Figure 5. Functional analysis of dissociated hippocampal-like neurons using electrophysiology and calcium imaging.

(a) A representative cell image for patch-clamp recording. Many neurons showed voltage-dependent Na–K current (b, n=30), action potential following injection of depolarizing currents (c, n=18) and spontaneous excitatory postsynaptic currents (d, n=9, 2.33±0.75 Hz), at day 136 (dissociated at day 83). The percentage of neurons recorded showing Na–K currents, action potentials and synaptic responses were 100% (30 out of 30), 72% (18 out of 25) and 75% (9 out of 12), respectively. (e–h) The data set of calcium imaging. (e) Representative image of active neurons (day 143, 8 weeks after dissociation), and their firing pattern shown by trace image of calcium response (f). (g) Pharmacological perturbation by TTX (**P<0.01, n=3, unpaired t-test with Welch's correction). (h) Time-dependent promotion of neuronal activity. Percentage of active neurons significantly increased at 8 weeks after dissociation (***P<0.001, n=3, one-way analysis of variance). (i) Synchronization analysis by cross-correlations of 100 neurons at 8 and 4 weeks after dissociation. Colours from red to blue indicate their cross-correlations from high (synchronized activity) to low. Cross-correlation was higher at 8 weeks after dissociation than 4 weeks. The data are representative of three independent experiments. (j) Histogram of correlation coefficients indicated a strong correlation at 8 weeks after dissociation. The data are representative of three independent experiments. (k) Histogram of average correlation coefficients of one hundred neurons (*P<0.05, n=3, unpaired t-test). Scale bars, 20 μm (a); 100 μm (e). Bars in graph, s.e.m.