Central glucostasis is a critical monitored variable in neuroendocrine regulation of pituitary LH secretion. Glucoprivic signals originating within the caudal hindbrain suppress LH. Septopreoptic mu opioid receptors (mu-R) function within neural pathways maintaining basal LH levels and mediate the effects of diverse physiological stimuli on hormone release. To identify potential sites in the septopreoptic area where ligand neuromodulatory actions may occur in response to hindbrain glucoprivic signaling, the present studies evaluated the distribution of mu-R-immunoreactive (-ir) neurons in the septopreoptic area that are genomically activated in response to caudal fourth ventricular (CV4) delivery of the glucose antimetabolite, 5-thioglucose (5TG). The effects of lateral ventricular pretreatment with the selective mu-R antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), on LH secretory and GnRH neuronal transcriptional responses to hindbrain glucoprivation were also evaluated. Estradiol benzoate- and progesterone-primed, ovariectomized female rats were treated by CV4 administration of 5TG or the vehicle, saline, at the onset of the afternoon LH surge. The inhibitory effects of hindbrain glucoprivation on mean plasma LH levels as well as colabeling of rostral preoptic GnRH neurons for Fos-ir were attenuated in animals pretreated by lateral ventricular delivery of CTOP. Dual immunocytochemical labeling for septopreoptic mu-R-ir and Fos-ir demonstrated a robust induction of Fos expression by receptor-positive neurons within discrete septopreoptic sites in response to CV4 5TG, a genomic response that was diminished by CTOP pretreatment. The current studies provide novel evidence for the transcriptional activation of neuroanatomically characterized, mu-R-expressing neurons by decreased hindbrain glucose utilization and show that the functional status of mu-R is critical for maximal induction of the Fos stimulus-transcription cascade in these cells by central glucoprivic signaling. The finding that receptor antagonist-mediated suppression of this genomic response is correlated with increased reproductive neuroendocrine output supports a role for these discrete mu-R-expressing neuron populations as substrates for ligand regulatory effects on the GnRH-pituitary LH axis during neuroglucopenia.

Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. O RPHANIN FQ (OFQ), also known as nociceptin, is a member of the family of endogenous opioid peptides, which also includes ␤-endorphin, enkephalins, and dynorphin (1). Whereas sharing some homology with dynorphin, OFQ does not exhibit appreciable binding affinity for classical opioid receptors (1, 2); rather it functions as an endogenous ligand for the opioid receptor like (ORL)-1 receptor (1). OFQ cells are present in the preoptic area, anterior hypothalamus, and arcuate nucleus of the hypothalamus of the rat (3) and human (4). Functionally, OFQ has been implicated in a variety of systems including pain (5), anxiety (6), cardiovascular function (7), food intake (8), circadian rhythms (9), and cognition (10). There is also evidence that OFQ may play a role in neuroendocrine function: intracerebroventricular delivery of OFQ in the rat stimulates GH and prolactin (11, 12) and has been reported to both stimulate (13) and inhibit (14) corticosterone secretion. Sinchak et al. (15) have shown that OFQ delivered into the ventromedial nucleus facilitates lordosis in female rats in a dose-dependent manner. However, there has been relatively little attention to the possible role of OFQ in reproductive neuroendocrine function, specifically in the control of GnRH secretion.GnRH neurons, and their projections to the median eminence, control the secretion of pituitary LH and thus comprise the final common pathway for the neuroendocrine control of reproduction (16). Endogenous opioid peptides are important regulators of the GnRH system, controlling both the preovulatory surge (17) and pulsatile secretion (18) of GnRH. OFQ has been shown to inhibit forskolin-induced GnRH secretion in a dose-dependent manner from rat hypothalamic fragments (19). OFQ cells in the preoptic area and anterior hypothalamus overlap the site of a majority of GnRH perikarya (20 -22). In addition, OFQ fibers and ORL-1 receptors are present in high abund...

It is now clear that seasonal breeding in ewes is due to an increase in response to estradiol (E(2)) negative feedback in the nonbreeding season (anestrus) that is mediated by the A15 group of dopaminergic (DA) neurons. Because A15 cells do not contain estrogen receptors, we have postulated the presence of estrogen-responsive afferents and recently reported evidence that input from neurons containing gamma-aminobutyric acid (GABA) contribute to the control of A15 activity by E(2). However, GABAergic afferents account for only a fraction of A15 synaptic input and do not appear to vary with season. We therefore investigated the possible role of stimulatory glutamatergic input to A15 neurons. In experiments 1 and 2, local administration into the A15 of either a N-methyl-D-aspartate (NMDA) receptor or a kainate/alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor antagonist stimulated episodic LH secretion in a dose-dependent manner in ovary-intact anestrous ewes. In experiment 3, we examined the number of glutamatergic close contacts onto A15 neurons using dual immunocytochemistry in tissue from E(2)-treated ovariectomized anestrous and breeding season ewes. All A15 DA neurons were contacted by glutamatergic vesicles, and the number of close contacts was significantly higher in anestrus than the breeding season. Finally, using a triple-label immunocytochemistry procedure, we did not observe any colocalization of markers for GABA and glutamate in vesicles contacting A15 neurons. These results support the hypothesis that glutamatergic afferents actively stimulate A15 DA neurons in ovary-intact anestrous ewes and raise the possibility that alterations in this input may contribute to increased A15 neural activity during anestrus.

The neurochemical mechanisms underlying hindbrain glucoprivic suppression of the luteinizing hormone (LH) surge are not known. A body of experimental evidence supports the view that gonadal steroid positive-feedback action on the reproductive neuroendocrine axis relieves tonic GABAergic inhibition of gonadotropin-releasing hormone neurons by diminishing preoptic release of this neurotransmitter. The present studies evaluated the hypothesis that hindbrain glucoprivic attenuation of the LH surge may be correlated with site-specific modifications in gonadal steroid suppression of γ-aminobutyric acid release in this region of the brain. Individual septopreoptic loci were microdissected from the brains of estrogen, progesterone-primed ovariectomized female rats injected with the glucose antimetabolite, 5-thioglucose (5-TG), or vehicle into the caudal fourth ventricle during the ascending phase of the surge, and analyzed by high-performance liquid chromatography. The data show that 5- TG administration increased GABA release within the rostral preoptic area (rPO), anteroventral periventricular nucleus (AVPV), and median preoptic nucleus (MEPO), relative to the vehicle-treated controls, but did not alter neurotransmitter release in other structures evaluated. The rate of GABA turnover in each brain site was equivalent between animals injected with the µ opioid receptor antagonist CTOP and 5-TG versus their vehicle-treated controls. These results constitute novel evidence for site-specific modulation of steroid positive-feedback suppression of this inhibitory neurotransmitter by caudal hindbrain signaling of glucose insufficiency, and support the need for neurochemical characterization of glucoprivic-sensitive afferent input to GABAergic neurons terminating within the rPO, AVPV, and MEPO, as well as the relevance of enhanced local GABA release for reproductive neuroendocrine function.

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.