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. 2013 Mar 6;33(10):4456–4467. doi: 10.1523/JNEUROSCI.3491-12.2013

Figure 4.

Figure 4.

Patch-clamp analysis of IHC presynaptic function. A, Ca2+current–voltage relationships from WTgt (black closed circles, n = 24 IHCs), Bsngt (black open circles, n = 26), WTΔEx4/5 (gray closed circles, n = 43), and BsnΔEx4/5 IHCs (gray open circles, n = 46). Peak Ca2+ currents were reduced to 71% of the value in WT littermates in BsnΔEx4/5 (p < 0.001) and to 80% in Bsngt (p < 0.01), respectively. B, representative original capacitance recordings (bottom) from IHCs illustrating a comparable reduction in capacitance jumps after 20 ms depolarizations in Bsn mutants compared with WT IHC (mean reduction of 40% in BsnΔEx4/5 and 44% in Bsngt). IHCs were depolarized to the membrane potential eliciting the maximum Ca2+ current (top). Replacing endogenous intracellular buffers (“endo” line) by 5 mm [EGTA]i (dashed lines) resulted in a comparable relative amount of suppression of exocytosis in all genotypes. C, RRP exocytosis (20 ms depolarizations) was reduced to a similar extent in both mutants (endogenous buffer, 3 wk; WTgt: n = 24 IHCs, Bsngt: n = 24 IHCs, WTΔEx4/5: n = 38 IHCs, BsnΔEx4/5 IHCs: n = 37 IHCs). Unaffected RRP exocytosis in heterozygous BsnΔEx4/5 IHCs argues against a dominant-negative effect of the 180 kDa bassoon protein fragment on vesicle fusion (see text; endogenous buffer, 8 wk; WTΔEx4/5: n = 7 IHCs, HzΔEx4/5: n = 10 IHCs, BsnΔEx4/5 IHCs: n = 9 IHCs). Similar relative reduction of exocytosis across genotypes in the presence of 5 mm [EGTA]i suggests comparable coupling of vesicle fusion to presynaptic Ca2+ influx in both mutants (EGTA 3 wk; WTgt: n = 10 IHCs, Bsngt: n = 9 IHCs, WTΔEx4/5: n = 17 IHCs, BsnΔEx4/5 IHCs: n = 16 IHCs). D, Sustained exocytosis was more severely affected in BsnΔEx4/5 than in Bsngt IHCs. Sustained exocytosis was assessed by (cell-wise) subtraction of capacitance jumps after 20 ms depolarizations from corresponding 100 ms counterparts (endogenous buffer, 3 wk; WTgt: n = 24 IHCs, Bsngt: n = 25 IHCs, WTΔEx4/5: n = 38 IHCs, BsnΔEx4/5 IHCs: n = 36 IHCs). [EGTA]i (5 mm) had comparable effects on both mutants (p > 0.05, EGTA 3 wk; WTgt: n = 8 IHCs, Bsngt: n = 9 IHCs, WTΔEx4/5: n = 11 IHCs, BsnΔEx4/5 IHCs: n = 11 IHCs). E, Paired pulse measurements showing that recovery from RRP depletion was slowed in both mutants, but to a lesser extent in Bsngt IHCs. Graph shows the grand average ratio of two capacitance jumps in response to two depolarization pulses of 20 ms duration at varying interpulse intervals (WTgt: n ≥ 20 IHCs, Bsngt: n ≥ 13 IHCs WTΔEx4/5: n ≥ 23 IHCs, BsnΔEx4/5 IHCs: n ≥ 20 IHCs, number provides minimum available for all intervals).