Fig. 2: VTA^DA neurons mediate the CES-induced anxiety-like behavior.

a–c Results of c-fos staining in single emotional stress (SES) model. a Experimental diagram for SES modeling. b The c-fos expression in VTA area in SES and control (CON) mice. Scale bar, 100 μm. c Statistical results of c-fos-positive cells in CON and SES mice (two-tailed unpaired t-test with Welch’s correction, P = 0.0045). n = 4 mice for each group. d–f Detection of the basic firing rate of putative VTA^DA neurons by in vivo electrophysiological technique in free-moving CES mice. d Experimental scheme. e Raster plot of spontaneous spikes of putative VTA^DA neurons. f Firing rate of putative VTA^DA neurons in CES and CON mice (two-tailed unpaired t-test, P = 0.5557). n = 11 neurons from 3 mice/group. g–k Detection of neuronal activity of VTA^DA neurons of wild-type C57BL/6 mice in anxiety-inducing events by in vivo calcium recording. g Schematic of viral injection and optical fiber implantation. h Experimental scheme of anxiety-inducing events. i Representative image of VTA-injection sites. Scale bar, 50 μm. j Trial-by-trial heatmap of anxiety-inducing event-evoked Ca²⁺ transients. The black dotted line represents the onset of anxiety-inducing event exposure. Each trail represents the target mice that observed their conspecific received attacks by CD1 aggressor. k Perievent plot of average Ca²⁺ transients (n = 4 mice). Black dotted line, the onset of emotional stress exposure (target mice observe a conspecific attacked by CD1 mice). Surrounding shaded areas indicate error bars (SEM). l–x Detection of neuronal activity of VTA^DA neurons of CES mice in anxiety-inducing contexts by in vivo calcium recording, n = 7 mice for each group. l Schematic of viral injection and optical fiber implantation. m Experimental scheme of CES modeling and behavioral tests. n–s Results of EPM. n Group of exploration traces in EPM. o Statistics of time spent in the open arm and closed arm of EPM (open arms: two-tailed unpaired t-test, P = 0.012; closed arms: two-tailed unpaired t-test, P = 0.0020). p The representative traces of Ca²⁺ fluorescence in CON and CES mouse. The transparent cyan background and papaya whip color display the mice located in the open arms and closed arms, respectively. q Group average of Ca²⁺ activities of VTA^DA neurons in EPM. r Statistics of average Ca²⁺ activities of VTA^DA neurons in the open arm and closed arm of EPM (open arms: two-tailed unpaired t-test, P = 0.0062; closed arms: two-tailed unpaired t-test, P = 0.4331). s Statistics of variation rate of Ca²⁺ fluorescence in the open arm and closed arm of EPM (open arms: two-tailed unpaired t-test, P = 0.0002; closed arms: two-tailed unpaired t-test, P = 0.5442). t–x Results of SIT. t Group of exploration traces in SIT. u Statistics of time spent in the social interaction zone of SIT (two-tailed unpaired t-test, P = 0.1043). v Group average of Ca²⁺ activities of VTA^DA neurons in SIT. w Statistics of average Ca²⁺ activities of VTA^DA neurons in the social interaction zone of SIT (two-tailed unpaired t-test, P = 0.8763). x Statistics of variation rate of Ca²⁺ fluorescence in the social interaction zone of SIT (two-tailed unpaired t-test, P = 0.8843). All data are shown as mean ± SEM. ns not significant, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001.