Figure 7.

iPN Inhibition Imposes a High-Pass Filter on ePN Synaptic Output

(A) Raw two-photon images of spH fluorescence in ePN projections to the LH at the indicated stimulation frequencies, in the absence (top) or presence (bottom) of 50 μM GABA. ePN axons were stimulated for 5 s by passing 1 ms pulses of current via an extracellular electrode.

(B) Average spH fluorescence changes in ePN projections to the LH, evoked by electrical stimulation at the indicated frequencies, in the absence (black) or presence (red) of 50 μM GABA (mean ± SEM, n = 5 flies).

(C) Average ratio of integrated spH fluorescence transients (areas under the fluorescence traces during 5 s electrical stimulation) in the presence and absence of 50 μM GABA (mean ± SEM, n = 5 flies). The ratios of ΔF/F at 0 versus 50 μM GABA differ across frequencies (p < 0.0001; one-way repeated measures ANOVA). Asterisks indicate significant differences between the presence and absence of 50 μM GABA at specific frequencies (^∗p < 0.05, ^∗∗p < 0.005; paired t test).

(D) Thermally evoked iPN activity has a similar effect on ePN synaptic release as bath application of 50 μM GABA. Flies carried GH146-QF, QUAS-spH, Mz699-GAL4, and UAS-dTRPA1 transgenes. spH fluorescence changes were measured at two electrical stimulation frequencies (40 and 130 Hz) while flies were held at 25°C and 32°C. Columns depict the ratios of the integrated spH fluorescence transients (areas under the fluorescence traces during 5 s electrical stimulation trains) between 32°C and 25°C (mean ± SEM, n = 7–8 flies). A ratio of 1 indicates no effect of thermally evoked iPN activity on ePN synaptic release, whereas a ratio <1 indicates that iPN activity inhibits ePN output. Red brackets denote significant differences (p < 0.05, with Bonferroni-corrected paired t tests to compare the 32°C:25°C ratios at 40 versus 130 Hz within genotypes and one-way ANOVA with a Tukey-Kramer post hoc test to compare the ratios of the 32°C:25°C ratios at 40 versus 130 Hz across genotypes).