Cell-site specific synapse formation in culture

通过对突触前和突触后神经元的细胞内荧光染色和对突触前终末的免疫细胞化学染色，研究了在大鼠小脑分离细胞培养中形成的浦肯野细胞上突触的空间分布。一个浦肯野细胞被全细胞电压夹住并充满德州红。然后用抗突触囊泡单克隆抗体FITC对其进行固定和染色。这一染色显示浦肯野细胞同时接受体细胞和树突的突触输入。同时对突触前小神经元和浦肯野细胞进行全细胞记录，并测定突触传递特性。同时，突触前神经元充满了路西法黄，浦肯野细胞充满了德克萨斯红。轴突肿胀被认为是兴奋性颗粒细胞的突触末梢，仅局限于浦肯野细胞的树突，而被认为是抑制性中间神经元的突触末梢在树突和体细胞上都被发现。同时用扫描电镜观察突触。培养针对谷氨酸受体mGluR1亚型的抗体，并用免疫细胞化学染色检测mGluR1在浦肯野细胞上的分布模式。在培养的浦肯野细胞树突上观察到mGluR1的点状标记。mGluR1主要分布在树突上，而不是细胞体上。mGluR1的这种分布模式与来自颗粒细胞的兴奋性谷氨酸突触的分布模式一致。因此，细胞位点特异性突触的形成和受体的细胞位点特异性分布已经在一个简单的培养系统中建立起来。

Spatial distribution of synapses on Purkinje cells formed in a dissociated cell culture of rat cerebellum, was studied by intracellular fluorescent stainings of pre- and postsynaptic neurons and by immunocytochemical staining of presynaptic terminals. A Purkinje cell was whole-cell voltage-clamped and filled with Texas red. Then, it was fixed and stained with an anti-synaptic vesicle monoclonal antibody using FITC.This staining revealed that a Purkinje cell receives synaptic inputs on both a soma and dendrites. Simultaneous whole-cell recordings were performed on a presynaptic small neuron and a Purkinje cell, and the property of synaptic transmission was determined. At the same time, the presynaptic neuron was filled with Lucifer yellow and the Purkinje cell was filled with Texas red. Axonal swellings presumed to be synaptic terminals of an excitatory granule cell, were exclusively localized along dendrites of a Purkinje cell, and presumed synaptic terminals of an inhibitory interneuron were found on both dendrites and a soma. Synapses were also observed with a scanning electron microscope. Antibodies were raised against one subtype of glutamate receptors, mGluR1, and distribution pattern of mGluR1 on a Purkinje cell was examined with immunocytochemical staining. Punctate labelings of mGluR1 were observed along dendrites of cultured Purkinje cells.mGluR1 were distributed more on dendrites than on a cell body. This distribution pattern of mGluR1 is consistent with that of excitatory glutamatergic synapses from granule cells. Thus, cell-site specific synapse formations and also cell-site specific distribution of the receptor have been established in a simple culture system.