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. 2023 Oct 5;131(10):107004. doi: 10.1289/EHP12532

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Figure 5A is a schematic diagram depicting the viral injection and optogenetics with the adeno-associated virus-calmodulin-dependent protein kinase 2-channelrhodopsin-2-mCherry-expressing medial geniculate body fibers within lateral amygdala and 473 nanometers of light Figure 5B is a stained tissue that displays the typical images of the viral injection in the medial geniculate body and an optical fiber track above the lateral amygdala with channelrhodopsin-2-expressing medial geniculate body fibers. Figures 5C, 5D, 5G, and 5H are clustered bar graphs, plotting time in center, ranging from 0 to 60 in increments of 20; time in open arms (second), ranging from 0 to 150 in increments of 50; time in center (second), ranging from 0 to 60 in increments of 20; and time in open arms (second), ranging from 0 to 150 in increments of 50 (y-axis) across mCherry and channelrhodopsin-2 (x-axis). Figure 5I is a timeline depicting the chemogenetic experiments including adeno-associated virus-calmodulin-dependent protein kinase 2-channelrhodopsin-2-mCherry-expressing lateral amygdala, medial geniculate, clozapine-N-oxide, auditory cortex. Between days 0 and 1, habituation was conducted. Between days 1 and 28, noise exposure was conducted. From day 14, daily clozapine-N-oxide injections were started. From day 28, open field tests or elevated plus mazes began. Figures 5J, 5K, and 5L are bar graphs, plotting time in center (second), ranging from 0 to 50 in increments of 10; total distance (centimeter), ranging from 0 to 4000 in increments of 1000; time in open arms (second), ranging from 0 to 120 in increments of 40 (y-axis) across mCherry and human M4 muscarinic receptor (x-axis). — Behavioral effects of optogenetic activation or chemogenetic inactivation of auditory inputs to the LA. Optogenetic activation was used to selectively excite auditory inputs to the LA to determine whether activating these inputs could mimic noise-evoked anxiety-like behaviors. Chemogenetic blocking was used to selectively silence auditory inputs to the LA during noise exposure to determine whether they were necessary for noise-evoked anxiety-like behavior. (A) Schematic for viral injection and optogenetics. (B) Typical images of the viral injection in the MG (left) and optical fiber track above the LA with ChR2-expressing MG fibers (right). Scale bars: $200 micrometers$ . (C and D) Summarized data for time spent in the center of OFT (C, $virus \times light$ interaction, $uppercase italic f begin subscript open parenthesis 1, 20 close parenthesis end subscript equals 52.22$ , $lowercase italic p less than 0.0001$ ; main effect of light, $uppercase italic f begin subscript open parenthesis 1, 20 close parenthesis end subscript equals 32.65$ , $lowercase italic p less than 0.0001$ ) and time in the open arms of the EPM (D, $virus \times light$ interaction, $uppercase italic f begin subscript open parenthesis 1, 20 close parenthesis end subscript equals 25.67$ , $lowercase italic p less than 0.0001$ ; main effect of light, $uppercase italic f begin subscript open parenthesis 1, 20 close parenthesis end subscript equals 23.41$ , $uppercase italic p less than 0.0001$ ) before (pre) and during (light) light stimulation of $MG \to LA$ fibers. (E) Schematic for viral injection and optogenetics. (F) Typical images of viral injection in the ACx (left) and optical fiber track above the LA with ChR2-experssing ACx fibers (right). Scale bars: $200 micrometers$ . (G and H) Summarized data for time spent in the center of OFT (G, $virus \times light$ interaction, $uppercase italic f begin subscript open parenthesis 1, 22 close parenthesis end subscript equals 64.09$ , $lowercase italic p less than 0.0001$ ; main effect of light, $uppercase italic f begin subscript open parenthesis 1, 22 close parenthesis end subscript equals 36.02$ , $lowercase italic p less than 0.0001$ ) and time in the open arms of the EPM (H, $virus \times light$ interaction, $uppercase italic f begin subscript open parenthesis 1, 18 close parenthesis end subscript equals 27.47$ , $lowercase italic p less than 0.0001$ ; main effect of light, $uppercase italic f begin subscript open parenthesis 1, 18 close parenthesis end subscript equals 32.77$ , $lowercase italic p less than 0.0001$ ) before (pre) and during (light) light stimulation of $ACx \to LA$ fibers. (I) Timeline for chemogenetic experiments. (J–L) Summarized data for time spent in the center (J, $lowercase italic t begin subscript open parenthesis 14 close parenthesis end subscript equals 8.097$ , $lowercase italic p less than 0.0001$ ) and total distance traveled (K, $lowercase italic t begin subscript open parenthesis 14 close parenthesis end subscript equals 0.7899$ , $lowercase italic p equals 0.4427$ ) in the OFT and time in the open arms of EPM (L, $lowercase italic t begin subscript open parenthesis 14 close parenthesis end subscript equals 4.386$ , $lowercase italic p equals 0.0006$ ). The data are expressed as the $mean \pm SEM$ . *** $uppercase italic p less than 0.001$ . Two-way ANOVA with Bonferroni post hoc analysis for (C), (D), (G), and (H). Unpaired t-test for (J), (K), and (L). For (C) $lowercase italic n equals 10$ mCherry mice, 12 ChR2 mice. For (D) $lowercase italic n equals 10$ mCherry mice, 12 ChR2 mice. For (G) $lowercase italic n equals 12$ mCherry mice, 12 ChR2 mice. For (H) $lowercase italic n equals 10$ mCherry mice, 10 ChR2 mice. For (J) $lowercase italic n equals 8$ mCherry mice, 8 hM4Di mice. For (K) $lowercase italic n equals 8$ mCherry mice, 8 hM4Di mice. For (L) $lowercase italic n equals 8$ mCherry mice, 8 hM4Di mice. The numerical data underlying this figure are shown in Excel Tables S1. Note: AAV, adeno-associated virus; ACx, auditory cortex; ANOVA, analysis of variance; CaM, calcium-calmodulin; CaMKII, CaM-dependent protein kinase II; ChR2, channelrhodopsin-2; CNO, clozapine-N-oxide; EPM, elevated plus maze; hM4, human M4 muscarinic; hM4Di, human M4 muscarinic receptor; LA, lateral amygdala; MG, medial geniculate body; NS, not significant; OFT, open-field tests; Pre, before light stimulation; SEM, standard error of the mean.