Date Published: June 8, 2015
Publisher: Public Library of Science
Author(s): Weiling Yin, Zengrong Sun, John M. Mendenhall, Deena M. Walker, Penny D. Riha, Kelsey S. Bezner, Andrea C. Gore, Andrew Wolfe.
The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E2 + VEH), or estradiol + progesterone (E2+P4). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.
Reproductive activity is regulated by the coordinated release of GnRH from secretory vesicles in neuroterminals located in the median eminence. The mechanisms by which GnRH terminals release the neuropeptide are complex, as they involve intrinsic processes within the GnRH neurons themselves (e.g., electrophysiological activity) together with the coordination of inputs from other neurotransmitters that may act upon GnRH cells through receptors and intracellular signaling mechanisms. Glutamate, an excitatory neurotransmitter in the hypothalamus, is one such neurotransmitter: it stimulates GnRH gene expression [1, 2], GnRH peptide release [3–5], and GnRH electrical activity [6, 7]. Glutamate is further involved in the reproductive life transitions of puberty [2, 8] and senescence [9–13]. These effects are mediated by glutamate receptors including NMDA and non-NMDA receptors, which are detectable on GnRH cell bodies and terminals [2, 4, 10, 14–16].
The pulsatile release of GnRH is crucial for maintaining reproductive function, and this pattern is altered during reproductive aging in a species-specific manner. Previously, using an aging rat model, we showed a number of ultrastructural changes in GnRH terminals, and their relationship to glia in the median eminence, that may be related to functional changes in GnRH release [24, 39]. Here we further tested the potential role of glutamate, and its relationship to GnRH terminals in the median eminence. We applied this question to aging female rats, and in addition, we investigated hormonal regulation of this phenomenon.
Our findings provide further evidence that glutamate plays an important role in GnRH release. To our knowledge, the large dense-core vesicle localization of glutamate transporters in the central nervous system has not been shown before and therefore, its functional importance is unknown and need further investigation. We speculate that vGluT2 transports and packages glutamate within the GnRH neural process and that glutamate in large dense-core vesicles may play a role in regulating GnRH terminal function instead of synaptic transmission. Our results suggest a potentially different role of glutamate in stimulating neuroendocrine terminals in the median eminence.