Fig. 2. Synchronous patterns of spontaneous network activity in the hippocampal CA1 region emerge after birth. (A-E) Correlation network analysis of the CA1 hippocampal region at E16. (A,B) Paired photomicrographs showing the same field viewed under fura-2 fluorescence (A) and DIC optics (B) illustrating dozens of cells loaded with the Ca²⁺ indicator. Black squares indicate cells with spontaneous changes of fura-2 fluorescence signal over time. (C) Raster plot representing the activity profile of each of the 12 spontaneously active cells shown in A and B over 800 seconds. The activity profiles of individual cells are represented by horizontal lines and each tick mark labels the onset of each Ca²⁺ transient. Dotted lines indicate simultaneous onset of [Ca²⁺]i oscillations of at least two cells of the plot. Note the small number of co-activations at E16 (e.g. cells no. 6 and no. 11). (D) Spatiotemporal correlation map illustrating all the active cells shown in A-C, in which pairs of cells with statistically significant correlation coefficients are linked by lines. Only 5 out of 12 active cells, located in the stratum oriens and in the pyramidal layer, show significant synchrony. The P value reflects the probability that the overall degree of synchronous correlation present in this network is caused by chance (see E). (E) Distribution of pair-wise correlations found in the real data (arrow) and in 1,000 simulations obtained by the Monte Carlo test (bell-shaped curve) of the active cells shown in A-D (see Materials and Methods). In this case, the number of correlated events in the real data set (arrow) does not exceed that expected by chance in simulated data, P=0.25. (F-I) Correlation network analysis of the CA1 hippocampal region at P1-P2. (F) CA1 region of a P2 hippocampal slice viewed with fura-2 fluorescence. Note the greater number of active cells (squares) than at E16 (A). (G) Raster plot illustrating the activity profile of each of the 70 cells shown in F. Highly synchronous activity patterns, in which network oscillations involve virtually the entire population of active neurons, are easily recognized. (H) Spatiotemporal correlation map of the CA1 region of a P1 slice illustrating complex synchronous networks, which recruit vast populations of active cells. Note the greater overall complexity than at E16. Furthermore, the lines connecting co-active cells are thicker than at E16, indicating higher degrees of correlation. The P value reflecting the probability that the overall degree of synchronous correlation present in this network is caused by chance is 0 (see I). (I) The number of simultaneous co-activations present in the network represented in H (arrow) exceeds the frequency distribution of random experiments created by Monte Carlo simulation. (J) Percentages of active cells involved in synchronous correlated networks at different ages (E16-P5). (K) Average of P values representing network correlation during development. The number of cases with significant synchronous correlated networks (P<0.05) in relation to the total number of cases analyzed is shown at the top of each bar. (L) Graph illustrating the correlation between the rise in network synchrony (P values, left, solid line) and the increase in the number of active cells (dotted line; see Fig. 1D) in the hippocampal CA1 region during development. Values in J and K are given as mean ± s.e.m. Scale bars, 40 µm. Abbreviations as in Fig. 1.

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