Random response fluctuations lead to spurious paired-pulse facilitation

We studied paired-pulse depression (PPD) of GABA(A) ergic IPSCs under conditions of reduced transmitter release (caused by Cd2+, baclofen, or reduced stimulus intensity) with whole-cell voltage clamp in CA1 pyramidal cells in vitro. The use-dependent model of paired-pulse responsiveness holds that a decrease in the probability of neurotransmitter release during the first stimulus will cause predictable changes in the paired-pulse ratio (PPR, the amplitude of the second IPSC divided by that of the first). However, the applicability of the use-dependent model to inhibitory synapses is controversial. Our results are inconsistent with this model, but are consistent with the hypothesis that random fluctuations in response size significantly influence PPR. PPR was sensitive to the extracellular stimulus intensity in all conditions. Changes in PPR were not correlated with changes in the first IPSC, but were correlated with changes in variability of the PPRs of individual traces. We show that spurious paired-pulse facilitation (PPF) can result from averaging randomly fluctuating PPRs because the method of calculating PPR as the mean of individual PPRs is biased in favor of high values of PPR. Spurious PPF can mask the intrinsic paired-pulse property of the synapses. Calculating PPR as the mean of the second response divided by the mean of the first avoids the error. We discuss a simple model that shows that spurious PPF depends on both the number of synapses recruited for release and the probability of release at each release site. The random factor can reconcile some conflicting published conclusions.