Sex differences in the control of neuronal nitric oxide synthase by GABA-A receptors in the developing rat diencephalon

The nitric oxide free radical (NO), which is synthesized by neuronal nitric oxide synthase (nNOS), is known to play an important morphogenetic role in the developing rat brain. In the cortex, the levels of nNOS are regulated by phosphorylated cAMP response element binding protein (pCREB) downstream of GABA-A receptor activation. During early stages of neonatal development, binding of GABA to its type A receptors leads to depolarization of the neuronal membrane. One of the developmental processes mediated through GABA-A receptors is the sexual differentiation of the brain. In the present work, we investigated the effect of GABA-A receptor activation on nNOS and pCREB immunoreactivity in the developing diencephalon of 5-day-old male and female rats. Our results showed that in the bed nucleus of the stria terminalis activation of GABA-A receptors leads to increased numbers of nNOS, and pCREB as well as nNOS-pCREB doubly immunopositive cells only in the males while in the posterior hypothalamus this effect is observed in both sexes. The GABA-A receptor-mediated increase in nNOS and pCREB is abolished when L-type voltage-gated Ca2+ channels are blocked. These results indicate that the following mechanism could be operating in a gonadal hormone-dependent and brain area-specific manner during neonatal rat brain development: Depolarization following GABA-A receptor activation leads to opening of L-type voltage-gated calcium channels, resulting in an increased Ca2+ influx, which in turn leads to phosphorylation, and thus activation of the transcription factor CREB; the phosphorylated CREB can then induce nNOS.