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

Institut de Médecine Prédictive et de Recherche Thérapeutique, Université du Droit et de la Santé Lille 2, Lille, Nord-Pas-de-Calais, France
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 07/2007; 27(23):6103-14. DOI: 10.1523/JNEUROSCI.5595-06.2007
Source: PubMed


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.

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    • "Both male and female pups were used for experiments. Even though, circulating testosterone at birth is already higher in males as compared to females (Amateau and McCarthy 2004), hormone exposure is too short to mediate masculinization of the P0 male brain and to promote irreversible changes in hormone-sensitive neurons (Finn et al. 1996). After decapitation and removal of the brain, the meninges and optic chiasm were discarded and the preoptic region was isolated under a binocular magnifying glass with Wecker's scissors (Moria, France). "
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    ABSTRACT: Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.
    Journal of Neurochemistry 03/2009; 109(1):214-24. DOI:10.1111/j.1471-4159.2009.05949.x · 4.28 Impact Factor
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    • "Interestingly, we have recently reported that estrogens promote cyclic fluctuations in the association between nNOS and the NMDA receptor subunit 2B (NR2B) in the preoptic region of the hypothalamus in female rats during the ovarian cycle (d'Anglemont de Tassigny et al. 2007). Thus, one mechanism involved in the regulation by estradiol of nNOS activity in neurons may reside in its increased coupling to PSD-95/NR2B complex to facilitate the access to the Ca 2+ -calmodulin located just underneath the NMDA receptor. "
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    ABSTRACT: The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N omega-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.
    Endocrinology 03/2008; 149(2):587-96. DOI:10.1210/en.2007-1260 · 4.50 Impact Factor
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