Figure 4
BNSTCRF Activity Changes with Repeated Noxious Heat Exposure. (A–B) Average z-score of BNSTCRF neurons by epoch for trials 1–4 in (A) male and (B) female subjects. Epochs are color-coded: Before (blue), Heat Start (red), Heat End (orange), and After (green) (Males = Two-way mixed-model ANOVA with Sidak’s post hoc: Epoch × Trial interaction [F(4.352, 208.9) = 8.148, P < 0.0001], main effect of Epoch [F(2.375, 114.0) = 16.66, P < 0.0001] and Trial [F(2.822, 135.4) = 10.63, P < 0.0001]; Females = Two-way mixed-model ANOVA with Sidak’s post hoc: Epoch × Trial interaction [F(3.150, 211.0) = 21.05, P < 0.0001], main effect of Epoch [F(1.679, 112.5) = 8.880, P = 0.0006] and Trial [F(2.491, 166.9) = 6.030, P = 0.0014]). (C–D) Average neuronal coactivity of BNSTCRF neurons by epoch for trials 1–4 in (C) male and (D) female subjects. Epochs are color-coded with the same system as (A–B): Before (blue), Heat Start (red), Heat End (orange), and After (green) (Males = Two-way mixed-model ANOVA with Sidak’s post hoc: no Epoch × Trial interaction [F(1.526, 3.052) = 1.046, P = 0.4252] or main effect of Epoch [F(1.419, 2.838) = 3.149, P = 0.1859] and Trial [F(1.178, 2.355) = 0.5741, P = 0.5447]; Females = Two-way mixed-model ANOVA with Sidak’s post hoc: no Epoch × Trial interaction [F(2.082, 8.329) = 0.9274, P = 0.4366] or main effect of Epoch [F(1.745, 6.981) = 2.102, P = 0.1935] and Trial [F(1.581, 6.326) = 1.041, P = 0.3871]). (E) (Left) Maximum z-score response to heat exposure by trial in male (purple) and female (magenta) subjects (Two-way mixed-model ANOVA with Sidak’s post hoc: Trial × Sex interaction [F(3, 345) = 14.96, P < 0.0001], main effect of Trial [F(2.609, 300) = 20.28, P < 0.0001] and Sex [F(1, 115) = 13.69, P = 0.0003]). (Right) Average maximum z-score response to heat in each trial by sex (unpaired t-test: t(115) = 3.70, P = 0.0003). (F) (Left) Latency of maximum z-score response to heat exposure by trial in male (purple) and female (magenta) subjects (Two-way mixed-model ANOVA with Sidak’s post hoc: Trial × Sex interaction [F(3, 345) = 16.90, P < 0.0001], main effect of Trial [F(2.717, 312.5) = 7.890, P < 0.0001], no main effect of Sex [F(1, 115) = 3.20, P = 0.0763]). (Right) Average latency of maximum z-score response to heat stimulus in each trial by sex (unpaired t-test: t(115) = 1.789, P = 0.0763). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.
