Figure 4. Spatiotemporal properties of cooperative spine Ca²⁺ signalling in distal compartments.

(a) Left: low-magnification z-stack of a CA1PC, stimulation site on oblique dendrite indicated by yellow box. Arrowhead points to dendrite tip. Middle and right: high-magnification z-stack of the stimulated segment, with clustered (middle) and distributed (right) arrangement of inputs. Distributed inputs were always activated distal to proximal. (b,c) Representative Ca²⁺ signals from spine #1 (upper) and spine #4 (lower) in clustered (b) and distributed (c) arrangement using the 4S protocol (ISI=0.1 ms). (d) Integrated somatic EPSPs corresponding to (b,c). In b–d, black traces represent calculated responses and red traces represent measured responses. (e) Spine Ca²⁺ nonlinearity (average of all four spines) in clustered versus distributed arrangement (P<0.05, Wilcoxon test). Grey lines represent experiments in individual dendrites. Inset: Ca²⁺ nonlinearity from spine #1 (first, most distal) and #4 (last, most proximal) in experiments with distributed input. (f) Dependence of Ca²⁺ nonlinearity on input synchrony using clustered arrangement in distal dendritic segments (n=9–14 spine sets in 17 dendrites, seven cells, multiple comparisons after Kruskal–Wallis test with P<0.001). Grey lines represent experiments on the same spine set. (g,h) Somatic EPSP nonlinearity as a function of the dendritic segment length for spatial distribution (g, P<0.05, Wilcoxon test), and of ISI (h, clustered stimulation, ISD<6 μm, no significant differences with multiple comparisons after Kruskal–Wallis test with P=0.044). (i) Left: single 2P image of a distal segment with two stimulated spines indicated. Right: Ca²⁺ signals in the two spines during synchronous stimulation. Cartoon depicts measurement of interspine distance (ISD). (j) Ca²⁺ cooperativity among spine pairs as a function of ISD (n=122 spines). Positive or negative ISD value represents spine sequence towards tip or soma, respectively. Symbols represent mean±s.e.m. for data binned in 0±2.5, 2.5–7.5 and 7.5–12.5 μm. Kruskal–Wallis test for 0±2.5 (n=62), 2.5–7.5 (n=27) and 7.5–12.5 μm (n=23) bins, P=0.003. (k) EPSP nonlinearity as a function of ISD (grouped by absolute ISD values binned as in j). Filled symbols and error bars represent mean±s.e.m. distr., distributed; nonlin., nonlinearity.