[Involvement of GABAergic projection from central amygdaloid nucleus to paraventricular nucleus of hypothalamus in electroacupuncture induced regulation of gastric function in GAD2-Cre mice].

Objective: To explore the effect of γ-aminobutyric acid (GABA)ergic neuronal circuit of the central amygdaloid nucleus (CeA) and the paraventricular nucleus of hypothalamus (PVN) on electroacupuncture (EA)-induced regulation of gastric function by way of CeA-PVN projection.

Methods: The present study included 3 parts: 1) C57BL/6 mice were randomly divided into control and EA groups (=6 in each group). EA was applied to right "Weishu"(BL21, Back-shu point) and "Zhongwan"(CV12, Front-mu point) for 20 min, followed by detecting the expression of c-fos in the CeA and PVN by using immunofluorescence staining; 2) Microinjection of anterograde tracer (rAAV-EF1α-DIO-mcherry-WPRE-pA) into the CeA was conducted in GAD2-Cre mice for confirming the projection of GABAergic neurons from CeA to PVN; 3) GAD2-Cre mice were randomly divided into rAAV-DIO-mcherry (intra-CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA), rAAV-DIO-hM3D（Gq）-mcherry(intra-CeA injection of rAAV-EF1α-DIO-hM3D(Gq)-mcherry-WPRE-pA) and rAAV-DIO-hM3D（Gq）-mcherry+EA groups(=6 in each group). The food intake and gastric empty were detected, and the concentration of GABA in the PVN was assayed by using high performance liquid chromatography on the 28 day after intra-CeA injection.

Results: 1) The expression of c-fos in the CeA and PVN was significantly increased in the EA group relevant to the control group(<0.01), suggesting an activation of neurons in both CeA and PVN after EA. 2) Following CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA, the densely expressed virus GABAergic neurons were found in CeA and large number of projection fibers found in the PVN, suggesting a direct connection between CeA and PVN. 3) After activating the GABAergic neurons of CeA, the concentration of GABA in the PVN was obviously increased (<0.01), the food intake and the gastric empty were considerably decreased relevant to the rAAV-DIO-mcherry group(<0.01). Following EA intervention，the concentration of GABA in the PVN was obviously decreased(<0.01), the food intake and the gastric empty were significantly increased relevant to the rAAV-DIO-hM3D（Gq）-mcherry group (<0.01).

Conclusion: EA of BL21 and CV12 (Back-shu and Front-mu acupoints) can increase food intake and gastric empty in GAD2-Cre mice, which may be achieved by suppressing the release of GABA in PVN through CeA-PVN GABAergic neural circuit.