1. Oxytocin is known to act on autoreceptors of oxytocin neurones in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor-mediated synaptic inputs on these cells. 2. GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurones in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics. 3. The effect of oxytocin was observed in 70% of the magnocellular neurones recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin-sensitive SON neurones. 4. The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurones. 5. This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium.6. Intracellulax perfusion of SON neurones with GTPyS mimicked the effect of oxytocin on IPSCs, while GDP/6S, similarly applied, abolished the effect of oxytocin. 7. Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred. 8. Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurones sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurones.

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.

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SUMMARY1. The putative neurotransmitter FMRFa (Phe-Met-Arg-Phe-amide) caused an inhibitory modulation of the voltage-gated sodium current (INa) in central neurones, the peptidergic caudo dorsal cells (CDCs) of the mollusc Lymnaea stagnal7is. FMRFa reduced INa at all command potentials tested (ranging from -35 to + 20 mV), but the amplitude of the effect of FMRFa was voltage dependent, inhibition being stronger at more negative potentials (50 + 5 % reduction at half-maximal INa activation versus 25 + 8 % at the peak of the I-V curve).2. INa current traces were well fitted by a Hodgkin & Huxley based model, using m3 activation kinetics and two time constants for inactivation.3. The steady-state inactivation curve of INa was characterized by half-maximal inactivation at -42-5 + 1-81 mV and a slope factor of 4-6 + 0-28 mV. The fastest time constant of inactivation ran from 100 + 5 to 0-8 + 0-32 ms and the slower time constant from 505 +45 to 4-8 + 1-40 ms in the range -40 to -5 mV.4. FMRFa had no significant effect on either component of inactivation, nor on the voltage dependence of steady-state inactivation, nor on the maximal conductance.5. FMRFa affected the activation of INa. The activation time constant was increased, ranging from 0-75 + 0-050 to 0-22 + 0-017 ms under control and from 0-91 + 0 043 to 0-31 + 0-038 ms with FMRFa in the voltage range -25 to + 5 mV. The steady-state activation curve was shifted to less negative potentials: half-maximal activation occurred at -26-5 + 1-2 mV under control and at 23-6 + 1-4 mV with FMRFa; the slope factor (4-6 + 1-4 mV in control experiments) was not affected. The combination of slower activation kinetics and a shift in the voltage dependence of activation in the Hodgkin & Huxley based model, adequately explained the reduction of INa by FMRFa.