AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures

Abstract

Ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the major mediators of fast synaptic neurotransmission. In this work, we used primary cortical cultures from rats as a model system to study AMPA receptor regulation during in vitro cell maturation and after synaptic activity modifications. The levels of AMPA receptor mRNA and protein, along with the alternative splicing and RNA editing of the AMPA receptor subunit (GluR1-4) mRNAs, were analyzed in immature (DIV5) and mature (DIV26) rat neuronal cultures. We observed an increase in the expression of all four AMPA receptor subunits during in vitro neuronal maturation. This finding might be due to the formation of new synapses between neurons during the development of a complex neuronal network. We also analyzed the effects of stimulation (KCl and glutamate) and inhibition (APV/TTX) on rat mature neuronal cultures (DIV26): stimulation with KCl led to an overall down-regulation of GluR1 and GluR3 AMPA receptor subunits and an up-regulation of the GluR2 subunit. Similarly, glutamate treatment induced a significant down-regulation of GluR1 together with an up-regulation of GluR2. In contrast, the chronic blockade of neuronal activity that resulted from APV/TTX treatment up-regulated GluR1 and GluR3 with a parallel down-regulation of GluR2 and GluR4. RNA editing at the R/G site increased during neuronal cell maturation for all AMPA receptors (from 8-39% at DIV5 to 28-67% at DIV26). Unexpectedly, all the treatments tested induced a marked reduction (ranging from -9% to -52%) of R/G editing levels in mature neurons, primarily for the mRNA flip variant. In summary, we showed that cultured rat cortical neurons are able to vary the stoichiometric ratios of the AMPA receptor subunits and to control post-transcriptional processes to adapt fast synaptic transmission under different environmental conditions.

Figure 1. Immunofluorescence analysis of rat cortical cultures.

Double labeling of rat cortical neurons, shown at 20× magnification. The left column shows the immunolabeling of immature cultures at DIV5, while the right column shows the localization of the same markers in mature cultures at DIV26. (A, B) Double labeling of dendrites with antibodies to MAP2 (green) and of axons with antibodies to neurofilament heavy chain subunit (≥200 KDa; red). (C, D) Double labeling of the glial population with antibodies to GFAP (green) and of neuronal precursor cells with antibodies to nestin (red). (E, F) Double labeling of dendrites with anti-MAP2 (green) and of neuronal precursor cells with anti-nestin (red). (G and H) Double labeling of dendrites with anti-MAP2 (green) and of glial cells with anti-GFAP (red). Scale bar: 50 µm.

Figure 2. mRNA and protein levels of AMPA receptor subunits during cell maturation.

(A) Western blot analysis of the four AMPA receptor subunits. A time course at four different stages of maturation of cultured neurons is shown. DIV5 protein levels of AMPA receptor subunits were set to 100%. GAPDH was used as loading control and the results are the mean ± S.E. of 3 independent experiments, performed in independent preparations. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001). (B) Normalized expression level of the four AMPA receptor subunit mRNAs analyzed by qGene software in immature (DIV5) and mature (DIV26) cortical cultures. Each value is normalized to a geometric mean of three house-keeping genes: GAPDH, RPLR2 and β-actin. The results are the mean ± S.E. of 3 independent experiments, performed in independent preparations. Statistical analysis was done using Student's t test.

Figure 3. AMPA receptor expression is predominant in neuronal cells.

Double labeling of rat cortical neurons at DIV5 and DIV26 (20×) with an antibody against the glial marker GFAP (green) and with antibodies against the four AMPA receptor subunits: (A, B) GluR1; (C, D) GluR2; (E, F) GluR3 and (G, H) GluR4 (red). The images were acquired from rare regions with high density of glial cells to show that the GFAP-positive cells do not express AMPA receptors. Scale bar: 50 µm.

Figure 4. GluR1-4 flip and flop isoform relative expression and GluR2-4 R/G site editing during cell maturation.

(A) The graphs represent the percentage of the flip isoform among the total flip + flop isoforms present during the maturation of cortical cultured neurons and in extracts of adult rat prefrontal/frontal cortex (P/FC). (B, C) Editing levels at the R/G site of the flip (B) and flop (C) variants of subunit GluR2-4 mRNAs. The results are the mean ± S.E. of 3 independent experiments, performed in independent preparations. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).

Figure 5. Protein levels of AMPA receptor subunits after acute and chronic treatment with KCl and APV/TTX.

GluR1-4 protein levels in primary cortical cultures (DIV26) after both acute and chronic treatment with KCl and APV/TTX. The results are expressed as the mean ± S.E. of 3 independent experiments performed in independent preparations and reported as percentage of non-treated control (CNT). Statistical analysis was performed by one-way ANOVA followed by the Dunnett test (*p<0.05, **p<0.01, ***p<0.001).

Figure 6. GluR1-4 splicing levels after acute and chronic treatment with KCl and APV/TTX.

Relative expression of the flip variant as a percentage of the total flip + flop for each of the four AMPA receptor subunits. The results are expressed as the mean ± S.E. of 3 independent experiments performed in independent preparations. Statistical analysis was performed by one-way ANOVA followed by the Dunnett test (*p<0.05, **p<0.01, ***p<0.001).

Figure 7. GluR2-4 R/G editing levels after acute and chronic treatment with KCl and APV/TTX.

Editing levels at the R/G site of subunits GluR2-4 for the flip and flop variants. The effects of KCl and APV/TTX treatments on the editing levels at the R/G site are shown separately for each variant. The results are expressed as the mean ± S.E. of 3 independent experiments performed in independent preparations. Statistical analysis was performed by one-way ANOVA followed by the Dunnett test (*p<0.05, **p<0.01, ***p<0.001).

Figure 8. Protein levels, flip/flop splicing and RNA editing at the R/G site of GluR1-4 after 24-hour treatment with glutamate.

(A) Western blot of the four AMPA receptor subunits after 24-hour treatment with glutamate on mature cortical cultures. The results are expressed as the mean ± S.E. of 3 independent experiments performed in independent preparations and reported as percentage of non-treated control (CNT). (B) The graphs represent the percentage of the flip isoform among the total flip + flop isoforms after glutamate treatment. (C) Editing levels at the R/G site of subunits GluR2-4 for the flip and flop variants. The results are expressed as the mean ± S.E. of 3 independent experiments performed in independent preparations. Statistical analysis was performed by unpaired Student's t-tests (*p<0.05, **p<0.01, ***p<0.001).