Extended Data Fig. 3: Two-photon imaging and fibre photometry.
From: Intercalated amygdala clusters orchestrate a switch in fear state
a, Schematic showing dual-colour in vivo 2-photon imaging through the implanted GRIN lens. ITCdm neurons were labelled by co-injection of an AAV expressing Cre-dependent tdTomato in Foxp2-Cre mice. A GRIN lens was implanted above ITCdm and surrounding BLA and CeA to record Ca2+ responses to aversive skin shocks (USs). b, Mean projected FOV. Green, GCaMP6; magenta, tdTomato. Dashed lines indicate the intermediate capsule surrounding ITCdm. c, Heatplot of Ca2+ responses (ΔF/F) to US presentations showing clustered activation of ITCdm neurons. d, Regions of interest (ROIs) corresponding to ITCdm (magenta) and BLA (blue) neurons. ROI numbers correspond to traces shown in e. e, Example Ca2+ traces from ITCdm and BLA neurons. Red lines indicate US presentations (1 s). We confirmed that face skin shock used in this experiment and footshock similarly activated ITCdm neurons (data not shown). f, Schematic illustrating in vivo fibre photometry in a freely moving mouse. g, ITCdm and ITCvm clusters were targeted with AAVs encoding Cre-dependent jRGECO1a and jGCaMP7f, respectively. Recording fibres were placed in BLA to simultaneously monitor axon terminal Ca2+ dynamics of ITCdm and ITCvm axons. Isosbestic control signals were recorded in the blue channel. h, Example traces of simultaneously recorded dual-colour Ca2+ signals and a control signal during a fear conditioning session. i, Cross-correlation traces between two simultaneously recorded Ca2+ signals. Dark grey lines represent five-trial averaged traces and light grey lines represent individual trials. j, Top, minimum peak values of cross correlation. Bottom, lags of the minimum peak points (n = 2 mice). k, l, Activity heatplot of trial-averaged responses (left, first 5 trials; right, last 5 trials) to footshock US omissions of all the recorded ITCdm (k; n = 271 neurons, from 9 mice) or ITCvm neurons (l; n = 372 neurons, from 6 mice) aligned by CS offset. Cells were sorted according to their averaged ΔF/F responses in the first five trials.
