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. 2023 Apr 17;14:2198. doi: 10.1038/s41467-023-37878-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b Repeated optogenetic activation of the BLA–ACC pathway did not induce anxiety-like behaviors in the LD (3 stim: ctrl: n = 20 mice; 114.8 ± 5.34; stim: n = 18 mice; 112.9 ± 6.75; P = 082.; 6 stim: ctrl: n = 15 mice; 104.0 ± 10.31; stim: n = 12 mice; 116.7 ± 15.37; P = 0.49; 9 stim: ctrl: n = 8 mice; 64.0 ± 6.73; stim: n = 10 mice; 68.0 ± 5.34; P = 0.64) nor NSF (3 stim: ctrl: n = 22 mice; 136.5 ± 11.65; stim: n = 20 mice; 142.6 ± 12.44; P = 0.72; 6 stim: ctrl: n = 12 mice; 137.8 ± 19.52; stim: n = 8 mice; 106.4 ± 14.51; P = 0.26; 9 stim: ctrl: n = 7 mice; 110.3 ± 8.36; stim: n = 5 mice; 123.8 ± 15.24; P = 0.42) tests. Three stimulations did not change grooming in the splash test (ST, ctrl: n = 15 mice; 84.20 ± 9.37; stim: n = 17 mice; 83.41 ± 5.33; P = 0.94), nest building (ctrl: n = 24 mice; stim: n = 24 mice; Chi-square=0.012; P = 0.91) nor immobility in the FST (ctrl: n = 6 mice; 150.8 ± 32.29; stim: n = 7 mice; 171.0 ± 2.89; P = 0.26). Six stimulations did not alter grooming (ctrl: n = 19 mice; 98.16 ± 7.37; stim: n = 20 mice; 86.10 ± 8.7; P = 0.51) nor nesting (ctrl: n = 20 mice; stim: n = 18 mice; Chi-square=2.81; P = 0.094), but increased immobility in the FST (ctrl: n = 15 mice; 131.1 ± 9.47; stim: n = 11 mice; 155.1 ± 8.71; P = 0.042). The latter increase was still present after 9 stimulations (ctrl: n = 18 mice; 120.3 ± 13.69; stim: n = 12 mice; 155.5 ± 9.15; P = 0.033), along with decreased grooming (ctrl: n = 29 mice; 94.79 ± 5.02; stim: n = 26 mice; 56.81 ± 3.96; P < 0.0001) and nest quality (ctrl: n = 20 mice; stim: n = 16 mice; Chi-square=7.35; P = 0.0067). c Emotionality z-scores across tests and timepoints: three stimulations had no effect (ctrl: n = 67 mice; 0.033 ± 0.11; stim: n = 64 mice; 0.023 ± 0.11; P = 0.97), while a tendency for a decrease emerged after 6 stimulations (ctrl: n = 40 mice; −0.004 ± 0.082; stim: n = 35 mice; −0.232 ± 0.109; P = 0.095) and became significant after 9 (ctrl: n = 59 mice; 0.042 ± 0.11; stim: n = 52 mice; −0.78 ± 0.12; P < 0.0001). d Representative RNAscope images of Slc17a7 (upper-left panel), Gad2 (upper-right), c-fos (lower-left) mRNAs and their co-localization (lower-right) in the ACC. Scales = 100 µm. e Proportions of Slc17a7 + /c-fos + (green) and Gad2 + /c-fos + (red) cells increased in stimulated animals (ctrl: n = 5 mice; stimulated: n = 5 mice; Gad2 + /c-fos + : ctrl: 2.52 ± 1.09; stim: 7.68 ± 1.48, P = 0.008; Slc17a7 + /c-fos + : ctrl: 12.28 ± 2.89; stim: 22.04 ± 2.64, P = 0.028). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-sided unpaired t test (LD, NSF, ST, z-score); chi-square test for trend (Nest); one-sided Mann–Whitney test (mRNA quantification). 24a/b: areas 24a/b of the ACC, II, III, V/VI ACC layers. Sagittal mouse brain cartoons (a) were created with Biorender.com. Source data are provided as a Source Data file.