 |
 Previous Article | Next Article 
The Journal of Neuroscience, November 15, 2002, 22(22):9721-9732
SynGAP Regulates ERK/MAPK Signaling, Synaptic Plasticity,
and Learning in the Complex with Postsynaptic Density 95 and NMDA
Receptor
Noboru H.
Komiyama1,
Ayako M.
Watabe2,
Holly J.
Carlisle3,
Karen
Porter1,
Paul
Charlesworth1,
Jennifer
Monti1,
Douglas J. C.
Strathdee1,
Colin M.
O'Carroll1,
Stephen J.
Martin1,
Richard G. M.
Morris1,
Thomas J.
O'Dell2, and
Seth G. N.
Grant1
1 Division of Neuroscience, University of Edinburgh,
Edinburgh EH8-9JZ, United Kingdom, and 2 Department of
Physiology and 3 Interdepartmental PhD Program for
Neuroscience, University of California Los Angeles School of Medicine,
Los Angeles, California 90095
 |
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.
Key words:
SynGAP; H-Ras; PSD-95; long-term potentiation; hippocampus; spatial learning
| |
INTRODUCTION |
At many excitatory synapses,
activation of postsynaptic NMDA-type glutamate receptors by different
patterns of synaptic activity can induce persistent increases and
decreases in the strength of synaptic transmission. Within the
postsynaptic density, NMDA receptors (NMDARs) exist as
multiprotein complexes (NRC, NMDA receptor complex) that contain
neurotransmitter receptors and cell adhesion proteins linked by
adaptors to signaling and cytoskeletal proteins (Husi et al., 2000 ).
The colocalization of multiple signaling pathways into a complex with
NMDA receptors might have several important implications for NMDA
receptor signaling (Migaud et al., 1998). For instance, the presence of
general signaling proteins, such as ERK or protein kinase A
(PKA), in these complexes might allow NMDA receptor activation
to regulate the function of these proteins within a spatially
restricted postsynaptic microdomain (Westphal et al., 1999; Hardingham
et al., 2001). In addition, these multicomponent signaling complexes
might also allow intracomplex signal interactions that enable
activation of distinct downstream pathways in response to different
patterns of neuronal activity. Properties of this type could have a
crucial role in NMDAR-dependent forms of synaptic plasticity, learning,
and developmental plasticity, which are dependent on many of the
proteins found within these complexes (Husi et al., 2000; Erzurumlu and
Kind, 2001).
One key component of NRCs is postsynaptic density 95 (PSD-95), an NMDA
receptor-associated protein that may function as an adapter protein
that couples various signaling molecules to NMDA receptors (Kornau et
al., 1995; Blackstone and Sheng, 1999; Garner et al., 2000). Although
the phenotype of PSD-95 mutant mice indicates an important role for
this protein in both synaptic plasticity and learning (Migaud et al.,
1998), the relevant PSD-95-dependent signaling pathways involved in
these processes have not yet been identified. One recently identified
candidate, however, is SynGAP, a protein that colocalizes with NMDA
receptors at excitatory synapses via direct interactions with the
PDZ domains of PSD-95 (Chen et al., 1998; Kim et al., 1998).
Both the structure and in vitro enzyme activity of SynGAP
indicate that it is a likely GTPase-activating protein for H-Ras.
SynGAP may thus be an important component of signaling pathways that
underlie NMDA receptor-dependent activation of Ras-dependent signaling
pathways such as the c-Raf1-MEK-ERK, PI3-kinase, and RalA
pathways (Gille and Downward, 1999).
To investigate the role of SynGAP in synaptic plasticity and learning,
we have begun a genetic dissection of the NRC using mice with single
and compound mutations in SynGAP, H-Ras, and PSD-95. Our results
indicate that SynGAP has an important role in both learning and
long-term potentiation (LTP) in the hippocampal CA1 region. Although
PSD-95 couples SynGAP to NMDA receptors (Chen et al., 1998; Kim et al.,
1998), we found that PSD-95 and SynGAP mutant mice exhibit distinct
physiological and behavioral phenotypes, suggesting that PSD-95 may
couple NMDA receptors to multiple downstream signaling pathways with
very different roles in LTP and learning.
| |
MATERIALS AND METHODS |
Gene targeting. An internal ribosomal entry site
upstream of a  -galactosidase reporter gene was introduced to monitor
the cellular expression pattern (Migaud et al., 1998). Targeting
constructs were electroporated into embryonic stem (ES) cells, and
Southern blot analysis indicated that homologous recombination had
occurred in 18 of 180 clones (10%) for SynGAP and 58 of 72 (81%) for
H-Ras and germline transmission of the mutations established
(see Fig. 1a,b). A 565 bp cDNA fragment that
encodes part of the SynGAP pleckstrin homology and C2 domains
was generated from mouse brain RNA extract by RT-PCR. This cDNA was
used as a probe to screen a 129/Ola mouse genomic library, and
overlapping  clones covering 22 kb of the SynGAP locus were
isolated. To construct a SynGAP targeting vector, an 8.1 kb
SmaI-XhoI fragment and a 3.8 kb
SpeI-SpeI fragment were used as 5' and 3'
homology arms, respectively. A coding sequence for hemagglutinin (HA)
epitope tag was inserted in frame at an XhoI site in the C2
domain (Chen et al., 1998; Kim et al., 1998) at the 3' end of the 5'
homology arm and followed by stop codons and internal ribosomal entry
site (IRES)-lacZ-polyA · MC1neo-polyA cassette (Friedrich and
Soriano, 1991). The resultant vector deletes exons encoding the C2 and
GAP domains. The Diphtheria toxin A gene (MC1-DT-A) was used as a
negative selection marker. The targeting vector was linearized and
electroporated into E14TG2a ES cells. Neomycin (neo)-resistant clones
were screened for homologous recombination by Southern blot analysis
using a 700 bp 3' flanking probe (EcoRI or XbaI
digest) and a 354 bp cDNA internal 5' probe (BglII digest).
The targeting vector for the H-Ras1 locus comprised 1.9 and 5.5 kb
bands of 5' and 3' DNA flanking a cassette (TAG3-IRES-geo-polyA) containing the positive selectable marker neo. The 5' end of this cassette contained stop codons in all reading frames to terminate translation of H-Ras at the SpeI site in exon 4, followed by
an IRES that allows a -geo reporter gene to be expressed under the control of the H-Ras1 promoter. The targeting construct was linearized and electroporated into HM1 ES cells. Neomycin-resistant clones were
screened for homologous recombination by Southern blot analysis using a
1.1 kb fragment (5' probe) flanking the homology region and a 1.2 kb
genomic DNA (3' probe). Chimeric mice were produced by injecting
targeted ES cells into C57BL/6 blastocysts, and heterozygous mutants
were generated as described previously (Papaioannou and Johnson, 2000).
All comparisons of SynGAP/+ and
wild-type (wt) mice were performed using littermates on an F2 MF1
genetic background. Animals were treated in accordance with the UK
Animal Scientific Procedures Act (1986) and National Institutes of
Health guidelines.
Biochemistry. The hippocampus was homogenized in 50 mM Tris-HCl, 1% sodium deoxycholate, 50 mM NaF, 20 mM
ZnCl2, 1 mM sodium orthovanadate, 0.5 mg/ml PMSF, and Protease Inhibitors Complete (Roche
Molecular Biochemicals). Proteins were separated by SDS-PAGE (25 µg
per lane), and standard procedures were used for Western blotting. The
antibodies used were SynGAP, NR2A (Upstate Biotechnology); Neurofibromin-1 (NF1), SynGAP, HA (Santa Cruz Biotechnology); p120RasGAP, H-Ras, NR1, NR2B, PSD-95, ERK, MEK (Transduction
Laboratories); panRas (Oncogene); phospho-MAPK (pERK) and phospho-MEK
(New England BioLabs).
Morphology. Mice were perfusion fixed via the heart with
heparinized saline followed by 2% paraformaldehyde, 2.5%
glutaraldehyde in 0.1 M sodium phosphate buffer,
pH 7.4. The brain was removed and placed in fixative for a further 2 hr. Coronal sections (70 µm) were taken through the anterior part of
the hippocampus using a vibrating microtome (Vibratome, Lancer). From
every three serial sections, one was taken for EM processing and the
remainder were used for section Golgi impregnation. Sections were
dehydrated and mounted in resin before viewing in the light microscope.
Consistent regions of dorsal CA1 were resectioned and viewed in an
electron microscope (Philips CM12) (Bolam, 1992; Morrison et al.,
1998). The stratum radiatum was examined, and the first 20 clearly
defined asymmetrical axospinous synapses were photographed.
5-Bromo-4-chloro-3-indolyl--galactoside staining. SynGAP
mice were fixed via cardiac perfusion with 0.1 M
sodium phosphate buffer, pH 7.4, followed by 4% paraformaldehyde in
0.1 M sodium phosphate buffer, pH 7.4. The brain
was removed and placed in fixative for a further 1 hr. Frozen sagittal
sections were cut at 48 µm and then stained overnight for
5-bromo-4-chloro-3-indolyl--galactoside (X-gal; Invitrogen) (Migaud
et al., 1998). H-Ras unfixed sagittal sections were cut at 20 µm and
then fixed using 0.01% glutaraldehyde in 0.1 M
sodium phosphate buffer, pH 7.4, for 15 min and then stained overnight
for X-gal (Invitrogen) (Migaud et al., 1998). Sections were dehydrated
and mounted in PDX mountant (BDH) before viewing in the light microscope.
Immunofluorescent staining. Brain tissue was fixed in 10%
buffered Formalin for 6 hr, dehydrated, and then wax embedded. Coronal sections (8 µm) were cut on a microtome. Standard procedures were followed for immunofluorescent staining, mounting, and coverslipping (Morrison et al., 1998). Primary antibodies were anti-synaptophysin monoclonal antibody (mAb) (1:100; Boehringer Mannheim) and MAP2B mAb
(1:100; Transduction Labs); secondary antibody was Cy3-conjugated antibody (1:200; Jackson Laboratory, Bar Harbor, ME).
Slice electrophysiology. Hippocampal slices (400 µm thick)
were prepared from 14- to 20-week-old mice using standard techniques and maintained in interface-type recording chambers perfused at a
constant rate (1-3 ml/min) with a warmed (30°C), oxygenated (95%
O2/5% CO2) artificial CSF
(ACSF) consisting of (in mM): 124 NaCl, 4.4 KCl,
25 Na2HCO3, 1 NaH2PO4, 1.2 MgSO4, 2 CaCl2, and 10 glucose. Low-resistance glass microelectrodes filled with ACSF were
placed into stratum radiatum of the hippocampal CA1 region to record
field EPSPs (fEPSPs) evoked by presynaptic stimulation pulses delivered
once every 50 sec to the Schaffer collateral/commissural fibers via a
bipolar nichrome wire stimulating electrode. At the beginning of each
experiment the intensity of presynaptic fiber stimulation was adjusted
to evoke fEPSPs that were approximately half of the maximal fEPSP
amplitude that could be evoked by strong intensity stimulation.
High-frequency stimulation-induced LTP was elicited using two trains of
100 Hz stimulation (1 sec in duration) delivered with an intertrain
interval of 10 sec. To elicit saturating levels of LTP, we delivered
six, 1-sec-long trains of 100 Hz stimulation with an intertrain
interval of 5 min. For statistical comparisons (unpaired t
tests, two-tailed; n = number of animals), we compared
the average amount of potentiation present 55-60 min after the first
high-frequency stimulation train or 40-45 min after low-frequency
trains of stimulation.
Whole-cell current-clamp recordings were used to study the induction of
LTP by low-frequency presynaptic fiber stimulation paired with
postsynaptic depolarization. In these experiments, slices with the CA3
region removed were bathed in a modified ACSF containing elevated
levels of CaCl2 and MgSO4
(4 mM each), lower levels of KCl (2.2 mM), and
100 µM picrotoxin. EPSPs evoked by 0.05 Hz presynaptic
fiber stimulation were recorded from individual CA1 pyramidal cells
using low-resistance (2-5 M ) patch-clamp electrodes filled with a
solution containing (in mM): 122.5 Cs-gluconate, 17.5 CsCl,
10 TEA-Cl, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.3 GTP, pH 7.2. Constant
current injections were used to hyperpolarize cells to between  80 and
85 mV, and a 50-msec-long 0.1 nA pulse of hyperpolarizing current was
injected 150 msec after each evoked EPSP to monitor input and access
resistance throughout the experiment. The intensity of presynaptic
fiber stimulation was set to evoke EPSPs between 5 and 10 mV in
amplitude. After a 10 min period of baseline recording, LTP was induced
by depolarizing the postsynaptic cell to near 0 mV using a tonic
injection of current through the recording electrode and pairing this
depolarization with 100 presynaptic fiber stimulation pulses delivered
at 5 Hz. Membrane potential was closely monitored during the pairing
protocol, and the amount of current injected through the recording
electrode was adjusted continuously to keep the postsynaptic membrane
potential near 0 mV. Unpaired t tests (two-tailed) were used
to compare the average amount of LTP induced 25-30 min after pairing
in cells from wild-type and mutant animals. Whole-cell voltage-clamp
recordings were used to examine the NMDA receptor-mediated component of
EPSCs across a range of postsynaptic membrane potentials. In these
experiments, slices were bathed in the modified ACSF described above,
and CA1 pyramidal cells were voltage clamped using patch-clamp
electrodes (2-4 M; access resistance ranged from 14 to 22 M)
filled with either the Cs-gluconate-based electrode-filling solution or
with a solution containing (in mM): 120 CsMeSO3, 20 CsCl, 8 NaCl, 0.2 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 GTP, pH 7.2. Similar results were obtained with both
internal solutions, and the results were combined. The AMPA and NMDA
receptor-mediated components of the EPSCs were estimated from the
amplitude of the EPSCs measured 5 and 50 msec, respectively, after the
onset of the EPSC. All experiments and initial data analysis were
performed blind except for experiments performed on three
H-Ras/
mutant mice. The results from these experiments did not differ from
additional experiments on these animals that were done in a blind
manner, and the results were therefore combined. All values are
reported as mean ± SEM.
Primary neuronal culture and electrophysiology. Standard
techniques were used to prepare primary cultures of forebrain neurons from individual embryonic day 18 (E18) mice that were subsequently genotyped. Briefly, forebrain was dissected, transferred to cold PBS,
trypsinized, and disaggregated by drawing up and down in a 1 ml pipette
or flamed Pasteur pipette. The resultant cell suspension was
centrifuged twice, and the pellet was resuspended in Neurobasal medium
supplemented with B27 (Invitrogen) and 0.5 mM
glutamine. Cells were plated at ~0.5-1 × 105/cm2 on
glass coverslips coated with poly-D-lysine and
laminin (Sigma, St. Louis, MO). NMDA receptor-mediated responses were
examined in wild-type, SynGAP/+, and
SynGAP/
neurons maintained in culture for 6-9 d. Before an experiment, cells
were bathed in an external solution that contained 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0 or 1 mM MgCl2, 10 mM glucose, 20 nM TTX, and
15 mM HEPES, pH 7.4. Whole-cell voltage-clamp
recordings were performed using low-resistance electrodes filled with a
solution containing (in mM): 120 CsMeSO3, 5 CsBAPTA, 4 MgATP, 0.4 Na2GTP, and 15 HEPES, pH 7.3. Currents were evoked by brief application of 100 µM NMDA (+ 10 µM
glycine) delivered by a solenoid valve-operated U-tube perfusion
system. Responses were recorded and analyzed using PClamp software
(Axon Instruments). For each cell, NMDA-evoked currents were elicited
at different holding potentials and then normalized to the maximal
current response. These values were then averaged across all cells
recorded from a given genotype.
Slice stimulation. Hippocampal slices were prepared and
maintained in the same interface chambers as those used for
electrophysiological experiments and were allowed to recover for at
least 2 hr. Slices (six at a time) were either left untreated (control
slices) or exposed to 100 µM NMDA (+ 10 µM glycine) for 2.5, 5, or 10 min. The slices
were removed rapidly from the chamber and immediately frozen in liquid
N2. Samples were homogenized in the same buffer as described above for Western blot analyses. Three separate
experiments were performed with tissue obtained from six wild-type and
six SynGAP/+ mice.
Behavior. We used an open-field water maze (2 m in diameter;
opaque water; 25 ± 1°C; automated swim path monitoring). In the cue task, mice were trained to a randomly located platform position marked with a cylindrical cue [four trials per day for 3 d; 30 cm
platform diameter; curtains drawn around the pool to occlude extra-maze
cues; maximum trial duration was 90 sec; intertrial interval (ITI), 10 min]. For spatial training, we used a hidden platform with the extra
maze cues visible (four trials per day for 5 d; 30 cm platform
diameter; platform/pool area was 1:44; 30 sec was spent on the
platform at the end of each trial; maximum trial duration 90 sec; ITI,
10 min). Transfer test 1 was conducted 10 min after the previous
training trial, and mice were placed in the pool for 60 sec (platform
absent; the start position was opposite to whatever training quadrant
had been used for each individual animal). Two measures of transfer
test performance were calculated: (1) percentage time spent in the
target quadrant of the pool and (2) time spent in a zone corresponding
to the area of the absent platform (radius = 15 cm), expressed as
a percentage of the total time spent in all four possible zones (see
Fig. 8d,f). Training to
criterion involved using a smaller hidden platform, (20 cm diameter;
platform area/pool area ratio was 1:100), again with the extra
maze cues visible, until the animals had completed 2 consecutive days
with each trial taking <20 sec, or until 32 trials had been completed.
On reaching criterion, and 10 min after the previous training trial,
individual mice were placed in the pool for 60 sec (platform absent;
transfer test 2). This training protocol was identical to that used for
PSD-95/
mice (Migaud et al., 1998), thus enabling a comparison of the performance of
PSD-95/
and SynGAP/+ mutant mice. Two
replications completed testing up to this point (SynGAP/+ total, n = 21;
wild type total, n = 21). Only the initial
replication underwent retention testing as follows
(SynGAP/+, n = 12; wild
type, n = 12). Two further transfer tests, or retention tests, were conducted 7 and 45 d after the mice had reached
criterion and had completed the second transfer test. Again, mice were
placed in the pool for 60 sec (platform absent; transfer test 3 and 4, respectively).
| |
RESULTS |
The murine genes encoding SynGAP and H-Ras were cloned and
characterized, knock-out targeting vectors were constructed (Fig. 1a,b), and mutant
mice were generated. Male and female
SynGAP/+ mice appeared normal, displayed
no sign of seizures, tremor, ataxia, or other neurological abnormality,
and were fertile. Genotypes of 3-week-old pups from
SynGAP/+ intercrosses showed no
surviving homozygote
SynGAP/
animals. Of 543 pups born, 325 reached weaning, of which 128 were wild
type and 197 SynGAP/+
( 2 = 8.417; p < 0.001). Eighty newborn pups were genotyped and showed normal mendelian
transmission of the mutation (23 wt, 39 SynGAP/+, 18 SynGAP/;
2 = 0.714; p > 0.05).
All
SynGAP/
pups were observed to breathe and feed and showed no gross anatomical abnormality, although most died within 48 hr, indicating that SynGAP is
essential for postnatal viability. In contrast,
H-Ras/+ intercrosses produced
H-Ras/
offspring at the expected mendelian ratio (323 pups at weaning, 93 wt,
152 H-Ras/+ ,79 H-Ras/;
2 = 0.293; p > 0.05).
These offspring were fertile and showed no signs of neurological
abnormality.
View larger version (35K):
[in this window]
[in a new window]
|
Figure 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, -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 of
EcoR1-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. -geo
consists of a -galactosidase gene and a neomycin-resistance fusion
gene. Right, Genomic Southern blots of
EcoR1-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.
|
|
The neuronal expression pattern of SynGAP is unknown, and X-gal
staining of SynGAP/+ brains revealed
highest expression in neurons of the hippocampus CA1 and dentate gyrus
and cortex and lower expression in striatum (Fig. 1c), where
PSD-95 is also expressed (Migaud et al., 1998). In contrast to SynGAP,
H-Ras was widely expressed in most brain regions (Fig. 1c).
Disruption of protein expression was confirmed using immunoblotting of
hippocampal extracts (Fig. 1d). SynGAP levels were reduced
by 50% in SynGAP/+ compared with
wild-type mice (p < 0.001; n = 12) using both N- and C-terminal antibodies. No truncated protein
products were detected using these antibodies or with antibodies to the
HA tag inserted into the locus. Immunoblotting of H-Ras showed no
detectable H-Ras in hippocampus extracts from
H-Ras/
mice. We tested for compensation by the two other mammalian GAP proteins, NF1 and p120RasGAP, in the hippocampus extracts of
SynGAP/+ mice and observed no increases
above wild-type levels (Fig. 1d). We used a Pan-Ras antibody
that detects all Ras isoforms and saw a reduction in total Ras levels
in H-Ras mutant mice. Finally, we examined levels of expression of NR1,
NR2A, NR2B, and PSD-95 in SynGAP/+ and
H-Ras/
mutant mice and saw no changes from wild-type levels (Fig.
1d).
To explore the role of SynGAP and H-Ras in the brain, we examined the
neuroanatomy at the light and electron microscopic level in mature
(6-8 weeks old) SynGAP/+ and
H-Ras/
mice (Fig. 2). There was no gross
detectable abnormality found using Nissl staining in the brain of
SynGAP/+ or
H-Ras/
mice. Both SynGAP and H-Ras were highly expressed in the CA1 region of
the hippocampus (Fig. 1c). In this region, the intensity and
distribution of Nissl staining and the distribution of the synaptic-terminal marker synaptophysin and of the dendritic marker MAP2
were the same in SynGAP/+,
H-Ras/,
and wild-type mice (Fig. 2). Dendritic architecture was further examined using Golgi staining and electron microscopy, revealing dendritic branching, spine distribution, and asymmetric synapse morphology, which were all normal. We prepared primary neuronal cultures from individual E18.5 embryos and found that neurons from
SynGAP/
mice extended neurites and formed morphological synapses, as shown by
immunostaining with the synaptic vesicle proteins synaptophysin and
dendritic protein MAP2, which were indistinguishable from wild-type
cultures. Electrophysiological evidence for synaptic transmission
between
SynGAP/
neurons was obtained using whole-cell patch clamping to record miniature EPSCs and showed typical kinetics of glutamatergic responses (data not shown). These data indicate that SynGAP was not essential for
synapse formation or synaptic transmission in vitro.
View larger version (82K):
[in this window]
[in a new window]
|
Figure 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/+ and
H-Ras/
mutants are indicated.
|
|
Synaptic transmission and NMDA receptor function in SynGAP
mutant mice
We next examined the role of SynGAP in synaptic transmission and
plasticity in the CA1 region of the hippocampus. Under normal experimental conditions, where extracellularly recorded fEPSPs are
mediated predominantly by AMPA-type glutamate receptors, we detected no
difference in the maximal fEPSP amplitude that could be recorded using
strong intensity presynaptic fiber stimulation (maximum fEPSPs were
8.4 ± 0.7 mV in wild-type slices and 8.3 ± 0.5 mV in slices
from SynGAP/+ mice; n = 23 animals for each). Moreover, the ratio of fiber volley amplitude to
fEPSP slope of responses evoked using presynaptic fiber stimulation
intensities that evoked fEPSPs that were 25, 50, 75, and 100% of the
maximal amplitude were similar in slices from
SynGAP/+ and wild-type mice (Fig.
3a). This indicates that the
number of presynaptic fibers needed to evoke an equivalent postsynaptic response in slices from wild-type and
SynGAP/+ mice was similar and suggests
that the activity of postsynaptic AMPA receptors (AMPARs) is unaltered
in SynGAP/+ mice. Finally, pairs of
presynaptic fiber stimulation pulses delivered at interpulse intervals
of 25, 50, 100, and 200 msec evoked nearly identical amounts of
paired-pulse facilitation in slices from wild-type and
SynGAP/+ mice (Fig. 3b).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 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 of
traces) 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).
|
|
To examine whether the NMDAR components of EPSCs were altered in CA1
pyramidal cells in SynGAP/+ slices, we
used whole-cell voltage-clamp techniques to record EPSCs at membrane
potentials of 80 and +40 mV and compared the contribution of NMDARs
to EPSCs in SynGAP/+ and wild-type cells
by measuring the NMDAR currents relative to the size of the AMPAR
currents recorded at holding potentials of 80 and +40 mV. As shown in
Figure 4a, the contribution of NMDARs to the synaptic current measured in this way was unaltered in
SynGAP/+ cells (Fig. 4a). At
holding potentials of +40 mV, where the NMDAR-mediated component of
EPSCs is most evident, the absolute current mediated by both AMPA and
NMDA receptors was also the same in cells from wild-type and
SynGAP/+ mice (in wild-type cells the
AMPAR component was 104 ± 8 pA and the NMDA component was 79 ± 13 pA in 16 cells from five animals, whereas the AMPAR and NMDAR
components of EPSCs were 101 ± 4 and 75 ± 5 pA,
respectively, in 15 cells from five
SynGAP/+ mice). Single exponential fits
to the decay of the synaptic currents measured at +40 mV were nearly
identical in cells from wild-type and
SynGAP/+ mice
( decay = 58.9 ± 6.5 msec in wild-type
cells, n = 16 cells from five animals; and 56.3 ± 6 msec in SynGAP/+ cells,
n = 15 cells from five animals), suggesting that the
time course of the NMDAR-mediated component of EPSCs is not altered in
SynGAP/+ cells. Finally, we also found
no difference between wild-type and
SynGAP/+ neurons in both the amplitude
and voltage dependence of NMDAR-mediated responses in whole-cell
voltage-clamp recordings from cultured forebrain neurons (Fig.
4b,c). NMDAR-mediated responses were also unaltered in cultures obtained from
SynGAP/
mice (Fig. 4b,c). Thus, we found no evidence for
changes in several aspects of normal synaptic physiology, including
NMDAR channel function, in SynGAP/+
mice.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 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.
|
|
Synaptic plasticity in SynGAP mutant mice
To examine whether NMDAR-dependent forms of synaptic plasticity
might be altered in SynGAP/+ mice, we
first investigated the effects of brief trains of high-frequency synaptic stimulation on synaptic strength at Schaffer
collateral/commissural fibers synapses onto pyramidal cells in the
hippocampal CA1 region. As shown in Figure
5a, 60 min after two,
1-sec-long trains of 100 Hz stimulation, synaptic transmission was
potentiated >80% in slices from wild-type mice (n = 7) but only 50% in slices from SynGAP/+
mice (n = 7; p < 0.01 compared with
wild type). The amount of LTP induced by high-frequency stimulation was
also significantly reduced (p < 0.005) in
slices from SynGAP/+ mice in experiments
in which six trains of high-frequency stimulation were delivered once
every 5 min to induce saturating levels of LTP (Fig. 5b).
Thus, although high-frequency synaptic stimulation induces significant
potentiation in slices from SynGAP/+
mice, the amount of potentiation induced by 100 Hz stimulation is
greatly reduced compared with that seen in wild-type slices.
View larger version (38K):
[in this window]
[in a new window]
|
Figure 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 set
of 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).
|
|
To explore further the diminished magnitude of LTP in
SynGAP/+ mutant slices, we examined the
ability of lower-frequency trains of synaptic stimulation to induce
persistent changes in synaptic strength (Fig. 5c). A 900 pulse train of 1 Hz stimulation that was below threshold for LTP
induction in slices from wild-type mice (fEPSPs were 98 ± 3% of
baseline 45 min after 1 Hz stimulation; n = 5) also had
little lasting effect on synaptic strength in slices from
SynGAP/+ mice (fEPSPs were 94 ± 3% of baseline; n = 9). Likewise, 900 pulses of
synaptic stimulation delivered at 5 Hz induced only a small amount of
potentiation in slices from wild-type mice (fEPSPs potentiated to
135 ± 9% of baseline; n = 5) and
SynGAP/+ mice (fEPSPs were 117 ± 5% of baseline; n = 9; not significantly different
from wild type; p = 0.07). When delivered at higher frequencies (10 and 20 Hz), 900 pulse trains of synaptic stimulation induced larger LTP in slices from wild-type mice (fEPSPs were potentiated to 153 ± 5%, n = 5, and 141 ± 4% of baseline, n = 7, respectively). In contrast,
these stimulation protocols had relatively little effect on synaptic
transmission in slices from SynGAP/+
mice (fEPSPs were potentiated to 116 ± 2%, n = 7, after 10 Hz stimulation, p < 0.001 compared with
wild type, and were 123 ± 7% of baseline, n = 7, after 20 Hz stimulation, p < 0.05 compared with wild type).
We also compared the induction of LTP in slices from
SynGAP/+ and wild-type mice in
experiments in which trains of presynaptic stimulation were delivered
at a fixed frequency (5 Hz) and the number of stimulation pulses in the
train was varied (Fig. 5d). Trains of 5 Hz stimulation that
consisted of just 25 pulses had little lasting effect on synaptic
transmission in slices from wild-type and
SynGAP/+ mice [45 min after 5 Hz
stimulation, fEPSPs were 115 ± 5 and 118 ± 5% of baseline
in slices from wild-type (n = 6) and
SynGAP/+ mice (n = 6),
respectively]. Longer duration trains consisting of 75 and 150 pulses
induced LTP in slices from wild-type mice [fEPSPs potentiated to
175 ± 6% (n = 5) and 194 ± 14%
(n = 4) of baseline, respectively] but had little
effect on synaptic transmission in slices from
SynGAP/+ mice [fEPSPs were 118 ± 5% (n = 6) and 117 ± 3% (n = 4)
of baseline; p < 0.01 compared with wild type].
Similarly, a 5 Hz train of synaptic stimulation consisting of 300 pulses that induced an intermediate amount of LTP in slices from
wild-type mice (fEPSPs potentiated to 143 ± 7% of baseline;
n = 5) also induced significantly less LTP in slices
from SynGAP/+ mice (fEPSPs were 106 ± 2% of baseline; n = 6; p < 0.01).
The postsynaptic responses evoked during all the trains of synaptic stimulation used to induce LTP in these experiments were similar in
wild-type and SynGAP/+ slices (data not
shown), suggesting that the LTP deficit observed in
SynGAP/+ mice is not caused by
alterations in presynaptic function.
To investigate whether changes in postsynaptic excitability and
inhibitory synaptic transmission contribute to the reduction in LTP
observed in SynGAP/+ mutant mice, we
blocked inhibitory synaptic transmission by including picrotoxin (100 µM) in the extracellular solution and examined the
induction of LTP by pairing low-frequency (5 Hz) presynaptic fiber
stimulation with depolarization of the postsynaptic cell produced by
current injected through an intracellular recording electrode. Although
this pairing protocol induced LTP in both wild-type and
SynGAP/+ cells, the average amount of
potentiation induced in pyramidal cells from
SynGAP/+ mice was significantly less
(p < 0.001) than that observed in cells from
wild-type animals (Fig. 5e,f). This
indicates that the LTP deficit observed in
SynGAP/+ mice is not caused by changes
in inhibitory synaptic transmission or excitability of the postsynaptic
CA1 pyramidal cells. In addition, because LTP was induced in these
experiments by low-frequency synaptic stimulation paired with
experimentally imposed postsynaptic depolarization, these findings
indicate that the LTP deficit observed in
SynGAP/+ mice is not caused by reduced
postsynaptic depolarization resulting from presynaptic changes in
synaptic transmission in SynGAP/+ slices
that might alter transmitter release during LTP-inducing trains of
synaptic stimulation. Together, these data indicate that SynGAP is part
of a signaling pathway that facilitates LTP induction.
SynGAP regulation of H-Ras and ERK signaling
Previous work suggests a number of possible mechanisms whereby
reduced levels of SynGAP might lead to impaired LTP. Because SynGAP
facilitates conversion of active, GTP-bound Ras to an inactive, GDP-bound form, one possibility is that the LTP deficits observed in
slices from SynGAP/+ mice are caused by
increased levels of Ras activation. Indeed, a recent study reported
that LTP is enhanced in H-Ras mutant mice, suggesting that H-Ras
activation stimulates pathways that inhibit LTP induction (Manabe et
al., 2000). Ras signaling may be an important component of the
signaling pathway underlying NMDA receptor-mediated activation of ERK
(Iida et al., 2001), and ERK activation is required for the induction
of LTP by some patterns of synaptic stimulation (English and Sweatt,
1997; Impey et al., 1998; Coogan et al., 1999; Winder et al., 1999;
Watabe et al., 2000; Sweatt, 2001). Thus, a second possibility is that
a deficit in NMDAR-dependent activation of the MAPK pathway might be
responsible for the LTP deficits observed in
SynGAP/+ mice.
To test these ideas, we first investigated whether basal levels of ERK
pathway activity were altered in
SynGAP/+ mice by immunoblotting
hippocampal extracts with antibodies to the phosphorylated active forms
of MEK and ERK (Fig.
6a,b). Consistent with the hypothesis that SynGAP regulates the Ras-ERK pathway, basal
levels of the phosphorylated, active form of MEK and ERK were increased
in SynGAP/+ extracts in the absence of a
detectable change in total levels of both proteins. We next examined
whether SynGAP also regulates NMDAR stimulation-induced activation of
ERK pathway and whether the elevated basal levels might preclude
further activation. Levels of phospho-ERK2 (pERK2) were examined in
extracts from hippocampal slices from wild-type and
SynGAP/+ mice that were either untreated
or exposed to 2.5, 5, or 10 min applications of 100 µM NMDA. As shown in Figure 6b, NMDA
strongly activated ERK in slices from both wild-type and
SynGAP/+ mice. Although peak levels of
pERK2 were higher at all three time points in extracts from
SynGAP/+ mice, the magnitude of ERK
activation relative to basal levels measured in untreated control
slices was similar in slices from SynGAP/+ and wild-type mice. Together
these results indicate that although SynGAP may be a regulator of
signaling through the Ras-ERK pathway, NMDA receptor activation still
induces robust ERK activation in the hippocampus of
SynGAP/+ mice.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 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 µM
glycine 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).
|
|
To examine whether the reduced LTP observed in
SynGAP/+ mice might be caused by
increased H-Ras activity, we compared levels of LTP induced in
wild-type, SynGAP/+,
H-Ras/,
and
SynGAP/+/H-Ras/
mice by a short train of synaptic stimulation (150 pulses delivered at
5 Hz) that induces robust, but nonsaturating, levels of LTP in
wild-type slices. Although the induction of LTP by high-frequency synaptic stimulation is enhanced in
H-Ras/
mice (Manabe et al., 2000), we found that a 5 Hz train of synaptic stimulation induced similar amounts of LTP in slices from
H-Ras/
and wild-type mice (Fig. 6c). Compared with wild-type
slices, however, a 150 pulse train of 5 Hz stimulation induced
significantly less LTP in slices from
SynGAP/+ mice (p < 0.02) (Fig. 6c). Moreover, the amount of LTP induced by
150 pulses of 5 Hz stimulation was also significantly reduced (p < 0.05) in slices from
SynGAP/+/H-Ras/
double mutant mice compared with that seen in
H-Ras/
mutant mice (Fig. 6c). On the basis of these findings, it
seems unlikely that changes in H-Ras activity are responsible for the LTP deficits observed in SynGAP/+ mice.
This conclusion is further supported by our finding that NMDAR-mediated
synaptic currents and responses in slices and cultured neurons from
SynGAP/+ mice were not different from
wild type (Fig. 4), whereas NMDAR-mediated responses in
H-Ras/
mutant mice are strongly upregulated (Manabe et al., 2000).
In these experiments we also tested whether H-Ras was required for
SynGAP regulation of basal levels of ERK pathway activation by
immunoblotting extracts from
H-Ras/
and
SynGAP/+/H-Ras/
double mutant mice. As shown in Figure 6a, levels of MEK and ERK phosphorylation in
H-Ras/
mice were similar to wild type and increased in
SynGAP/+/H-Ras/
double mutant mice to levels similar to that observed in
SynGAP/+ mutants. These results indicate
that signaling through H-Ras is not required for elevated basal levels
of ERK activation in SynGAP/+ mutants.
Genetic dissection of SynGAP and PSD-95 pathways
In addition to investigating the role of H-Ras as a potential
component of the downstream pathways underlying the role of SynGAP in
LTP, we also used a genetic approach to examine the relationship
between SynGAP and its potential upstream partner PSD-95. Because
SynGAP and PSD-95 are both required for NMDAR-dependent plasticity, we
can use genetic tools to determine their relationship within pathways.
One possibility is a signaling pathway that leads from NMDAR to PSD-95
to SynGAP. A second possibility is that SynGAP is not downstream of
PSD-95, as in the first scenario, but instead is in a separate parallel
pathway downstream of the NMDAR. A genetic test that can distinguish
these possibilities is to examine LTP in
SynGAP/+/PSD-95/
double mutant mice. If they are in distinct parallel pathways, then the
predicted magnitude of LTP will be intermediate between the enhanced
LTP in
PSD-95/
and reduced LTP in SynGAP/+ mutants.
Alternatively, if PSD-95 is upstream of SynGAP, then the magnitude of
LTP in the double mutant may resemble that seen in
PSD-95/
mutants. As shown in Figure 7, the amount
of potentiation induced by a 150 pulse train of 5 Hz stimulation in
slices from
SynGAP/+/PSD-95/
mutants was identical to that seen in
PSD-95/
mice, supporting the model that there is a signaling pathway from NMDA
to PSD-95 to SynGAP. We also found that a 900 pulse train of 5 Hz
stimulation, which induces little or no LTP in slices from wild-type
mice (Thomas et al., 1996) and large (more than twofold) LTP in PSD-95
mutant slices (Migaud et al., 1998), also induced large LTP (more than
twofold) in slices from
SynGAP/+/PSD-95/
mutant mice (data not shown). Thus, the LTP phenotype in
SynGAP/+/PSD-95/
double mutants is consistent with previously reported hypotheses suggesting a pathway from NMDARs to SynGAP via PSD-95 (Chen et al.,
1998; Kim et al., 1998).
View larger version (36K):
[in this window]
[in a new window]
|
Figure 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).
|
|
SynGAP regulates rate of learning
PSD-95/
mutants show a severe impairment in the performance of a spatial
learning task (Migaud et al., 1998), suggesting that PSD-95-dependent
signaling pathways have a crucial role in spatial learning. Thus, to
assess the potential role of SynGAP in the PSD-95-associated signaling
pathways involved in learning, we examine spatial learning in
SynGAP/+ mutant mice. To afford a
comparison, we deliberately chose a water maze training protocol that
was identical to that used in the study of
PSD-95/
mutant mice. Both wild-type (n = 21) and heterozygous
SynGAP/+ mutants (n = 21) were trained in cued and spatial versions of the water maze, with
the experimenter blind to genotype throughout.
In the NMDA receptor-independent cue task, both wild-type and
SynGAP/+ mutant mice learned to approach
a randomly located platform marked by a visible cue with an equivalent
(F <1) and progressive reduction in path length over
successive trials (Fig. 8a).
In the NMDA receptor-dependent spatial task, both groups showed a
decrease in path length over the course of 20 trials (mutants:
F(19,380) = 1.91; p < 0.05; wild types: F(19,380) = 2.74;
p < 0.001), with the swim paths of
SynGAP/+ mutants being slightly longer
overall than those of wild types (F(1,40) = 6.75; p < 0.05). All mice were then subject to the first transfer test (platform
removed). On the conventional measure of time in the training quadrant,
which revealed a striking impairment in
PSD-95/
mutants (Migaud et al., 1998, their Fig. 7d), the difference between SynGAP/+ mutants and wild types
approached but did not reach significance (t(40) = 1.98; 0.10 > p > 0.05). However, using the more sensitive measure
of the proportion of time spent in a zone centered around the target
platform relative to time in all four possible platform locations
(Moser et al., 1993), the
SynGAP/+ mutants showed significantly
less focused swimming (Fig. 8c) (t(40) = 3.08; p < 0.005). When
PSD-95/
mutants were given overtraining on the spatial task to reach a strict
performance criterion, only 2 of 12 mutant mice were successful
compared with 9 of 9 wild types (Migaud et al., 1998) (2 = 14.3; p < 0.001).
In contrast, 11 of 21 SynGAP/+ mice
(compared with 20 of 21 wild types) reached this criterion. Although
worse than wild types (2 = 9.98;
p < 0.005), significantly more
SynGAP/+ mice reached criterion than
PSD-95/
mice (2 = 5.22; p < 0.05). Consistent with this result, when tested in the second transfer
test immediately after reaching criterion, SynGAP/+ mice showed moderately focused
searching in the target zone (Fig. 8c). This result
contrasts with the performance of
PSD-95/
mutants, who remained at chance in both transfer tests (Fig. 8e). Comparison of the two mutant strains revealed that
SynGAP/+ mice were significantly less
impaired than
PSD-95/ |
TOP
ABSTRACT
INTRODUCTION
