Impaired presynaptic functions in cultured afadin-deficient hippocampal neurons.

<p>(A, B) Labeling of functional synapses in cultured live hippocampal neurons. (A) Recycling synaptic vesicles labeled by incubation of cultured live hippocampal neurons at 14 DIV with the synaptotagmin I luminal domain Ab. Scale bars, 1 µm. (B) Quantification of the total integrated intensity of the internalized synaptotagmin I Ab. Sixty punctae for each genotype were analyzed. (C–E) Modulation of presynaptic vesicular release by afadin in cultured hippocampal neurons. (C) Examples of postsynaptic responses evoked by paired action potentials in a presynaptic cell with a 50-ms interval in control and <i>afadin</i> cKO neurons. Superimposed images for 15 recordings of unitary EPSC (top), average unitary EPSC (middle), and action potentials (bottom). A vertical scale bar denotes 20 pA for the top and middle rows and 5 mV for the bottom row; a horizontal scale bar denotes 20 ms. (D) The average unitary amplitudes (n = 10 and 12 in control and <i>afadin</i> cKO neurons, respectively). (E) paired-pulse ratios (n = 8 and 11 in control and <i>afadin</i> cKO neurons, respectively). Error bars, s.e.m. Control, <i>afadin</i>^+/f; cKO, <i>afadin</i>^f/f;nestin-Cre.