Extended Data Fig. 4: Injury sensitizes lPBN Y1R neurons.
From: A parabrachial hub for need-state control of enduring pain
(a) ΔF/F of GCaMP6s signal from lPBN Y1R neurons during paw stimulation with a 0.07 g von Frey filament before and after SNI. Dark lines represent mean and lighter, shaded areas represent SEM. (b) Average ΔF/F (5 s bins) of the GCaMP6s signal shown in (a) (n = 11 mice, repeated measures two-way ANOVA, main effect of SNI p = 0.0009, SNI x time interaction p = 0.0046). (c-d) Mean (c) and maximum (d) ΔF/F of the GCaMP6s signal after the stimulus shown in (a) (n = 11 mice, paired two-sided t-test, p = 0.0008 (c), p = 0.0044 (d)) (e) ΔF/F of GCaMP6s signal from lPBN Y1R neurons during paw stimulation with a 1.0 g von Frey filament before and after SNI. (f) Average ΔF/F (5 s bins) of the GCaMP6s signal shown in (e) (n = 11 mice, repeated measures two-way ANOVA, SNI x time interaction p = 0.0239). (g-h) Mean (g) and maximum (h) ΔF/F of the GCaMP6s signal after the stimulus shown in (e) (n = 11 mice, two-sided Wilcoxon matched pairs signed-rank test, n.s. (g), paired two-sided t-test, n.s. (h)) (i) ΔF/F of GCaMP6s signal from lPBN Y1R neurons during paw stimulation with a cold stimulus (acetone) before and after SNI. (j) Average ΔF/F (5 s bins) of the GCaMP6s signal shown in (i) (n = 11 mice, repeated measures two-way ANOVA, main effect of SNI p = 0.0294, SNI x time interaction p = 0.0008). (k-l) Mean (k) and maximum (l) ΔF/F of the GCaMP6s signal after the stimulus shown in (i) (n = 11 mice, two-sided Wilcoxon matched pairs signed-rank test, p = 0.0049 (k), p = 0.0322 (l)) (m) ΔF/F of GCaMP6s signal from lPBN Y1R neurons during paw pin prick before and after SNI. (n) Average ΔF/F (5 s bins) of the GCaMP6s signal shown in (m) (n = 11 mice, repeated measures two-way ANOVA, SNI x time interaction p = 0.0015). (o-p) Mean (o) and maximum (p) ΔF/F of the GCaMP6s signal after the stimulus shown in (m) (n = 11 mice, two-sided Wilcoxon matched pairs signed-rank test, n.s.) (q) Npy1r-Cre mice were injected with a Cre-dependent fluorophore to label lPBN Y1R neurons. Mice were then injected with CFA and acute brain slices were prepared 24 h later. (r) Example traces from a whole-cell voltage clamp recording from an lPBN Y1R neuron from a naïve mouse (top, grey) and a mouse 24 h after a paw CFA injection (bottom, blue). (s-t) sEPSC frequency (c) and amplitude (d) in cells from naïve mice or mice 24 h after CFA injection (n = 16–17/group, unpaired t-test, p < 0.01 (c), p < 0.001 (d)). (u) Npy1r-Cre mice were injected with a Cre-dependent inhibitory DREADD that also expresses mCherry to label lPBN Y1R neurons. (v) Representative trace showing a decrease in firing of an lPBN Y1R neuron after bath application of the DREADD ligand CNO (1μM). Data are expressed as mean ± SEM. Grey dots and lines represent individual mice (a-p) or cells (s,t). BL, baseline. T-test and post-hoc comparisons: *p < 0.05, **p < 0.01, ***p < 0.001. ANOVA main effect of group: #p < 0.05, ###p < 0.001. ANOVA interaction: †p < 0.05, ††p < 0.01, †††p < 0.001.
