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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 the morphological localization and synaptic connections of striatal neurons, and to prove the practicability and effectiveness of neural tracing techniques in this field. Methods The experiments were performed with stereotactic injection of tracer and immunohistochemistry and immunoelectron microscopy. The experimental data were statistically analyzed by SPSS 10.0 software. Results (1) Neurotracer BDA showed better directional transport characteristics, with sensitive and ideal labeling effect. Retrograde single labeling experiments with tracer BDA injection showed no significant morphological differences in the size of the soma and the number of branches of the striatum-NSr and -GPe projection neurons. Positive dendrites and trees Spines and their formation of excitatory synaptic connections. Light microscopy showed that axon terminals of PFN neurons labeled with BDA were densely distributed in the striatum and the synaptic connections between these positive terminals and striatal neurons were clearly visible under electron microscope. (2) Neurological tracing combined with immunohistochemical double labeling showed that the number of striatum-NSr and -GPe neurons were less located in the interchromic region, and there was no obvious qualitative relationship between the striatum Parv intervening neurons Visible dense distribution of TH-positive fibers in the brain. At the electron microscopic level, the parallel BDA-positive PFn-positive terminals formed typical excitatory synaptic connections with the striatal-direct pathway and striatal-indirect pathway neurons, respectively, but with both, and There was no significant difference in the percentage of synaptic connections between dendrites and dendritic spines (P> 0.05, P> 0.05). (3) Fluorescence double labeling showed that immunophenotypic dorsal thalami-striatum-positive terminals of BDA-labeled PFn-positive terminal and VGlut2 antibodies showed 95.4% coexistence in the striatum, and the latter was more marked full. At the same time, it was also confirmed that striatal-NSr and -GPe two types of projection neurons do not show the visible coexistence relationship with each other. Conclusions BDA is a highly sensitive neurotracer that can mark the target neurons and synaptic structures more conveniently and adequately. There was no significant difference in the morphological structure and distribution of striatal-Srr and -GPe projection neurons. There is a morphological relationship between midbrain and dorsal thalamic neurons and striatum neurons, especially typical dorsal thalamic neurons and striatum-direct pathway and striatal-indirect pathway neurons Excitatory synaptic connections suggest that it may have a wide range of excitatory effects on striatal neurons.