SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor

Abstract

At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

Fig. 1.

SynGAP and H-Ras mutant mice. a, Targeted disruption of SynGAP gene. Top, SynGAP gene with restriction enzyme sites (B,BglII; E, EcoRI;Sm, SmaI; Sp,SpeI; X, XhoI;Xb, XbaI); black boxes, exon; thick horizontal lines, homologous regions used in the targeting vector; Southern blot probes are indicated (3′).Bottom, SynGAP targeting vector. HA, Hemagglutinin sequence; IRES, internal ribosomal entry site; lacZ, β-galactosidase gene; neo, neomycin-resistance gene. The arrow below the SynGAP gene indicates arginine 312 (or 470) (Chen et al., 1998; Kim et al., 1998), which is highly conserved in RasGAP proteins and necessary for GAP activity and was deleted in the targeting vector.Right, Genomic Southern blots ofEcoR1-digested DNA from littermates of a heterozygote intercross and probed with the 3′ probe. Wild type (+/+), heterozygote (−/+), homozygote (−/−) are indicated. b, Targeted disruption of H-Ras gene. Top, H-Ras gene with restriction enzyme sites (abbreviations as above with the addition of the following: Bg, BglI;S, SphI; F,FspI); all other detail as in a.Bottom, Targeting vector. β-geoconsists of a β-galactosidase gene and a neomycin-resistance fusion gene. Right, Genomic Southern blots ofEcoR1-digested DNA from littermates of a heterozygote intercross and probed with the 5′ probe. Wild type (+/+), heterozygote (−/+), homozygote (−/−) are indicated. c, Expression patterns of SynGAP and H-Ras using X-gal staining of whole-mount sagittal brain sections. BS, Brain stem;C, cortex; CB, cerebellum,H, hippocampus. Representative SynGAP^−/+ and H-Ras^−/−sections are shown. Scale bar, 1 mm. d, Immunoblots comparing different protein levels of wild-type mice, SynGAP^−/+, and H-Ras^−/−mutants in hippocampus extracts. Left panels, Immunoblot analysis of GAP proteins. SynGAP, NF-1, and Ras-GAP were detected in wild-type (wt), SynGAP^−/+, and H-Ras^−/−extracts. A reduced amount of SynGAP protein was observed in SynGAP^−/+ mutants compared with wild type.Middle panels, Immunoblot analysis of Ras proteins. H-Ras was detected in wild-type mice and was normal in SynGAP^−/+ mutants. Pan-Ras antibodies, which recognize all Ras isoforms, revealed lower total Ras levels in H-Ras^−/−mutants and normal levels in SynGAP^−/+ mutants.Right panels, Immunoblot analysis of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95. Equivalent levels were observed in wild type (wt), SynGAP^−/+, and H-Ras^−/−extracts.

Fig. 2.

Neuroanatomy of hippocampus CA1 region in SynGAP^−/+and H-Ras^−/−mutant mice. a, Nissl; cresyl violet stain of CA1 pyramidal cells. b, Synaptophysin; immunohistochemistry for synaptic vesicle marker protein. c, MAP2; immunohistochemistry for dendritic marker protein. d, Golgi; montaged images of CA1 apical dendrites from Golgi-impregnated pyramidal neurons in the distal region of the stratum radiatum.e, EM; electron micrograph images of asymmetric axospinous synapses in the stratum radiatum of the CA1 region. Scale bars: a–c, 50 μm; d, 10 μm;e, 0.5 μm. SR, Stratum radiatum;SP, pyramidal cell body layer; wt, wild type. SynGAP^−/+ andH-Ras^−/−mutants are indicated.

Fig. 3.

Basal properties of synaptic transmission are unaltered in SynGAP mutant mice. a, Input–output curves were generated by comparing the fiber volley amplitude and slope of fEPSPs elicited by presynaptic stimulation intensities that evoked fEPSPs that were 25, 50, 75, and 100% of the maximal fEPSP amplitude that could be generated in each slice. No differences across all four stimulation intensities were evident in slices from SynGAP^−/+ (●, n = 18 slices from 5 animals) and wild-type (○, n = 19 slices from 5 animals) mice. Inset shows overlaid traces (each an average of 3 responses) evoked in a slice from a wild-type (left set of traces) and SynGAP^−/+ mutant (right set oftraces) mouse. Calibration: 5 msec, 1 mV.b, Pairs of presynaptic stimulation pulses delivered with an interpulse interval of 25, 50, 100, or 200 msec elicits similar amounts of paired-pulse facilitation in slices from SynGAP^−/+ (●, n = 16 slices from 5 mice) and wild-type mice (○, n = 16 slices from 5 mice).

Fig. 4.

NMDAR-mediated currents are unaltered in SynGAP mutant mice. a, The magnitude of the NMDA receptor-mediated component of EPSCs was estimated by the amplitude of the synaptic currents measured 50 msec after the start of the EPSC and expressed relative to the size of the AMPA receptor component estimated by the size of the EPSC measured 5 msec after the start of the EPSC. The size of the NMDA receptor-mediated component of the EPSCs in pyramidal cells from SynGAP^−/+ mutant mice (filled bars, n = 5 mice, 15 cells) was not different from that observed in wild-type pyramidal cells (open bars, n = 5 mice, 16 cells) at holding potentials of −80 mV (where the NMDA component is mostly blocked by extracellular Mg²⁺) and +40 mV. The inset shows example EPSCs (average of 3 responses) recorded at −80 and +40 mV in cells from SynGAP^−/+and wild-type mice. Calibration: 50 pA, 25 msec. b, Peak current densities (picoamperes/picofarads) for currents evoked by application of 100 μm NMDA (+10 μm glycine) in the absence of extracellular Mg²⁺ in cultured neurons from SynGAP^−/+ cells (black bar, n = 11), SynGAP^−/−(gray bar, n = 10), and wild-type mice (open bar, n = 11). Currents were recorded at a holding potential of −70 mV.c, Current–voltage relationships for NMDA receptor-mediated currents (normalized to maximal current) evoked by application of 100 μm NMDA applied in the absence (right plot) and presence (left plot) of 1.0 mm extracellular Mg²⁺ in cultured neurons from wild-type (open symbols,n = 11 cells), SynGAP^−/+ mice (black symbols, n = 11 cells), and SynGAP^−/−mice (gray symbols, n = 10). Calculated junction potential was +15 mV and is not corrected for in Figure 3C. Values correspond to mean ± SEM.

Fig. 5.

LTP is reduced in SynGAP^−/+mutants. a, After a 20 min period of baseline recording, two trains (1 sec duration) of 100 Hz stimulation were delivered with an intertrain interval of 10 sec at time 0. Although this protocol induced robust LTP in slices from wild-type animals (○, fEPSPs potentiated to 188 ± 9% of baseline, n = 11 slices from 7 animals), it induced significantly less LTP in slices from SynGAP^−/+ mutant mice (●, fEPSPs potentiated to 150 ± 10% of baseline, n = 11 slices from 7 animals; p < 0.01 compared with wild-type LTP).b, Six trains of 100 Hz stimulation (each 1 sec in duration) were delivered with an intertrain interval of 5 min beginning at time = 0 to induce saturating levels of LTP. In wild-type slices (○, n = 7 slices from 4 animals), fEPSPs were potentiated to 257 ± 25% of baseline but potentiated to only 154 ± 3% of baseline in slices from SynGAP^−/+ mutant mice (●, n = 11 slices from 5 animals; p < 0.005 compared with wild type). c, Summary graph showing the amount of LTP present 40–45 min after 900 pulse trains of 1, 5, 10, or 20 Hz stimulation in slices from wild-type (○, n = 5, 5, 5, and 7 animals, respectively, for each frequency) and SynGAP^−/+ mutant mice (●, n = 9, 7, 7, and 7 animals, respectively, for each frequency). Although 1 and 5 Hz trains of synaptic stimulation had similar effects on synaptic transmission in slices from SynGAP^−/+ and wild-type mice, 10 and 20 Hz trains of stimulation induced significantly less LTP in slices from SynGAP^−/+ mice (**p < 0.001, *p < 0.05).d, Summary graph showing the amount of LTP present 40–45 min after trains of 5 Hz stimulation containing 25, 75, 150, and 300 stimulation pulses in slices from wild-type (○,n = 5, 5, 4, and 5 animals, respectively, for each point) and SynGAP^−/+ mutants (●,n = 6, 6, 4, and 6 animals, respectively). Although a 25 pulse train of 5 Hz stimulation had similar effects on synaptic transmission in slices from wild-type and SynGAP^−/+mice, significantly less LTP was induced in SynGAP^−/+ slices by trains of 5 Hz stimulation containing 75, 150, and 300 pulses (*p < 0.01).e, LTP induced by low-frequency (5 Hz) presynaptic fiber stimulation paired with postsynaptic depolarization is reduced in CA1 pyramidal cells from SynGAP^−/+ mice. EPSPs were paired with postsynaptic depolarization at time = 0. EPSPs recorded between 25 and 30 min after pairing were potentiated to 262 ± 15% of baseline in cells from wild-type slices (○,n = 13 cells from 7 animals) and to 185 ± 9% of baseline in cells from SynGAP^−/+ mice (●,n = 12 cells from 8 animals; p< 0.001 compared with wild-type). The inset shows EPSPs (average of 3 responses) recorded during baseline and 30 min after pairing in cells from a wild-type animal (left set of traces) and SynGAP^−/+ mutant animal (right setof traces). Calibration: 5 mV, 25 msec.f, Cumulative probability distribution showing the amount of pairing-induced LTP seen in all cells represented by the average results shown in e (○, cells from wild-type animals; ●, cells from SynGAP^−/+ mice).

Fig. 6.

The role of H-Ras and SynGAP in LTP and ERK signaling. a, Basal levels of ERK pathway activation in SynGAP, H-Ras, and SynGAP/Ras mutant hippocampus extracts. Immunoblots measuring MEK and ERK phosphorylation (pMEK,pERK) and protein levels (MEK,ERK) are shown for extracts from wild-type mice (wt), SynGAP^−/+, SynGAP^−/+/ H-Ras^−/−, and H-Ras^−/−mutants. Phosphorylated forms of MEK and ERK were increased in SynGAP^−/+ and SynGAP^−/+/H-Ras^−/−mutants. b, NMDA induced activation of ERK in hippocampal slices from wild-type and SynGAP^−/+mice. Immunoblots show pERK levels in hippocampal slices from wild-type and SynGAP^−/+ mice that were either untreated (control) or exposed to 100 μm NMDA + 10 μmglycine for 2.5, 5, or 10 min. The top blot shows the levels of pERK2 (arrowhead) and pERK1 (top faint band). The bottom blot shows the total protein level of ERK2. The graph shows the change in ERK2 activation as measured using pERK antibody. For each experiment, pERK2 levels were normalized relative to ERK2 and control slices from wild type and then quantified using image analysis software (NIH Image version 1.62) and represented graphically. Mean and SEM are indicated, and significant difference after stimulation is shown (*p < 0.05). Note that although basal levels of pERK2 are clearly elevated in SynGAP^−/+ slices, NMDA induces further increases in pERK. ○, Wild type; ●, SynGAP^+/−. c, Summary of the amount of potentiation seen 45 min after a 150 pulse train of 5 Hz stimulation in wild-type, SynGAP^−/+, H-Ras^−/−, and SynGAP^−/+/H-Ras^−/−double mutants. After 5 Hz stimulation, synaptic transmission potentiated to 178 ± 14% of baseline in slices from wild-type animals (n = 12 slices from 6 animals) but to only 129 ± 5% of baseline in slices from SynGAP^−/+ mice (n = 10 slices from 5 animals; **p < 0.02 compared with wild type). The amount of LTP induced by this protocol was also reduced significantly in SynGAP^−/+/H-Ras^−/−double mutants (fEPSPs were 120 ± 4% of baseline;n = 9 slices from 5 animals; *p< 0.05) compared with that seen in H-Ras^−/−mutant mice (fEPSPs potentiated to 157 ± 13% of baseline;n = 12 slices from 6 animals).

Fig. 7.

LTP in single and double mutants of SynGAP and PSD-95. A brief train of 5 Hz stimulation (150 pulses, delivered at time = 0) induces robust LTP in wild-type slices (○,n = 12 slices from 6 animals) but induces only a small potentiation in slices from SynGAP^−/+ mice (●, n = 10 slices from 5 animals) (data from the same experiments summarized in Fig. 5c). In slices from both PSD-95^−/−and SynGAP^−/+/PSD-95^−/−double mutant mice, 150 pulse trains of 5 Hz stimulation induce large LTP. Forty-five minutes after 5 Hz stimulation (delivered at time = 0), synaptic transmission was potentiated to 258 ± 33% of baseline in PSD-95^−/−slices (■, n = 6 slices from 4 animals) and was potentiated to 244 ± 7% of baseline in slices from SynGAP^−/+/PSD-95^−/−mutant (▪, n = 9 slices from 4 animals).

Fig. 8.

Learning and memory in SynGAP mutant mice.a, Path length (mean ± SEM) across 3 d of training to a visible cue (4 trials per day) in SynGAP^−/+ (n = 21) and wild-type (n = 21) mice. Both groups performed equally well. b, Path length across 5 d of spatial training to a hidden platform. SynGAP mutants were slightly impaired relative to wild types. c, Time spent in the target zone on transfer tests 1 and 2, expressed as a percentage of the total time spent in all four zones. On transfer test 1, SynGAP mutants were significantly impaired relative to wild types, but after training to a performance criterion, mutants searched almost as accurately as wild types on transfer test 2. d, Representative swim paths of individual SynGAP^−/+ and wild-type mice on transfer tests 1 and 2. Dashed circles indicate the four zones, with the target zone filled in gray. Platform locations were counterbalanced, but swim paths have been rotated for display purposes such that the target zone always appears in the northeast quadrant of the pool. The wild-type mouse searched in the correct location in both transfer tests, whereas the mutant performed at chance on transfer test 1 but searched almost as accurately as the wild type on transfer test 2. e, Time spent in the target zone on transfer tests 1 and 2 for PSD-95^−/−mutants (n = 12) and wild types (n = 9). Wild types performed well in both transfer tests, but PSD^−/−mutants performed at chance throughout. f, Representative swim paths of a PSD-95^−/−mutant and a wild-type mouse. Note that the mutant fails to search in the target location even after extensive training (Transfer test 2). g, A possible framework for interpreting the transfer test data. Wild-type mice learn rapidly, soon reaching an asymptote. SynGAP mutants learn slowly, but gradually begin to approach an asymptote equivalent to that reached by wild types. PSD-95 mutants, in contrast, learn little, regardless of the amount of training that they receive.