Supplementary Figure 14: Acute optogenetic activation of VTA-targeting CRH terminals does not alter anxiety. | Nature Neuroscience

Supplementary Figure 14: Acute optogenetic activation of VTA-targeting CRH terminals does not alter anxiety.

From: Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety

Supplementary Figure 14

Optogenetic activation of CRH-positive terminals within the VTA was performed in Crh-ires-Cre mice bred to ChR2-expressing Ai32 mice. This enabled targeting of all VTA-projecting CRH fibers without having to perform multiple viral injections and optic fiber placements in the BNST and CeA. Lack of local CRH-expressing cell bodies in the VTA ensured the predominant activation of VTA-targeting CRH fibers. (a) Electrophysiological confirmation of optogenetic activation of a subset of ChR2-expressing CRH neurons of the CeA in acute brain slices. Current-clamp recordings show that ChR2-expressing CRH neurons fire an action potential to single blue laser pulses (488 nm, 2 msec, 5 mW/mm², 1 Hz) and can follow 10 Hz and 20 Hz laser stimulation (representative of n = 4 animals). (b) Representative coronal image showing the optic fiber tract (white dashed line) above the VTA. (c) Individual, bilateral placements of optic fiber tips in the VTA of Crh-ires-Cre mice bred to ChR2-expressing Ai32 animals (green) and Crhr-ires-Cre mice bred to Ai9 reporter mice (red). (d-f) VTA-targeting CRH fibers were stimulated by delivering blue (460 nm) light (20 Hz, 15 ms pulses) via bilateral optical fibers implanted over the VTA.. (d) Photostimulation of VTA-projecting CRH fibers in the ChR2 group did not affect anxiety in the DaLi (light ON during the entire test) or (e) cued fear conditioning (1 min light epoch shown in blue) compared to tdTom-expressing controls (unpaired two-tailed t-test for DaLi – lit zone time (%) t(18) = 0.82, p = 0.43; no. lit zone entries t(18) = 0.84, p = 0.41; n = 9 Ctrl, 11 ChR2 / RM ANOVA for FC – genotype x time interaction F(20,340) = 0.75, p = 0.78; genotype F(1,17) = 0.30, p = 0.59; n = 8 Ctrl, 11 ChR2). (f) 15 min session in the EPM with alternating OFF-ON-OFF illumination epochs (5 min each). No significant effect of light stimulation was detected in ChR2 mice for open arm entries or open arm time (RM ANOVA / open arm time (%): group x epoch interaction F(2,34) = 0.22, p = 0.81; group F(1,17) = 1.1, p = 0.31; epoch F(2,34) = 2.7, p = 0.08: open arm entries (%): group x epoch interaction F(2,34) = 0.5, p = 0.61; group F(1,17) = 0.45, p = 0.51; epoch F(2,34) = 4.7, *p = 0.014; n = 8 Ctrl, 11 ChR2). Both, Ctrl and CrhR2 mice appeared to exhibit time-dependent habituation to the EPM, as seen by a gradual decrease in distance travelled/exploration already during the first 5 min (RM ANOVA: group x time interaction F(14, 238) = 2.2, **p = 0.008; genotype F(1,17) = 0.7, p = 0.42; time F(14,238) = 8.7, ***p < 0.0001; n = 8 Ctrl, 11 ChR2). Error bars represent s.e.m.

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