Figure 3 |. Activation or inhibition of CeA_GA neurons bi-directionally modulated pain-related behaviors in naïve mice and acute pain models.

a, Schematic of the responses to von Frey filaments applied to the whisker pad, including head withdraw and face wiping.

b, Quantification of optogenetic manipulation of CeA_GA neurons induced changes in the withdrawal frequency to 8 different von Frey filaments in the contralateral and ipsilateral whisker pad (to the right CeA). (Ipsilateral, control, n=7 animals (0 ± 0 (.008g), 0 ±0 (.02g), 0 ± 0 (.04g), 0 ± 0 (.07g), 0.29 ± 0.57 (.16g), 0.14 ± 0.26 (.40g), −0.29 ± 0.29 (.60g), −0.29 ± 0.18 (1.0g)), ChR2, n=8 animals (0 ± 0 (.008g), 0 ± 0 (.02g),0 ± 0 (.04g), −0.5 ± 0.38 (.07g), −4.13 ± 0.85 (.16g), −6.13 ± 0.79 (.40g), −6.0 ± 0.42 (.60g), −1.13 ± 0.64 (1.0g)), eArch, n=7 animals 0.57 ± 0.57 (.008g), 1.29 ± 0.52 (.02g), 1.71 ± 1.04 (.04g), 2.71 ± 0.84 (.07g), 3.14 ± 0.46 (.16g), 1.29 ± 0.7 (.40g), 0.57 ± 0.43 (.60g), 0.43 ± 0.43 (1.0g)); two-way ANOVA; ****P<0.0001, ***P<0.001, and *P<0.05; Contralateral, control, n=7 animals (0 ± 0 (.008g), 0 ± 0 (.02g), 0 ± 0 (.04g), 0 ± 0 (.07g), 0.29 ± 0.57 (.16g), 0 ± 0.22 (.40g), −0.43 ± 0.30 (.60g), 0.29 ± 0.29 (1.0g)), ChR2, n=8 animals (0 ± 0 (.008g), 0 ± 0 (.02g), 0 ± 0 (.04g), −0.63 ± 042 (.07g), −3.13 ± 0.44 (.16g), −4.25 ± 0.62 (.40g), −5.38 ± 0.98 (.60g), −1.63 ± 0.63 (1.0g)), eArch, n=7 animals (0 ± 0 (.008g), 0.57 ± 0.30 (.02g), 0.86 ± 0.63 (.04g), 1.0 ± 0.58 (.07g), 2.43 ± 1.0 (.16g), 1.43 ± 0.72 (.40g), 0.57 ± 0.30 (.60g), 0.29 ± 0.29 (1.0g)); two-way ANOVA; ****P<0.0001, **P<0.01, and *P<0.05; F_14,152=11.40 (ipsi) F_14,152=8.680 (contra).

c, Optogenetic manipulation of CeA_GA induced changes in the withdrawal threshold in response to electronic von Frey applied to the paw (control, n=7 animals (0.07 ± 0.33g (ipsi), 0.21 ± 0.35g (contra)), ChR2, n=8 animals (3.12 ± 0.49g (ipsi), 3.23 ± 0.47g (contra)), eArch, n=7 animals (−2.09 ± 0.46g (ipsi), −0.86 ± 0.53g (contra)); two-way ANOVA; ****P<0.0001, **P<0.01; F_2,38=49.51). Data are mean ± s.e.m..

d and e, Quantification of the optogenetics induced change in withdrawal latency (t2-t1) for Hargreaves heat (control, n=7 animals (0.42 ± 0.47s (ipsi), −0.67 ± 0.54s (contra)), ChR2, n=8 animals (7.0 ± 1.19s (ipsi), 3.28 ± 0.64s (contra)), eArch, n=6 animals (−4.76 ± 1.02s (ipsi), −4.33 ± 1.29s (contra)); two-way ANOVA; ****P<0.0001, **P<0.01, and *P<0.05; F_2,36=57.56) and Cold dry ice test (control, n=8 animals (0.04 ± 0.10s (ipsi), −0.51 ± 0.38s (contra)), ChR2, n=7 animals (3.29 ± 0.48s (ipsi), 4.76 ± 0.88 (contra)), eArch, n=7 animals (−1.62 ± 0.22s (ipsi), −0.81 ± 0.35s (contra)); two-way ANOVA; ****P<0.0001, *P<0.05; F_2,38=71.37). Data are mean ± s.e.m..

f, Example images of coping behaviors such as licking hind paw (top) or wiping whisker pad (bottom) to separate injections of formalin on different days.

g, Quantification of self-caring behaviors (total licking duration (sec)) per 2-minute bins with off and on light stimulation. (Hind paw formalin injection, control, n=8 animals (0.51 ± 0.09 (off1), 0.31 ± 0.12 (on1), 0.34 ± 0.06 (off2), 0.31 ± 0.04 (on2), 0.33 ± 0.09 (off3), 0.22 ± 0.11 (on3), 0.28 ± 0.06 (off4), 0.19 ± 0.06 (on4), 0.24 ± 0.09 (off5), 0.19 ± 0.07 (on5), 0.21 ± 0.13 (off6), 0.19 ± 0.09 (on6)); ChR2, n=9 animals (0.31 ± 0.05 (off1), 0.01 ± 0.01 (on1), 0.34 ± 0.06 (off2), 0.01 ± 0 (on2), 0.35 ± 0.07 (off3), 0 ± 0 (on3), 0.22 ± 0.08 (off4), 0 ± 0 (on4), 0.15 ± 0.07 (off5), 0.01 ± 0.01 (on5), 0.08 ± 0.06 (off6), 0 ± 0 (on6)); eArch, n=7 animals (0.33 ± 0.07 (off1), 0.25 ± 0.05 (on1), 0.30 ± 0.03 (off2), 0.45 ± 0.06 (on2), 0.31 ± 0.06 (off3), 0.25 ± 0.06 (on3), 0.47 ± 0.05 (off4), 0.48 ± 0.07 (on4), 0.13 ± 0.04 (off5), 0.20 ± 0.08 (on5), 0.20 ± 0.07 (off6), 0.14 ± 0.07 (on6)); two-way repeated measure ANOVA; **P<0.01, ••P<0.01 and *P<0.05; F_5,75=4.81; Whisker pad formalin injection, control, n=7 animals (0.46 ± 0.07 (off1), 0.22 ± 0.08 (on1), 0.25 ± 0.06 (off2), 0.44 ± 0.11 (on2), 0.33 ± 0.06 (off3), 0.27 ± 0.08 (on3)), ChR2, n=9 animals 7animals (0.48 ± 0.05 (off1), 0.01 ± 0.01 (on1), 0.41 ± 0.06 (off2), 0 ± 0 (on2), 0.38 ± 0.07 (off3), 0 ± 0 (on3)); two-way repeated measure ANOVA; ****P<0.0001, *P<0.05; F_5,70=8.825). Data are mean ± s.e.m..

h, Quantification of total wiping and licking behaviors during the first phase after formalin injection comparing off and on stimulation (2 minute bins with off and on light stimulation, total 6 minutes of stimulation) (ChR2, n=6 animals (55.00 ± 21.56s (licking-off), 10.33 ± 8.6s5 (licking-on), 92.33 ± 17.84s (wiping-off), 11.00 ± 8.33s (wiping-on)); one-way ANOVA; ***P<0.001 and ****P<0.0001; F_3,20=33.55).

i, Example heat map of CPP/CPA experiment.

j, Quantification of the percent of time (%) naïve mice spent on the stimulated side (control, n=8 animals (46.52 ± 0.80 (pre), 54.16 ± 2.45 (post), ChR2, n=8 animals (39.05 ± 2.70 (pre), 51.54 ± 3.20 (post), eArch, n=10 animals (61.16 ± 2.17 (pre), 34.39 ± 5.11 (post),; two-way repeated measure ANOVA; ****P<0.0001 and *P<0.05; F_2,23=29.66). Data are mean ± s.e.m..