Fig. 4. BDNF-overexpressing hippocampi show increased spontaneous correlated network activity at E18. (A-H) Representative examples of network activity in the hippocampal CA1 region in control (A-D) and transgenic (E-H) E18 embryos. (A) Fura-2-loaded control hippocampal slice showing few active neurons (squares). (B) Plot of F/F over time illustrating spontaneous [Ca²⁺]i changes in a control neuron. The initiation of [Ca²⁺]i transients is indicated by bars on the x axis. (C) Raster plots of all active cells shown in A. Horizontal lines represent the activity profile over 800 seconds of each active neuron and vertical thick lines mark the onset of [Ca²⁺]i oscillations. (D) Correlation map illustrating a significant (P<0.01) spatio-temporal co-activation among all cell pairs shown in A and C assayed with ² contingency tables. The thickness of the lines is proportional to the degree of significance. The P value indicated at the bottom (P=0.15) reflects the overall level of co-activation obtained by Monte Carlo simulation, and represents the probability that the co-activations present in this field are caused by chance. (E) Fura-2-loaded transgenic hippocampal slice illustrating more active neurons (squares) than in controls. (F) Representative plot of F/F over time showing spontaneous [Ca²⁺]i oscillations in a hippocampal neuron from a transgenic embryo. (G) Raster plot of all active neurons shown in E, illustrating the activity profile of each of the 49 cells over 800 seconds. Note the greater number of active neurons, [Ca²⁺]i transients and co-activations than in C. (H) Correlation map showing the spatio-temporal co-activations present in the transgenic CA1 region shown in E. BDNF-overexpression increases the complexity of spontaneous correlated networks in the hippocampus. Furthermore, the overall synchrony of this network also increases in BDNF transgenic embryos, as indicated by the P value through the Monte Carlo simulation test (P=0.001). (I-K) Histograms showing significant increases in the percentage of active neurons out of the total of fura-2-loaded cells (I), the activation rates (Ca²⁺ transients/cell/10⁴ seconds) (J) and in number of active cells involved in correlated networks (K), in BDNF-overexpressing hippocampi (black bars) compared to controls (white bars). (L) The average of P values obtained by Monte Carlo simulation in co-active networks of transgenic slices (black bars) is much lower than in controls (white bars). *P<0.01, **P<0.001 Scale bars: 50 µm.