Fife regulates homeostatic plasticity. (A–F) Representative traces of EJPs and mEJPs recorded in 0.6 mM Ca²⁺ with (B, D, and F) or without (A, C, and E) pretreatment in HL3 containing 20 µM PhTx at wild-type (A and B), Fife^AC/Df (C and D), and Fife^AC/Fife^ex (E and F) NMJs. Stimulus artifacts have been removed for clarity. (G) Pretreatment in HL3 containing 20 µM PhTx significantly reduces mEJP amplitude in wild type, Fife^AC/Df, and Fife^AC/Fife^ex (wild type, 0.95 ± 0.06 mV, n = 22 NMJs; wild type + PhTx, 0.61 ± 0.03 mV, n = 14 NMJs, P < 0.0001; Fife^AC/Df, 1.15 ± 0.07 mV, n = 12 NMJs; Fife^AC/Df + PhTx, 0.60 ± 0.02 mV, n = 14 NMJs, P < 0.0001; Fife^AC/Fife^ex, 0.94 ± 0.08 mV, n = 11 NMJs; Fife^AC/Fife^ex + PhTx, 0.51 ± 0.03 mV, n = 12 NMJs, P < 0.0001). (H) Pretreatment in HL3 containing 20 µM PhTx significantly reduces EJP amplitude in Fife^AC/Df and Fife^AC/Fife^ex, but not wild type (wild type, 30.98 ± 1.71 mV, n = 22 NMJs; wild type + PhTx, 27.83 ± 0.1.83 mV, n = 14 NMJs, P = 0.23; Fife^AC/Df, 15.50 ± 1.97 mV, n = 12 NMJs; Fife^AC/Df + PhTx, 7.61 ± 0.76 mV, n = 14 NMJs, P = 0.0017; Fife^AC/Fife^ex, 17.65 ± 1.54 mV, n = 11 NMJs; Fife^AC/Fife^ex + PhTx, 9.83 ± 1.37 mV, n = 12 NMJs, P = 0.0010). (I) Pretreatment in HL3 containing 20 µM PhTx significantly increases quantal content in wild type but not in Fife^AC/Df or Fife^AC/Fife^ex (wild type, 34.74 ± 2.45, n = 22 NMJs; wild type + PhTx, 47.33 ± 4.25, n = 14 NMJs, P = 0.0092; Fife^AC/Df, 13.99 ± 1.88, n = 12 NMJs; Fife^AC/Df + PhTx, 12.98 ± 1.37, n = 14 NMJs, P = 0.53; Fife^AC/Fife^ex, 19.28 ± 1.66, n = 11 NMJs; Fife^AC/Fife^ex + PhTx, 19.39 ± 2.67, n = 12 NMJs, P = 0.83). Statistical significance represents comparison to untreated in the same genotype. ns, not significant; **, P < 0.01; ****, P < 0.0001; Student’s t test with Welch’s correction if unequal variance; Mann–Whitney test for non–normally distributed datasets. Error bars represent SEM. (J) Model of Fife function. At wild-type active zones, vesicles of the readily releasable pool (RRP) are docked at the presynaptic membrane and molecularly primed (red) within nanometer distance of clustered Ca²⁺ channels (green) clustered beneath the active zone cytomatrix. Loss of Fife function impairs the size and molecular organization of the active zone cytomatrix and, through modulation of the number of readily releasable synaptic vesicles and their coupling to clustered Ca²⁺ channels, the probability of neurotransmitter release and its homeostatic modulation.