Characterization of the differentiated neurons in indirect contact co-cultures.

In indirect co-cultures we differentiated the human neurons in close proximity of rat astrocyte layers (Line B1 and B2). (A, B) Bright field images of indirect cultures during differentiation day 26 and 56 are shown. (C-F) We observed the expression of MAP2, VGAT, synaptophysin1 and (G-J) VGLUT1 and HOMER1 co-localizing puncta using immunocytochemistry at day 56. We then analyzed (K) spontaneous mEPSCs and mIPSCs (holding potential at -70mV in voltage clamp mode) recorded in 1 μM TTX supplemented with 40 μM Bicuculline or 50 μM AP5 + 10 μM DNQX respectively. (L) Quantification of amplitude, area, time to decay and frequency of mEPSCs and mIPSCs was also analyzed (n = 2). We then recorded calcium traces of (M, N) neurons loaded with Fluo-5 AM ester from n = 2. (O) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on M and N respectively; upon TTX addition and 20 minutes after TTX was washed away (P) or upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 1(M, O) and ROI 8 (N, P)) are shown. (Q) Analysis of activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also S5 Fig for indirect contact plate preparation method.