Figure 4: Ca²⁺ cluster activity in the neonatal occipital neocortex in vivo.

(a) Experimental arrangement. (b) GCaMP3 fluorescence overlaid with area plots of four spatially confined cluster events (colour-coded). Binary area plots were calculated from ΔF/F₀ sequences (averaged over 10 frames) as maximum intensity projections thresholded at 25% of the peak amplitude. Scale bar, 200 μm. (c) Sample traces (ΔF/F₀) from individual ROIs. Colour code refers to clusters in b. Inset corresponds to field of view as shown in b, and indicates positions of ROIs from which sample traces were obtained. Note the presence of several spatiotemporally distinct Ca²⁺ clusters. Scale bars, 0.25 ΔF/F₀, 2 s. (d) Top: Raster plot indicating times of peak of Ca²⁺ transients from all individual ROIs. The time period shown in c is highlighted in grey. Bottom: time-aligned plot of the fraction of active ROIs, obtained by summing peaks over moving windows 10 frames in length. (e) In vivo view of a craniotomy (∼1.2 mm²) over the occipital cortex. The field of view during recording (shown in f) is indicated by a dotted rectangle (trans, transverse sinus; sag, superior sagittal sinus; ch, margin of recording chamber). Scale bar, 500 μm. (f) GCaMP3 fluorescence image after positioning of an extracellular electrode (EC). Scale bar, 200 μm. (g) Time-aligned sample traces of GCaMP3 fluorescence (average of 25 ROIs shown in f high-pass filtered at 0.01 Hz) and local field potential (LFP, band-pass-filtered at 0.1–100 Hz). Scale bars, 0.2 ΔF/F₀, 200 μV, 50 s. LED synchronization pulses were clipped for clarity. (h) Time-aligned GCaMP3 fluorescence (top, overlaid for 25 ROIs shown in f), wide-band extracellular signal (0.1–6,000 Hz), multiunit activity (200–3,000 Hz) and Morlet wavelet spectrum for the three time intervals indicated in g. Scale bars, 0.2 ΔF/F₀, 100 μV, 1 s. The asterisk in event 1 indicates a single blanked LED synchronization pulse.