Extended Data Fig. 6: PAC alters the excitation and inhibition balance of rACC→Pn neurons.
a, Strategy to label Pn-projecting rACC neurons for electrophysiological recording. b, Time mice spent in chamber 2 during days 3–6 of PAC. n = 7, 8 in Ctrl and Cond groups. c, Boxplots of the resting membrane potential (RMP; left; P = 0.37) and the input resistance (right; P = 0.44) of rACC→Pn neurons from Ctrl and Cond mice. n = 14, 16 neurons in Ctrl and Cond groups, respectively. d, Boxplots of the peak amplitude (left; P = 0.85) and half-duration of the action potentials (right; P = 0.39). n = 14, 16 neurons in Ctrl and Cond groups, respectively. e, Action potential firing pattern of rACC→Pn neurons from littermate control (cyan) and PAC-conditioned mice (red). f, Action potential firing frequency evoked by different levels of injected current. n = 14 neurons/group. g, Percentage of rACC→Pn neurons displaying different numbers of spikelets in the first action potential. n = 14 neurons in Ctrl and 16 in Cond. h, Traces of the action potential firing pattern evoked by 1-s current injection (black, bottom) in tdTomato-negative (non rACC→Pn) neurons from Ctrl (green, top) and Cond (purple, middle) mice. i, Plot of the action potential firing frequency evoked by different levels of injected current (two-way ANOVA, Tukey post-hoc test, F(1,255) = 10.61, P = 0.001; n = 11, 22 neurons in Ctrl and Cond groups, respectively). j, Example traces of sEPSCs in a rACC→Pn neuron holding at −70 mV from a Ctrl mouse (cyan, top) and a Cond mouse (red, bottom). k, Cumulative histograms of sEPSC frequency from Ctrl (cyan) and Cond (red) mice. Inset: boxplot of sEPSC frequency (P = 0.56; n = 11 neurons in each group). l, Cumulative histograms of sEPSC amplitude from Ctrl (cyan) and Cond (red) mice. Inset: boxplot of the sEPSC peak amplitude (P = 0.028; n = 11 neurons in each group). m, Example traces of two EPSCs evoked at 50-ms intervals from Ctrl (cyan, top) and Cond (red, bottom) mice. n, Paired-pulse ratio as a function of Δt (20, 50, 100, 200, 500 ms) between two stimuli (two-way ANOVA, Tukey post-hoc test, F(1,87) = 0.37, P = 0.54) of the inputs to rACC→Pn neurons from Ctrl (cyan) and Cond (red) mice. n = 8, 11 in Ctrl and Cond groups. o, Recordings of mixed EPSCs and IPSCs while holding rACC→Pn neurons at −30 mV from Ctrl (cyan, top) and Cond (red, bottom) mice. Because the holding potential is between the reversal potentials for excitatory and inhibitory events, EPSCs are inwardly directed and IPSCs outwardly directed. p, Strategy to express excitatory opsin (ChR2) in PV+ interneurons and label the Pn-projecting rACC neurons for electrophysiological recording. q, Example trace of the action potential firing from a PV+ interneuron in the rACC (top) evoked by current injection (bottom). r, Whole-cell recording configuration to analyse the feedforward inhibition from PV+ interneurons to rACC→Pn neurons. A blue light (494 nm, 1 ms) was given to evoke neurotransmitter release from PV+ interneurons. s, Example trace showing a light-evoked IPSC (blocked by 10 μM SR-95531) in one rACC→Pn neuron from a control mouse. Blue bar indicates the time point of light stimulation. t, Boxplots of 20–80% rise time (left; P = 0.96) and half-duration (right; P = 0.65) of IPSCs from Ctrl (cyan) and Cond (red) mice. n = 8 neurons in each group. u, Paired-pulse ratio as a function of Δt between two light stimulations (20, 50, 100, 200, 500 ms) of the inhibitory inputs from PV+ interneurons to rACC→Pn neurons (two-way ANOVA, Tukey post-hoc test, F(1,65) = 0.018, P = 0.89) from Ctrl (cyan) and Cond (red) mice. n = 7 for each group. v, Light-evoked individual IPSCs (grey), and average IPSC (cyan or red) from Ctrl (top) and Cond (bottom) mice. Blue bar indicates the time point of light stimulation and dashed line indicates the IPSC onset of the neuron from the Ctrl group. Inset: average IPSCs at expanded time scale. w, Boxplots of the amplitude (left; P = 0.02) and latency (right; onset to onset; P = 0.01) of light-evoked IPSCs from Ctrl (cyan) and Cond (red) mice. n = 8 cells in each group. Two-sided Wilcoxon rank-sum test was used in (c), (d), (i), (j), (f) and (w). In boxplots, horizontal lines represent median; boxes, quartiles; whiskers, most extreme data points ≤ interquartile range from box edges; and single points, data from individual cells. Data in (b, f, i, n, u) are mean ± SEM.
