated that VMH lesions can affect feeding, but do so indirectly by increasing insulin secretion [9]. Thus, the neuroanatomical basis for lesion-induced changes in body weight homeostasis has remained unclear. However, VMH neurons have been shown to express leptin receptors 14, 19. Also, it was shown that some populations of VMH neurons are activated by glucose [30]. These observations suggest roles of VMH neurons in energy homeostasis.
On the other hand, identification of the leptin signaling pathway and recent molecular genetic studies about several hypothalamic neuropeptides are unveiling the molecular mechanisms involved in energy homeostasis. These studies have revealed a number of central regulatory pathways mediated by various neuropeptides 12, 17, 18, 21, 23, 29, 32, 39, 44, 45.
Leptin has been thought to act as a hormonal feedback signal to regulate body adiposity via hypothalamic mechanisms controlling food intake and metabolic rate. The site of leptin's action has been thought to exist in the medial part of the hypothalamus. Many neuropeptides involved in the regulation of energy homeostasis are known to exist mainly in the medial/periventricular part of the hypothalamus and to be regulated by leptin [16]. Especially, the arcuate nucleus (Arc), located in the mediobasal hypothalamus, is critical in the regulation of body weight and metabolism, as it contains neurons that express leptin receptors, neuropeptide Y (NPY), α-melanin stimulating hormone (α-MSH) and agouti-related peptide (AgRP).
Many other neuropeptides localized in the ventral/medial part of the hypothalamus are also thought to be involved in the regulation of food intake, such as glucagon-like peptide-1 (7–36) amide (GLP-1) [39], galanin [24]and cocaine- and amphetamine-regulated transcript (CART) [23]. Appetite may be regulated by reciprocal and/or cooperative interactions of these neuropeptides.
Recently, several neuropeptides produced by neurons specifically localized in the lateral part of the hypothalamus have been shown to be implicated in feeding behavior. Melanin-concentrating hormone (MCH) is an attractive candidate in the LHA for regulating food intake, as feeding is increased by intracerebroventricular (i.c.v.) administration of MCH. It was shown that targeted disruption of MCH gene in mice results in decreased food intake and body weight [36].
Orexin (ORX)-A and -B are also specifically localized within and around the LHA, and were shown to increase feeding after i.c.v. administration. ORX-A is a 33-amino acid residue peptide with two intrachain disulfide bonds, while ORX-B is a linear 28-residue peptide. They are derived from the same precursor, prepro-orexin, by proteolytic processing. They bind and activate two closely related G protein-coupled receptors (OX1R and OX2R). ORX-A binds to both receptors equally, while ORX-B has a higher affinity to OX2R. A previous study showed that prepro-orexin mRNA and immunoreactive ORX-A are specifically localized in neurons within and around the lateral hypothalamic area [34]. These observations suggest that ORXs play an important role in the regulation of feeding behavior.
We report here the projection of ORX-containing neurons to provide a general overview of the organization of the ORX neuronal system. The LHA has been thought to contain neurons that innervate the entire neuraxis, including monosynaptic projections to several regions of the cerebral cortex, limbic system, and brain stem, suggesting an important role of the LHA in integrating the complex physiological responses underlying feeding behavior. Our present study suggests that ORX neurons are included in this projection, which may be a fundamental neuronal pathway in the regulation of feeding behavior in the mammalian brain.
Section snippets
Animals
Male Wistar rats weighing 250–350 g were obtained from Charles River (Kanagawa, Japan). They were housed in a climate-controlled, 12 h light/12 h dark room, and received water and food ad libitum.
Antiserum
Anti-ORX-A antiserum was raised in rabbits by immunization with synthetic ORX-A 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, CRLYELLHGAGNHAAGILTL-amide, conjugated to keyhole limpet hemocyanin (Carbiochem, La Jolla, CA) using 3-maleimidobenzoic acid N-hydroxysuccimide
Antiserum specificity
To check the specificity of the ORX antiserum, we used a cell expression system and preabsorption of anti-ORX antiserum with ORX-A, ORX-B or unrelated peptide (NPY or angiotensin II). Prepro-orexin cDNA transfected cells were positively stained with anti-ORX antiserum, while mock transfected cells were not stained (Fig. 1A, B). Staining for ORX-immunoreactivity (ir) in rat brain sections was completely abolished by preincubation of the antiserum with 5 μM ORX-A or ORX-B, but was not abolished
Discussion
ORX were originally found as ligands for orphan G protein-coupled receptors (GPCRs), termed OX1R and OX2R. They stimulate food intake when administered i.c.v. A previous study showed that ORX-containing neuronal perikarya were localized within and around the LHA [34]. These areas are classically regarded as the regions implicated in feeding and drinking behavior [16].
In the present study, we performed a further investigation of the distribution and organization of ORX neurons in the adult rat
Acknowledgements
This study was supported by a grand-in-aid for scientific research from Ministry of Education, Science and Culture of Japan, University of Tsukuba Project Research, and Uehara Memorial Foundation. We would like to thank Dr. W.A. Gray for reading our manuscript.
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Published on the World Wide Web on 17 March 1999.