Extended Data Fig. 1: Activity labeling system for labelling prefrontal engrams for long term fear recall and neurons responsive to foot shock stimulation and tonic pain.
From: Prefrontal engrams of long-term fear memory perpetuate pain perception
(a) Doxycycline-controlled, activity-evoked expression of protein tags under c-fos promoter following viral injection of tagging constructs in the murine prelimbic cortex. Shown is an example of neurons tagged with mCherry expression in an activity-dependent manner. (b) Control of tagging by presence of Doxycyline (Dox ON, 1 mg/mL Dox for 16 days) in comparison to Doxycline-free time window (Dox OFF) is demonstrated in baseline home-cage conditions. Example images show mCherry reporter expression (red) on the background of Hoechst-stained cell nuclei (blue). Magnified images are shown in lower panels. Quantitative summary is shown on the right; unpaired t-test; * p < 0.05; Dox OFF: n = 2 mice/group, Dox ON: n = 4 mice/group. (c, d) Examples (panel c) and quantitative summary (panel d) of prelimbic neuronal labeling upon foot shock, fear memory recall at 28 days post-conditioning or paw injection of capsaicin. In panel d, one-way ANOVA with Dunnet’s multiple comparisons; * p < 0.05; Homecage: n = 7 mice/group, Fear Recall: n = 9 mice/group, Capsaicin: n = 6 mice/group, Footshock: n = 4 mice/group. (e) Typical examples showing dual labeling of neuronal activity over two discrete events of the same behavioral state or two distinct behavioral states via mCherry and Fos immunohistochemistry. Arrow-heads: double-labelled neurons. Scale bars represent 0.5 mm in panel a, 250 µm in upper images in panel b and 50 µm lower images in panel b, 100 µm in panel c and 50 µm in panel e. (f) Distribution of labelled neurons across layers of the prelimbic cortex. All data are shown as mean + /− S.E.M. All p and F values are shown in Supplementary Table 1.
