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Prenatal exposure to diazepam (DZ), a positive allosteric modulator of the γ-aminobutyric acidA (GABAA) receptor complex, exerts profound effects that become more evident during puberty and in many cases are sex-specific, suggesting that such exposure interferes with the activity of steroid hormones. Apart from their well known effects on the genome, the reduced metabolites of many steroid hormones also interact directly with membrane receptors, including those for N-methyl- d -aspartate (NMDA). In this study, we compared the effects of several neurosteroids on NMDA receptors from normal rats and those exposed in utero to DZ (1.25 mg/kg per day) from the 14th through the 20th day of gestation. In superfused rat hippocampal synaptosomes, activation of the NMDA receptor stimulates the basal release of [ 3 H ]noradrenaline ([ 3 H ]NA), which was used in our study as an index of receptor function. [ 3 H ]NA release was evoked in a concentration-dependent manner by NMDA (100 μM) plus glycine (GLY). The maximal increase (68.23±3.86%) with respect to basal release was achieved with a GLY concentration of 10 μM, and the EC50 for GLY was 0.1 μM. Release stimulated by 100 μM NMDA+0.1 μM GLY was not modified by any of the neurosteroids tested, with the exception of pregnenolone sulfate (PREG-S), which produced a 78.57±3.94% reduction in release at the maximal concentration used (0.3 μM). In synaptosomes from animals exposed in utero to DZ, the inhibitory effect of PREG-S was reduced by 46.55±2.33%. Given the important roles played by NMDA receptors in physiological and pathological processes within the central nervous system (CNS), characterization of NMDA receptor modulation is an important objective. The fact that this modulation can be altered by exposure in utero to DZ indicates that the behavioral abnormalities observed in exposed animals might be partially attributed to an altered sensitivity of NMDA receptors to the modulatory effects of neurosteroids.

The pH fluctuations have been often interpreted as an insufficient regulation or as a consequence of the onset of pathological events, such as ischemia, in which a significant decrease in pH levels occurs. Neurotransmitter release appears to be affected by pH drop significantly. In this study, we investigated the effect of an extracellular and an intracellular acidification on tritiated dopamine release ([3H]-DA release), from superfused rat hypothalamic synaptosomes. When compared to basal release, extracellular acidification, due to a reduction in the external pH of the nominally carbonic-free superfusion media, provoked a significant increase in [3H]-DA release that showed a sensitiveness to calcium omission. Intraterminal acidification, obtained blocking the Na(+)/H(+) exchanger by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and 5-(N,N-dimethyl)-amiloride (DMA), induced a significant increase in [3H]-DA outflow which occurred in a calcium-dependent manner (80% inhibition in absence of calcium from superfusion media). To further promote an intraterminal acidification through a H(+) inner accumulation, the proton ionophore nigericin was used. At every dose employed (10 microM), this compound induced a significant increase in [3H]-DA outflow, compared to basal release. Nigericin-evoked [3H]-DA release showed a 50% decrease when calcium was omitted from superfusion media. When BAPTA-AM, a chelator of intracellular calcium, was added, nigericin-evoked [3H]-DA was completely abolished. These data indicate that [3H]-DA release can be induced by extracellular acidification due to a lowering of external pH and by an intraterminal acidification due to an internal proton accumulation. The mechanism that can trigger this exocytotic process appears to depend on calcium presence, and in particular, on an increased intraterminal calcium availability.