Extended Data Fig. 5: Non-Y1R lPBN neurons do not exhibit tonic neural activity responses after formalin.
From: A parabrachial hub for need-state control of enduring pain
(a) Injection strategy to confirm specificity of Cre-Off GCaMP (AAV.DO.GCaMP6s) expression in an Npy1r-Cre mouse. (b) Representative image showing Cre-Off GCaMP and Cre-dependent tdTomato expression in the PBN. Scale bar, 200 μm. (c) (top) Number of cells expressing only tdTomato, only GCaMP, or both in the PBN. (bottom) Average number of neurons expressing tdTomato, GCaMP, and both. Grey dots represent individual sections. (d) Time spent licking paw (5 min bins) after an injection of formalin while recording from Y1R or non-Y1R neurons (n = 9 Y1R, n = 6 non-Y1R mice, two-way ANOVA, n.s.) (e) Measuring calcium dynamics of lPBN Vglut2-expressing neurons. Inset, representative image showing GCaMP6s expression in lPBN Vglut2 neurons below a fiber optic track. Scale bar, 200 μm. (f) Average ΔF/F of GCaMP6s signal from lPBN Vglut2 neurons after a formalin injection. Blue, 470 nm; grey, 405 nm. Dark lines represent mean and lighter, shaded areas represent SEM. (g) Mean ΔF/F of lPBN Vglut2 neurons (n = 7 mice, one-way ANOVA, p = 0.0031). (h) Time spent licking paw (5 min bins) after an injection of formalin while recording from Y1R neurons or Vglut2 neurons (n = 9 Y1R, n = 7 Vglut2 mice, two-way ANOVA, n.s.). (i) Fiber photometry was used to measure calcium dynamics of lPBN Pdyn-expressing neurons. Inset, representative image showing GCaMP6s expression in lPBN Pdyn neurons below a fiber optic track. Scale bar, 200 μm. (j) Average ΔF/F of GCaMP6s signal from lPBN Pdyn neurons after a formalin injection. (k) Mean ΔF/F of lPBN Pdyn neurons (n = 6 mice, one-way ANOVA, p < 0.05). (l) Time spent licking paw (5 min bins) after an injection of formalin while recording from Y1R neurons or Pdyn neurons (n = 9 Y1R, n = 6 Pdyn mice, two-way ANOVA, n.s.). (m) Measuring calcium dynamics of lPBN Penk-expressing neurons. Inset, representative image showing GCaMP6s expression in lPBN Penk neurons below a fiber optic track. Scale bar, 200 μm. (n) Average ΔF/F of GCaMP6s signal from lPBN Penk neurons after a formalin injection. (o) Mean ΔF/F of lPBN Penk neurons (n = 6 mice, repeated measures one-way ANOVA, n.s.). (p) Time spent licking paw (5 min bins) after an injection of formalin while recording from Y1R neurons or Penk neurons (n = 9 Y1R, n = 6 Penk mice, two-way ANOVA, n.s.). Data are expressed as mean ± SEM. Grey lines represent individual mice. Post-hoc comparisons: *p < 0.05. **p < 0.01. ANOVA main effect of group: #p < 0.05, ##p < 0.01.
