腹侧被盖区多巴胺神经元全脑神经环路示踪

Whole Brain Tracing of Dopamine Neurons in the V entral Tegmental Area

目的:研究小鼠中脑腹侧被盖区(VTA)多巴胺能神经元接受的全脑输入性上游投射及其输出性下游投射,解析其全脑上下游神经环路连接。方法:用立体定位仪将辅助病毒AAV-EF1a-DIO-GT和AAV-EF1a-DIO-G的混合液(1:1)注射到DAT-cre转基因小鼠的VTA脑区,2周后将重组狂犬病毒(RV)EnVA-RV-mCherry微注射到VTA脑区,1周后RV病毒完成逆向跨突触感染并充分表达荧光蛋白,全脑冰冻切片,用全自动扫描荧光显微镜全脑拍片。用立体定位仪将顺行示踪病毒AAV-EF1a-DIO-GFP微注射到DAT-cre转基因小鼠的VTA脑区,2周后待病毒及荧光蛋白充分表达后,全脑冰冻切片,VTA区脑片用TH抗体行免疫荧光染色,全自动扫描荧光显微镜全脑拍片。结果:狂犬病毒逆向跨单级突触示踪结果显示,全脑许多脑区核团神经元表达RV病毒携带的红色荧光蛋白,主要包括前脑皮层、纹状体、伏隔核、下丘脑视前区、外侧下丘脑、下丘脑室旁核、杏仁核、腹侧被盖区、黑质、中缝背核、臂旁核、缰核。顺行示踪病毒结果显示,表达绿色荧光蛋白的纤维投射主要集中在内侧前额叶皮层、纹状体、伏隔核、背外侧隔核、杏仁核、外侧下丘脑几个脑区。结论:VTA多巴胺能神经元的上游输入性投射广泛的分布于全脑,包括前脑皮层、基底神经节区、下丘脑区、边缘系统、中脑的许多核团都向其发出纤维投射。VTA多巴胺神经元的下游输出性投射主要集中在基底神经节的伏隔核和纹状体,内侧前额叶皮层及下丘脑也有一定投射。

Objective:To investigate the whole brain input and output projection of dopamine neuron in the ventral tegmental areal(VTA),analyse the whole brain upstream and downstream neural circuits connections.Methods:The mixture of helper viruses AAV-EFla-DIO-GT and AAV-EFla-DIO-G(1:1)was injected into the VTA of DAT-cre transgenic mice under stereotaxic apparatus.After two weeks,recombinant rabies virus(RV)EnVA-RV-mCherry was microinjected into VTA,One week later,the RV virus completed retrograde transsynaptic infection and fully expressed the fluorescent protein,prepare whole brain frozen section,scan the sections with an automatic scanning fluorescence microscope.The anterograde tracing virus AAV-EFla-DIO-GFP was microinjected into the VTA of DAT-cre transgenic mice under stereotaxic apparatus.After two weeks,virus and fluorescent protein were fully expressed,prepare whole brain frozen sections,the sections containing VTA were immunofluorescence stained with(tyrosine hydroxylase)TH antibody,scan the sections with an automatic scanning fluorescence microscope.Results:RV retrograde transsynapse tracing results shows,the neurons expressed red fluorescent protein carried by RV virus distributed in many region of whole brain.Mainly includes the anterior cortex,striatum,nucleus accumbens(NAc),preoptic area of hypothalamus(POH),lateral hypothalamus(LH),paraventricular nucleus of hypothalamus(PVN),amygdala,ventral tegmental area(VTA),substantia nigra(SN),dorsal raphe(DR),parbrachial nucleus(PBN)and habenula(HB).Anterograde tracing results shows,the fibers expressing GFP were mainly concentrated in the medial prefrontal cortex(mPFC),striatum,nucleus accumbens(NAc),lateral septal nucleus(LS),amygdala,and lateral hypothalamus(LH).Conclusion:The upstream input projections of VTA dopamine neurons were widely distributed in the whole brain,including the anterior cortex,basal ganglia area,hypothalamus area,limbic system,and many nuclear in the midbrain.The downstream output projections of VTA dopamine neurons were mainly concentrated in the nucleus accumbens(NAc)and striatum of the basal ganglia,but also some projection in the medial prefrontal cortex(mPFC)and hypothalamus.