IMMUNOCYTOCHEMICAL COLOCALIZATION OF GABA-B RECEPTOR SUBUNITS IN GONADOTROPIN-RELEASING HORMONE NEURONS OF THE SHEEP J. H. SLIWOWSKA, a H. J. BILLINGS, a R. L. GOODMAN b AND M. N. LEHMAN a1 * a Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, USA b Department of Physiology, West Virginia University Health Sciences Center, Morgantown, WV 26506-9229, USA Abstract—GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype me- diates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B re- ceptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these sub- units in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the per- centage of GnRH neurons expressing GABA-B receptor sub- units tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B recep- tors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: sheep, gonadotropin releasing hormone, GABA receptor, preoptic area, hypothalamus, seasonality. Neurosecretion of gonadotropin-releasing hormone (GnRH) represents the final common pathway for regulation of reproduction in mammals. Sheep, and other seasonally breeding mammals, display an annual cycle of fertility that is associated with both structural and functional plasticity in the activity of GnRH and other neurons (Lehman et al., 2002). In the ewe, seasonal reproductive transitions are due to a striking increase in the responsiveness of the GnRH system to the negative feedback action of estradiol during anestrus. Thus, low concentrations of estradiol, which do not inhibit the frequency of GnRH pulses during the breeding season can markedly suppress pulsatile GnRH and luteinizing hormone (LH) secretion during anestrus (Legan et al., 1977, Karsch et al., 1993, 1984). To understand the neuronal events that lead to this change in responsiveness to estradiol negative feedback, it is nec- essary to identify the phenotype of steroid-sensitive neu- ronal populations that seasonally alter the activity of the GnRH neurons. This action of estrogen is unlikely to occur directly within GnRH neurons, since GnRH neu- rons lack estrogen receptor (ER)- protein (Herbison et al., 1993; Lehman and Karsch, 1993) and ER  agonists are unable to mimic the negative feedback effects of estradiol in anestrous ewes (Hardy et al., 2003). There- fore, some other steroid-sensitive neuronal system must relay this information to GnRH neurons. Manipulation of brain regions or terminals that are afferent to GnRH neurons can block the inhibitory effects of estradiol on GnRH secretion during anestrus; this strongly implicates GnRH afferents as having a role in seasonal changes in responsiveness to estradiol negative feedback (Lehman et al., 2002). GABA neurons have been viewed as one candidate for this action. GABA is the primary inhibitory neurotransmitter in the brain and acts via three different types of receptor: GABA-A, GABA-B and GABA-C (Bormann, 2000; Chebib and Johnston, 1999). GABA-A and GABA-C receptors are ionotropic receptors that gate Cl - channels, whereas the GABA-B receptor is a metabotropic receptor that couples to Ca +2 and K + channels via G proteins and second messenger systems (Bormann, 1988; Bowery and Enna, 2000; Hammond, 2001). GABA neurons expressing ER are found in the ovine preoptic area (POA) (Herbison et al., 1993), the site of the majority of GnRH cell bodies. More- over, close contacts between GABAergic terminals and preoptic GnRH cell bodies were identified in sheep (Her- bison et al., 1993) and GABAergic synapses onto GnRH neurons in other mammals have been observed (Leranth et al., 1985). Numerous studies have shown that adminis- tration of GABA, or GABA agonists and antagonists, alters LH secretion both in ewes (Clarke and Scott, 1993; Jack- son and Kuehl, 2002, 2004; Scott and Clarke, 1993; To- 1 Present address: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada. *Correspondence to: M. N. Lehman, Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada N6A 5C1. Fax: +1-519-850-2906. E-mail address: Michael.Lehman@schulich.uwo.ca (M. N. Lehman). Abbreviations: ER, estrogen receptor; GnRH, gonadotropin-releasing hormone; ir, immunoreactivity; LH, luteinizing hormone; MBH, medial basal hypothalamus; NDS, normal donkey serum; POA, preoptic area. Neuroscience 141 (2006) 311–319 0306-4522/06$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.03.039 311