Fig. 5: The involvement of 5-HT was required in the EA-mediated effect on PSD.

A Left: Representative images of immunofluorescent staining of 5-HT⁺ neurons (green) in the DRN from Control, PSD, PSD+sham-EA, and PSD+EA groups. Right: The graphical plot of the density of 5-HT. (one-way ANOVA test, N = 9 per group). Scale bar, 50 μm. B Left: Confocal images showing TPH2⁺ fibers in mPFC from DRN. Right: The intensity of TPH2⁺ terminal in mPFC from DRN was significantly reduced by PSD induction, and this impairment could be recovered by EA treatment. (Kruskal-Wallis test, N = 12 slices per group). Scale bar, 50 μm. C Diagram of rAAV-hsyn-taCasp3-TEVp-p2A-EGFP-WRPEs or rAAV-hsyn-taCasp3-p2A-EGFP-WRPEs virus was injected into DRN (green); blue, DAPI. Scale bar, 100 μm. D Left: The number of NeuN⁺ neurons was ablated significantly in the Casp3+PSD+EA group compared with the EGFP+PSD+EA group. Right: The density of NeuN⁺ neurons was measured within the infected area. (one-way ANOVA test, N = 11 slices per group). Scale bar, 50 μm. E–H Behavioral effects of PSD mice after virus-mediated ablation of DRN neurons in OFT (E), FST (F), TST (G), and SPT (H). I The timeline shown upper demonstrates the experimental procedure. Schematic diagram showing the strategy used to determine whether the calcium signaling in CaMKIIα or Vgat1 neurons in mPFC was modulated by chemogenetic activation or inhibition of TPH2-positive neurons in the DRN. rAAV-CaMKIIα-GCaMP6s/rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus was injected into the mPFC, rAAV-TPH2-DIO-mCherry, rAAV-TPH2-DIO-hM3Dq-EGFP or rAAV-TPH2-DIO-hM4Di-mCherry virus was injected into the DRN. J Left: Representative heatmaps illustrating the Ca²⁺ signal changes of CaMKIIα⁺ neurons in the mPFC from the following groups: the Control group (Control+DIO, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, and injection of rAAV-CaMKIIα-GCaMP6s+rAAV-retro-cre virus into the mPFC), the PSD group (PSD+DIO, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, and injection of rAAV-CaMKIIα-GCaMP6s+rAAV-retro-cre virus into the mPFC), the PSD with EA treatment group (PSD+DIO+EA, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, injection of rAAV-CaMKIIα-GCaMP6s+rAAV-retro-cre virus into the mPFC, and treated with EA), the PSD with activation of the TPH2-positive neurons group (PSD+hM3Dq+CNO, injection of rAAV-TPH2-DIO-hM3Dq-EGFP activation virus into the DRN, injection of rAAV-CaMKIIα-GCaMP6s+rAAV-retro-cre virus into the mPFC, and CNO intraperitoneal injection), the PSD with EA after inhibition of TPH2-positive neurons group (PSD+hM4Di+CNO+EA, injection of rAAV-TPH2-DIO-hM4Di-mCherry inhibition virus into the DRN, injection of rAAV-CaMKIIα-GCaMP6s+rAAV-retro-cre virus into the mPFC, and treated with EA after CNO intraperitoneal injection). Right: The statistical analysis of peak △F/F. The Ca²⁺ signaling in excitatory neurons was increased in both the PSD+DIO+EA and the PSD+hM3Dq+CNO group compared to the PSD+DIO group. After inhibiting the TPH2-positive neurons in the PSD+hM4Di+CNO+EA group, the effect of EA on the increase of calcium signaling in CaMKIIα⁺ neurons in the mPFC could be blocked. K Left: Representative heatmaps illustrating the Ca²⁺ signal changes of Vgat1⁺ neurons in mPFC from the following groups: the Control group (Control+DIO, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, and injection of rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus into the mPFC), the PSD group (PSD+DIO, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, and injection of rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus into the mPFC), the PSD with EA treatment group (PSD+DIO+EA, injection of rAAV-TPH2-DIO-mCherry control virus into the DRN, injection of rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus into the mPFC, and treated with EA), the PSD with activation of the TPH2-positive neurons group (PSD+hM3Dq+CNO, injection of rAAV-TPH2-DIO-hM3Dq-EGFP activation virus into the DRN, injection of rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus into the mPFC, and CNO intraperitoneal injection), the PSD with EA after inhibition of TPH2-positive neurons group (PSD+hM4Di+CNO+EA, injection of rAAV-TPH2-DIO-hM4Di-mCherry inhibition virus into the DRN, injection of rAAV-Vgat1-GCaMP6s+rAAV-retro-cre virus into the mPFC, and treated with EA after CNO intraperitoneal injection). Right: The statistical analysis of peak △F/F. In the inhibitory neurons, the Ca²⁺ signaling showed a significant increase in the PSD + DIO group compared to the Control+DIO group. However, the PSD+DIO+EA group and activation of the TPH2-positive neurons in the PSD+hM3Dq+CNO group can attenuate this increased Vgat1⁺ neuronal activity. Data are presented as mean ± SEM. P values are set to *P < 0.05, **P < 0.01, ****P < 0.0001.