GLUTAMATE-IMMUNOREACTIVE NEURONS AND THEIR GONADOTROPIN-RELEASING-HORMONE NEURONAL INTERACTIONS IN THE MONKEY HYPOTHALAMUS

Glutamate (Glu) is the most prevalent excitatory neurotransmitter in t he brain and has been implicated in the regulation of GnRH secretion i n several mammalian species, including the monkey. To investigate the neuroanatomical basis for Glu-GnRH interactions, we performed an immun ocytochemical study at both the light and electron microscopic levels on the brains of four female and five male macaques. Initially, we det ermined the location of Glu-immunoreactive (-ir) elements using a mono clonal antibody specific for glutaraldehyde-fixed Glu (Glu-2) and 3,3' -diaminobenzidine-4-HCl (DAB). Glu-ir was observed in the cytoplasm an d to a variable degree in the nuclei of neurons in the diencephalon. C ytoplasmic staining was particularly intense in numerous neurons in th e arcuate nucleus, supraoptic nucleus, and many paraventricular nucleu s neurons. Short Glu-ir processes were evident in these and other hypo thalamic regions and were extremely dense in the infundibular stalk an d median eminence. Prior absorption of the Glu-2 antibody with a Glu-g lutaraldehyde-BSA conjugate completely abolished all immunostaining in both neuronal nuclei and cytoplasm. Double label Glu-GnRH immunostain ing for light microscopy was performed using Glu-2 and DAB without enh ancement, and a polyclonal antibody (LR(1) or LR(2)) with silver-enhan ced DAB for Glu and GnRH, respectively. Glu-ir interactions with GnRH- ir cell bodies were not apparent, but a few Glu-ir axons seemed to con tact GnRH-ir dendrites in the organum vasculosum of the lamina termina lis, medial septum, and arcuate nucleus regions. Reciprocal interactio ns occurred more frequently, however, in which GnRH-ir axons and dendr itic fibers engaged Glu-ir cell bodies en passant, particularly toward the medial and posterior hypothalamus. For ultrastructural analyses, Glu-ir elements were stained with the Glu-2 antibody and 15 nm immunog old or DAB. Electron microscopy demonstrated that Glu-ir was associate d with clear microvesicles within the neuronal cytoplasm. Glu-ir proce sses made classical asymmetrical synapses with one another and receive d asymmetrical synapses from unlabeled afferents. In sections double l abeled for Glu with immunogold and for GnRH with DAB, axe-somatic inte ractions were not observed. However, axo-dendritic Glu-GnRH synapses w ere seen, which usually exhibited Glu-ir labeling of terminal vesicles and inconsistent postsynaptic densitites, with GnRH-ir neurosecretory granules sometimes congregated in the apposing dendrite or spine. Sur prisingly, reverse GnRH-Glu interactions were observed more frequently . These included well defined axo-dendritic synapses with GnRH-ir micr ovesicles near the synaptic density as well as axo-dendritic and dendr o-dendritic contacts in which GnRH-ir granules abutted the terminal me mbrane, but with few other specializations of the interacting profiles . The findings show that Glu neurons are present in the monkey hypotha lamus and demonstrate for the first time in any species that the GnRH neuronal network receives synaptic input from this excitatory neurotra nsmitter.