Kirchhoff F, Mülhardt C, Pastor A, Becker C M, Kettenmann H
Institute for Biochemistry, University of Erlangen-Nürnberg, Erlangen, Germany.
J Neurochem. 1996 Apr;66(4):1383-90. doi: 10.1046/j.1471-4159.1996.66041383.x.
We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.
我们先前已证明，抑制性神经递质甘氨酸可在大鼠脊髓的神经胶质细胞中诱导膜电流。在本研究中，膜片钳技术与逆转录介导的聚合酶链反应相结合，以分析3至18日龄大鼠单个神经胶质细胞中甘氨酸受体亚基的表达。使用全细胞模式的膜片钳技术，通过其膜电流模式识别神经胶质细胞，并检测其对甘氨酸的反应性。随后，收集细胞质，接着对总细胞质RNA进行逆转录。亚基特异性cDNA片段经扩增后，通过琼脂糖凝胶电泳、Southern印迹法和测序进行分析。在所研究的所有细胞类型中，均检测到α1亚基的转录本，但未检测到α2或α3亚基的转录本。此外，约一半被分析的神经胶质细胞含有β亚基mRNA。这些结果表明，与培养的神经胶质细胞不同，大鼠脊髓的神经胶质细胞表达功能性甘氨酸受体。神经胶质细胞与突触区域紧密接触，这使得这些非神经元受体可能在甘氨酸能传递过程中被激活，并可能在神经胶质细胞中引发未知的反应。
