Central Actions of GHS-25/Bailey et al. Vol. 14, No. 1 15 Author to whom all correspondence and reprint requests should be addressed: Alex Bailey, Eurosterare Administrator, Centre for Reproductive Biology, Edinburgh EH3 9ET. E-mail: alex.bailey@obg93.obg.ed.ac.uk Endocrine, vol. 14, no. 1, 15–19, February 2001 0969–711X/01/14:15–19/$11.25 © 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 15 Growth hormone secretagogues (GHSs) increase the activity of hypothalamic arcuate nucleus neurons thought to be involved in controlling the release of growth hormone (GH). The GHS receptor is also found in hypothalamic regions not associated with the release of GH, suggesting that GHSs may influence other hypothalamic systems. This study utilized double-labeling immunocytochemical techniques to examine the hypothalamic actions of a novel non- peptide GHS, GHS-25. In common with other GHSs, GHS-25 induced significant amounts of Fos immunore- activity in the arcuate nucleus of conscious male rats. However, unlike other GHSs, GHS-25 also induced Fos immunoreactivity in the supraoptic nucleus. Double labeling revealed that approx 66% of supraop- tic nucleus cells that were Fos positive after the admin- istration of GHS-25 were also immunoreactive for oxytocin. Thus, in addition to its actions on the GH axis, GHS-25 may influence the release of neurohypo- physeal hormone. Key Words: Hypothalamus; arcuate nucleus; supraop- tic nucleus; oxytocin; vasopressin. Introduction Growth hormone (GH) release can be elicited directly from the anterior pituitary gland by growth hormone secre- tagogues (GHSs), such as GHRP-6 and MK-0677 (1,2). In addition to inducing GH release directly from the pituitary, GHSs act centrally to increase Fos protein expression in the hypothalamic arcuate nucleus and induce a prolonged in- crease in the electrical activity of neurosecretory arcuate nucleus neurons (3,4). Within the arcuate nucleus, the main populations of GHS-responsive cells are thought to include both GH-releasing hormone (GHRH) and neuropeptide Y (NPY) cells (5). Indeed, GHS mRNA has recently been localized to GHRH and NPY arcuate nucleus neurons (6,7). However, GHS receptor mRNA is also found in several other hypothalamic nuclei (e.g., the supraoptic nucleus [SON] and suprachiasmatic nucleus [SCN]), and in other discrete regions of the brain, including the CA2 and CA3 regions of the hippocampus, the dentate gyrus, and sub- stantia nigra (8). This had led to speculation that the GHS receptor (GHS-R), and therefore its endogenous ligand, may play a role in processes that are not generally associ- ated with the control of GH release. The cloning of the GHS-R has provided a context in which agonist/receptor interactions can be investigated sys- tematically. This has revealed that several structurally diverse compounds are capable of binding to the receptor and that different classes of GHSs can bind to different regions of the ligand-binding pocket of the receptor (9). This, in turn, has led to the generation of several novel GHSs that may activate intracellular pathways different from those by conventional GHSs, possibly leading to a broader range of physiologic actions. Recent studies have demonstrated that some GHSs are capable of eliciting the release of vasopressin from hypo- thalamic explants (10), and vasopressin release is thought to, at least partially, underlie the hypothalamo-pituitary- adrenal axis activation induced by GHSs (11). Cell bodies of vasopressin neurons are located in the SON, SCN, and paraventricular nucleus (PVN) of the hypothalamus (12), and all these nuclei express GHS-R mRNA (8). However, previous studies have shown that GH-releasing peptide-6 (GHRP-6) and MK-0677 do not elicit Fos immunoreactivity within these regions (3,4). As within the hypothalamic- neurohypophyseal system, the use of Fos immunocyto- chemistry as a marker of neuronal activity is considered a reliable and intensity-dependent stimulus (13); it appears that GHRP-6 and MK-0677 do not activate cells in the SON or PVN. However, because novel GHSs may interact with the GHS-R in a different manner from GHRP-6 or MK- 0677, the apparent disparities between these studies may reflect activation of different regions of the GHS-R. Therefore, in the present study, we employed the immu- nocytochemical detection of Fos protein in the rat to com- Central Actions of the Nonpeptide Growth Hormone Secretagogue GHS-25 Alex R. T. Bailey, 1 Louise Gilliver, 2 Gareth Leng, 2 and Roy G. Smith 1 1 Huffington Center on Aging, Baylor College of Medicine, Houston, TX; 2 Department of Biomedical Sciences, University Medical School, Edinburgh, EH8 9AG, UK.