病毒示踪法研究小鼠中缝背核5‐羟色胺能神经元的传出投射差异

Differences in efferent projections from 5‑hydroxytryptamine neurons in the dorsal raphe nucleus through virus tracing in mice

目的研究小鼠中缝背核(DRN)中5‐羟色胺(5‐HT)能神经元的下游投射特征和DRN中投射向不同脑区5‐HT能神经元的解剖分布差异。方法将特异性顺向示踪病毒rAAV2/9‐Ef1α‐DIO‐mCherry注射至Sert‐Cre小鼠(3只)的DRN,病毒表达4周后将小鼠灌注取脑,并将全脑切成厚度为40μm的冠状脑片,进行全玻片免疫荧光成像扫描,观察DRN中5‐HT能神经元投射向下游的脑区分布。将特异性逆向示踪病毒rAAV2/retro‐EF1α‐DIO‐BFP、rAAV2/retro‐EF1α‐DIO‐mCherry和rAAV2/retro‐EF1α‐DIO‐EGFP分别注射到Sert‐Cre小鼠(3只)的伏隔核(NAc)、中央杏仁核(CeA)和腹侧被盖区(VTA),病毒表达4周后将小鼠灌注取脑,制备包含NAc、CeA、VTA和DRN的脑切片于共聚焦荧光显微镜下观察病毒标记的5‐HT能神经元在DRN中解剖分布情况。结果DRN的5‐HT能神经元向全脑发出广泛投射,包括梨状皮质、内侧前额叶皮质、NAc、基底前脑、CeA、外侧僵核、外侧下丘脑、VTA等。其中,投射向NAc的5‐HT能神经元主要分布在DRN的背侧中央区和外侧区,投射向CeA的5‐HT能神经元主要位于DRN的背外侧区,投射向VTA的5‐HT能神经元主要位于DRN的腹侧束间部。结论投射向不同脑区的5‐HT能神经元在DRN的解剖学分布中存在差异,提示DRN内的5‐HT能神经元存在相对独立的多个平行的亚系统。

Objective To investigate the characteristics of efferent projections from 5‐hydroxytryptamine(5‐HT)‐ergic neurons in the dorsal raphe nucleus(DRN)in mice,and to explore the differences in anatomical distribution of 5‐HT‐ergic neurons that project to different brain regions in the DRN.Methods A specific anterograde tracing virus rAAV2/9‐Ef1α‐DIO‐mCherry was injected into the DRN of Sert‐Cre mice(n=3).After virus expression for four weeks,the mice underwent transcardiac perfusion,and the brains were removed and cut into 40μm‐thick coronal slices.The whole‐slide immunofluorescence imaging scans were performed to observe the distribution of 5‐HT‐ergic neurons in the downstream projection brain regions of the DRN.The specific retrograde tracing viruses rAAV2/retro‐EF1α‐DIO‐BFP,rAAV2/retro‐EF1α‐DIO‐mCherry,and rAAV2/retro‐EF1α‐DIO‐EGFP were injected into the nucleus accumbens(NAc),central nucleus of the amygdala(CeA)and ventral tegmental area(VTA)of Sert‐Cre mice(n=3),respectively.After virus expression for four weeks,the mice underwent transcardiac perfusion and the brains were collected.Then,slices containing the NAc,CeA,VTA and DRN were prepared for observing the anatomical distribution of virus‐labeled 5‐HT‐ergic neurons in the DRN under a confocal fluorescence microscope.Results 5‐HT‐ergic neurons in the DRN extensively projected to the whole brain,including the piriform cortex,medial prefrontal cortex,NAc,basal forebrain,CeA,lateral sclerotic nucleus,lateral hypothalamus,and VTA.Notably,5‐HT‐ergic neurons projecting to the NAc were mainly distributed in the dorsal central and lateral regions of the DRN,5‐HT‐ergic neurons projecting to the CeA were mainly located in the dorsal lateral region of the DRN,and 5‐HT‐ergic neurons projecting to the VTA were mainly located in the ventral interfascicular region of the DRN.Conclusions The anatomical distribution of 5‐HT‐ergic neu‐rons in the DRN that project to different brain regions are different,indicating the existence of relatively independent multiple parallel subsystems of 5‐HT‐ergic neurons within the DRN.