Figure 2.

Spontaneous network bursts and onset time analysis. A, An area CA3 steady-state fluorescence (F₀) image obtained by fast confocal microscopy of an immature hippocampal slice stained with the calcium indicator OGB-1 AM. B, Synchronous network activity recorded from area CA3. ΔF/F₀ traces for 10 cells (top) and raster plot of color-coded fluorescence versus time (bottom) for 64 cells that exhibited synchronous network activity. The synchronous spontaneous network events occurred repeatedly. Note that there is nonsynchronous isolated activity between the synchronous events. C, Comparison of the utility of various image acquisition speeds in capturing neuronal activation onset times during spontaneous network bursts. ΔF/F₀ raster plots of grayscale-coded fluorescence change versus time for eight neurons that exhibited spontaneous synchronous activity. Images were captured at 70 ms per frame (C1), a typical speed with full-frame (512 × 512 pixel) resolution, and 11.2 ms per frame (C2), the maximum speed that can be achieved by 4 × 4 binning with our EM-CCD camera. The faster acquisition speed was required to resolve differences in onset times among cells (compare cells with *). D, ΔF/F₀ traces (dots) for three different cells within a synchronous network event, together with an onset time estimation by sigmoid curve fitting (line). Dotted vertical lines depict differences in calculated cell onset times for the three neurons. E1, Cellular onset latency distribution of spontaneous network bursts (four bursts). Median onset time within a network burst was defined as zero. If a cell activated earlier than the median value, negative values were assigned to the cell, while positive onset value was assigned to the cells that activated later than median timing. The majority of cells distributed within ∼200 ms, with an SD of 54 ms. E2, Cellular onset latency distribution of evoked synchronous events for the same cells shown in E1. The distribution became narrower, with the majority of cells responding within ∼150 ms, with an SD of 35 ms.