Fig. 6

Reduced calcium influx in the boutons of NHE9 KO mice. a, b Cultured hippocampal neurons were loaded with Fluo5F-AM and stimulated at either 10 Hz for 60 s (a) or once (b). Left panels show the mean response for all coverslips and the middle panel the mean fluorescence during the final 10 s of stimulation. The decay for each curve was fit to a single exponential and the mean time constant for each coverslip plotted in the right panel. Bars indicate mean ± s.e.m. *p < 0.05, ***p < 0.001, and ****p < 0.0001 by unpaired two-tailed Student’s t test. n = 22 coverslips/3 cultures/genotype for a and n = 90–100 action potentials averaged from 30 boutons/coverslip across 14–15 coverslips and 3 cultures/genotype for b. c The same boutons shown in b were stimulated twice at a 50 ms interval. Left panel shows sample traces, middle panel the mean peak fluorescence, and right panel the ratio of paired to single action potential responses (b). Bars indicate mean ± s.e.m. ****p < 0.0001 by unpaired two-tailed Student’s t test. n = 90–100 responses averaged from 30 boutons/coverslip across 14–15 coverslips and 3 cultures/genotype. d Hippocampal neurons were stimulated to produce single action potentials and bouton Ca²⁺ entry monitored with Fluo5F in the presence of first ω-Agatoxin TK (300 nM) to block P-/Q-type Ca²⁺ channels and then omega Conotoxin GVIA (1 μM) to block N-type channels as well. NHE9 KO neurons show less presynaptic Ca²⁺ entry through P-/Q- and N-type channels than WT neurons (right panel). Left panel depicts the contribution of each Ca²⁺ channel subtype to the total response. Bars indicate mean ± s.e.m. ****p < 0.0001, **p<0.01 by paired two-way ANOVA. n = 48 paired action potentials averaged from 30 boutons/coverslip across 12 coverslips and 3 cultures/genotype