**Fig. 4. **Optogenetic stimulation of LSd^{Sst} neurons does not affect stress-related behaviors or autonomic functions. (A) Schematic of patch-clamp recording from ChR2-expressing LSd^{Sst} neurons. The LS area was illuminated with blue light for optogenetic stimulations. Right, example images of a LSd^{Sst} neuron expressing ChR2-eYFP during recording. DIC, differential interference contrast. (B) Representative traces showing the responses of LSd^{Sst} neurons upon the delivery of light pulse trains in 15 Hz or 30 Hz. (C) 15 Hz blue laser-stimulation evokes action potentials (AP) with high fidelity, whereas 30 Hz stimulation shows 50~60% of success rate in inducing action potentials (n=4 cells from 2 animals). (D) Schematics showing Cre-dependent expression of ChR2 and fiberoptic cannula implantation for bilateral optogenetic stimulations of LSd^{Sst}. Representative confocal image shows LSd-restricted expression of ChR2. Scale bar, 200 µm. (E~K) Optogenetic stimulation of LSd^{Sst} neurons did not affect time spent in open arms of the elevated plus maze (n=11 ChR2, n=12 Ctrl, p=0.339) (E), time spent in the center (n=11 ChR2, n=11 Ctrl, two-way repeated measures ANOVA interaction, F(1,20)=0.097, p=0.759) (F) or velocity (n=11 ChR2, n=11 Ctrl, two-way repeated measures ANOVA interaction, F(1,20)=7.787, p=0.011) (G) in an open field arena, struggling time in the tail suspension test (n=4 ChR2, n=7 Ctrl, two-way repeated measures ANOVA interaction, F(2,18)=0.778, p=0.474) (H), nor real-time place preference (n=11 ChR2, n=12 Ctrl, p=0.347) (I). Dashed line indicates 50% preference. This manipulation also did not affect heart rate (n=12, one-way repeated measures ANOVA interaction, F(2,22)=0.199, p=0.821) (J) and respiratory rate (n=12, one-way repeated measures ANOVA interaction, F(2,22)=1.114, p=0.339) (K), Data are represented as mean±s.e.m. Asterisks indicate significance levels for comparisons in each panel using Bonferroni post-tests following two-way repeated measures ANOVA or Wilcoxon rank-sum test.

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