Quantifying G Protein-coupled Estrogen Receptor Expression in the Rat Ventral Cervical Spinal Cord Across the Estrus Cycle
17β-estradiol (E2) is the most abundant, neuroactive form of estrogen. Serum E2 concentrations fluctuate in female rats in a similar fashion as humans, with high serum E2 occurring during the proestrus phase, and low serum E2 occurring during the estrus phase of the normal 4-5 day rat estrous cycle. E2 exerts physiological effects on phrenic motor neurons (PhMNs), located between levels C3-C6 of the ventral spinal cord in mammals, that innervate the diaphragm to control breathing. Prior studies from our laboratory demonstrated that female rats only express neuroplasticity in the phrenic motor circuit during the proestrus phase (when E2 levels are high). The G protein-coupled estrogen receptor (GPER) is expressed in spinal cord motor neurons and has been implicated in fast-acting E2 intracellular signaling processes. Our prior work suggests that fast-acting E2 mechanisms are sufficient to restore respiratory neuroplasticity in ovariectomized rats with low serum E2, potentially implicating GPER in phrenic motor function and neuroplasticity. GPER expression fluctuates relative to available E2 in other areas of the central nervous system including the hippocampus, with higher expression during proestrus. Thus, we hypothesized that GPER expression would also be higher during proestrus compared to estrus in the rat ventral cervical spinal cord. We quantified GPER expression using Western Blot analysis of isolated ventral cervical spinal cord segments containing C3-C6 (location of phrenic motor neurons) in young adult female Sprague Dawley rats during proestrus and estrus. Our data indicate that GPER expression is similar (p>0.05) between the proestrus and estrus phases of the cycle, suggesting that GPER expression remains somewhat constant across the estrous cycle. These data are being confirmed with immunohistochemical analysis. Overall, our findings indicate that serum E2 levels do not influence GPER expression in the female rat ventral cervical spinal cord. Future studies will dissect GPER intracellular signaling pathways potentially involved in restoring respiratory neuroplasticity in conditions of reduced E2, as well as quantify GPER protein expression in PhMNs across the estrus cycle.
Anne Davis, Jessica Grittner, MS, CCC-SLP, Edgar Juarez Lopez, Brendan Dougherty, PT, PhD