Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: An immunohistochemical study using an antibody against vesicular glutamate transporter-2.

Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors, including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion by means of E-responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST), and A1 and A2 cell groups in the brainstem. In three ewes, E receptor-alpha was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA, and 37-58% of neurons in the BnST. E injection (i.m. or i.v.) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P < 0.01 and P < 0.001, respectively). In six ewes, injection of the retrograde tracer Fluoro-Gold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labeling of varicosities in the POA showed colocalization of VGluT2 in 12.5 +/- 3% of dopamine beta-hydroxylase-immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brainstem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E-responsive glutamatergic neurons arising from the brainstem, the BnST, and ARC/VMH provide input to the POA and may be involved in the regulation of GnRH secretion.