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. 2016 Jul 20;36(29):7580–7588. doi: 10.1523/JNEUROSCI.4425-15.2016

Figure 1.

Figure 1.

Optogenetic activation of dPAG neurons evokes defensive behaviors. A, Schematic showing virus injection (top) and implantation of optical fiber (bottom) into the dPAG. B, Top, Visualization of ChR2-mCherry expression in the dPAG (DAPI, blue; ChR2-mCherry, red). Bottom, Overlay of virus expression (n = 13 mice). Color bar, the number of mice with virus expressed in that area. C, Placement of optical fiber tips (red circles; n = 13 mice). D, Top, Representative immunostaining images showing virally induced mCherry expression under the CaMKIIα promoter and endogenous CaMKIIα expression immunolabeled with anti-CaMKIIα antibody. Bottom, Higher-magnification images of the boxed area in the top panels. Arrowhead indicates aqueduct (Aq). E, Percentage of overlap between dPAG CaMKIIα+ and mCherry+ cells (n = 3 mice, 3 sections for each mice). F, Activity of an example unit in response to laser stimulation (blue shading; 5 mW, 20 Hz, 1 ms pulse). Left, Top, Spike raster, with each row corresponding to a single trial and each tick to a single spike. Left, Bottom, Peri-stimulus time histogram calculated by averaging firing rates across trials. Gray shading, SEM. Right, Spike raster showing latency following first laser pulse stimulation (3.07 ± 0.08 ms; mean ± SD). G, H, Velocity during photostimulation (G) and freezing percentage during ITI (H) at different pulse frequencies (power, 5 mW) and laser intensities (frequency, 20 Hz) in the ChR2 and control groups (n = 5 per group). Two-way ANOVA with Bonferroni's post hoc analysis. I, J, Latencies from laser stimulation to flight (I) and freezing (J) onset at different pulse frequencies (power, 5 mW) and laser intensities (frequency, 20 Hz) in the ChR2 group (n = 5). I, Left, One-way ANOVA, F(2,8) = 18.89, p = 0.0009, Tukey's post hoc. I, Right, Paired t test, t(4) = 4.659, p = 0.0096. J, Left, One-way ANOVA, F(3,12) = 12.06, p = 0.0006, Tukey's post hoc. J, Right, One-way ANOVA, F(2,8) = 0.3364, p = 0.7239, Tukey's post hoc. K, Locomotion tracks of a representative ChR2-expressing mouse during real-time place-aversion test. Blue bar, Laser delivery; color bar, time; time 0, laser onset. L, Withdrawal velocity (left, t test, t(8) = 5.9824, p < 0.001) and withdrawal latency (right, t test, t(8) = −5.8712, p < 0.001) in response to laser illumination in the real-time place-aversion test in the ChR2 and control groups (n = 5 per group). M, Percentage of time spent in stimulation chamber during baseline and laser epochs in the ChR2 and control groups (group × epoch interaction, F(1,8) = 66.74, p < 0.001; two-way ANOVA, Bonferroni's post hoc.; n = 5 per group). N, Schematic of conditioned place-aversion test (left). Time that ChR2 (n = 8) and control (n = 7) groups spent in the stimulation-paired chamber on the pretest and test days (right; group × day interaction, F(1,13) = 10.24, p = 0.007; two-way ANOVA, Bonferroni post hoc). O, Freezing percentage during the baseline and tone presentation periods on the training (F(1,18) = 55.35, p < 0.0001) and test (F(1,18) = 21.31, p = 0.0002) days in the ChR2 and control groups (two-way ANOVA, Bonferroni's post hoc.; n = 10 per group). On both the training and test days, the ChR2 group showed significantly higher level of freezing during tone presentation compared with the baseline period (p < 0.0001 for training day; p < 0.0001 for test day; two-way ANOVA, Bonferroni's post hoc.) and the control group (p < 0.0001 for training day; p < 0.0001 for test day; two-way ANOVA, Bonferroni's post hoc.). Error bars, SEM; n.s., nonsignificant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.