Mogensen H S, Jorgensen O S
Laboratory of Neuropsychiatry, Department of Pharmacology, University of Copenhagen, Rigshospitalet-6102, 2100, Copenhagen, Denmark.
Int J Dev Neurosci. 2000 Feb;18(1):61-8. doi: 10.1016/s0736-5748(99)00105-7.
Cultured mouse cerebellar granule cells differ from their rat counterparts in that they survive well when grown in non-depolarising medium (5 mM K(+)). However, when chronically stimulated by added glutamate agonists, including (RS)alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), rat cerebellar granule cells also survive well in non-depolarising medium. We hypothesised that the relatively good survival of mouse cerebellar granule cells in the absence of added glutamate agonists might reflect AMPA receptors resistant to desensitisation. These receptors might be stimulated by endogenous glutamate. We tested this hypothesis by comparing cultured mouse and rat cerebellar granule cells grown in depolarising (25 mM K(+)) and non-depolarising (5 mM K(+)) medium. We studied the AMPA-induced increase in intracellular Ca(2+) concentration (Ca(2+)), using the fluorescent Ca(2+) chelator, Fluo-3, and the relative concentrations of mRNAs for the four AMPA receptor subunits, GluR1-4. GluR1-4 mRNAs were measured by restriction enzyme analysis of a PCR product containing cDNA with a composition proportional to the four subunit mRNAs. We found that the Ca(2+)-response to AMPA receptor activation in cultured cerebellar granule cells is determined mainly by the desensitisation properties of the AMPA receptors rather than by their ion permeability. We also found that mouse cerebellar granule cells express AMPA receptors which are more resistant to desensitisation than the corresponding rat AMPA receptors. Thus, relatively slow AMPA receptor desensitisation kinetics may contribute to the survival of mouse cerebellar granule cells in non-depolarising medium.
培养的小鼠小脑颗粒细胞与其大鼠对应细胞不同，在于它们在非去极化培养基（5 mM K⁺）中生长时存活良好。然而，当用添加的谷氨酸激动剂，包括（RS）α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）进行慢性刺激时，大鼠小脑颗粒细胞在非去极化培养基中也能良好存活。我们推测，在未添加谷氨酸激动剂的情况下，小鼠小脑颗粒细胞相对良好的存活可能反映了对脱敏有抗性的AMPA受体。这些受体可能受到内源性谷氨酸的刺激。我们通过比较在去极化（25 mM K⁺）和非去极化（5 mM K⁺）培养基中培养的小鼠和大鼠小脑颗粒细胞来验证这一假设。我们使用荧光钙螯合剂Fluo-3研究了AMPA诱导的细胞内钙浓度（[Ca²⁺]i）的增加，以及四种AMPA受体亚基GluR1 - 4的mRNA相对浓度。通过对包含与四种亚基mRNA组成成比例的cDNA的PCR产物进行限制性酶切分析来测量GluR1 - 4 mRNA。我们发现，培养的小脑颗粒细胞中[Ca²⁺]i对AMPA受体激活的反应主要由AMPA受体的脱敏特性决定，而非其离子通透性。我们还发现，小鼠小脑颗粒细胞表达的AMPA受体比相应的大鼠AMPA受体对脱敏更具抗性。因此，相对较慢的AMPA受体脱敏动力学可能有助于小鼠小脑颗粒细胞在非去极化培养基中的存活。
