Figure 2.

Hyper-innervation elicits a homeostatic decrease in presynaptic release probability. (A) Schematic illustrating a reduction in readily releasable pool (RRP) size and functional release site number on hyper-innervated muscle 6. (B) Failure analysis reveals no significant change in failure rate on muscle 6 in M6 >FasII, consistent with unaltered quantal content on this target. (C) Representative paired-pulse EPSC traces at 0.4 mM extracellular Ca²⁺ with an interstimulus interval of 16.7 ms in the indicated genotypes. Increased paired-pulse facilitation (PPF) was observed on hyper-innervated targets, consistent with reduced release probability. (D) Quantification of PPF ratio (EPSC2/EPSC1). (E) Representative paired-pulse EPSC traces at 1.5 mM extracellular Ca²⁺ with an interstimulus interval of 16.7 ms in the indicated genotypes. Reduced paired-pulse depression (PPD) was observed on hyper-innervated targets, consistent with a reduced probability of release. (F) Quantification of PPD ratio (EPSC2/EPSC1) shows an increase. (G) Representative EPSC recordings of 30 stimuli at 3 mM extracellular Ca²⁺ during a 60 Hz stimulus train in the indicated genotypes. Insets represent the average cumulative EPSC plotted as a function of time. A-line fit to the 18^th–30^th stimuli was back-extrapolated to time 0. (H) The estimated size of the RRP is unchanged on muscle 6 in M6 >FasII compared with wild type, suggesting reduced RRP per bouton. (I) Scatter plot EPSC distribution of recordings on muscle 6 from wild type and M6 >FasII in the indicated extracellular Ca²⁺ concentrations. (J) Variance-mean plots for the indicated genotypes. Dashed lines are the best-fit parabolas to the data points. (K) The estimated number of functional release sites (N#) obtained from the variance-mean plots in (J) showing no significant difference between the genotypes. Error bars indicate ±SEM (n ≥ 9; one-way ANOVA; Supplementary Table S2). **p < 0.01; ns, not significant.