Synaptic strength is stabilized despite enhanced synaptic growth and quantal size in endo mutants. (A) Representative images of third-instar larval muscle 4 NMJs immunostained with antibodies against the neuronal membrane marker HRP. Wild-type NMJs (w¹¹¹⁸) serve as the control condition. endo mutants (endo: w;endo¹/endo^Δ4) and neuronal knockdown of endo (endo^RNAi: w;OK6-Gal4;UAS-endo-RNAi) result in synaptic overgrowth. Neuronal expression of endo in endo mutant backgrounds (endo rescue: c155-Gal4;+;endo¹) rescues the synaptic overgrowth phenotype. Presynaptic overexpression of vGlut (vGlut-OE: w;OK371-Gal4/UAS-vGlut) shows no significant change in synaptic growth. (B) The number of synaptic boutons, a measure of synaptic growth, is significantly increased in endo and endo^RNAi (n ≥ 13; one-way ANOVA; Table S1). (C) Schematic and representative electrophysiological traces in the indicated genotypes. Neurotransmitter released by SVs is meant to illustrate evoked release. In addition to increased bouton number in endo, note the increased vesicle size schematized in endo, endo^RNAi, and vGlut-OE, which induces PHD. (D–E) mEPSP amplitudes are significantly increased in endo, endo^RNAi, and vGlut-OE (E), while EPSP amplitudes remain unchanged compared with wild type (D), leading to a homeostatic reduction in presynaptic neurotransmitter release (quantal content; F). (G) Quantification of mEPSP amplitude and quantal content, normalized to wild-type values, demonstrates PHD in endo, endo^RNAi, and vGlut-OE, with increased mEPSP amplitude but a compensatory reduction in quantal content. Error bars indicate ± SEM (n ≥ 9; one-way ANOVA; Table S1). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.