Fig. 3: Low Cacna1c dosage is correlated with a reduction of spine Ca2+ signals elicited by backpropagated action potentials in CA1 pyramidal cells.
A CA1 pyramidal neuron during spine Ca2+ imaging (Z-projected pseudo-colour image, Alexa Fluor 594 channel; white square: spine imaging region; e: recording electrode; scale: 50 µm). B Imaging of spine Ca2+ transients elicited by backpropagated action potentials (APCaTs). Top-to-bottom: imaging region in (A) with spines (s1, s2), parent dendrite (d1, d2) and line-scan trajectory (red); line-scan images, Alexa Fluor 594 (Alexa) and Fluo 5F (Fluo) channels, during five somatic APs (Vm); APCaTs during the 5 APs (grey: ΔF/A, change in fluorescence intensity in Fluo channel, relative to Alexa channel; black: double-exponential fit). Scales, vertical: 2 µm, 20 mV, 0.1 arbitrary units (a.u.); horizontal: 0.2 s. C Example APCAT waveforms elicited by 1–5 somatic AP (Vm) in spines within three distance zones. Scale: 20 mV, 0.02 a.u., 0.2 s. APCaTs have smaller amplitude (D) and time integral (E) in Cacna1c+/− versus Cacna1c+/+ neurons (pairwise comparisons of 5 AP APCaTs, in distance zones (µm) <150: amplitude p = 0.0473, time integral p = 0.414; 150–250: both measures p < 0.0001; 250–400: both measures p < 0.0001). F, G Summation of APCaT amplitude (F) and time integral (G) with the number of APs is reduced in Cacna1c+/− versus Cacna1c+/+ neurons (pairwise comparisons for APCaTs at 150–250 µm, for 2 AP: normalized amplitude p = 0.15, normalized time integral p < 0.0001, 3 AP: both measures p < 0.0001; 5 AP: both measures p < 0.0001). Data were expressed as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 determined by two-way repeated measures ANOVA followed by Tukey adjustment of p values for contrasts. Statistical analysis results and sample sizes are given in Supplementary Table S2 and Fig. S8; all p values for pairwise comparisons are given in Supplementary Table S3 (colour figure online).
