Extended Data Fig. 5: Additional characterization of optical-pacing effects on mouse behaviour.
a–c, A hot-plate test was performed to assess for potential effects on thermal pain thresholds from optical pacing (n = 17 (control), 16 (ChRmine)) and the following were quantified: time to first rear (unpaired two-tailed t-test, p = 0.53) (a); rears per minute (unpaired two-tailed t-test, p = 0.64) (b); and time to first jump (unpaired two-tailed t-test, p = 0.22) (c). Note no statistical significance in thermal thresholds was observed with cardiac pacing. d–f, To assess behavioural differences between control and virally transduced ChRmine-expressing mice, the following comparisons were performed: time spent in one chamber during baseline day (no light delivery) (n = 16 per group, unpaired two-tailed t-test p = 0.21) (d); time spent in the open arm of the EPM test during the first 5-min epoch with no light delivery (n = 16 per group, unpaired two-tailed t-test p = 0.61) (e); and time spent in the centre of the OFT during the first 3-min epoch with no light delivery (n = 5 (control), 9 (ChRmine), unpaired two-tailed t-test p = 0.15) (f). Note no statistical significance in behaviour was observed from viral-transfection. g,h, To assess for effects of light stimulation alone on mouse behaviour, the following comparisons were performed: time spent in the open arms by control (saline injected) mice during the 15 min EPM assay, where intermittent light delivery (10-ms pulse width, 900 bpm for 500 ms every 2 s) was delivered during the 5 min ON epoch (n = 16, one-way repeated-measures ANOVA with Bonferroni post-hoc test: condition F(1.44,21.62)=2.942, p = 0.088, individual F(15,30)=1.61, p = 0.13. Post-hoc: OFF vs. ON p = 0.99, OFF vs. OFF p = 0.9, ON vs. OFF p = 0.27) (g); and time spent in the centre by control mice during the 9 min OFT, where intermittent light delivery was delivered during the 3 min ON epoch (n = 5, one-way repeated-measures ANOVA with Bonferroni post-hoc test: condition F(1.84,7.45)=1.67, p = 0.25, individual F(4,8)=1.51, p = 0.29. Post-hoc: OFF vs. ON p = 0.99, OFF vs. OFF p = 0.99, ON vs. OFF p = 0.41) (h). Note no statistical significance in behaviour was observed from light stimulation alone. i,j, To assess for effects from optical pacing (10-ms pulse width, 900 bpm for 500 ms every 2 s) on anxiety-like behaviour in female mice, the following behavioural assays were measured: time spent in the open arms during the EPM (n = 8 (control) and 7 (ChRmine) female mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,26)=1.91, p = 0.17, group (opsin) effect F(1,13)=5.1, p = 0.042; time effect F(2,26)=4.24, p = 0.026. Bonferroni post-hoc: ON epoch ChRmine vs Control *p = 0.033) (i); and time spent in the centre during the OFT (n = 7 (control) and 7 (ChRmine) female mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,24)=0.37, p = 0.70; group (opsin) effect F(1,12)=7.47, p = 0.018; time effect F(2,24)=6.9, p = 0.0043. Post-hoc: ON epoch ChRmine vs Control *p = 0.031) (j). k,l, To determine whether cardiac pacing was aversive in female mice, we also measured the percentage of time spent on baseline and stimulation day for control (grey) and ChRmine-expressing (red) mice during the RTPP assay (n = 7 female mice per group, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x treatment interaction F(1,12)=0.68, p = 0.42; group (opsin) effect F(1,12)=0.11, p = 0.91; treatment effect F(1,12)=0.35, p = 0.57. Post-hoc: Baseline vs Stimulation for Control: p = 0.67, ChRmine: p = 0.99) (k); and the average velocity on the optically paced side during RTPP (n = 7 female mice per group, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x treatment interaction F(1,12)=0.088, p = 0.77; group (opsin) effect F(1,12)=3.69, p = 0.079; treatment effect F(1,12)=0.12, p = 0.73. Post-hoc: Stimulation day Control vs Chrmine: p = 0.59) (l). m,n, To determine whether increased heart rate variability can affect anxiety-like behaviour, we introduced constant 660 bpm stimulation with a Poisson distribution in mice and measured the time spent in the centre during the OFT (n = 12 (control) and 14 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,48)=1.29, p = 0.28; group (opsin) effect F(1,24)=5.80, p = 0.024; time effect F(1.36,32.6)=0.47, p = 0.55. Post-hoc ChRmine vs Control: OFF p = 0.61, ON p = 0.28, OFF p = 0.12) (m); and the time spent in the open arms during the EPM (n = 14 (control) and 14 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,52)=1.75, p = 0.18; group (opsin) effect F(1,26)=3.3, p = 0.082; time effect F(1.84,47.8)=5.157, p = 0.011. Post-hoc ChRmine vs Control: OFF p = 0.25, ON p = 0.20, OFF p = 0.99) (n). o,p, To determine whether intermittent tachycardia at a rhythm below 900 bpm can affect anxiety-like behaviour, we introduced intermittent 660 bpm stimulation (10-ms pulse width, 660 bpm for 500 ms every 2 s) in mice and measured the time spent in the centre during the OFT (n = 6 (control) and 10 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,28)=0.13, p = 0.88; group (opsin) effect F(1,14)=0.25, p = 0.63; time effect F(2,28)=1.1, p = 0.35. Post-hoc ChRmine vs Control: OFF p = 0.99, ON p = 0.99, OFF p = 0.99) (o); and the time spent in the open arms during the EPM (n = 6 (control) and 10 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,28)=0.52, p = 0.60; group (opsin) effect F(1,14)=0.03, p = 0.86; time effect F(2,28)=2.23, p = 0.12. Post-hoc ChRmine vs Control: OFF p = 0.99, ON p = 0.99, OFF p = 0.99) (p). q, Schematic overview of the chronic stimulation experiment. Mice were stimulated with intermittent optical pacing (900 bpm for 500 ms every 2 s) for 1 h every other day for two weeks before performing the OFT and EPM behavioural assays. r, Time spent in open arm during the EPM test for control (grey) and ChRmine (red) mice subjected to chronic optical stimulation (n = 6 (control) and 9 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,26)=0.87, p = 0.43; group (opsin) effect F(1,13)=0.71, p = 0.41; time effect F(1.65,21.4)=0.96, p = 0.38. Post-hoc ChRmine vs Control: 0–5 min p = 0.99, 5–10 min p = 0.89, 10–15 min p = 0.84). s, Time spent in centre during the OFT test for control (grey) and ChRmine (red) mice subjected to chronic optical stimulation (n = 6 (control) and 9 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,26)=0.25, p = 0.78; group (opsin) effect F(1,13)=0.097, p = 0.76; time effect F(1.78,23.2)=0.49, p = 0.60. Post-hoc ChRmine vs Control: 0–3 min p = 0.99, 3–6 min p = 0.99, 6–9 min p = 0.99). t, Average velocity (cm/s) of mice in the OFT test for control (grey) and ChRmine (red) mice subjected to chronic optical stimulation (n = 6 (control) and 9 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,26)=0.29, p = 0.78; group (opsin) effect F(1,13)=0.005, p = 0.94; time effect F(1.34,17.4)=17.3, p = 2.6e-4. Post-hoc ChRmine vs Control: 0–3 min p = 0.99, 3–6 min p = 0.99, 6–9 min p = 0.99). u, Total distance travelled (cm) mice during the OFT test for control (grey) and ChRmine (red) mice subjected to chronic optical stimulation (n = 6 (control) and 9 (ChRmine) mice, two-way repeated-measures ANOVA with Bonferroni post-hoc test: group (opsin) x time interaction F(2,26)=0.29, p = 0.75; group (opsin) effect F(1,13)=0.005, p = 0.94; time effect F(1.34,17.4)=17.3, p = 2.6e-4. Post-hoc ChRmine vs Control: 0–3 min p = 0.99, 3–6 min p = 0.99, 6–9 min p = 0.99). Data represent mean ± s.e.m.
