BDA神经示踪研究证实大鼠纹状体神经通路连接

Confirmation of Neural Pathway Connections by BDA Neural Tracing in Rat Striatum

目的为了观察证实纹状体神经元的形态定位和突触连接,以及由此证实神经示踪技术在此领域的实用性和有效性。方法实验借助示踪剂立体定位注射,并结合免疫组化和免疫电镜超微结构技术,实验数据以SPSS10.0软件统计处理。结果 (1)神经示踪剂BDA显示较好的定向运送特性,具有敏感和理想的标记效果。借助示踪剂BDA注射的逆行单标记实验结果显示纹状体-SNr和-GPe两类投射神经元的胞体大小和分支数量无明显的形态学差异,在免疫电镜下明显可见阳性树突和树突棘及其所形成的兴奋性突触连接。光镜下可见BDA顺行标记的PFn神经元的轴突终末密集分布于纹状体,并在电镜下清晰可见这些阳性终末与纹状体神经元之间的突触连接。(2)神经示踪结合免疫组化双标记显示纹状体-SNr和-GPe神经元均较少定位于Patch间区,而且均与纹状体Parv中间神经元之间无明显定性关系,但可见中脑TH阳性纤维的密集分布。在电镜超微结构水平可见BDA顺行标记的PFn阳性终末分别与纹状体-直接通路和纹状体-间接通路神经元形成典型的兴奋性突触连接,但与两者之间,及其所属的树突和树突棘之间的突触连接百分率均没有明显的统计学差异(P>0.05,P>0.05)。(3)荧光双标记结果显示BDA标记的PFn阳性终末和VG lut2抗体的免疫标记的背侧丘脑-纹状体阳性终末,在纹状体内呈现95.4%的共存率,而且后者标记更充分。同时也证实纹状体-SNr和-GPe两类投射神经元相互之间不显示可见的共存关系。结论 BDA为高度敏感的神经示踪剂,能够较为方便和充分地标记目的神经元和突触结构。纹状体-SNr和-GPe两类投射神经元的形态结构、分布形式无明显的差异。中脑和背侧丘脑神经元均与纹状体神经元之间存在形态学联系,尤其是背侧丘脑神经元与纹状体-直接通路和纹状体-间接通路神经元之间形成典型的兴奋性突触连接,提示其对纹状体神经元可能具有广泛的兴奋作用。

Objective To observe and confirm morphologic localization and synaptic contact with striatal neurons, and to confirm the practicability and efficacy of pathway tracing technology in this field. Methods The stereotaxis injection technology of pathway tracer was combined with immunochemistry and immunoelectron microscopy in this research. All the experimental data were statistically analyzed with SPSS10.0 software. Results （1）BDA pathway tracer displayed as a sensitive and ideal agent of orientation transport and labeling. With the BDA retrograde labeling, the experimental data revealed that the variances of soma diameter and the number of primary dendrites were not statistical significant between the striato - SNr and - GPe neurons. Positive dendrites and spines and their excitatory connections were clearly observed at the immunoelectron microscopic level. At the light microscopic level, the BDA -labeled PFn terminals densely local- ized in rat striatum and at the electron microscopic level, these positive terminals synapsed with striatal neurons. （2）Double labeling of pathway tracing and immunohistochemistry revealed that few striato - SNr and - GPe neurons which had no obvious correlation with the stiatal PV interneurons localized in the Patch compartment, but the positive mesocephalic TH fibers were in dense arrangement. At electron microscopic level, the BDA - labeled PFn terminals form typical excitatory synapsed with the neurons of striato - direct and - indirect pathway respectively. But there were no statistically significant differences in PFn - positive synaptic percentage of whole postsynaptic profile. dendrites and spines between striato - SNr ＋ and - GPe ＋ （ P 〉 0. 05,Pa 〉 0. 05, Ps 〉 0. 05 ）. （3）Data on double immunofluores- cence revealed that BDA -labeled PFn -positive terminals had 94.5% of coexistence with thalamo -striatal positive terminals immunolabeled by VGluT2 while the latter was more sufficiently labeled. Simuhaneously, immunoflurescence images revealed that the coexistence was not observed between striatal - SNr and - GPe projection neurons. Conclusion BDA is a highly sensitive neural pathway tracer, which can yield reliably and exquisitely detailed labeling of target neurons and synoptic structures. The variances of morphologic structures attd localization do not show statistical significance between striato - SNr and - GPe projection neurons. Mesencephalic and thalamic neu- rons are correlated with striatat neurons in morphology. Especially, the latter ones have typical excitatory synapses with striato - direct and - indirect neurons, suggesting that thalamic neurons may extensively excite striatal neurons.