Fig 4. SL- and RQ,SL-expressing NMJs display hyperexcitability in evoked neurotransmission.

(A) Average EPSP amplitudes at 0.4 mM [Ca²⁺]_e for non-transgenic control (w¹¹¹⁸) or Cac-GFP-expressing lines (** p < 0.01 by one-way ANOVA with Tukey’s post-hoc vs. w¹¹¹⁸; or # p < 0.05 and ### p < 0.001 vs. WT; n ≥ 12 for all genotypes). (B) Average quantal content (QC, estimated as EPSP/mEPSP) at 0.4 mM [Ca²⁺]_e (p > 0.15 by one-way ANOVA with Tukey’s post-hoc for all genotypes, compared to both w¹¹¹⁸ and WT controls). (C) Log-log plots of extracellular calcium concentration vs. QC corrected for non-linear summation (NLS QC). There are no statistically significant differences in calcium cooperativity between genotypes (p = 0.16, linear regression analysis). (D, E) Example electrophysiological traces of (D) normal and (E) abnormal EPSP waveforms. (F) Effect of genotype on EPSP waveforms in response to 30 presynaptic pulses. “RQ only” signifies larvae with a null endogenous cac mutation rescued to viability by the RQ-expressing transgene. (G) Effect of genotype on number of extra discharges observed per 30 presynaptic pulses (* p < 0.05 and *** p < 0.001 vs. WT by one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc). (H) Penetrance and (I) severity of RQ,SL-associated extra discharge waveform dysfunction in low extracellular Mg²⁺ (6 mM). (J) NMJ recordings of 2 min spontaneous neurotransmission with an intact CNS. Measurements assessed: continuous trains of spontaneous activity > 2 sec in duration at any point in the recording; trains with postsynaptic events > 4 mV; trains with postsynaptic events > 10 mV; any observed postsynaptic event (trains or not) > 10 mV; any recording that was continuous trains of throughout (n = 9 for WT, n = 10 for RQ,SL; * p < 0.05, ** p < 0.01 by Fisher’s exact Test). All genotypes abbreviated (WT, RQ, SL, RQ,SL) are elaV(C155)-Gal4/Y; UAS-cac-eGFP^(X)/+ or w¹¹¹⁸ for non-transgenic wild type. Data bars represent the average value and error bars +/- SEM.