Scattered GnRH neurons in the primate hypothalamus perform different functions and engage in selective interactions. To establish which interactions are relevant to neuroendocrine regulation, (1) We will identify neuroendocrine GnRH neurons in the primate, and determine the afferent inputs involved in their control. We will identify neuroendocrine GnRH neurons in pre-ovulatory female primates by retrograde labeling from the median eminence (ME) and immunocytochemical (ICC) staining for GnRH with. DAB. We will use ICC staining with colloidal gold (red) to identify their specific afferents, e.g. dopamine (DA), norepinephrine (NE), and serotonin (5-HT), the peptide beta-endorphin (b-E), gamma-aminobutyric acid (GABA), and the excitatory amino acids (EAAs) glutamate and aspartate. Synapses will be characterized at the electron microscopic (EM) level and quantified. (2) We will determine which afferents mediate estrogen (E2) and progesterone (P4) feedback on neuroendocrine GnRH neurons. Since GnRH neurons lack E2 receptors, inhibitory feedback is thought to occur through afferent DA, b-E, or GABA neurons, which concentrate E2 and in some cases P4. We will use ICC staining with heavy metal-intensified DAB (black) to reveal neuronal nuclei containing E2 receptors in pre-ovulatory female primates, and identify neuroendocrine GnRH neurons with standard DAB (brown). We will perform ICC staining for DA, b-E, or GABA neurons with colloidal gold to identify which afferents mediate E2 feedback. Types and numbers of synapses will be evaluated by EM and quantified. Using the same protocol, we will perform ICC staining for P4 receptors and determine which afferents mediate P4 feedback. We will repeat these studies in early follicular and mid-luteal phase animals to examine feedback sites and effects of changing steroid levels. Puberty in primates involves reactivation of the dormant prepubertal GnRH pulse generator. While this implies that a competent GnRH neuronal system is present in juveniles, neuroanatomical changes which ma , enhance pulsatile GnRH secretion have yet to be assessed. (3) We will determine if GnRH neuronal connections or afferent synapses are altered during the peripubertal period. We will perform double EM ICC staining for GnRH. neurons (with DAB) and their afferents (with colloidal gold) in prepubertal and adult male monkeys. We will quantify and compare the types, characteristics, and numbers of GnRH neuronal interactions and afferent synapses. Using serial plasma LH measurements (by RIA), we will identify peripubertal (nocturnal LH surge) animals, perform ICC staining, and evaluate participation of the neural changes observed. In subsequent studies, we will examine prepubertal and peripubertal female monkeys and quantify their GnRH neurointeractions. We will compare the data to that previously obtained in adult, cycling females, and correlate the observed changes with associated peripubertal events. Together, these studies will elucidate the nature of GnRH neuronal control, the generation of pulsatile GnRH secretion, and assess neuroanatomical changes contributing to the onset of puberty.
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