Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing gamma-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABA A or GABA B receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABA A and GABA B receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABA A-alpha1 and the GABA B-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.

Ovine reproduction is characterized by a breeding season and an anestrous season. Seasonal anestrus is induced by increased responsiveness of the GnRH neurosecretory system to estrogen (E) negative feedback. Previous studies have shown that inhibitory A15 dopamine neurons mediate this increased sensitivity to E, however, these neurons lack E receptors. In this thesis we performed three experiments. For experiment 1 we looked at the specific sites of actions that thyroid hormones act to cause neuronal plasticity of A15 DA neurons, utilizing dual-labeled immunocytochemistry. The results of this study demonstrated no change in dendrite morphology or the number of close contacts of A15 DA neurons. Because of the inconclusive data observed in Experiment 1, the final two experiments investigated the role of GABA as an estrogen-responsive afferent to A15 DA neurons. Studies have shown that GABA neurons express E receptors and local administration of a GABA A receptor agonist in the A15 elevates LH levels in anestrous ewes. In experiment 2, we tested if this effect of the GABA A agonist occurs directly on A15 neurons by determining whether A15 dopamine neurons express GABA A receptors and if the expression of the GABA A receptor increases in the breeding season, utilizing dual labeling immunocytochemistry. Approximately 30 percent of A15 DA neurons express GABA A receptors, whereas 40 percent and 80 percent colocalization was seen in A14 DA neurons and A12 DA neurons respectively. There was no seasonal difference in receptor expression in any dopamine populations investigated. The third experiment investigated the whether administration of both GABA A and GABA B receptor antagonists into the A15 would cause a suppression in LH in OVX, anestrus season ewes and if administration of a dopamine receptor antagonist reverses this suppression. Administration of the antagonists locally into the A15 caused a significant decrease in LH pulse frequency and LH concentrations, however LH amplitude was unaffected. The dopamine receptor antagonist treatment caused a significant increase in mean LH concentrations. These data are consistent with a role for GABA in mediating estrogen negative feedback in anestrus.