CaV3 channels and CaV2.1/2.2 exhibit distinct functional distributions. A, Schematic of 2PLSM point scan imaging protocol. Points were imaged in sets of 5, with each point separated by 0.5 µm. The laser was parked at a single diffraction-limited point for 25 ms preceding and 100 ms following an AP and calcium influx was measured with OGB-5N. Points were scanned in the sequence 2, 4, 1, 3, 5 and each point sampled a single AP. Data were averaged over 20–50 repetitions. B, Isochronal calcium peaks from neighboring point sets. Calcium influx at each point is color coded as in panel A. B1 shows a point set with a hotspot at point 5. B2 is the point set immediately adjacent to B1 and shows equivalent calcium influx across all points. Gray bar indicates the calcium transient onset and offset. C, Average of calcium influx from a set of five points (bottom), its time derivative (middle), aligned to somatic voltage recording (top). Gray bar defines transient onset/offset, which corresponds to deviation of time derivative above baseline noise. D, Distribution of point sets containing hotspots. Peak calcium influx at the brightest point was divided by the isochronal calcium influx at the point(s) 1 µm away. A total of 46 of 59 sites imaged fell within a normal distribution, while 13 sites exhibited higher relative calcium influx. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Red line indicates the fit of a normal distribution. Total distribution fit for normality (Shapiro–Wilk test p = 0.0016). E, Calcium influx at hotspots was approximately 2× higher than calcium influx at non-hotspot points. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± SD. F, Comparison of the flanks of point sets with a local hotspot and those without. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± SD for each 0.5-µm increment from the brightest point of the set. G, Influence of selective CaV antagonists or store depletion on peak calcium influx during point scan imaging. Circles represent single point sets. Black, control; cyan, TTA-P2; red, NiCl2; green, ω-conotoxin MVIIC; orange, CPA; *p < 0.001, Kruskall–Wallis test with rank-sum post hoc. H, Influence of selective CaV antagonists or store depletion on calcium hotspots. Hotspots were classified as points >1.5 times brighter than the point(s) 1 µm away. Dotted gray line represents the distinction between point sets with a local hotspot (above) and those without (below). Color code as in panel F; *p < 0.001, Kruskall–Wallis test with rank-sum post hoc.