(A) Schematic showing experimental design in B-E. (B) Averaged fIPSP paired pulse representative traces at an interstimulus interval of 100 ms in 0.5 mM Ca2+ (pulse one black, pulse two gray), 1 mM Ca2+ (pulse one pink, pulse two light pink) or 2 mM Ca2+ (pulse one blue, pulse two light blue). (C) Paired pulse ratio (PPR) (P2/P1) was lower in 2 mM extracellular Ca2+ than in 0.5 mM Ca2+ or 1 mM Ca2+ and lower in 1 mM Ca2+ than 0.5 mM Ca2+, indicating that extracellular Ca2+ concentration is positively associated with presynaptic release probability (repeated measures two-way ANOVA F(2,48) = 45.96, p<0.0001, Tukey’s post hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.0145, 0.5 mM Ca2+ vs 2 mM Ca2+ p<0.0001, 1 mM Ca2+ vs 2 mM Ca2+ p<0.0001, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (D) Quantification of baseline peak amplitudes confirming that greater extracellular Ca2+ concentration increases presynaptic release probability and is associated with greater peak amplitude of fIPSPs (one-way ANOVA F(2,14) = 13.85, p=0.0005, Tukey’s post-hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.0072, 0.5 mM Ca2+ vs 2 mM Ca2+ p=0.0005, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (E) Time course showing block of 0.1 Hz evoked fIPSPs following PTX application in 0.5 mM Ca2+ (black), 1 mM Ca2+ (pink) or 2 mM Ca2+ (blue). Greater presynaptic release probability via increased extracellular Ca2+ is associated with faster block of GABAARs (non-linear regression single exponential fit, Sum-of-Squares F test F(6, 2048)=571.3, p<0.0001, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (F) Schematic showing experimental design in G-I. (G) Time course showing evoked fIPSP response to 0.1 Hz stimulation before (black) and following application of PTX at rest for 0 min (gray), 5 min (red) or 10 min (blue). Treatment of PTX for increasing amounts of time resulted in a lesser remaining response upon continuation of 0.1 Hz stimulation (n = 6 for 0 min, n = 7 for 5 min, n = 8 for 10 min). (H) Averaged fIPSP representative traces at baseline (black) and first fIPSP response following 0 min (gray), 5 min (red) or 10 min (blue) of PTX administration at rest. (I) Quantification of unblocked GABAAR mediating response remaining after 0, 5 or 10 min perfusion of PTX at rest, adjusted for upper and lower boundaries of fIPSP response. Following PTX administration at rest, the unblocked evoked response is 103.12 ± 3.82% of baseline after 0 min (gray), 88.0 ± 4.89% of baseline after 5 min (red) and 34.05 ± 5.37% of baseline after 10 min (blue) (one-way ANOVA F(2,18) = 56.30, p<0.0001, Tukey’s post-hoc testing 0 min vs 10 min p<0.0001, 5 min vs 10 min p<0.0001, n = 6 for 0 min, n = 7 for 5 min, n = 8 for 10 min). Thus a response to evoked stimulation remains following 10 min of GABAAR block by spontaneous neurotransmission, indicating partial segregation. Graphs are mean ± SEM. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.