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AMPA receptor subunit mRNAs and intracellular [Ca(2+)] in cultured mouse and rat cerebellar granule cells
1Laboratory of Neuropsychiatry, Department of Pharmacology, University of Copenhagen, Rigshospitalet-6102, 2100, Copenhagen, Denmark. helle.smidt@forensic.ku.dk
Abstract:
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+)](i)), 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+)](i)-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.
Insights
Mouse cerebellar granule cells survive better due to AMPA receptors resistant to desensitization. These receptors, stimulated by endogenous glutamate, show slower desensitization kinetics, aiding survival in non-depolarizing media.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Cultured mouse cerebellar granule cells exhibit robust survival in low K+ medium, unlike rat cells.
- Rat cerebellar granule cells survive well in low K+ medium when exposed to glutamate agonists like AMPA.
- Mouse cell survival in non-depolarizing conditions may stem from AMPA receptors resistant to desensitization, potentially activated by endogenous glutamate.
Purpose of the Study:
- To investigate the hypothesis that mouse cerebellar granule cells possess AMPA receptors with reduced desensitization.
- To compare the AMPA receptor-mediated calcium responses and subunit mRNA expression in mouse and rat cerebellar granule cells.
- To determine the role of AMPA receptor desensitization properties versus ion permeability in cerebellar granule cell responses.
Main Methods:
- Cultured mouse and rat cerebellar granule cells were maintained in depolarizing (25 mM K+) and non-depolarizing (5 mM K+) media.
- AMPA-induced intracellular calcium ([Ca2+]i) increase was measured using the fluorescent indicator Fluo-3.
- Relative mRNA concentrations for AMPA receptor subunits (GluR1-4) were quantified via restriction enzyme analysis of a PCR product.
Main Results:
- The intracellular calcium response to AMPA receptor activation is primarily governed by receptor desensitization kinetics, not ion permeability.
- Mouse cerebellar granule cells express AMPA receptors that exhibit greater resistance to desensitization compared to rat cerebellar granule cells.
- Slower desensitization kinetics of AMPA receptors in mouse cells likely contribute to their enhanced survival in non-depolarizing medium.
Conclusions:
- AMPA receptor desensitization properties are a key determinant of cerebellar granule cell responses to glutamate agonists.
- Mouse cerebellar granule cells possess AMPA receptors with inherently slower desensitization rates.
- These findings elucidate a mechanism for the differential survival of mouse and rat cerebellar granule cells in specific culture conditions.
