Fig. 3: Chemogenetic activation of DRN GABAergic neurons resulted in a loss in both the tube test and the warm spot test.
From: GABAergic neurons in the dorsal raphe nucleus regulate social hierarchy in mice
a Schematic representation of the injection site in the DRN for the virus AAV2/9-GAD67-hM3D(Gq)-mCherry-WPRE. b Representative photomicrographs of the DRN depicting hM3Dq (red, left) with the enlarged pictures for the boxed area showing hM3Dq expression (red, second subplot), GAD67+ (green, third subplot), and their co-localization with DAPI (right). c Example of the ranking positions for a cage of mice tested daily over a period of 7 days, indicating that the first-ranked mouse injected with the hM3Dq virus was relegated to the last position at 6-8 h post-CNO injection. d Summary of rank changes in DRN-hM3Dq mice before and after CNO injection. Each line represents an individual animal. e Difference in average rank change for DRN-hM3Dq mice following CNO injection (Wilcoxon signed rank test; at 1-1.5 h: Z5 = -1.414, p = 0.157; at 3-5 h: Z5 = -2.646, p = 0.008; at 6-8 h: Z5 = -1.342, p = 0.180; at 24 h: Z5 = -1.342, p = 0.180; at 48 h: Z5 = -0.816, p = 0.414; at 72 h: Z5 = -0.816, p = 0.414). f Behavioral annotations of two tube test trials between the same pair of mice before and after CNO injection. g Chemogenetic activation of DRN GABAergic neurons significantly increased both the number (Mann-Whitney U-test: U = 90, p = 0.043) and duration (U = 86, p = 0.040) of stillness, as well as the percentage of time spent retreating (U = 2, p < 0.001). However, no significant changes were observed in the number (U = 129, p = 0.512) and duration (U = 142, p = 0.864) of push-initiation, the number (U = 134, p = 0.551) and duration (U = 133, p = 0.522) of push-back, or the percentage of time for resistance (U = 146, p = 0.968). “n” refers to the number of occurrences of the specific fine behaviors being measured. h Picture for the warm spot test, wherein four mice competed for occupancy of a warm corner situated on an ice-cold floor. i Duration of warm spot occupancy (top; One-way repeated measures ANOVA with Bonferroni correction: F2,18 = 0.142; p = 0.868; n = 10) and rank in the warm spot test (bottom; Friedman test with Wilcoxon signed-rank test for multiple comparisons: χ2 = 0.677; p = 0.717; n = 10) for DRN-hM3Dq mice are reported for 1 day before, 2 h after, and 2 days after saline injection. Each gray folded line represents an individual animal, while the colored line indicates the mean. Animals of the same rank are arranged in parallel to facilitate the presentation of results. j Duration of warm spot occupancy (top; F2,14 = 11.785; p = 0.007; n = 8) and rank in the warm spot test (bottom; χ2 = 8.538; p = 0.014; n = 8) for DRN-hM3Dq mice are presented for 1 day before, 2 h after, and 2 days after CNO injection. *p < 0.05, **p < 0.01, ***p < 0.001.
