Extended Data Fig. 2: Ca2+ imaging from ITCdm and ITCvm.
From: Intercalated amygdala clusters orchestrate a switch in fear state
a–c, Histological validation of GCaMP6f expression. Wild-type mice (not used for in vivo imaging; n = 2) were injected with AAV-CaMK2-GCaMP6f and killed after 1 month of expression. Thin slices (120 μm) were cut and stained with an anti-FOXP2 antibody. In addition to BLA and CeA neurons, most of the FOXP2-positive ITCdm neurons expressed GCaMP6f. Blue arrow shows a putative large, FOXP2-negative ITC neuron. Scale bars, 500 μm (a), 100 μm (b), and 10 μm (c). d, Example Ca2+ traces from neurons in the CeA, ITCdm, and BLA that were simultaneously imaged with a miniature microscope during fear conditioning. Data obtained from the same mice as in Fig. 1d–g. Grey shading indicates CS presentation (30 s); red line indicates footshock US presentation (1 s). e, f, Histological validation of GCaMP6f expression in ITCvm neurons. Foxp2-Cre mice were injected with an AAV encoding Cre-dependent GCaMP6f, implanted with a GRIN lens, and killed after behavioural experiments. Thin slices (120 μm) were cut and stained with an anti-FOXP2 antibody. Scale bars, 200 μm (e), 50 μm (f). Similar results were obtained with all six mice. g, Summary of histologically confirmed GRIN lens implantation locations for ITCvm recordings. Animals with off-target implantations were excluded from analysis. h, Example Ca2+ traces from neurons in the ITCvm cluster during fear conditioning. Grey shading indicates CS presentation (30 s); red line indicates footshock US presentation (1 s). Images of GCaMP6f expression from the same mouse are shown in e, f. i, j, Correlation matrices of all simultaneously recorded neuron pairs in CeA, ITCdm, and BLA (i), or in ITCvm (j) from representative animals. The entire recording session (11 min) was used. k, Distributions of correlation coefficients from CeA–CeA, ITCdm–ITCdm, and BLA–BLA pairs. Arrowheads indicate medians of the distributions. l, Distribution of correlation coefficients from ITCvm–ITCvm pairs. Arrowhead indicates median of the distribution. m, Summary of medians of correlation coefficient distributions shown in k. Solid lines indicate individual animals in which CeA, the ITCdm cluster, and BLA were simultaneously imaged (n = 3). Dotted lines indicate animals in which only the ITCdm cluster and BLA were simultaneously imaged (n = 5). *P = 0.007, one-way ANOVA followed by Tukey–Kramer test. n, Medians of correlation coefficient distribution. The same analysis as in m was applied to data from home-cage recording sessions. ITCdm neurons also show a trend towards a higher correlation in the absence of CS or US stimulation. *P = 0.12, one-way ANOVA.
