Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones - PubMed (original) (raw)
Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones
Zhanyan Fu et al. J Physiol. 2005.
Abstract
We investigated the role N-methyl-d-aspartate (NMDA) receptor subunits play in shaping excitatory synaptic currents in cultures of cerebellar granule cells (CGCs) from NR2A knockout (NR2A-/-) and wild-type (+/+) mice. Cultures were maintained in a condition that facilitates the occurrence of functional synapses, allowing us to record NMDA-miniature excitatory postsynaptic currents (mEPSCs) in addition to NMDA receptor-mediated whole-cell currents at three ages in vitro. Whole-cell NMDA current density decreased with development in both strains though currents from NR2A-/- neurones demonstrated greater sensitivity to CP101 606, an NR2B subunit specific blocker. Sensitivity to Mg(2+) blockade decreased with age in vitro in +/+ but not in NR2A-/- CGCs. Immunocytochemistry revealed that dendrites and somas displayed distinct NR1 and NR2A subunit clusters which became increasingly colocalized in +/+ neurones. Qualitatively the overall NR2B subunit staining pattern was similar in +/+ and NR2A-/- neurones throughout development, suggesting that the NR2B subunit distribution is not mediated by the NR2A subunit. In addition, staining with markers for excitatory synapses showed that expression of NR2A subunit (but not NR2B) increases at both synaptic and extrasynaptic sites in +/+ neurones during development. In parallel, NMDA-mEPSCs were faster in +/+ compared with NR2A-/- neurones at all time points studied, suggesting that the NR2A subunit begins to replace NR2B-rich NMDA receptors even at early stages of development. Many NR2A-/- neurones were devoid of NMDA-mEPSCs at the later time point, and transfection of the NR2A subunit in these neurones restored fast decay and the occurrence of NMDA-mEPSCs. Taken together, our results indicate that the NR2A subunit is mainly responsible for the developmental changes observed in the maturation of excitatory synapses.
Figures
Figure 1. Changes of NMDA current density and blockade by a selective NR1/NR2B receptor antagonist in +/+ and NR2A−/− CGCs with age in vitro
A, whole-cell currents were elicited in CGCs from NR2A−/− and +/+ mice by application of a saturating concentration of NMDA (200 μ
m
). Application duration is indicated by solid bars above traces. B, summary of the results from the experiments shown in A, indicating a similar developmental reduction in current density in the NR2A−/− and +/+ CGCs during development. Data derived from >30 cells from four different cultures. C, representative traces from whole-cell recordings of currents elicited by 200 μ
m
NMDA in +/+ and NR2A−/− CGCs at DIV8 in the presence or absence of CP101 606 (10 μ
m
). D, summary of the data obtained from experiments as in C, showing that the whole cell responses from NR2A−/− CGCs are more sensitive to the NR1/NR2B blocker than +/+ CGCs. Data are shown as percentage reduction of NMDA whole-cell current by CP101 606 (10 μ
m
) normalized to control conditions derived from >6 cells in each experimental group. **Significant difference compared to DIV6–8 (P < 0.01, two-tailed, unpaired Student's t test); *Significant difference compared to +/+ at the same age (P < 0.05; two-tailed, unpaired Student's t test); error bars:
s.e.m.
Figure 2. Developmental changes in NMDA current sensitivity to Mg2+ are observed in +/+ but not in NR2A−/− CGCs in vitro
A, whole-cell currents elicited in CGCs from +/+ mice at DIV7 (left) and DIV14 (right) by application of a saturating NMDA dose (200 μ
m
) in the presence of MgCl2 at different concentrations as indicated. Application duration is indicated by solid bars above traces. B, summary of the results obtained from experiments as in A, illustrating a rightward shift in Mg2+ blockade in +/+ but not in NR2A−/− CGCs with age in vitro. Data derived from >20 cells in two sets of cultures. IC50s are presented in parentheses.*Significantly different in comparison to DIV7+/+ (P < 0.05); error bars:
s.e.m.
Figure 3. Immunostaining illustrating NR1 subunit colocalization with NR2A or NR2B subunits in +/+ and NR2A−/− CGCs at two distinct days in vitro
A_–_D, example images illustrating an increase in NR2A staining intensity as the neurones mature, that is concomitant with a quantitative increase in NR1/NR2A colocalization. Large panels show granule cells double labelled for NR1 (green) and NR2A (red) subunits using fluorescently tagged antibodies. Exposure and thresholding for all images in the figure are identical, allowing for a qualitative appreciation of the findings. Fluorescent images are shown overlaying the matching Nomarski DIC images. Dendritic regions indicated by the white boxes in the larger panels are magnified to the right, illustrating the colocalization of NR1 and NR2A receptor subunit clusters (arrows). The separate channels are shown individually in the magnified panels (top, middle) and as an overlaid composite (bottom). Neurones from both +/+ (A and B) and NR2A−/− (C and D) mice are shown at DIV7 (A and C) and DIV14 (B and D). Arrowheads indicate clusters which are not colocalized. Note the very faint, background staining for NR2A in the NR2A−/− CGCs (C and D), illustrating the specificity of the antibody used. Scale bar 10 μm and 3 μm for insets. E_–_H, example fluorescence images illustrating that a decrease in NR2B staining intensity is correlated with culture age but not genotype. Legend as in A_–_D, but granule cells are double labelled for NR2B (red) and NR1 (green) subunits. Neurones from both +/+ (E and F) and NR2A−/− (G and H) mice are shown at DIV7 (E and G) and DIV14 (F and H).
Figure 4. Quantification of immunostaining for NMDA receptor subunits in +/+ and NR2A−/− CGCs with age in vitro
A, a comparison of the linear cluster density measurements (clusters(10 μm of dendrite)−1) of NR2A, NR2B or NR1 subunits in CGCs at DIV7 and DIV14 from both genotypes. Images were analysed with Metamorph Software. Antibody-positive clusters were defined as clusters of fluorescence that were at least twice the background fluorescence of the image. B, summary of quantitative results examining the colocalization of NR1 and NR2 subunits. Colocalization was based on the mean centres of fluorescent clusters being separated by <1 μm. Data were derived from at least 24 dendritic segments, in at least 18 neurones per subunit tested and 3 distinct culture preparations in each group. **Significantly different in comparison to DIV7 (P < 0.01); ⋄⋄ significantly different in comparison to +/+ at the same age (P < 0.01); error bars:
s.e.m.
Figure 5. Changes of NMDA-mEPSCs in +/+ and NR2A−/− CGCs with age in vitro
A and B, representative traces from NMDA-mEPSCs recorded from +/+ and NR2A−/− CGCs at DIV7 (A) and DIV14 (B). NMDA-mEPSCs were pharmacologically isolated using bicuculline metabromide (25 μ
m
), TTX (0.5 μ
m
) and NBQX (5 μ
m
) in a Mg2+-free solution. Each panel illustrates example traces of NMDA-mEPSCs on the left with their equally scaled fitted averages (superimposed in grey) and an indication of the respective weighted time constant (_τ_w) on the right. C and D, summary of the amplitude and kinetics from the analysis of synaptic currents in CGCs illustrating that the NMDA-mEPSCs become larger and faster with development in +/+ but not in NR2A−/− cells. Data derived from >15 cells in each experimental group from at least four different culture preparations. E, summary of the percentage of CGCs that exhibit NMDA-mEPSCs in +/+ and NR2A−/− groups at different ages showing the considerable reduction in events for NR2A−/− CGCs at later time points. Data derived from >40 cells in each experimental group from at least four different culture preparations.**Significant difference in comparison to DIV6–7 (P < 0.01); *significant difference in comparison to DIV6–7 (P < 0.05); ⋄⋄ significant difference in comparison to +/+ at the same age (P < 0.01); error bars:
s.e.m.
Figure 6. Distinct developmental changes in sensitivity of NMDA-mEPSCs to CP101 606 and spermine in CGCs from +/+ and NR2A−/− mice
A, example traces of NMDA-mEPSCs recorded from +/+ and NR2A−/− CGCs at DIV7 and DIV12 in the presence and the absence of 10 μ
m
CP101 606, a specific antagonist of NR1/NR2B receptors. B, summary of the action of 10 μ
m
CP101 606 on the frequency of NMDA-mEPSCs recorded from CGCs at different ages in culture and in distinct genotypes. Data derived from >8 cells in each experimental group from two culture preparations. Data on NMDA-mEPSCs amplitude and decay are summarized in the Results section. *Significant difference in comparison to +/+ at the same age (P < 0.05). C, summary of the action of 300 μ
m
spermine on the amplitude (left) and weighted decay time constant (right) of NMDA- mEPSCs recorded from CGCs at different DIV and in distinct genotypes. Data derived from >10 cells in each experimental group from two culture preparations. *Significant difference compared to control (P < 0.05; two-tailed, paired Student's t test); error bars:
s.e.m.
Figure 7. NMDA-mEPSCs properties are restored in NR2A−/− neurones transfected with plasmid encoding the NR2A subunit
A, NMDA-mEPSCs recorded from −/− CGCs at DIV7, which have been transfected with a plasmid that contains either pEGFP alone (top panels, pEGFP) or pEGFP together with NR2A subunit (bottom panels, NR2A and pEGFP). To the right, each panel also illustrates the respective fitted averages of NMDA-mEPSCs (superimposed in grey) with an indication of the respective weighted time constant of decay (_τ_w). B, summary of the amplitude (left) and kinetics (middle) illustrating that the NMDA-mEPSCs become larger and faster when NR2A−/− CGCs are transfected with the NR2A subunit. Data derived from >11 cells. Summary of the percentage of cells (right) with NMDA-mEPSCs (% CGCs) in +/+ and NR2A−/− groups. Data derived from >25 cells in each experimental group. **Significant difference in comarison to pEGFP-transfected CGCs (P < 0.01); error bars:
s.e.m.
Figure 8. Developmental changes in colocalization of vGlut and glutamate receptor subunits in CGCs from +/+ and NR2A−/− mice
A_–_D, large panels show examples of +/+ CGC cultures double labelled for vGlut-1 and vGlut-2 (vGlut, green) and NR2 (red) subunits using fluorescently tagged antibodies at DIV7 (A and C) and DIV14 (B and D). Images are shown with the matching Nomarski DIC image. Dendritic regions indicated by the white boxes in the larger panels are magnified to the right to illustrate the matching of vGlut and NR2 receptor subunit clusters. The separate channels are shown individually in the magnified panels (top, middle) or overlaid (bottom). Exposure and thresholding for all images in the figure are identical, allowing for a qualitative appreciation of the increase in synapses facing NR2A subunit clusters and decrease in those facing the NR2B clusters in +/+ CGCs with age in vitro. In all panels, arrows indicate synaptic receptors while arrowheads indicate extrasynaptic receptors. Scale bar 10 μm and 6 μm for insets. E, summary of data obtained from colocalization experiments using a combination of vGlut1 and 2 (VGlut) as a marker of excitatory synapses. The left panel compares the percentage of vGlut-positive boutons that face NR2 or Glur2/3 subunit clusters in CGCs (%synapses versus subunit) from different genotypes and at different ages in culture, with an indication of statistical significance with respect to wild-type (**) and DIV7 (⋄⋄). The right panel compares the percentage of NR2 subunit clusters that face vGlut positive boutons (% subunits versus synapses) in CGCs from different genotypes and at different ages in culture with an indication of statistical significance (**) with respect to DIV7. Colocalization was based on the mean centres of fluorescent clusters being separated by <1.6 μm. Data for NR2 subunit from >40 cells in three separate cultures and for Glur2/3 subunit from >12 cells in one culture. **Significantly different in comparison to +/+ at the same age (P < 0.01); ⋄⋄ significantly different in comparison to DIV7 (P < 0.01); error bars:
s.e.m.
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