Figure 1. AHN stimulation induces escape-associated behaviors.

(a) Schematic illustration of optogenetic activation in the AHN (green circle depicts the AAV infusion). (b) An example of histological confirmation showing the expression of ChR2 and placement of optic fiber in the AHN. (c) Schematic describing optogenetic stimulation paradigm. (d) Three different escape conditions where the effects of AHN stimulation was examined. Top: open field arena with short transparent walls (condition 1, easy). Middle: tall opaque walls (condition 2, hard). Bottom: physical restraint tube (condition 3, impossible). (e) Condition 1: speed increase from the light OFF epoch to ON epoch (GFP N=7, ChR2 N=6 unpaired t-test, two-tailed, t=4.119, df=11, **p=0.0017). (f) Condition 1: freezing time during the light ON epoch (GFP N=7, ChR2 N=6, unpaired t-test, two-tailed, t=6.695, df=11, ****p<0.0001). (g) Condition 1: number of jumps during the light ON epoch (unpaired t-test, two-tailed, t=2.308, df=11, *p=0.0414). (h) Condition 2: speed increase from the light OFF epoch to ON epoch (GFP N=7, ChR2 N=6, unpaired t-test, two-tailed, t=3.778, df=11, **p=0.0031). (i) Condition 2: freezing time during the light ON epoch (GFP N=7, ChR2 N=6, unpaired t-test, two-tailed, t=4.259, df=11, **p=0.0013). (j) Condition 2: number of jumps during the light ON epoch (GFP N=7, ChR2 N=6, unpaired t-test, two-tailed, t=3.796, df=11, **p=0.003). (k) Condition 3: struggle movement during the 30 min of physical restraint (GFP N=4, ChR2 N=6, unpaired t-test, two-tailed, t=26.05, df=366, ****p<0.0001). All results reported are mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001. Scale bar=1 mm.

Figure 1—figure supplement 1. Compilation of viral expression and optic fiber implantation sites for optogenetic manipulation experiments.

(a,b) AHN soma activation. (N=6, each pair of colored circles shows the optic fiber tip placements in one animal). Viral spread in all injection areas (AHN, HPC) are depicted by the green blots across coronal plates of mouse brain atlas. Color bar, the number of mice with viral expression in the area (DAPI, blue; ChR2 and ArchT, green). Scale bar=200 µm.

Figure 1—figure supplement 2. The effects of low vs high-frequency AHN stimulation.

(ChR2 N=6, GFP N=7). (a) Schematic illustration of open field box where the low- and high-frequency stimulation occurred. (b) Testing paradigm for with low- vs. high-frequency AHN stimulation. (c–g) Behavioral changes induced by low-frequency (6 Hz) AHN stimulation. (c) No jumping observed upon light stimulation. (d) Freezing was increased upon AHN stimulation (two-way RM ANOVA, light x genotype, F(2,22)=13.13, ***p=0.0002, light effect, F (1.610, 17.71) = 16.95, ***p=0.0002, genotype effect, F (1, 11) = 35.33, ****p<0.0001, Sidak’s multiple comparison test, ON *p=0.0167, Off 2 **p=0.0098). (e) Speed did not change (two-way RM ANOVA, light x genotype, F(2,22)=0.9463, p=0.4034, NS,), light effect, F (1.868, 20.54) = 1.426, p=0.2622, NS, genotype effect, F (1, 11) = 0.2379, p=0.6352, NS. (f) Grooming was increased after AHN stimulation during the second light OFF period. (two-way RM ANOVA, light x genotype, F(2,22)=2.430, p=0.1113, NS, Sidak’s multiple comparison test, Off 2, *p=0.0164). (g) Rearing was decreased (two-way RM ANOVA, F(2,22)=2.407, p=0.1135, NS, F (1.691, 18.60) = 21.68, ****p<0.0001, F (1, 11) = 13.98, **p=0.0033, Sidak’s multiple comparison test, ON, *p=0.0317, the second light OFF period, *p=0.0297). (h–l) Behavioral changes induced by high frequency (20 Hz) AHN stimulation. (h) Jumping was increased upon AHN stimulation (two-way RM ANOVA, light x genotype, F (2, 22) = 5.328, *p=0.0130, light effect, F (1.000, 11.00) = 5.328, *p=0.0414, genotype effect, F (1, 11) = 5.328, *p=0.0414). (i) Freezing was increased upon AHN stimulation. (two-way RM ANOVA, light x genotype, F (2, 22) = 5.432, *p=0.0121, light effect, F (1.772, 19.50) = 8.363, **p=0.0031, genotype effect, F (1, 11) = 9.633, *p=0.01, Sidak’s multiple comparison test, ON, *p=0.0286). (j) Speed was increased upon AHN stimulation, light x genotype, F (2, 22) = 15.55, ****p<0.0001, light effect, F (1.119, 12.30) = 15.12, **p=0.0017, genotype effect, F (1, 11) = 2.073, p=0.1777, NS. (k) Grooming was increased during the second light OFF period. (two-way RM ANOVA, light x genotype, F (2, 22) = 4.473, *p=0.0234, light effect, F (1.153, 12.69) = 3.175, p=0.0949, genotype effect, F (1, 11) = 6.429, *p=0.0277) (l) Rearing is reduced upon AHN stimulation (two-way RM ANOVA, light x genotype, F (2, 22) = 5.564, *p=0.0111, light effect, F (1.406, 15.47) = 4.790, *p=0.0339, genotype effect, F (1, 11) = 6.128, *p=0.0308, Sidak’s multiple comparison test, *p=0.0146). All results reported are mean ± s.e.m. Sidak’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.