NMDAR1 mRNA expression and glutamate receptor stimulated increase in cytosolic calcium concentration in rat and mouse cerebellar granule cells

doi:10.1016/0197-0186(96)00018-6

Neurochemistry International

Volume 29, Issue 5, November 1996, Pages 497-506

https://doi.org/10.1016/0197-0186(96)00018-6 Get rights and content

Abstract

We have previously reported that, unlike their rat counterparts, the survival of mouse cerebellar granule cells is independent of chronic stimulation whether owing to elevated K⁺-induced depolarization or NMDA (N-methyl-d-aspartate) receptor activation. One explanation could be that during the critical period mouse granule cells are very sensitive to NMDA receptor stimulation by endogenous glutamate released in the cultures. If so, this might be reflected by an increased expression of NMDA receptors or an increased response to their activation. We tested this hypothesis by measuring (a) the concentration of mRNA for the obligatory NMDA receptor subunit, NMDAR1, and (b) the glutamate/NMDA stimulated increase in cytosolic Ca²⁺-ion concentration in cultures at physiological or elevated K⁺-ion concentration. The expression of NMDAR1 mRNA was measured by competitive PCR of reversely transcribed mRNA and was normalized to that of the constitutively expressed H3.3 histone mRNA. The glutamate and NMDA stimulated increase in cytosolic Ca²⁺-ion concentration was measured using the fluorescent Ca²⁺-chelator Fluo3. In contrast to the hypothesis, we found NMDAR1 mRNA expression to be lower in mouse than in rat granule cells cultured for 4 days at physiological K⁺-ion concentration. However, the NMDA stimulated increase in cytosolic Ca²⁺-ion concentration did not differ in 4-day rat and mouse cultures. Although the glutamate-stimulated increase in cytosolic Ca²⁺-ion concentration in 2-day cultures was higher in mouse granule cells than in rat granule cells, the developmental profile of the glutamate-stimulated increase in cytosolic Ca²⁺-ion concentration was the same in both cases. In conclusion, we found no obvious evidence for increased NMDA receptor activity in mouse cerebellar granule cells cultured at physiological K⁺-ion concentration.

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Citation Excerpt :
The low AMPA response in cells grown in K25 is not in agreement with previous reports, which show that growing cells in 25 mM K+ increases the AMPA-induced 45Ca2+ influx as compared to cells grown in 10 mM K+[5]. However, this discrepancy could be explained by the different methods applied [25]. Furthermore, Condorelli et al. measured the AMPA-induced 45Ca2+ influx only at 7 DIV, where the K5 rat cultures are partly degenerated.
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)α-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 Ca²⁺ concentration ([Ca²⁺]_i), using the fluorescent Ca²⁺ 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 [Ca²⁺]_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.
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NMDAR1 mRNA expression and glutamate receptor stimulated increase in cytosolic calcium concentration in rat and mouse cerebellar granule cells

Abstract

Neuroscience

Neuroscience

Neuron

Nature

Neurosci. Lett.

C. Adv. Exp. Med. Biol.

Int. J. Devel. Neurosci.

J. Neurosci.

Neurochem. Int.

Neurochem. Res.

Int. J. Devl. Neurosci.

Neuron

Science

Neuroreport

J. Neurochem.

FEBS Lett.

Combinatorial RNA splicing alters the surface charge on the NMDA receptor

FEBS Lett.

Neurobiology of excitatory amino acids

Neurobiology of excitatory amino acids

NMDA treatment and K+-induced depolarization selectively promote the expression of a NMDA-preferring class of the ionotropic glutamate receptor in cerebellar granule cells

Neurosci. Lett.

NMDA treatment and K⁺-induced depolarization selectively promote the expression of a NMDA-preferring class of the ionotropic glutamate receptor in cerebellar granule cells