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NR2B signaling regulates the development of synaptic AMPA receptor current - PubMed

  • ️Mon Jan 01 2007

Comparative Study

NR2B signaling regulates the development of synaptic AMPA receptor current

Benjamin J Hall et al. J Neurosci. 2007.

Abstract

The postnatal maturation of glutamatergic synapses involves a change in composition and functional contribution of postsynaptic receptors. Developing cortical synapses are dominated by NMDA receptors (NMDARs) containing NR2B subunits and are characterized by a low ratio of AMPA/NMDA receptor-mediated current. Synapse maturation is marked by the incorporation of NR2A-containing NMDA receptors and an increase in the AMPA/NMDA current ratio. We show here that NMDARs containing the NR2B subunit regulate glutamatergic transmission at developing synapses by negatively influencing the synaptic incorporation of AMPA receptors (AMPARs). Genetic removal of NR2B leads to increased surface expression and synaptic localization of AMPA receptor subunits and a corresponding increase in AMPAR-mediated synaptic current. Enrichment of synaptic AMPARs, in the absence of NR2B signaling, is associated with increased levels of transmembrane AMPAR regulatory protein (TARP) expression and is blocked by expression of a dominant-negative TARP construct (gamma-2deltaC). These observations suggest that NR2B signaling limits AMPA receptor incorporation at developing synapses by negatively regulating TARP expression and provide a mechanism to explain the maintenance of low AMPA/NMDA ratio at immature glutamatergic synapses.

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Figures

Figure 1.
Figure 1.

Development of spontaneous synaptic activity in rat cortical cultures correlates with surface and synaptic trafficking of AMPA receptor subunits GluR1 and GluR2. A, Representative traces of spontaneous synaptic activity in sister cultures of rat E18 cortical neurons at subsequent DIV, measured by whole-cell voltage clamp at a holding potential of −65 mV. Network-driven spontaneous synaptic activity develops slowly in culture, increasing dramatically after the first week in vitro, with a rapid increase in unitary event amplitude and frequency between 10 and 14 DIV. B, Live labeling the surface pool of GluR1 and GluR2 subunits in these cultures revealed a strong increase in the surface expression of AMPAR subunits between 6 and 14 DIV. Data shown are example fields of view for cultures live labeled with anti-GluR1 (top images) and anti-GluR2 (middle images) antibodies and merged images with the dendritic marker MAP2 (bottom images) at 6,10, and 14 DIV. C, Surface biotinylation and Western blot analysis of receptors in culture at increasing days in vitro confirms the surface immunofluorescence data showing the increase in GluR1 and GluR2 surface expression over this time period. D, Cumulative data from multiple cultures are presented showing the increase in synaptic localization of these GluR puncta measured by costaining with an excitatory synaptic marker (VGlut1 and VGlut2) (mean ± SEM).

Figure 2.
Figure 2.

Increase in AMPA receptor-mediated current in the absence of NR2B protein. A, Lysates from WT (+) and NR2B null (−) mouse cortical cultures (12 DIV) blotted and probed for NR2A protein (top) and reprobed for NR2B (bottom) show that NR2A protein expression is unchanged in the absence of NR2B. B, Pooled data showing the similarity in levels of the main NMDAR subunits in NR2B knock-out cultures (mean ± SEM). C, Recordings from mouse cortical cultures revealed that the generation of spontaneous synaptic currents was similar to that seen in rat culture, arising ∼1 week in vitro (wild type; top traces). Example traces from knock-out cultures (20 s in duration at −65 mV) show this developmental pattern and the enhancement of synaptic current in NR2B−/− cultures as early as 8/9 DIV. D, Cumulative data (mean ± SEM) showing the increase in mean sEPSC amplitude in NR2B−/− cultures compared with controls. This increase in amplitude was significant between genotypes. E, Control (wild-type or heterozygous) cultures were generated from heterozygous matings and compared with sister cultures from homozygous knock-out embryos. An example PCR genotyping from one of these crosses is shown. The knock-out allele results from a PGK-neo cassette insertion that removes the first ATG in the initial coding exon of the NR2B gene. The wild-type reaction primes a 422 bp segment of this same exon.

Figure 3.
Figure 3.

Increase in AMPAR-mediated current at individual synapses in the absence of NR2B. A, B, Example whole-cell voltage-clamp traces from NR2B sister cultures (WT and NR2B−/−) recorded in the presence of gabazine, TTX, and cyclothiazide to isolate mEPSCs demonstrate the increase in “miniature” AMPAR-mediated EPSC amplitudes in the absence of NR2B protein. C, Cumulative data showing the range and mean amplitude ± SEM of mEPSCs in the two conditions; recordings from sister cultures are delineated by the connecting lines. D, The same dataset plotted by cumulative histogram for all the recorded events from each genotype showing the right shift, toward higher amplitude, in the knock-out neurons. E, Ensemble average mEPSCs from single neurons of each genotype (at 11 DIV) demonstrate this increased amplitude in the absence of any change in current kinetics.

Figure 4.
Figure 4.

NR2B negatively regulates surface and synaptic localization of the AMPAR subunit GluR1. A, B, Cultured cortical neurons from NR2B−/− embryos (right) and WT littermates (left) were live labeled with anti-GluR1 antibodies and subsequently fixed and stained for MAP2 at 14 DIV. These data show the dramatic increase in surface localization of this receptor subunit in the absence of NR2B. Scale bars; A, 5 μm; B, 2 μm. C, D, Cultures at 14 DIV were live labeled for GluR1 and then fixed and stained for MAP2 and the synaptic marker VGlut1 and VGlut2. C, Quantification of the immunostaining data showing the surface expression of GluR1 puncta per length of dendrite was increased in NR2B−/− neurons compared with WT neurons. D, The integrated pixel density of GluR1 staining in VGlut1- and VGlut2-positive synapses was also significantly increased in NR2B−/− neurons versus control neurons (350 synapses per animal). All histograms show mean ± SEM.

Figure 5.
Figure 5.

Enhancement of PSC amplitude in NR2B−/− neurons is the result of a cell-autonomous mechanism. A, B, NR2B WT neurons from GFP-expressing animals (β-actin GFP; WT-GFP) were cocultured with either WT or NR2B−/− cells (50%/50% cocultures). This allowed acquisition of simultaneous recordings from WT-GFP neurons and nearby neurons, either NR2B WT or NR2B−/− in culture (position of recording pipettes is highlighted in A). Simultaneous whole-cell recordings of AMPA-mediated sEPSCs (V hold, −65 mV) are shown for a neuron pair in a WT:WT-GFP coculture (C) and an NR2B−/−:WT-GFP coculture (D). E, Cumulative data from coculture recordings showed a significant increase in the amplitude of sEPSCs in NR2B−/− neurons recorded simultaneously with WT neurons in the same cultures (mean ± SEM). F, Unitary sEPSC event traces with collective average traces overlaid show the strong increase in amplitude of NR2B−/− responses (black) compared with simultaneously recorded WT-GFP responses (green) in the absence of any change in response kinetics (inset).

Figure 6.
Figure 6.

Suppression of NR2B protein in individual cortical neurons leads to enrichment of synaptic AMPARs. A, Experimental design: rat cortical neurons were cultured from E18 embryos and transfected at 5 or 6 DIV with siRNA against NR2B (and GFP as a transfection marker) or treated with NMDAR antagonists starting at 5 DIV. Recordings were made 7 d later at 12 or 13 DIV. B, Efficacy of the NR2B-siRNA construct was shown by stimulating neurons with local, somatic application of NMDA plus

d

-serine in the presence of TTX, gabazine, and

d

-serine. Example traces show average responses from a siRNA-expressing and nontransfected control neuron. These traces are averages of five trials taken from two neurons within a single field of view in culture, and therefore stimulated under identical conditions. The I–V relationship for the NMDAR-evoked current in the nontransfected neuron is shown (inset). Calibration: 200 pA, 200 ms. C, The NMDA-evoked response recorded at +50 mV was significantly suppressed in neurons expressing siRNA against NR2B compared with nontransfected neurons in the same cultures (mean ± SEM). D, The cumulative distribution of mEPSC events recorded under each experimental condition are shown compared with the nontransfected event population (thick line). Transfection of neurons with siRNA against NR2B led to an increase in mEPSC amplitude (solid line) compared with nontransfected control cells in the same dish (thick line). Neither GFP alone nor scrambled siRNA (two conditions combined, dotted line) mimicked the effect of NR2B siRNA. This effect was confirmed by analysis of the mean event amplitude in cells expressing the siRNA (E). The transfection conditions for this experiment are shown in E. F, Cultures were treated from 5 DIV with either 100 μ

m

APV or 3 μ

m

ifenprodil (dotted lines). AMPA-mediated mEPSCs were recorded between 11 and 13 DIV. Neither the cumulative histograms (F) nor comparison of cell means (G) revealed a difference in mEPSC amplitude after chronic antagonist block. In F, the siRNA event population distribution is shown for comparison.

Figure 7.
Figure 7.

Negative regulation of AMPAR-mediated current by NR2B requires synaptic localization but not PDZ domain function. A, The experimental design is identical to Figure 6 except that cells were cotransfected with NR2B siRNA as well as GFP and either wild-type or a mutated NR2B construct. Rescue constructs are shown schematically in A: the wild-type construct with intact C terminus, a PDZ mutant (ΔPDZ) that suppresses the synaptic incorporation of NR2B, and a double mutant construct (ΔPDZ/ΔAP2) that retains synaptic localization (via the Y1472A mutation) but lacks a functional PDZ domain. B, Representative ensemble mEPSC averages are shown for a nontransfected cell and cells transfected with NR2B siRNA plus the WT receptor, the PDZ mutant, or the double mutant construct. C, Cumulative distribution of mEPSCs are plotted and show that WT NR2B expression rescued the effect of NR2B-siRNA (dashed line). D, The double mutant construct ΔPDZ/ΔAP2 was also able to rescue the effect of siRNA (dashed line), although the single ΔPDZ point mutant was unable to block the effect of siRNA (thin line). E, The significance of the effects in C and D are shown comparing average mEPSC amplitude from individual cells under each transfection condition.

Figure 8.
Figure 8.

Changes in cortical TARP levels correlate with altered AMPAR regulation in NR2B null neurons, and the effect of siRNA is blocked by coexpression of a dominant-negative TARP. A, B, Relative TARP levels in WT (+) and NR2B−/− (−) cultures at 16 DIV detected by Western blot analysis using a pan-TARP antibody. C, The increase in mEPSC amplitude seen in single cultured neurons in response to transfection with NR2B-siRNA is blocked by coexpression of the TARP dominant-negative construct γ-2ΔC. D, Analysis of the cumulative data showed a significant difference in the mean amplitude of AMPA-mediated mEPSCs in neurons expressing NR2B-siRNA and γ-2ΔC compared with those expressing NR2B siRNA alone.

Figure 9.
Figure 9.

Model of how NR2B signaling negatively regulates synaptic AMPARs at developing cortical synapses. NR2B-containing receptors act to limit cortically expressed TARP protein and thereby limit AMPAR incorporation at synapses, through exclusion of GluR1 and GluR2 subunits, providing a mechanism that maintains low AMPAR:NMDAR ratio at developing synaptic sites.

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