PROJECT SUMMARY/ABSTRACT The respiratory control system displays a remarkable capacity for neuroplasticity, imparting flexibility of breathing in response to changing physiological or environmental conditions across the lifespan. Gonadal hormones (estrogens, progestins, androgens) exert powerful modulatory effects and directly influence the development of neuroplasticity in other neural control areas. Yet, the role of gonadal hormone signaling in the development respiratory neuroplasticity has not been clearly defined. The overarching hypothesis guiding this proposal is that gonadal hormone signaling within the spinal cord is necessary to enable expression of respiratory neuroplasticity through regulation of spinal microglia. Five important preliminary findings support this hypothesis. First, while estrogens and progestins are typically associated as the principal gonadal hormones in females, whereas androgens are considered the primary gonadal hormone in males, it is in fact estradiol in both sexes that is required to permit respiratory neuroplasticity. Second, testosterone is aromatized to estradiol directly within the spinal cord to elicit plasticity in males. Third, the estrogen receptor isoforms (ERα, ERβ and GPER) essential for enabling respiratory plasticity are unique in females and males. Fourth, estradiol supplementation or the pharmacological activation of spinal estrogen receptors is sufficient to rescue plasticity following removal of the gonads in both sexes. Fifth, treating the spinal cord with a localized anti-inflammatory, or reducing the population of CNS microglia (using a CSF1R inhibitor), is sufficient to restore respiratory neuroplasticity in rats of both sexes following removal of the gonads, indicating a role for spinal microglia in the estrogen-induced recovery of plasticity. Using rigorous neurophysiologic measures of respiratory neuroplasticity in combination with estrogen receptor pharmacology, targeted gene manipulation by siRNA knockdown, flow cytometry, mass spectroscopy, and protein biochemistry, we will dissect the role of spinal estrogen receptor signaling for expression of respiratory neuroplasticity. Three specific hypotheses will be tested: 1) Spinal ER signaling is necessary for induction of respiratory neuroplasticity in female and male rats; 2) Spinal estrogen signaling is sufficient to restore respiratory plasticity when sex steroid levels are systemically reduced; and 3) Estrogen permits respiratory plasticity through modulation of spinal cord microglia. These studies address a critical gap in our basic biological understanding of respiratory neural function; how sex hormone signaling enables development of respiratory neuroplasticity. In addition, our results will directly inform ongoing translational studies targeting mechanisms of respiratory neuroplasticity for therapeutic benefit.