Postsynaptic receptor occupancy during evoked transmission at striatal GABAergic synapses in vitro

The effect of benzodiazepines (BZs) on GABA(A)-ergic synaptic responses depends on the control receptor occupancy: the BZ-induced enhancement of receptor affinity can lead to greater peak amplitudes of quantal responses only when, under normal conditions, receptors are not fully saturated at peak. Based on this fact, receptor occupancy at the peak of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) has been assessed in various mammalian neuronal preparations. To use the same principle with compound (or multiquantal), action potential-evoked IPSCs, complications introduced by quantal asynchrony in conjunction with the BZ-induced increase in the decay time of the quantal responses have to be overcome. We used a simple analytic convolution model to calculate expected changes in the rise time and amplitude of postsynaptic currents when the decay time constant, but not the peak amplitude, of the underlying quantal responses is increased, this being the expected BZ effect at saturated synapses. Predictions obtained were compared with the effect of the BZ flunitrazepam on IPSCs recorded in paired pre- and postsynaptic whole cell voltage-clamp experiments on striatal neurons in cell culture. In 22 pairs, flunitrazepam (500 nM) reliably prolonged the decay of IPSCs (49 +/- 19%, mean +/- SE) and in 18 of 22 cases produced an enhancement in their peak amplitude that varied markedly between 3 and 77% of control (26.0 +/- 5.3%). The corresponding change in rise time, however (+0.38 +/- 0.11 ms, range -0.8 to +1.3 ms) was far smaller than calculated for the observed changes in peak amplitude assuming fixed quantal size. Because therefore an increase in quantal size is required to explain our findings, postsynaptic GABA(A) receptors were most likely not saturated during impulse-evoked transmission at these unitary connections. The peak amplitudes of miniature IPSCs in these neurons were also increased by flunitrazepam (500 nM, +26.8 +/- 6.6%), and their decay time constant was increased by 26.3 +/- 7.3%. Using these values in our model led to a slight overestimate of the change in compound IPSC amplitude (+28 to +30%).