Fig. 3: Two-photon Ca²⁺ imaging with Cal-520 and single-cell electrophysiology in trained, awake behaving animals.

a Example of combined recording in a trained, behaving mouse. Light grey dotted lines illustrate the position of a micropipette (for loose-patch recording) targeting the cell marked by a red dot (pseudo-coloured response amplitude by two-photon Ca²⁺ imaging with Cal-520). b Combined recordings on an example cell (Cell 1 in (a)); upper traces (Cal-520 imaging) and lower traces (loose-patch, recorded in voltage-clamp mode) are displayed on the same temporal scale. c An enlarged view of the loose-patch recording with ~200 ms of data around the stimulation timepoints (same data as in (b)). d One example response event (left subpanel) and 27 overlaid example events (three consecutive events per cell, nine cells, right subpanel) with exponential fitting of data points of spike peaks (appearing as troughs in the recorded traces). Black lines: fitting to individual events, green line: average of all fittings. e Spike raster plot of all trials for 9 bursting responsive cells (cells sorted by their average response strength). f Inter-spike interval (ISI) histogram for all the response events as in (e). g Box plots show the timing of burst onset, lick onset and burst end. P = 1.28e−7 (Lick onset versus Burst onset), P = 0.98 (Lick onset versus Burst end), two-sided Wilcoxon rank-sum test, Bonferroni corrected, Burst n = 239 events, Lick n = 11 events, ***P < 0.001, n.s., P > 0.05. h Side-view projection of the 3D reconstruction of an example bursting responsive cell. i Box plots showing relations between single-trial Ca²⁺ response amplitude and the number of spikes per response event, n = 21, 20, 29, 25, 10, 29, 26, 26, 28, 48 events, respectively, involving response events pooled from the nine cells shown in (e–g) and another seven non-bursting cells. The red dashed line indicates Δf/f = 1.5. In this figure, violet shade: sound stimulation time, light blue shade: water pumping time. Boxes represent 25th and 75th percentiles (Q1 and Q3), i.e., interquartile range (IQR), central bars indicate the median, and whiskers indicate Q1-1.5 × IQR and Q3 + 1.5 × IQR.