脑立体定位斜插法在小鼠小脑蚓部第Ⅸ小叶浦肯野细胞纤维投射研究中的应用

Application of Stereotaxic Techniques with Oblique Injection in the Study of Tracing Fiber Projection of Purkinje Cells from Cerebellar Vermis Lobule IX in Mice

目的 探讨脑立体定位斜插法靶向注射小鼠小脑蚓部第Ⅸ小叶的可行性,并观察该方法对小鼠小脑蚓部第Ⅸ小叶浦肯野细胞纤维的神经投射。方法 利用脑立体定位直插法(进针方向与水平方向垂直)或斜插法(进针方向与水平方向的垂直线呈30°夹角)标记Pcp2-Cre小鼠小脑蚓部第Ⅸ小叶,对比两种手术方式对小鼠颈部上方肌肉组织的影响;选择梯度剂量(500、300、150 nL)的顺行示踪病毒,探索适宜的病毒注射剂量;通过斜插法在小鼠小脑蚓部第Ⅸ小叶注射重组酶依赖性顺行示踪病毒,观察该小叶浦肯野细胞的传出神经投射。结果 相较于脑立体定位直插法,斜插法中小鼠头部皮肤切口更小,且不易破坏小鼠颈部上方肌肉;在前囟后6.00 mm、中线旁开0.00 mm、深4.08 mm坐标处,采用斜插法能靶向定位到小脑蚓部第Ⅸ小叶;同时,150 nL低剂量的病毒可较好的标记小脑蚓部第Ⅸ小叶浦肯野细胞,且不发生泄漏;通过病毒顺行示踪,在小鼠小脑顶核中间部、前庭小脑核、前庭内侧核巨细胞部以及前庭下核均检测到来自第Ⅸ小叶浦肯野细胞的神经投射纤维。结论 脑立体定位斜插法能在一定程度上减轻手术对小鼠颈部上方肌肉的破坏,通过该方法注射顺行示踪病毒能精确靶向小脑蚓部第Ⅸ小叶,并观察到该小叶浦肯野细胞的传出神经投射。

Objective To explore the feasibility of targeted injection into the lobule IX of the vermis in mice using stereotactic slanted insertion method,and to use this method for anterograde tracing of Purkinje cells in the lobule IX of the vermis to observe their fiber projections.Methods The vertical(needle insertion perpendicular to the horizontal plane)and oblique(needle insertion at a 30°angle to the vertical line of the horizontal plane)injection techniques were used to label the lobule IX of the cerebellar vermis.And the effects of these two surgical approaches on the muscle tissue above the neck in mice were compared.By choosing varying doses of anterograde tracer viruses(500 nL,300 nL,and 150 nL),the research identified the optimal virus injection dose.By applying the oblique injection technique,recombinant enzyme-dependent anterograde tracer viruses were injected into the lobule IX of the cerebellar vermis in Pcp2-Cre mice,revealing the efferent neural projections of Purkinje cells in that lobule.Results Compared with the vertical injection method,the oblique injection technique resulted in smaller incision of the head skin in mice and almost no damage to the muscles above the cerebellum.Utilizing the oblique injection technique at the specific coordinates(6.00 mm posterior to bregma,0.00 mm lateral,and 4.08 mm depth),we accurately targeted the lobule IX of the cerebellar vermis.Analysis revealed that a low dose of 150 nL virus effectively labeled the Purkinje cells in the lobule IX of the cerebellar vermis without any leakage.Through anterograde viral tracing techniques in Pcp2-Cre mice,projections from Purkinje cells of the lobule IX were identified in the medial cerebellar nucleus,vestibulocerebellar nucleus,medial vestibular nucleus magnocellular part,and the spinal vestibular nucleus.Conclusion The application of the oblique injection in stereotaxic techniques moderates the impact of surgery on the muscle tissue above the neck in mice.And through this approach,anterograde tracer viruses are precisely delivered to the lobule IX of the posterior vermis,enabling the identification of efferent neural projections from Purkinje cells in this lobule.