Objective: To observe the effect of electroacupuncture (EA) stimulation of "Zusanli"(ST36)on gastric function (food consumption and gastric emptying rate) and excitability of hippocampal glutamatergic neurons in mice, so as to explore its mechanism underlying enhancing gastrointestinal function.
Methods: The present study includes 2 parts. 1) C57BL/6 mice were randomly divided into normal and EA groups (n=12 in each group). EA (2 Hz/15 Hz, 1-3 mA) was applied to bilateral ST36 for 20 min, once daily for 7 days. In each group, 6 mice were used to measure the food consumption and gastric emptying rate, and the other 6 mice used to detect the hippocampal glutamate secretion content by using in vivo microdialysis and high performance liquid chromatography. 2) Thirty CaMKIIα-Cre mice received microinjection of a recombinant adeno-associated viral vector containing inhibitory designer receptor exclusively activated by a designer drug (DREADD, AAV-DIO-hM4Di-eYFP) into the hippocampus. Twenty-one days later, 3 mice were selected to observe the expression of eYFP-labeled hM4Di by immunohistochemistry, and 15 mice employed to observe the electrical activities of hM4Di-eYFP positive neurons exposed in chemogenetic activating drug Clozapine N-oxide (CNO) perfusion conditions (n=3) and without CNO in the recording chamber (n=6 in the control and EA groups) by using whole cell patch clamp. The rest 12 CaMKII-Cre mice were equally randomized into AAV-DIO-hM4Di-eYFP+CNO group and AAV-DIO-hM4Di-eYFP+CNO+EA group, and CNO was given by intraperitoneal injection for observing the effect of EA on gastric function.
Results: 1) In C57BL/6 mice, compared with the normal group, the food consumption, gastric emptying rate, and the glutamate content in the hippocampus were obviously increased in the EA group (P<0.01). 2) In CaMKIIα-Cre mice, the hM4Di-eYFP positive neurons distributing in the hippocampus showed an obvious increase of firing rates in the EA group relevant to the control group (P<0.01), and a hyperdepolarization potential after application of CNO. No significant changes were found between the AAV-DIO-hM4Di-eYFP+CNO and AAV-DIO-hM4Di-eYFP+CNO+EA groups in the food consumption and gastric emptying rate (P>0.05), suggesting an elimination of EA effect after acute DREADD-mediated activation of the CaMKIIα-positive hippocampal excitatory neurons.
Conclusion: EA at ST36 can promote food intake and gastric emptying in normal mice but not in CaMKIIα-Cre mice with activated hippocampal hM4Di receptors，suggesting a contribution of the CaMKIIα-positive hippocampal excitatory neurons (glutamatergic neurons in particular) to the enhanced gastrointestinal function of EA at ST36.
目的：观察电针“足三里”对正常小鼠进食量及胃排空的影响，聚焦海马谷氨酸能神经元探讨电针“足三里”调节胃功能的中枢机制。方法：实验分两部分进行：第一部分选取24只C57BL/6小鼠，随机分为正常组和电针组(电针双侧“足三里” ，20 min/次，1次/ d，连续7 d)，每组12只，每组随机选取6只测定进食量及胃排空率，另外6只用在体微透析联合高效液相色谱技术检测海马谷氨酸含量。第二部分选取30只CaMKII-Cre小鼠，海马注射化学遗传抑制病毒(AAV-DIO-hM4Di-eYFP)，病毒表达21 d后，随机选取3只用于荧光显微镜观察病毒表达情况，3只用于脑片膜片钳验证病毒是否具有功能性，将剩余小鼠随机分为4组：对照组、电针组、化学遗传抑制病毒组和化学遗传抑制病毒+电针组，每组6只。采用脑片膜片钳技术记录比较对照组和电针组CaMKII-Cre小鼠海马谷氨酸能神经元兴奋性；化学遗传抑制病毒组［腹腔注射氯氮平-N-氧化物(CNO)］和化学遗传抑制病毒+电针组(腹腔注射CNO+电针“足三里”)用于小鼠进食量、胃排空率检测。结果：1)与正常组比较，电针组C57BL/6小鼠进食量、胃排空率明显增加(P<0.01)，海马谷氨酸含量升高(P<0.01)；2)与对照组比较，电针组CaMKII-Cre小鼠海马谷氨酸能神经元兴奋性显著提高(P<0.01)；3)与化学遗传抑制病毒组比较，化学遗传抑制病毒+电针组CaMKII-Cre小鼠进食量和胃排空率变化不明显(P >0.05)。结论：电针“足三里”可以促进胃功能，其机制可能与激活海马中的谷氨酸能神经元有关。.
Keywords: CaMKIIα-positive excitatory neurons; Electroacupuncture; Gastric function; Hippocampus.