Supplementary Figure 3: In vivo validation of ChR2 in CeAHtr2a neurons and supporting data from CeAHtr2a-neuron optogenetic activation experiments.
From: Central amygdala circuits modulate food consumption through a positive-valence mechanism
a, Representative expression of ChR2-eYFP in the CeA of an Htr2a-Cre mouse. b,c, Intracranial photostimulation (20 Hz, 10 ms pulse length, 20 minutes) induced cfos in the CeA of CeAHtr2a::ChR2 mice compared to controls (n = 3 sections per mouse, 3 mice, two-tailed unpaired t test: t(2) = 7.05, P = 0.0196). d, Approximate optic fiber locations for photostimulation experiments (Fig. 1k-p, Fig. 4a-h, Supplementary Fig. 3, Supplementary Fig. 6a-d). e, Photostimulation of CeAHtr2a neurons at 10 Hz and 20 Hz increased food intake (n = 8 eYFP mice, n =9 ChR2 mice. For eYFP vs ChR2 ON 20 Hz, two-tailed unpaired t test: t(15) = 2.87, P = 0.0118. For ChR2 OFF vs ChR2 ON 20 Hz, two-tailed paired t test: t(15) = 3.45, P = 0.0087. For eYFP vs ChR2 ON 10 Hz, two-tailed unpaired t test: t(15) = 2.29, P = 0.0366. For ChR2 OFF vs ChR2 ON 10 Hz, two-tailed paired t test: t(8) = 2.10, P = 0.0688. For eYFP vs ChR2 ON 5 Hz, two-tailed unpaired t test: t(14) = 1.24, P = 0.2364. For ChR2 OFF vs ChR2 ON 5 Hz, two-tailed paired t test: t(7) = 1.27, P = 0.2443). f, Photostimulation of CeAHtr2a neurons at 20 Hz increased the total percentage of time spent feeding (n = 7 mice per group, 20 Hz, two-tailed unpaired t test: t(12) = 2.72, P = 0.0186. n = 5 eYFP mice, n= 5 ChR2 mice, 10 Hz, two-tailed unpaired t test: t(9) = 1.43, P = 0.1873. n = 5 eYFP mice, n = 6 ChR2 mice, 5 Hz, two-tailed unpaired t test: t(9) = 0.50, P = 0.6277). g, CeAHtr2a::ChR2 mice did not exhibit a change in latency to eat after the onset of photostimulation (n = 6 eYFP mice, n= 5 ChR2 mice, two-tailed unpaired t test: t(12) = 0.80, P = 0.4369). h, CeAHtr2a::ChR2 mice spent significantly more time engaging in appetitive motor sequences (n = 7 mice per group, 20 Hz, Mann Whitney test: P = 0.0209. n = 5 eYFP mice, n = 6 ChR2 mice, 10 Hz, two-tailed unpaired t test: t(9) = 2.83, P = 0.0196. n = 5 eYFP mice, n = 6 ChR2 mice, 5 Hz, two-tailed unpaired t test: t(9) = 0.10, P = 0.2999).
Line graph values are mean ± SEM. Box–whisker plots display median, interquartile range and fifth to ninety-fifth percentiles of the distribution. * P < 0.05, ** P < 0.01. Scale bars: 100 μm.
