Coupling of Neuronal Nitric Oxide Synthase to NMDA Receptors via Postsynaptic Density-95 Depends on Estrogen and Contributes to the Central Control of Adult Female Reproduction

Abstract

Considerable research has been devoted to the understanding of how nitric oxide (NO) influences brain function. Few studies, however, have addressed how its production is physiologically regulated. Here, we report that protein-protein interactions between neuronal NO synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic preoptic region of adult female rats is sensitive to cyclic estrogen fluctuation. Coimmunoprecipitation experiments were used to assess the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that nNOS strongly interacts with NR2B at the onset of the preovulatory surge at proestrus (when estrogen levels are highest) compared with basal-stage diestrous rats. Consistently, estrogen treatment of gonadectomized female rats also increases nNOS/NR2B complex formation. Moreover, endogenous fluctuations in estrogen levels during the estrous cycle coincide with changes in the physical association of nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95 synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous cyclicity, a process requiring coordinated activation of neurons containing gonadotropin-releasing hormone (the neuropeptide controlling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult mammalian brain to control NO release under physiological conditions.

Nitric Oxide as Key Mediator of Neuron-to-Neuron and Endothelia-to-Glia Communication Involved in the Neuroendocrine Control of Reproduction

“…Intriguingly, in contrast with previous studies suggesting that estrogen could modulate hypothalamic nNOS gene expression [58,81,82,83,84], these changes in NO secretion seen during the estrous cycle are not associated with changes in nNOS protein synthesis but rather with changes in nNOS activity [62,85]. Estradiol induces the formation of a NMDA-R-nNOS complex in neurons of the preoptic region [62,85], thus enhancing NO secretion [86] by coupling nNOS to its main stimulatory calcium influx pathway [12,14] (box).…”

“…Intracerebroventricular injection of NOS antisense oligodeoxynucleotides was shown to block the LH surge in steroid-primed ovariectomized rats [60], whereas intracerebral infusion of N G -nitro- L -arginine methyl ester ( L -NAME, a NOS inhibitor) either within the preoptic region (fig. 1) or the median eminence caused a marked disruption of rat estrous cyclicity [62,63]. Intriguingly, in mutant mice, the first targeted disruption of the nNOS gene in exon 2 did not markedly alter fertility [64].…”

“…Infusion starts at P0 (arrow pointing up) and ends 7 days later (arrow pointing down), when the pump content is exhausted. Di = Diestrus; Pro = proestrus; Es = estrus [adapted from [62], [73],with permission]. …”

“…Amperometric measurements performed with a NO-specific probe on rat hypothalamus explants showed that NO production varies during the estrous cycle in the preoptic region and that the amplitude of NO effluxes is significantly higher in proestrus (when estrogen levels are highest) than in diestrus [62] (fig. 1d).…”

“…Concurrently, the microscopic visualization of NOS catalytic activity using immunohistofluorescence to localize L -citrulline, which is formed stoichiometrically with NO [12] showed that the proportion of nNOS neurons producing NO is significantly higher in proestrus than in diestrus II in the preoptic region (average citrulline/nNOS co-expression, Di 16h 78 ± 5%, Pro 16h 90 ± 2%; t test, p = 0.049, n = 6 per stage of the estrous cycle; fig. 1c) [62]. …”