Santi M R, Ikonomovic S, Wroblewski J T, Grayson D R
Fidia-Georgetown Institute for the Neurosciences, Georgetown University, Washington, D.C.
J Neurochem. 1994 Oct;63(4):1207-17. doi: 10.1046/j.1471-4159.1994.63041207.x.
Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 mM than in 25 mM KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 mM KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 mM KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.
原代培养的小脑颗粒神经元表达与磷酸肌醇水解刺激和环磷酸腺苷（cAMP）形成抑制偶联的代谢型谷氨酸受体（mGluRs）。为了评估在不同去极化条件下颗粒神经元中表达哪些mGluR mRNA，我们在含有10或25 mM KCl的条件下，测量了培养3 - 13天的神经元中选定受体mRNA的绝对量。构建了与mGluR1a、mGluR2、mGluR3、mGluR4和mGluR5独特区域相对应的mGluR特异性引物对和内标，并用于竞争性PCR衍生测定以定量相应的mRNA水平。对于与磷酸肌醇偶联的受体，mGluR1a mRNA的绝对含量比mGluR5 mRNA的含量高3至10倍。mGluR5 mRNA的表达在体外培养至9天时增加，且在10 mM KCl中比在25 mM KCl中高得多。对于与cAMP偶联的受体，有大量的mGluR4 mRNA，而mGluR3和mGluR2 mRNA的含量则少得多。在10 mM KCl中体外培养颗粒神经元会增加体外培养9天和13天时编码mGluR2和mGluR4的mRNA的绝对量。相反，在25 mM KCl中培养的神经元中，mGluR3 mRNA的含量始终较高。这些数据与以下可能性一致：在小脑神经元的原代培养中，磷酸肌醇反应可能主要由mGluR1a而非mGluR5介导，并且cAMP抑制优先涉及mGluR4和mGluR3。
