Wouterlood Floris G, Böckers Tobias, Witter Menno P
Department of Anatomy, Graduate School of Neurosciences, Research Institute Neuroscience Vrije Universiteit Medical Center, 7, van der Boechorststraat, 1081 BT Amsterdam, Netherlands.
J Neurosci Methods. 2003 Sep 30;128(1-2):129-42. doi: 10.1016/s0165-0270(03)00171-7.
The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.
中枢神经系统中神经元的轴突及其精细的分支模式和终扣，可以通过传统神经解剖学技术以高度的准确性进行可视化。已识别的终扣是否与由另一种不同标记物识别的靶神经元形成突触联系，这需要超越传统光学显微镜所能提供的分辨率来解决。与突触连接相关的形态学元件由称为突触前和突触后致密物的特殊突触前和突触后连接复合体组成。对这些连接复合体进行电子显微镜检查会消耗大量时间和资源。为了提高我们分析神经元网络的速度，我们开发了一种高分辨率三重荧光方法，包括神经解剖学追踪、免疫荧光、共聚焦激光扫描和三维计算机重建，以在光学显微镜水平上确定参与突触连接的三个要素：传入纤维及其终扣、与通过荧光标记物的存在而识别的神经元紧密并置，以及夹在中间的突触后致密物标记物。我们使用形态学标准来检测轴突终末（纤维上的膨体）。针对ProSAP2/Shank3（一种与突触后致密物相关的支架蛋白）的抗体被用于确定突触连接的位置。结果显示存在三明治样结构：突触前纤维、ProSAP2/Shank3、突触后神经元。因此我们认为，我们可以将对电子显微镜的需求降至最低（甚至可能完全消除），从而显著提高神经解剖学追踪和网络分析的整体效率。
