The Journal of Neuroscience, July 30, 2003, 23(17):6703-6712
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Impaired NMDA Receptor-Mediated Postsynaptic Function and Blunted NMDA Receptor-Dependent Persistent Pain in Mice Lacking Postsynaptic Density-93 Protein
Yuan-Xiang Tao,1 *
Gavin Rumbaugh,2 *
Guo-Du Wang,3 *
Ronald S. Petralia,4
Chengshui Zhao,1
Frederick W. Kauer,5
Feng Tao,1
Min Zhuo,3
Robert J. Wenthold,4
Srinivasa N. Raja,1
Richard L. Huganir,2
David S. Bredt,5 and
Roger A. Johns1
1Department of Anesthesiology and Critical Care
Medicine and 2Howard Hughes Medical Institute,
Department of Neuroscience, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287, 3Washington University Pain
Center, Departments of Anesthesiology, Anatomy and Neurobiology, and
Psychiatry, Washington University School of Medicine, St. Louis, Missouri
63110, 4Laboratory of Neurochemistry, National
Institute on Deafness and Other Communication Disorders, National Institutes
of Health, Bethesda, Maryland 20892, and 5Department
of Physiology, University of California San Francisco, San Francisco,
California 94143-0444
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Abstract
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Modification of synaptic NMDA receptor (NMDAR) expression influences
NMDAR-mediated synaptic function and associated persistent pain. NMDARs
directly bind to a family of membrane-associated guanylate kinases (MAGUKs)
that regulate surface and synaptic NMDAR trafficking in the CNS. We report
here that postsynaptic density-93 protein (PSD-93), a postsynaptic neuronal
MAGUK, is expressed abundantly in spinal dorsal horn and forebrain, where it
colocalizes and interacts with NMDAR subunits NR2A and NR2B. Targeted
disruption of the PSD-93 gene reduces not only surface NR2A and NR2B
expression but also NMDAR-mediated excitatory postsynaptic currents and
potentials, without affecting surface AMPA receptor expression or its synaptic
function, in the regions mentioned above. Furthermore, mice lacking PSD-93
exhibit blunted NMDAR-dependent persistent pain induced by peripheral nerve
injury or injection of Complete Freund's Adjuvant, although they display
intact nociceptive responsiveness to acute pain. PSD-93 appears to be
important for NMDAR synaptic targeting and function and to be a potential
biochemical target for the treatment of persistent pain.
Key words: PSD-93; NMDA receptors; surface expression; persistent pain; spinal cord, forebrain
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Introduction
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Neurotransmission requires spatial and functional assembly of signal
transduction machinery at the plasma membrane. The postsynaptic density (PSD),
an electron-dense cytoskeletal structure beneath the plasma membrane of
excitatory synapses, is one site in which receptors, channels, and effectors
organize to mediate signaling. Recent studies indicate that a family of
membrane-associated guanylate kinases (MAGUKs) in the PSD may play a major
role in synaptic localization of channels, signaling enzymes, and adhesion
molecules (Craven and Bredt,
1998
; Garner et al.,
2000). Neuronal MAGUK proteins include PSD-93/chapsyn-110
(Brenman et al., 1996;
Kim et al., 1996),
PSD-95/synapse-associated protein (SAP)-90
(Cho et al., 1992;
Kistner et al., 1993),
SAP-97/hdlg (Muller et al.,
1995), and SAP102 (Lau et al.,
1996), all of which consist of three tandem PDZ domains at the
N-terminal side, an Src homology region 3 domain in the middle, and a
guanylate kinase-like domain at the C-terminal end. PDZ domains of MAGUKs are
motifs of 
90 amino acid repeats that have been recognized to mediate
protein-protein interactions and to bind to short amino acid motifs at the C
termini of interacting proteins at synapses
(Sheng, 1996;
Kornau et al., 1997).
The NMDA receptors (NMDARs) are MAGUK-interacting proteins
(Kornau et al., 1995;
Brenman et al., 1996;
Kim et al., 1996;
Lau et al., 1996) that
regulate multiple functions in physiological and pathological processes,
including the transmission and/or processing of pain signaling in the CNS
(Dougherty and Willis, 1991;
Basbaum and Woolf, 1999;
Hewitt, 2000). Functional
NMDARs are heteromeric complexes mainly consisting of NR1 and NR2 (NR2A-D)
subunits. The NR2 can determine synaptic localization and function of the
receptor, because deletion of the C-terminal tail of NR2 results in impaired
NMDAR-mediated synaptic activity (Mori and
Mishina, 1995; Sprengel et
al., 1998; Cull-Candy et al.,
2001). C-terminal tails of the NR2 subunits directly bind to MAGUK
proteins via PDZ domain interaction at synapses
(Kornau et al., 1995;
Brenman et al., 1996;
Kim et al., 1996;
Lau et al., 1996). PSD-95
enhances NMDAR clustering at synapses and inhibits NR2B-mediated
internalization (Roche et al.,
2001). Coexpression of PSD-95 with NMDARs increases surface
expression of NMDARs and enhances synaptic NMDAR function
(Carroll and Zukin, 2002). It
appears that MAGUKs as molecular scaffold proteins cluster and bind to NMDARs
at synaptic membranes and modulate their synaptic function. Interestingly,
PSD-95 mutant mice exhibit intact synaptic localization and postsynaptic
function of NMDARs, although they reveal enhanced long-term potentiation and
impaired spatial learning (Migaud et al.,
1998). Thus the physiological and pathological functional
significance of the NMDAR being targeted and clustered at synapses by MAGUK
proteins in vivo is unclear.
In this paper we demonstrated that PSD-93 was critical for surface NMDAR
expression, NMDAR-mediated postsynaptic function, and NMDAR-dependent
persistent pain. It appears that the deficiency of PSD-93 results in impaired
NMDAR-mediated postsynaptic functions and in blunted NMDAR-dependent
persistent pain by the mechanism of surface NMDAR alteration.
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Materials and Methods
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Reverse transcriptase-PCR. The cDNA sequences encoding a portion
of the PSD-93 gene were amplified by using the following synthetic
oligo-nucleotide primers: PSD1 (5'-AGTACTGTGCTGAGAATGAC-3') and
PSD2 (5'-GAAGCAGGCTCTATTGTTCG-3') for amplification of PSD-93
codon positions 514-1085. RNA samples (1 µg) were reverse transcribed to
generate first-strand cDNA. The PCR reactions were performed for 35 cycles.
Each cycle included 35 sec at 94°C, 45 sec at 55°C, and 1 min at
72°C. The PCR products were cloned directly into the TA cloning vector
(Invitrogen, San Diego, CA) and verified by automatic DNA sequencing.
Immunocytochemistry. Mice were perfused with 4% paraformaldehyde
in 0.1 M PBS. The spinal cord was harvested and postfixed at
4°C for 4 hr and cryoprotected in 30% sucrose overnight. Sections (30
µm) were cut on a cryostat and then blocked for 1 hr in PBS containing 10%
goat serum and 0.3% Triton X-100. Primary rabbit polyclonal anti-PSD-93
(1:1000) (Brenman et al., 1996,
1998;
Roche et al., 1999;
Sans et al., 2000;
McGee et al., 2001) or NR2A/2B
(1:100; Chemicon, Temecula, CA) (Petralia
et al., 1994; Follesa and Ticku,
1996a,b)
was diluted into blocking reagent and incubated with the sections overnight.
The sections then were incubated in biotinylated goat anti-rabbit IgG (1:200;
Vector Labs, Burlingame, CA) for 1 hr at 37°C, followed by
avidin-biotin-peroxidase complex (1:100; Vector Labs) for 1 hr at 37°C.
The immune reaction product was visualized by catalysis of
3,3-diaminobenzidine by horseradish peroxidase in the presence of 0.01%
H2O2. Control sections lacking primary antiserum were
stained in parallel.
Western blot analysis. The soluble protein or PSD fraction was
prepared as described (Tao et al.,
2000; Tao and Johns,
2002). The proteins were loaded onto 4% stacking/7.5% separating
SDS-polyacrylamide gels and then were transferred electrophoretically onto
nitrocellulose membrane. The membrane was blocked with 2% nonfat dry milk and
subsequently was incubated for 1 hr with polyclonal rabbit anti-PSD-93
antibody (1:500) or with polyclonal rabbit anti-NR2A antibody (1:500;
Chemicon) or with polyclonal rabbit anti-NR2B antibody (1:500) (Follesa and
Ticku,
1996a,b;
Shi et al., 1997). The
proteins were detected by using horseradish peroxidase-conjugated anti-rabbit
secondary antibodies and visualized by chemiluminescence reagents provided
with the ECL kit (Amersham Biosciences, Piscataway, NJ) and exposure to film.
The intensity of blots was quantified with densitometry.
Coimmunoprecipitation. The affinity-purified rabbit PSD-93
antiserum with or without preincubation with excess PSD-93 fusion protein (100
µg/ml) was incubated with 100 µl of a 1:1 slurry of protein A-Sepharose
for 1 hr, and the protein-antibody complex was spun down at 2000 rpm for 4
min. The solubilized PSD fraction (400 µg) then was added to the Sepharose
beads, and the mixture was incubated for 2-3 hr at 4°C. The mixture was
washed once with 1% Triton X-100 in immunoprecipitation buffer [containing (in
mM): 137 NaCl, 2.7 KCl, 4.3 Na2HPO4, 1.4
KH2PO4, 5 EGTA, 1 sodium vanadate, 10 sodium
pyrophosphate, 50 NaF, and 0.1 phenylmethylsulfonyl fluoride plus 20 U/ml
Trasylol], twice with 1% Triton X-100 in immunoprecipitation buffer plus 300
mM NaCl, and three times with immunoprecipitation buffer. The
proteins were separated by SDS-PAGE and detected by PSD-93, NR2A, or NR2B
antibody as described above. As a positive control (Input), 40 µg of the
solubilized PSD fraction was loaded.
Electron microscopy. The postembedding immunogold labeling was
done as described (Petralia and Wenthold,
1999; Sans et al.,
2000). Briefly, male mice were perfused with 4% paraformaldehyde
plus 0.5% glutaraldehyde. Sections 300 µm each were prepared with a
vibratome, frozen in liquid propane, and embedded in Lowicryl. Ultrathin
sections were cut from blocks and processed for immunogold staining. For
double labeling the first primary antibody and corresponding
immunogold-conjugated (10 nm of gold) antibody were applied. Then the
ultrasections were exposed to paraformaldehyde vapor at 80°C for 1 hr, and
the second primary and secondary (5 nm) antibodies were applied the next day.
Most of the primary antibodies were characterized previously
(Petralia and Wenthold, 1999;
Sans et al., 2000). Because
individual NR2A or NR2B antibody that we used in the Western blotting
described above is not recommended for immunogold staining
(Sans et al., 2000), the
primary antibodies used were PSD-93 (1.7 µg/ml), NR1 (4 µg/ml), and
NR2A/2B (4 µg/ml). Thus electron microscopy showed a synaptic relationship
of PSD-93 with NR1 or NR2A/2B (but not individual NR2A or NR2B), whereas
coimmunoprecipitation and surface expression assays revealed the interaction
of PSD-93 with individual NR2A or NR2B. Controls included an absence of
primary antibody for single labeling and an absence of the second primary
antibody for double labeling. Controls always showed little or no gold
labeling.
Behavioral testing. All behavioral experiments were performed with
the approval of the Animal Care Committee at the Johns Hopkins University and
were consistent with the ethical guidelines of National Institutes of Health
and the International Association for the Study of Pain. To test baseline pain
responses, we assessed mechanical withdrawal frequencies by application of
different forces of calibrated von Frey monofilaments (mN: 0.24, 1.47, 4.33,
8.01, 23.69, 40.31) (Stoelting, Wood Dale, IL) to the plantar hind paw
surface, and measured thermal withdrawal latencies after the application of
radiant heat to the plantar hind paw surface
(Mansikka et al., 2000;
Tao et al., 2000). In Complete
Freund's Adjuvant (CFA)-induced inflammatory pain, the mice received a 20
µl intraplantar injection of CFA solution (1 mg/ml). Mechanical withdrawal
frequencies were assessed by applying calibrated von Frey monofilaments 0.24
and 4.33 mN to the plantar hind paw surface as described
(Fairbanks et al., 2000;
Mansikka et al., 2000). To
quantify the inflammatory response, we measured the paw thickness with a
caliper after CFA injection. To test neuropathic pain, we isolated the fifth
lumbar nerve root, ligated it with a 6-0 silk suture, and transected it just
distal to the ligature in halothane-anesthetized mice. Control groups included
naive or sham-operated (without nerve ligation) mice (n = 5 for each
group). Mechanical withdrawal frequencies were assessed by applying calibrated
von Frey filaments 0.24 and 4.33 mN to the plantar hind paw surface. To
observe the effect of MK-801 on CFA- or nerve injury-induced mechanical
hyperalgesia, we gave MK-801 (1.0 nmol/5 µl; Research Biochemicals, Natick,
MA) (Zushida and Kamei, 2002)
intrathecally on day 3 after CFA injection or on day 6 after nerve injury as
previously described. Mechanical withdrawal frequencies on both ipsilateral
and contralateral sides were measured before drug injection and at 30 min
after drug injection. In all of the behavioral studies described above, the
observers were blind to the genotype of the mice.
Electrophysiological recordings. For patch-clamp recording of
cultured neurons, spinal dorsal horn neuronal cultures were prepared as
previously described (O'Brien et al.,
1997). In brief, the neurons from embryonic day 14 mouse dorsal
spinal cord were plated at 200,000 cells/ml. Recordings were performed at 9 d
in vitro (9 DIV) from at least two platings for each genotype.
Neurons were bathed in artificial CSF (ACSF) consisting of (in mM):
145 NaCl, 10 HEPES, 3 KCl, 2 CaCl2, 10 glucose, 0.1 glycine, 0.001
TTX, 0.1 picrotoxin, and 0.002 strychnine. MgCl2, an open channel
blocker of NMDARs, was omitted purposely from the recording solutions. The pH
was adjusted to 7.4, and the osmolarity was adjusted to 305-310. Intracellular
saline consisted of (in mM): 135 cesium methane-sulfonate, 10 CsCl,
5 EGTA, 4 ATP-Na, 0.4 GTP-Na, 10 HEPES, and 1 MgCl2. The pH was
adjusted to 7.2, and the osmolarity was adjusted to 290-295. Patch electrodes
were fire polished and typically had resistance <5 M
. After the
whole-cell configuration was achieved, the cell was allowed to stabilize for
2 min at a holding potential of -60 mV. Data then were acquired
continuously for 10-15 min at a sampling frequency of 10 kHz with low-pass
filtering at 1 kHz. Miniature EPSCs (mEPSCs) were selected and subsequently
aligned with Mini Analysis software (Synatosoft, Decatur, GA). The average of
at least 100 events for each cell was exported and then fit with a
double-exponential function in the form:
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where Ifast and Islow are the
amplitudes of the fast and slow decay components, and 
fast and
slow are their respective decay time constants used to fit the
data. Curve fitting was performed with Clampfit 8.1 (Axon Instruments, Union
City, CA).
For slice recordings the adult mice (8-12 weeks) were anesthetized with
1-2% halothane, and brain or spinal cord was isolated. Recordings from cortex
were done as described (Wei et al.,
2001). Briefly, transverse cortical slices were maintained in an
interface chamber at 28°C, where they were subfused with ACSF and bubbled
with 95% O2/5% CO2. Slices were kept in the recording
chamber for at least 2 hr before experiments. A bipolar tungsten stimulating
electrode was placed in layer V, and extracellular field potentials were
recorded with a glass microelectrode (3-12 M, filled with ACSF) placed
in layer II/III. Experiments were performed blind, and the placement of
stimulating and recording electrodes was kept consistent in different groups
of mice. To block synaptic responses mediated by AMPA/kainate receptors, we
applied 20 µM CNQX throughout the experiments. As reported
previously (Wei et al., 2001),
the residual EPSPs were blocked completely and reversibly by the bath
application of 100 µM aminophosphonopentanoic acid (AP-5).
Stimulation at different intensities was applied, and test responses were
elicited at 0.02 Hz. The measurement of field NMDAR-mediated EPSPs was
performed blind to the wild-type or knock-out genotype. For recording from
spinal cord, transverse spinal slices (450-500 µm) with attached dorsal
roots (7-12 mm) were prepared. Intracellular recordings of synaptic responses
were performed from neurons located in the dorsal horn lamina I and II with a
3 M potassium chloride-filled glass microelectrode (DC impedance,
75-200 M). The synaptic responses were activated by electrical
stimulation in dorsal roots with a bipolar electrode. EPSPs of the neurons
were evoked with stimulation at various intensities (10-40 V, 0.2 msec),
recorded through a high-input impedance bridge circuit amplifier (Axoclamp
2B), and stored with pClamp software (Axon Instruments). The ACSF was
oxygenated with 95% O2/5% CO2. The temperature and
perfusion rate of recording were kept at 34°C and 2-5 ml/min,
respectively. The AMPA/kainate receptor antagonist CNQX (20 µM)
was included in the bath solution throughout the experiments. To reduce the
magnesium blockade of NMDARs, we used 0.1 mM magnesium during the
recordings of NMDAR-mediated responses. In all experiments bicuculline
methiodide (10 µM) and strychnine hydrochloride (1
µM) were added to the perfusion solution to block inhibitory
synaptic transmission.
Surface expression assays. Biotinylation experiments were
performed as previously described (Roche
et al., 2001; Snyder et al.,
2001). Briefly, high-density cultured spinal dorsal horn neurons
were prepared as mentioned above and incubated with ACSF containing 1.5 mg/ml
sulfo-NHS-LC-biotin (Pierce, Rockford, IL) for 45 min on ice. Cultures were
rinsed in ACSF to quench the biotin reaction and lysed in 300 µl of
modified RIPA buffer [containing 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic
acid, and (in mM): 50 NaPO4, 150 NaCl, 2 NaF, 10 sodium
pyrophosphate, and 1 PMSF plus 1 mg/ml leupeptin]. The homogenates were
centrifuged at 14,000 x g for 15 min at 4°C, and the
supernatant was harvested. After the measurement of protein concentration, 20
µg of the supernatant was removed to measure total NR2A, NR2B, or GluR1.
Then 200 µg of the supernatant was incubated with 100 µl of 50%
NeutrAvidin-agarose (Pierce) for 3 hr at 4°C and washed three times with
RIPA buffer. Biotinylated proteins were resuspended in 40 µl of SDS sample
buffer and boiled. Quantitative Western blots were performed on both total and
biotinylated (surface) proteins with anti-NR2A, anti-NR2B, and anti-GluR1
antibodies as described above. The surface/total ratio was calculated.
Cross-linking experiments were performed as previously described
(Clayton et al., 2002;
Grosshans et al., 2002). In
brief, transverse spinal slices (400 µm) from adult wild-type (n =
5) and PSD-93 knock-out (n = 5) mice (10-12 weeks) were prepared as
mentioned above and recovered by perfusion for 1 hr. Then the slices were
placed into either ice-cold ACSF or cold ACSF containing 1 mg/ml
BS3 [bis-(sulfosuccinimidyl) suberate, Pierce] for 45 min at
4°C. Four slices per control or treatment group were included for each
animal. To quench the remaining BS3, we washed the slices three
times in cold ACSF containing 20 mM Tris, pH 7.6. After
homogenization the protein concentrations were determined. Surface expression
was determined after semi-quantitative Western blot analysis and after a
comparison of treated samples with nontreated controls.
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Results
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PSD-93 expression and its interaction with NMDARs in the spinal cord
and forebrain
We first examined the expression of PSD-93 messenger RNA and protein in the
pain-related regions of the nervous system. RNA extracted from the dorsal root
ganglion, spinal cord, and forebrain was probed by reverse transcriptase-PCR
(RT-PCR) analysis. The PCR product was highly detected in the dorsal horn of
spinal cord and forebrain areas (Fig.
1A). In contrast, it was detected very weakly or not at
all in the ventral horn of spinal cord and dorsal root ganglion
(Fig. 1A). The PCR
product then was cloned directly into the pCR2.1-TOPO vector and verified as
PSD-93 by automatic DNA sequencing. Immunoblot analysis further revealed
abundant PSD-93 protein expression in the dorsal horn of spinal cord and
forebrain areas, but not in the ventral horn of spinal cord or dorsal root
ganglion (Fig. 1B).
Using immunocytochemistry, we found that PSD-93 immunoreactivity occurred at a
higher density in the superficial laminas and at a lower density in other
laminas of spinal dorsal horn (Fig.
1C) and in the ventral horn. Moreover, the density of
PSD-93 immunoreactivity in the superficial laminas was unaffected after
unilateral sectioning of spinal dorsal nerve root or bilateral dorsolateral
fasciculi lesions. Finally, we characterized the subcellular localization of
PSD-93 under electron microscopy. In sections of the superficial dorsal horn
or the anterior cingular cortex of forebrain, immunogold labeling with a
PSD-93 antibody was associated with the postsynaptic membrane in neuronal
synapses (Fig. 1D).
These findings indicate that PSD-93 in the superficial dorsal horn and
anterior cingular cortex, to a great extent, is intrinsic to these areas.
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Figure 1. Expression and localization of PSD-93 in the major pain-related regions of
the CNS. A, RT-PCR analysis showed that a 571 bp product from PSD-93
was highly detected in the dorsal horn (DH) of spinal cord and forebrain (FB),
whereas the mRNA was detected very weakly or not detected in the ventral horn
(VH) of spinal cord and dorsal root ganglion (DRG). PCR product was cloned
directly into the TA cloning vector and verified as PSD-93 by automatic DNA
sequencing.  -Actin mRNA was used as a loading control. B,
Western blot analysis showed that PSD-93 protein was expressed abundantly in
the PSD fraction of the forebrain and dorsal horn, weakly in ventral horn, and
not detected in the dorsal root ganglion. Tubulin was used as a loading
control. C, PSD-93 immunoreactivity was distributed mainly in the
superficial dorsal horn of wild-type (WT) mice. There was no immunostaining
with the PSD-93 antibody in the dorsal horn of PSD-93 knock-out (KO) mice.
Scale bar, 125 µm. D, The subcellular localization of PSD-93 in
the superficial laminas of dorsal horn (DH) and the anterior cingular cortex
(CO) of forebrain. Immunogold labeling of PSD-93 was localized most commonly
in the postsynaptic membrane (arrows). Pr, Presynaptic; Po, postsynaptic.
Scale bar, 0.2 µm.
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The NMDAR subunits including NR2A and NR2B are expressed in the neurons of
the cortex and the superficial dorsal horn
(Yung, 1998;
Sun et al., 2000;
Hagemann et al., 2003). Under
electron microscopy, immunogold labeling with an NR1 or NR2A/2B antibody was
associated with the postsynaptic membrane in the superficial dorsal horn and
the anterior cingular cortex of forebrain
(Fig. 2A). In the
spinal dorsal horn the distribution of PSD-93 is similar to that of NR2A/2B
(Fig. 2B). These data
indicate that PSD-93 might colocalize and interact with the NMDARs in these
areas. Indeed, in sections from these areas labeled with both PSD-93 and
NR2A/2B antibodies, neuronal synapses showed labeling for both antibodies
interspersed along the postsynaptic membrane
(Fig. 2C). This was
confirmed further with the use of coimmunoprecipitation, showing that PSD-93
antibody was able to immunoprecipitate not only itself but also NR2A and NR2B
in the postsynaptic density fraction from the dorsal horn and forebrain
(Fig. 2D). However,
NR2A and NR2B could not be immunoprecipitated when PSD-93 antibody was
preincubated with excess PSD-93 fusion protein
(Fig. 2D). PSD-93 was
not immunoprecipitated with a glutamate receptor 1 (GluR1; an AMPA receptor
subunit) antibody (data not shown). As a molecular scaffold protein, PSD-93
targets and clusters NR2A and NR2B to synapses
(Brenman et al., 1996;
Kim et al., 1996). PSD-93
interaction with NMDARs at the synapses in these two regions suggests that
PSD-93 might be required for surface NMDAR expression.
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Figure 2. Identification of a complex assembled by PSD-93 and NMDARs in the spinal
cord and forebrain. A, The subcellular localization of NR1 in the
superficial laminas of dorsal horn (DH) and that of NR2A/2B in the anterior
cingular cortex (CO) of forebrain. Immunogold labeling of NR1 in postsynaptic
structures in the superficial laminas was associated almost exclusively with
the postsynaptic membrane (arrow). In the CO the immunogold labeling for
NR2A/2B was associated mainly with the postsynaptic membrane (arrow) but also
was prominent in the postsynaptic spine apparatus in some spines (data not
shown). Pr, Presynaptic; Po, postsynaptic. Scale bar, 0.2 µm. B,
Similar distribution of PSD-93 and NR2A/2B in the spinal dorsal horn. Scale
bar, 125 µm. C, Colocalization of PSD-93 and NR2A/NR2B in the
superficial laminas of dorsal horn (DH) and the anterior cingular cortex (CO)
of forebrain. Sections were double-labeled with PSD-93 antibody (10 nm gold)
and NR2A/2B antibody (5 nm gold). Arrows indicate the postsynaptic membrane.
Scale bar, 0.1 µm. D, Binding of PSD-93 and NR2A or NR2B in the
spinal cord and forebrain. PSD-93 antibody was able to immunoprecipitate (IP)
not only itself but also NR2A or NR2B in the soluble PSD fraction. However,
PSD-93, NR2A, and NR2B could not be immunoprecipitated when PSD-93 antibody
was preincubated with the PSD-93 fusion protein. The amount of sample loaded
for the input was 10% of that for the immunoprecipitation. IB,
Immunoblotting.
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PSD-93 deletion alters surface NR2A and NR2B expression
PSD-93 mainly binds to the C termini of NMDAR subunits NR2A and NR2B
(Brenman et al., 1996;
Kim et al., 1996). The
expression of NR2A and NR2B in total extracts of the spinal cord and forebrain
was examined. Western blotting revealed that the expression of total NR2A or
NR2B in the spinal cord as well as in the forebrain of knock-out mice was
similar to that in wild-type or heterozygous mice
(Fig. 3A).
Immunocytochemistry showed that the staining density and localization of
NR2A/2B immunoreactivity in the spinal cord of knock-out mice were similar to
those in wild-type mice (Fig.
3B). We further compared the surface expression of NR2A
and NR2B in cultured spinal dorsal horn neurons of wild-type and PSD-93
knock-out mice (McGee et al.,
2001). The surface receptors were labeled with biotin and then
precipitated. The ratio of surface to total NR2A or NR2B was determined by
quantitative Western blotting. The biochemical analysis showed that surface
NR2A and NR2B in knock-out mice were reduced by 64 and 50%, respectively, of
the value in wild-type mice, although total cellular NR2A and NR2B protein
remained similar (Fig.
3C,D). To rule out the possibility of nonspecific changes
in the postsynaptic neurons, we also monitored change of surface GluR1. In
contrast to the effect on NR2A and NR2B, the deficiency of PSD-93 had no
effect on surface GluR1 expression (Fig.
3E). Finally, we defined the surface expression of NR2A
and NR2B in adult spinal cord neurons by using a membrane-impermeable
cross-linking reagent (BS3), which cross-links proteins only on the
surface of cells (Clayton et al.,
2002; Grosshans et al.,
2002). Because the cross-linked product is unable to enter
polyacrylamide gels, the intracellular pool of receptor was measured directly.
Equal amounts of total protein were loaded so that changes in surface
expression were reflected in alteration in the levels of intracellular
proteins. As shown in Figure 3, F
and G, a significant decrease was seen in surface
expression of spinal cord NR2A and NR2B of PSD-93 knock-out mice compared with
that in wild-type mice (n = 5 in each case; p < 0.05),
which confirmed the results obtained from the cultured spinal neurons
described above. As a control, the expression of an uncross-linked
intracellular protein, 
-actinin, in the spinal cord of PSD93 knock-out
mice was similar to that in wild-type mice
(Fig. 3F,G). Thus
PSD-93 deletion specifically affects surface NR2A and NR2B expression. This
might alter NMDAR-mediated excitatory postsynaptic function.
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Figure 3. Expression of NR2A and NR2B in the spinal cord and forebrain of PSD-93
knock-out mice. A, Immunoblots of PSD-93, NR2A, and NR2B proteins in
total extracts of spinal cord and forebrain of wild-type (+/+), heterozygous
(+/-), and knock-out (-/-) mice. PSD-93 protein is undetectable in knock-out
mice. There was no significant difference in the density of NR2A or NR2B bands
among wild-type, heterozygous, and knock-out mice (p > 0.05;
n = 5 for each group). Tubulin was used as a loading control (data
not shown). B, The expression and distribution of NR2A/2B
immunoreactivity in the spinal cord of wild-type (+/+) and PSD-93 knock-out
(-/-) mice. The density and localization of NR2A/2B immunoreactivity in the
spinal cord of wild-type mice are similar to those in knock-out mice. Scale
bar, 50 µm. C-E, Effect of PSD-93 deletion on surface NR2A and
NR2B expression in the cultured neurons of spinal dorsal horn. The amount of
sample loaded for the total (T) was 10% of that for the surface (S).
C, The top panel depicts a representative Western blot that shows
samples of total and biotinylated surface NR2A in wild-type (WT) and PSD-93
knock-out (KO) mice. The bottom panel shows the statistical summary of the
densitometric analysis. Average surface NR2A levels in knock-out mice were
reduced by 64% of the value in wild-type mice (*p <
0.05). D, The top panel depicts a representative Western blot that
shows samples of total and biotinylated surface NR2B in wild-type (WT) and
PSD-93 knock-out (KO) mice. The bottom panel shows the statistical summary of
the densitometric analysis. Average surface NR2B levels in knock-out mice were
reduced by 50% of the value in wild-type mice (*p <
0.05). E, The top panel depicts a representative Western blot that
shows samples of total and biotinylated surface GluR1 in wild-type (WT) and
PSD-93 knock-out (KO) mice. The bottom panel shows the statistical summary of
the densitometric analysis. There was no significant difference in surface
GluR1 expression between wild-type and knock-out mice (p > 0.05).
F, G, Effect of PSD-93 deletion on surface NR2A and NR2B expression
in the spinal cord neurons from adult mice. Surface expression was assayed by
using a membrane-impermeable cross-linking agent (BS3). The
cross-linked product is unable to enter polyacrylamide gels, so only the
intracellular pool is resolved by Western blotting. Changes in surface
expression are detected by observing changes in this indirectly detected
intracellular pool. F, A representative Western blot that shows
samples of total and intracellular NR2A, NR2B, and -actinin,
respectively, in the untreated control and BS3-treated groups of
wild-type (WT) and PSD-93 knock-out (KO) mice. G, The statistical
summary of the densitometric analysis. Average percentages of intracellular
NR2A and NR2B levels in knock-out (KO) mice were increased by 190 and 194%,
respectively, of the value in wild-type (WT) mice (*p <
0.05). In contrast, the average percentage of intracellular -actinin
level in knock-out mice was decreased by 0.92% of the value in wild-type
mice.
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PSD-93 deletion reduces NMDAR-mediated postsynaptic function
To test directly the possibility that PSD-93 deletion alters postsynaptic
NMDAR function, we measured mEPSCs in cultured spinal dorsal horn neurons of
PSD-93 knock-out mice. mEPSCs were best described by a dual-exponential
function when individual events were aligned by their fast rising peak and
subsequently were averaged. This is typical of mEPSCs mediated by vesicular
glutamate release (Bekkers and Stevens,
1989). The addition of 100 µM AP-5 to the
extracellular solution completely abolished the slow exponential
(Fig. 4A), whereas the
addition of AP-5 and NBQX (100 µM) inhibited all mEPSCs.
Therefore, the slower of the two exponentials was considered a result of NMDAR
activation and the faster AMPAR activation. Most mEPSCs from neurons of
wild-type mice had obvious NMDAR components
(Fig. 4B). In
contrast, neurons from knock-out mice exhibited less obvious and sometimes
absent NMDAR responses (Fig.
4B). When individual events were averaged, the NMDAR
component from knock-out mice was reduced greatly (11.28 ± 1.02 pA,
n = 21 for wild type; 7.21 ± 1.56 pA, n = 19 for
knockout; p < 0.005) (Fig.
4C,D). As expected, the peak AMPA currents (46.68
± 3.20 pA, n = 21 for wild type; 44.36 ± 1.10 pA,
n = 19 for knock-out), NMDA decay times (55.86 ± 8.52 msec,
n = 21 for wild type; 54.46 ± 5.58 msec, n = 19 for
knock-out), and AMPA decay times (1.75 ± 0.13 msec, n = 21 for
wild type; 1.96 ± 0.15 msec, n = 19 for knock-out) were not
significantly different (Fig.
4C,D). In addition, mEPSC frequency (0.721 ± 0.11
Hz, n = 21 for wild type; 0.561 ± 0.05 Hz, n = 19 for
knock-out), average input resistance (373.1 ± 27.6 M, n
= 21 for wild type; 375.9 ± 26.32 M, n = 19 for
knock-out), and series resistance (17.42 ± 0.532 M, n =
21 for wild type; 18.05 ± 0.586 M, n = 19 for
knock-out) for each culture genotype were not changed statistically. These
data indicate that the deficiency of PSD-93 does affect NMDAR-mediated
postsynaptic function while having little effect on AMPA receptor
function.
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Figure 4. PSD-93 deletion reduced NMDAR-mediated mEPSCs from the cultured neurons of
spinal dorsal horn. A, mEPSCs were acquired in the presence or
absence of 100 µM AP-5. The addition of 100 µM
AP-5 to the extracellular solution abolished the slow exponential, whereas the
addition of AP-5 plus NBQX (100 µM) completely blocked miniature
events. One hundred events were selected before (control ACSF) and during AP-5
perfusion. After AP-5 perfusion the decay time of the fast component was not
altered significantly (before AP-5, fast = 1.98 ± 0.20
msec; during AP-5, fast = 1.96 ± 0.19 msec; n
= 20). Therefore, the amplitude of the second exponential was taken as the
NMDAR peak current. The amplitude of the averaged mEPSC was taken as the peak
AMPA current. B, Four consecutive 1 sec traces from a cultured neuron
representative of wild-type (WT) or PSD-93 knock-out (KO) mice. C,
Trace averages from the neurons illustrated in B. Selected from each
neuron were 120 events, and the responses were aligned by their peaks. The
neurons in wild-type and knock-out mice were fit by a double-exponential
function, and their individual components were displayed. D, The top
plot compares the average current ratio for NMDARs and AMPA receptors between
pooled wild-type and knock-out mice responses (*p <
0.005). The bottom plot compares the ratio of decay times for the AMPA
receptors and NMDARs between pooled wild-type and knock-out mice
responses.
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To observe further the effect of PSD-93 deletion on NMDAR-mediated
postsynaptic function, we recorded intracellular EPSPs in the spinal cord
slices from adult knock-out mice. EPSPs in the superficial dorsal horn neurons
were evoked by dorsal root stimulation
(Wei et al., 2001). After
blockade of AMPA and kainate receptors by CNQX (20 µM), we
observed a slow EPSP that could be blocked completely and reversibly by a
selective NMDAR antagonist, AP-5 (100 µM;
Fig. 5A). To enhance
sensitivity in NMDAR-mediated EPSPs, we injected current through the recording
electrode to depolarize neurons and relieve any voltage-dependent
Mg2+ blockade at a holding potential of -50 mV. Slow
EPSP slopes in knock-out mouse slices were reduced significantly as compared
with wild-type mouse slices (Fig.
5A). In contrast, fast AMPA and kainate receptor-mediated
EPSPs were similar in spinal cord slices of wild-type mice (5.17 ± 0.29
mV/msec; n = 20) and knock-out mice (4.94 ± 0.77 mV/msec;
n = 25). Resting neuronal membrane potentials were not different in
wild-type (-68.9 ± 2.7 mV; n = 20) and knock-out mouse (-69.5
± 2.7 mV; n = 25) slices.
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Figure 5. PSD-93 deletion attenuated NMDAR-mediated excitatory postsynaptic
responses. A, Traces of EPSPs recorded in the presence or absence of
CNQX (20 µM) or CNQX and AP-5 (100 µM) from the
neurons of the superficial dorsal horn in adult wild-type (WT) and knock-out
(KO) mice. Slow NMDAR-mediated synaptic responses were compared at four dorsal
root stimulation intensities (10, 20, 30, and 40 V), revealing significant
differences between wild-type (n = 6-8) and knock-out mice
(n = 8-10; p < 0.05). B, Traces of
NMDAR-mediated fEPSPs recorded in the presence of 20 µM CNQX
from the anterior cingular cortex of forebrain in adult wild-type (WT) and
knock-out (KO) mice. Similar traces also were found in insular cortex of
forebrain (data not shown). NMDAR-mediated synaptic responses were compared at
four stimulation intensities (40, 60, 80, and 100 V) in the anterior cingular
cortex (WT, n = 8; KO, n = 10) and insular cortex (WT,
n = 8; KO, n = 10), revealing significant differences
between wild-type and knock-out mice (p < 0.05).
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We also examined NMDAR-mediated postsynaptic function in the neurons of the
anterior cingular cortex. We prepared brain slices from adult mice and
recorded field EPSPs (fEPSPs) after local electron stimulation
(Sah and Nicoll, 1991).
Consistent with the findings above in the dorsal horn, slow AP-5-sensitive
fEPSPs, isolated in the presence of CNQX (20 µM), were decreased
dramatically in the anterior cingular cortex neurons of PSD-93 knock-out mice
(Fig. 5B). To test
further whether alteration in NMDAR-mediated postsynaptic function also
appears in other regions of forebrain, we recorded fEPSPs in the insular
cortex. As before, knock-out mice, compared with wild-type mice, displayed
reduced NMDAR-mediated fEPSP in the neurons of this area
(Fig. 5B). No
significant changes in AMPAR-mediated fEPSP were observed in either region of
forebrain (data not shown). These findings provide evidence that PSD-93
deletion influences NMDAR-mediated postsynaptic response in the spinal cord
dorsal horn and forebrain of adult mice.
PSD-93 deletion reduces NMDAR-dependent persistent pain
The spinal cord dorsal horn and forebrain are major pain-related regions in
the nervous system. We next asked whether the reduction of surface NMDAR
expression and its synaptic function by PSD-93 deletion affected the animals'
behavioral responses to acute noxious stimuli or peripheral inflammation/nerve
injury. Both male and female mice were viable and fertile, with normal
appearance and locomotor activity (McGee
et al., 2001). We first examined baseline responses of knock-out
mice to mechanical or thermal stimulation. The different intensities of von
Frey monofilaments (mechanical stimuli) and high-intensity radiant heat
(thermal stimulus) were applied to the plantar sides of both hind paws. No
significant differences in paw withdrawal latencies (in response to thermal
stimulus) or frequencies (in response to mechanical stimuli) were observed
among wild-type, heterozygous, and knock-out mice
(Fig. 6A,B). This
indicates that acute nociceptive transmission is not altered significantly in
knock-out mice. We then studied an inflammatory pain model produced by hind
paw injection of CFA. Subcutaneous injection of CFA produces long-lasting
inflammation and long-term mechanical pain hypersensitivity. In wild-type mice
the application of both 0.24 mN (low intensity) and 4.33 mN (moderate
intensity) von Frey filaments to the dorsum of the injected hind paw elicited
significant mechanical pain hypersensitivity, which appeared on the first day
after CFA injection and persisted for 9 d or longer
(Fig. 6C,D). In
contrast to the wild-type mice, there were no differences in paw withdrawal
frequencies in response to mechanical stimuli in PSD-93 knock-out mice as
compared to baseline (Fig.
3C,D). No obvious difference in the degree of hind paw
edema was found at day 9 between wild-type (paw thickness, 2.77 ± 0.07
mm; n = 10) and PSD-93 knock-out (2.54 ± 0.11; n =
10) mice (p > 0.05, Student's t test). We also determined
the possible role of PSD-93 in neuropathic pain, a common cause of chronic
pain treated in clinical practice. We tightly ligated the fifth lumbar spinal
nerve root and then completely transected it just distal to the ligature in
wild-type and knock-out mice. This nerve injury produces a persistent pain
syndrome characterized by a significant and long-lasting increase in paw
withdrawal frequency to mechanical stimulation on the injured side
(Kim and Chung, 1992;
Mansikka et al., 2000). In the
wild-type mice the mechanical response frequencies to both 0.24 and 4.33 mN
mechanical stimuli were increased significantly by the second day after spinal
nerve injury. This increased responsiveness persisted for 14 d or longer
(Fig. 6E,F). The
PSD-93 knock-out mice showed only modest increases in response frequencies to
low-intensity mechanical stimuli after surgery
(Fig. 6E). However,
the magnitude of the change in the knock-out mice was much less than what we
recorded in the wild-type mice (Fig.
6E). Moreover, the knockout mice exhibited no significant
increases in withdrawal frequency in response to moderate-intensity mechanical
stimulation after spinal nerve injury (Fig.
6F). Withdrawal frequencies on the contralateral side
essentially were unchanged after CFA injection and after nerve injury (data
not shown).
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Figure 6. Role of PSD-93 in pain behavioral responses. A, Baseline response
frequencies of paw withdrawal to mechanical stimulation that were elicited by
various forces of von Frey monofilaments in intact mice. Withdrawal
frequencies were similar in wild-type (+/+), heterozygous (+/-), and knock-out
(-/-) mice (n = 15 for each group). B, Baseline withdrawal
latency in response to thermal stimulation that was elicited by high-intensity
radiant heat applied to the plantar sides of left and right hind paws in
intact mice. Thermal thresholds of knock-out mice were not different from
those of wild-type or heterozygous mice (n = 12 for each group).
C, D, Effect of PSD-93 deletion on CFA-induced inflammatory pain. CFA
produced a significant increase in paw withdrawal frequencies in response to
0.24 mN (low intensity) and 4.33 mN (moderate intensity) mechanical stimuli on
the injured side in wild-type (WT) mice (repeated measures ANOVA, p
< 0.001; n = 5), but not in knock-out (KO) mice (p >
0.05; n = 5). Asterisks indicate a significant difference on the
injured side between wild-type and knock-out mice (*p <
0.05 and **p < 0.01, Bonferroni post-test). E,
F, Effect of PSD-93 deletion on neuropathic pain. In wild-type mice (WT)
the nerve injury produced a significant increase in paw withdrawal frequencies
to 0.24 and 4.33 mN mechanical stimuli on the injured side (p <
0.0001; n = 14 for each group). This increase was significantly
different from the paw withdrawal frequencies in the PSD-93 knock-out (KO)
mice. Asterisks indicate a significant difference on the injured side between
wild-type and knock-out mice (*p < 0.05 and
**p < 0.01). The knock-out mice also exhibited a modest
but significant mechanical hypersensitivity in response to low-intensity
mechanical stimulation on days 8, 10, and 14 after surgery (E;
p < 0.01), but not to moderate-intensity mechanical stimulation
(F; p > 0.05).
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Consistent with the previous studies
(Ren et al., 1992;
Mao et al., 1993;
Chaplan et al., 1997;
Jasmin et al., 1998),
intrathecal NMDA receptor antagonist MK-801 significantly attenuated CFA-or
nerve injury-induced mechanical hyperalgesia on the ipsilateral side, without
detectable effect on the behavioral responses on the contralateral side, in
wild-type mice. On day 3, when CFA-induced mechanical hyperalgesia was near
peak severity, MK-801 injection (1.0 nmol) reduced the
ipsilateral/contralateral differences in paw withdrawal frequencies to 24.4
± 2.7% (in response to 0.24 mN) and 27.5 ± 6.1% (in response to
4.33 mN) of predrug differences (n = 5; p < 0.05).
Similarly, the ipsilateral/contralateral differences in paw withdrawal
frequencies on postnerve injury day 6 (when mechanical hyperalgesia reached
the peak) were reduced by intrathecal administration of MK-801 32.3 ±
1.5% (in response to 0.24 mN) and 18.5 ± 3.6% (in response to 4.33 mN)
of predrug differences (n = 6; p < 0.05). In contrast,
paw withdrawal responses of PSD-93 knock-out mice (which exhibited blunted
responses to mechanical stimuli as described above) were unchanged by
intrathecal administration of MK-801. The equivalent values were 95.2 ±
2.3% (0.24 mN) and 91.8 ± 4.1% (4.33 mN) for the CFA model (n
= 5; p > 0.05) and 93.4 ± 4.2% (0.24 mN) and 90.7 ±
3.2% (4.33 mN) for the nerve injury model (n = 6; p >
0.05 in each case), respectively. Taken together with the observations above,
these results indicate that the deficiency of PSD-93 reduces NMDAR-dependent
persistent pain.
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Discussion
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The current work provides strong genetic evidence that the deficiency of
PSD-93 protein reduces NMDAR synaptic function and associated persistent pain
by the mechanism of surface NMDAR alteration. PSD-93 seems to be similar to
another PDZ domain protein, Drosophila disc-large protein (DLG), the
mutants of which altered localization and function of Shaker type
K+ channels (Tejedor et al.,
1997). Our study is the first identification of an interacting
protein required for normal synaptic NMDAR function and its dependent pain
signaling in the vertebrate CNS. PSD-93 may play an important role in coupling
NMDAR activation to central pain signaling.
Under normal conditions NMDARs are expressed stably in the postsynaptic
membrane (Luscher et al.,
1999; Ehlers,
2000; Lin et al.,
2000). However, recent experimental evidence demonstrates that the
surface expression of NMDARs is regulated tightly. For example, long-term
potentiation leads to rapid surface expression of NMDARs
(Grosshans et al., 2002),
whereas metabotropic glutamate receptor-stimulated long-term depression
produces rapid internalization, that is, loss of NMDARs from synapses
(Snyder et al., 2001).
Furthermore, the internalization of NMDARs is determined by the C terminus of
NR2B (Roche et al., 2001).
PSD-95, which binds to the C-terminal binding motif of NR2, inhibits
NR2B-mediated internalization, and the deletion of the PDZ-binding domain of
NR2B increases internalization in neurons
(Roche et al., 2001). We found
that genetic deletion of PSD-93 resulted in the reduction of surface NR2A and
NR2B expression in spinal dorsal horn neurons. These results indicate that
surface expression of NMDARs (at least NR2A and NR2B subunits) could be
regulated by the C-terminal motif-binding PDZ proteins.
The change of NMDAR expression on synaptic membranes influences
NMDAR-mediated synaptic function and associated pain signaling. Transgenic
mice that overexpress NR2B on central neurons displayed not only enhanced
NMDAR-mediated synaptic response but also enhanced pain responsiveness to
peripheral inflammation (Wei et al.,
2001). In vitro coexpression of PSD-95 protein with NMDA
receptors increased surface expression and channel-opening rates of NMDA
receptors (Carroll and Zukin,
2002). Our study demonstrated that targeted disruption of the
PSD-93 gene reduced not only surface NR2A and NR2B expression but also
NMDAR-mediated EPSCs in spinal dorsal horn neurons. Moreover, PSD-93 knock-out
mice exhibited blunted NMDAR-mediated excitatory postsynaptic responses and
dependent persistent pain. Interestingly, the mice with mutant PSD-95 protein
displayed normal synaptic NMDAR expression and NMDAR-mediated EPSCs in
hippocampal neurons, although they showed altered long-term potentiation and
impaired learning (Migaud et al.,
1998). The reason for the discrepancy between these two transgenic
mice is unclear but may be related to the targeted sites of PDZ domains. The
coding sequence of the second PDZ domain of PSD-93 is deleted completely in
PSD-93 knock-out mice (McGee et al.,
2001), whereas PSD-95 transgenic mice carry a targeted mutation in
the PSD-95 gene that leaves the first two PDZ domains intact by introducing a
stop codon into the third PDZ domain and replacing downstream sequences with
an internal ribosome entry site (Migaud et
al., 1998). The second PDZ domain of PSD-95 and PSD-93 is critical
for binding and anchoring of NMDARs at synaptic membrane. The deletion of the
second PDZ domain of PSD-95 or PSD-93 not only disrupts interaction between
NMDARs and PSD-95 or PSD-93 but also reduces NMDAR clustering at cellular
membranes (Kornau et al.,
1995; Brenman et al.,
1996; Kim et al.,
1996). That the first two PDZ domains were not detected in
synaptosome subfractions of PSD-95 mutant mice might be related to the
specificity of the antibody, because this antibody also did not detect the
first two PDZ domains of full-length PSD-95 in synapto some subfractions of
wild-type mice (Migaud et al.,
1998). Thus with the use of a suitable antibody the first two PDZ
domains may be detected in synaptosome subfractions of PSD-95 mutant mice.
These two PDZ domains may be involved in maintaining normal synaptic
localization and postsynaptic function of NMDARs in PSD-95 mutant mice. We
also found that PSD-93 deletion did not affect significantly the surface AMPA
receptor expression or AMPA receptor-mediated postsynaptic function in the
neurons of the spinal cord and forebrain cortex. This result is inconsistent
with recent in vitro studies in which the expressional alteration or
palmitoylation of the MAGUK proteins like PSD-95 regulates surface and
synaptic AMPA receptor-mediated trafficking via stargazin, an AMPA
receptor-interacting protein (Chen et al.,
2000; El-Husseini et al.,
2000,
2002;
El-Husseini and Bredt, 2002;
Schnell et al., 2002). It has
been noted that synaptic targeting/insertion and synaptic stabilization of
AMPA receptors may be mediated by several mechanisms. The delivery of AMPA
receptors to synapses also is related to other clustering proteins such as
GRIP/ABP or PICK1, which are important for synaptic targeting or synaptic
surface accumulation of AMPA receptors
(Sheng and Pak, 1999;
Braithwaite et al., 2000;
Garner et al., 2000;
Osten et al., 2000;
Barry and Ziff, 2002). We found
that PSD-93 knock-out mice displayed normal GRIP/ABP and PICK1 expression in
the spinal cord and forebrain (data not shown). It is very likely that these
clustering proteins maintain normal surface AMPA receptor expression and its
associated function in PSD-93 transgenic mice. This view is supported by other
studies (Migaud et al., 1998;
Hashimoto et al., 1999)
showing that surface and synaptic AMPA receptor trafficking is intact in
hippocampal neurons of PSD-95 or stargazin mutant mice.
PSD-93 is homologous to three other neuronal MAGUKs, PSD-95, SAP102, and
SAP97 (Kornau et al., 1997;
Craven and Bredt, 1998;
Garner et al., 2000), but
PSD-93 has some unique characteristics. For example, both PSD-93 and PSD-95
are palmitoylated N-terminally, but, unlike PSD-95, palmitoylation is not
necessary for the PSD-93 postsynaptic targeting
(Firestein et al., 2000). In
some regions of the CNS the expression and distribution of the four neuronal
MAGUK proteins are different. In cerebellar Purkinje neurons only PSD-93 is
expressed (McGee et al.,
2001). We have reported that PSD-95 and SAP102 also are expressed
in the spinal cord but are distributed mainly in lamina I and outer lamina II
(Tao et al., 2000). SAP97
protein was undetectable in the PSD fraction of the spinal cord (data not
shown). Compared with PSD-95 and SAP102, PSD-93 is expressed uniquely in inner
lamina II of the spinal cord. It is well known that the postsynaptic neurons
in inner lamina II differ considerably from those in lamina I and outer lamina
II with respect to forming synaptic architecture with primary afferent
terminals (Hunt et al., 1992;
Chen et al., 1995). These
results suggest that PSD-93, at least in the spinal dorsal horn, plays a
distinct role in targeting of NMDARs to the synapses, which may not be
compensated for completely by other related neuronal MAGUK proteins.
Our study implicates a molecular mechanism by which alteration of the
expression of NMDAR-interacting proteins could modulate NMDAR-mediated
synaptic function and behavioral responses. This might provide a potential
novel biochemical target for the prevention and therapy of persistent
pain.
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Footnotes
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Received Mar. 4, 2003;
revised Jun. 4, 2003;
accepted Jun. 10, 2003.
This work was supported by the Blaustein Pain Research Fund (Y.-X.T.),
National Institutes of Health Grants R01 GM 49111 and NS 44219 (R.A.J.) and
NS360017 (D.S.B.), Howard Hughes Medical Institute (R.L.H.), National
Institute on Drug Abuse Grant NIDA10833 (M.Z.), National Institute of
Neurological Disorders and Stroke Grant NINDS38680(M.Z.), and the Christopher
Reeves Paralysis Foundation (D.S.B.). We thank Dr. R. J. O'Brien, C. F.
Levine, Dr. Y.-X. Wang, and Dr. P. Mao for technical assistance and Dr. R. A.
Meyer for helpful discussion.
Correspondence should be addressed to Yuan-Xiang Tao, Department of
Anesthesiology and Critical Care Medicine, Johns Hopkins University School of
Medicine, 355 Ross, 720 Rutland Avenue, Baltimore, MD 21205. E-mail:
ytau{at}jhmi.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236703-10$15.00/0
* Y.-X.T., G.R., and G.-D.W. contributed equally to this work. 
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Abstract
Introduction
