LONG-TERM ALTERATION IN BODYWEIGHT AND FOOD RESTRICTION DOES NOT AFFECT THE GENE EXPRESSION OF EITHER PREPROOREXIN OR PRODYNORPHIN IN THE SHEEP J. IQBAL,* B. A. HENRY, S. POMPOLO, A. RAO AND I. J. CLARKE Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia Abstract—Various hypothalamic neuropeptides are involved in central regulation of food intake and expression of genes encoding these peptides changes with alterations in the bodyweight/metabolic status/nutritional status. Orexin(s) and dynorphin have been implicated in the regulation of appetite and neuroendocrine systems, but the function of these pep- tides is not well understood. We have employed in situ hy- bridization to examine the effects of long-term alterations in the bodyweight on expression of mRNA for preproorexin and prodynorphin in the putative feeding centers of the ovine hypothalamus. Expression of preproorexin was localized to the dorsomedial hypothalamic nucleus, perifornical area and lateral hypothalamic area. Cells expressing prodynorphin were localized to the periventricular, supraoptic, paraven- tricular, ventromedial hypothalamic nuclei and the thalamus. Small numbers of single scattered cells were seen in other brain areas. A few scattered prodynorphin-expressing cells were found in the lateral hypothalamic area but, in contrast to observations in the rat, there was no colocalization with pre- proorexin. Long-term alterations in the bodyweight did not influence the level of expression of preproorexin or pro- dynorphin. These findings suggest that orexin and dynorphin may not play a direct role in appetite regulation in sheep, although regulation at the level of the receptors for these peptides remains a possibility. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: in situ hybridization, mRNA expression, appetite regulation, neruoendocrine regulation, periventricular hypo- thalamic nucleus. A number of neuropeptides, produced in various hypotha- lamic nuclei regulate appetite/food intake and neuroendo- crine function (Schwartz et al., 2000). Changes in body- weight and/or nutritional status can affect the gene expres- sion in rodents (Schwartz et al., 2000). In sheep, recent studies have shown that reduction in bodyweight results in altered expression of neuropeptide Y (NPY), melanin con- centrating hormone (MCH), enkephalin (ENK) (Henry et al., 2000), somatostatin and growth hormone releasing hormone (Henry et al., 2001a), agouti-gene related protein and cocaine- and amphetamine-regulated transcript (CART) (Henry et al., 2001b). Immunohistochemistry shows that the level of NPY protein in the arcuate nucleus (ARC) is increased with reduction in the bodyweight of sheep (Barker-Gibb and Clarke, 1996). The orexin (ORX) peptides A and B (Sakurai et al., 1998) also known as hypocretins (de Lecea et al., 1998), have been implicated in the regulation of feeding behavior in rodents (Sakurai et al., 1998; Sakurai, 1999) and in neuroendocrine function (van den Pol et al., 1998; Date et al., 1999). ORX receptors are found throughout the rat brain (Hervieu et al., 2001; Marcus et al., 2001), suggest- ing multiple functions. In rats, central injections of ORX enhance food intake (Dube et al., 1999; Haynes et al., 1999) suppress prolactin release (Russell et al., 2000) and may either inhibit (Tamura et al., 1999) or stimulate (Pu et al., 1998) luteinizing hormone secretion. Intracerebroven- tricular injections of ORX-B has a short-term effect to stimulate feeding in sheep (Sartin et al., 2001) and intra- muscular injection causes a modest rise in food intake in young pigs (Dyer et al., 1999). ORX peptides also play an integral role in arousal in rodents (Kilduff and Peyron, 2000; Chemelli et al., 1999; Estabrooke et al., 2001). ORX- producing cells are exclusively localized to the dorsome- dial hypothalamic nucleus (DMH), zona incerta (ZI), lateral hypothalamic area (LHA) and perifornical area (PFA) in the rat and sheep brain (Sakurai et al., 1998; de Lecea et al., 1998; Iqbal et al., 2001a). Fasting up-regulates prepro- ORX (ppORX) mRNA expression in the hypothalamus of the rat (Sakurai et al., 1998), and leptin treatment modifies this response (Lopez et al., 2000). We have shown that almost all ORX-containing cells of the sheep brain express the long form of the leptin receptor (Iqbal et al., 2001a,b) further suggestive of a role in the regulation of food intake. Opioid peptides and their receptors have also been implicated in the regulation of appetite/food intake as well as neuroendocrine and autonomic function (Gosnell et al., 1986; Glass et al., 1999). Dynorphin (DYN), which pre- dominantly binds to the -receptor subtype of opioid re- ceptor, stimulates feeding in sheep when injected in the lateral cerebral ventricle (Baile et al., 1987). Earlier immu- nohistochemical studies in sheep reported that DYN cell bodies are predominantly localized to the suprachiasmatic *Corresponding author. Tel: +61-03-9594-4392; fax: +61-03-9594- 6125. E-mail address: javed.iqbal@med.monash.edu.au (J. Iqbal). Abbreviations: AHA, anterior hypothalamic area; ARC, arcuate nucleus; CART, cocaine- and amphetamine-regulated transcript; DIG, digoxigenin; DMH, dorsomedial hypothalamic nucleus; DYN, dynorphin; ENK, enkephalin; LHA, lateral hypothalamic area; MCH, melanin concentrating hormone; NPY, neuropeptide Y; ORX, orexin; OVX, ovariectomized; PB, phosphate buffer; Pe, periventricular hypo- thalamic nucleus; PFA, perifornical area; PHA, posterior hypothalamic area; POMC, pro-opiomelanocortin; ppORX, preproorexin; pDYN, prodynorphin; PVN, paraventricular hypothalamic nucleus; SCN, su- prachiasmatic nucleus; SON, supraoptic nucleus; TEA, triethanolamine; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta. Neuroscience 118 (2003) 217–226 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00815-1 217