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

Glutamate (Glu) is the most prevalent excitatory neurotransmitter in the brain and has been implicated in the regulation of GnRH secretion in several mammalian species, including the monkey. To investigate the neuroanatomical basis for Glu-GnRH interactions, we performed an immunocytochemical study at both the light and electron microscopic levels on the brains of four female and five male macaques. Initially, we determined the location of Glu-immunoreactive (-ir) elements using a monoclonal antibody specific for glutaraldehyde-fixed Glu (Glu-2) and 3,3'-diaminobenzidine-4-HCl (DAB). Glu-ir was observed in the cytoplasm and to a variable degree in the nuclei of neurons in the diencephalon. Cytoplasmic staining was particularly intense in numerous neurons in the arcuate nucleus, supraoptic nucleus, and many paraventricular nucleus neurons. Short Glu-ir processes were evident in these and other hypothalamic regions and were extremely dense in the infundibular stalk and median eminence. Prior absorption of the Glu-2 antibody with a Glu-glutaraldehyde-BSA conjugate completely abolished all immunostaining in both neuronal nuclei and cytoplasm. Double label Glu-GnRH immunostaining for light microscopy was performed using Glu-2 and DAB without enhancement, and a polyclonal antibody (LR(1) or LR(2)) with silver-enhanced 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 contact GnRH-ir dendrites in the organum vasculosum of the lamina terminalis, medial septum, and arcuate nucleus regions. Reciprocal interactions occurred more frequently, however, in which GnRH-ir axons and dendritic 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 immunogold or DAB. Electron microscopy demonstrated that Glu-ir was associated with clear microvesicles within the neuronal cytoplasm. Glu-ir processes made classical asymmetrical synapses with one another and received asymmetrical synapses from unlabeled afferents. In sections double labeled for Glu with immunogold and for GnRH with DAB, axe-somatic interactions were not observed. However, axo-dendritic Glu-GnRH synapses were 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. Surprisingly, reverse GnRH-Glu interactions were observed more frequently. These included well defined axo-dendritic synapses with GnRH-ir microvesicles near the synaptic density as well as axo-dendritic and dendro-dendritic contacts in which GnRH-ir granules abutted the terminal membrane, but with few other specializations of the interacting profiles. The findings show that Glu neurons are present in the monkey hypothalamus and demonstrate for the first time in any species that the GnRH neuronal network receives synaptic input from this excitatory neurotransmitter.