Group Title: Molecular Pain 2005, 1:25
Title: Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation
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Title: Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation
Series Title: Molecular Pain 2005, 1:25
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Creator: Caudle RM
Perez FM
Del Valle-Pinero AY
Iadarola MJ
Publication Date: 38597
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Molecular Pain Bioed Central


Research


Spinal cord NRI serine phosphorylation and NR2B subunit
suppression following peripheral inflammation
Robert M Caudle*1,2, Federico M Perez', Arseima Y Del Valle-Pinero2 and
Michael J Iadarola3


Address: 'Department of Oral and Maxillofacial Surgery and Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610,
USA, 2Department of Neuroscience, University of Florida College of Medicine, McKnight Brain Institute, Gainesville, FL 32610, USA and 3Pain
and Neurosensory Mechanisms Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD
20892, USA
Email: Robert M Caudle* caudle@ufl.edu; Federico M Perez fperez@dental.ufl.edu; Arseima Y Del Valle-Pinero delvalle@ufl.edu;
Michael J Iadarola miadarola@dir.nidcr.nih.gov
* Corresponding author



Published: 02 September 2005 Received: 22 July 2005
Molecular Pain 2005, 1:25 doi: 10.1 186/1744-8069-1-25 Accepted: 02 September 2005
This article is available from: http://www.molecularpain.com/content/1/1/25
2005 Caudle et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Spinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the
development and maintenance of central sensitization. However, the mechanisms mediating the
altered function of the NMDA receptors are not well understood. In this study the role of
phosphorylation of NRI splice variants and NR2 subunits was examined following hind paw
inflammation in rats. We further examined the level of expression of these proteins following the
injury.
Results: Lumbar spinal cord NRI subunits were found to be phosphorylated on serine residues
within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NRI
serine phosphorylation reversed within six hours. No phosphorylation on NRI threonine or
tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on
serine, threonine or tyrosine residues. An analysis of NRI and NR2 protein expression
demonstrated no change in the levels of NRI splice variants or NR2A following the inflammation.
However, spinal cord NR2B expression was depressed by the hind paw inflammation. The
expression of NR2B remained depressed for more than one week following initiation of the
inflammation.
Conclusion: These data suggest that NRI serine phosphorylation leads to an initial increase in
NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B
expression suggests compensation for the enhanced nociceptive activity. These data indicate that
spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery
from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA
receptors should be designed for the exact configuration of NMDA receptor subunits and post-
translational modifications present during specific stages of the disease.






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Background
Central sensitization is a form of plasticity in the spinal
cord that alters the input/output relationship of the neu-
ronal pain processing circuitry. Central sensitization is
symptomatically expressed as allodynia, pain to normally
non-painful stimuli, and hyperalgesia, an enhanced sen-
sation of pain to typically painful stimuli. When an indi-
vidual is injured central sensitization encourages the
protection of the injured area by enhancing the pain expe-
rience. The individual is then motivated to guard the dam-
aged tissue until it is healed. As a rule, central sensitization
will be reversed as the injury heals. However, on occasion
it fails to resolve and becomes the patient's primary dis-
ease. This disease is referred to as chronic pain. Thus, the
molecular processes that induce and reverse central sensi-
tization are important to understanding, preventing and
treating chronic pain.

Recent work on pain processing has highlighted the cen-
tral role of N-methyl-D-aspartate (NMDA) receptors in
central sensitization. NMDA receptors were found to play
a major role in hyperalgesia, allodynia, and expanded
receptive fields when central sensitization had been
induced by peripheral injury [1-5]. These findings using
NMDA receptor antagonists indicated that NMDA recep-
tors initiated events that lead to neuronal plasticity in the
spinal cord and that the NMDA receptors themselves par-
ticipated in the maintenance of central sensitization. Cen-
tral sensitization is the result of an increase in intracellular
calcium, which enhances synaptic inputs from primary
nociceptors. NMDA receptors conduct much of this cal-
cium from the extracellular space through their iono-
phore. The net effect of the increased calcium is an
increased number of effective synapses on dorsal horn
neurons and enhanced neuronal excitability [1,6,7].

Central sensitization, it must be noted, is distinct from the
frequently studied phenomenon of windup, which is rap-
idly reversed when the peripheral stimulus ceases. Win-
dup is produced by the well documented voltage
dependent magnesium block of the NMDA receptor's ion
channel. The magnesium block enables the receptor to
integrate nociceptive signals that arrive in the spinal cord
via C-fibers. The net result of the integration is that the
later stimuli in a series produces greater responses in dor-
sal horn neurons even when the stimuli are identical to
the first event [8-10]. Windup does not lead to a pro-
longed enhancement of dorsal horn neuronal excitability
like central sensitization, but may induce central sensitiza-
tion by increasing intracellular calcium levels. Thus,
although NMDA receptors are involved in both central
sensitization and windup their role in the two processes is
distinct [10].


Recently, Zou and colleagues examined the role of NMDA
receptor subunit phosphorylation in the development of
central sensitization [11]. These investigators found that
capsaicin injection into the hind paw of rats resulted in an
ipsilateral accumulation of phosphorylated NR1 subunits
in spinothalamic tract neurons. Zou detected the phos-
phorylation using an antibody that was selective for phos-
phorylated serine 897 on the NR1 subunit.
Phosphorylation of serine 897 on NR1 results in the accu-
mulation of NMDA receptors in synapses [12]. Zou et al.
further demonstrated that PKA mediated the phosphor-
ylation of serine 897 and that the enhanced activity of spi-
nothalamic tract neurons was sensitive to PKA
inhibitors[13]. Similarly, Brenner and colleagues demon-
strated that noxious heat applied to the hind paw of rats
produces an increase in serine 896 phosphorylation of
NR1 [14]. This phosphorylation was demonstrated to be
mediated by activation of protein kinase C (PKC). Previ-
ously, it was demonstrated that PKC phosphorylation of
serine 896 acts in concert with PKA phosphorylation of
serine 897 to induce membrane insertion of NMDA recep-
tors [15]. The findings from these two groups indicate that
a relatively brief nociceptive stimulus in the periphery
might induce the migration of new NMDA receptors to
synapses between the primary and secondary nociceptive
afferents. Presumably, the newly inserted NMDA recep-
tors participate in enhancing synaptic activity and in
inducing central sensitization. In contrast to the NR1 ser-
ine phosphorylation it was notable that these short dura-
tion stimuli did not induce any detectable change in NR1
protein expression.

In an excitotoxity induced spinal cord injury model, we
examined NR1 phosphorylation and found that an
increase in NR1 serine phosphorylation was associated
with spontaneous behaviors that indicate the expression
of hyperalgesia and allodynia [16]. We further found that,
in contrast to the short term peripheral nociception mod-
els, NR1 protein was up regulated in the spinal cord fol-
lowing the excitotoxic injury. These data suggest that
sustained injuries produce not only changes in NMDA
receptor phosphorylation, but also changes in protein
expression. The changes in NMDA receptor protein could
lead to a more extended period of central sensitization
than the readily reversed NMDA receptor
phosphorylation.

In addition to NR1 subunit phosphorylation, it was
recently demonstrated that NR2B subunits in the spinal
cord were phosphorylated on tyrosine residues following
either hind paw inflammation with Complete Freund's
Adjuvant (CFA) or mustard oil [17]. The NR2B phospho-
rylation was blocked by inhibitors of Src type kinases.
Interestingly, NR2A was reported not to be phosphor-
ylated following the CFA injury. In addition, these


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NR1 splice variant
0 0 0
o o 5 o -
exon 5 P


exon 21 -


+ exon 22 -.


- exon 22'


L E
E a)
vo E
a


- .. 121kD
-81kD


Figure I
Selectivity of rabbit polyclonal antibodies raised against splice
variants of the NRI subunit of the NMDA receptor. Anti-
bodies were raised against synthetic peptides and used for
western blots. The peptide sequences used to raise the anti-
bodies were SKKRNYENLDQLSYDNKRGPC, DRKS-
GRAEPDPKKKATFRAC, PRRAIEREEGQLQLC, and
QYHPTDITGPLNLSDPS for exons 5 (NI), 21 (C ), 22 (C2)
and 22 minus (C2') respectively. The antibodies were purified
with affinity columns and tested against cloned splice variants
of the NR I subunit transfected into COS-7 cells. The three
numbers at the top of the columns identify the splice variant
clones. The first number indicates the presence (I) or
absence (0) of N I, the second CI and the third C2. The pro-
tein was compared to protein taken from non-transfected
cells and rat brain homogenates.





investigators reported no change in the expression of NR2
protein in the spinal cord following the CFA treatment.

Overall, the NMDA receptor phosphorylation studies sug-
gest that phosphorylation of spinal cord NR1 and NR2B
subunits leads to enhanced NMDA receptor function and
central sensitization. However, it is unclear if extended
periods of central sensitization are maintained by NMDA
receptor phosphorylation or if changes in NMDA receptor
subunit protein expression contribute to the continuance
of the sensitized state as was observed in the spinal cord
injury model [16]. In the present study we examined
phosphorylation on serine, threonine and tyrosine resi-
dues, as well as protein expression, in both NR1 and NR2
subunits following carrageenan induced hind paw inflam-
mation. The goal was to determine if there is a transition
from NMDA receptor subunit phosphorylation to
changes in NMDA receptor subunit protein expression


during an extended period of nociceptive input and the
relationship of these changes to central sensitization.

Results
Characterization of NRI Splice Variant Selective
Antibodies
Four antibodies were raised in rabbits against four regions
of the NR1 subunit for identifying specific splice variants
containing the N1, C1, C2 and C2' cassettes [18]. NR1
splice variant clones were transfected into COS-7 cells and
the protein was harvested and run on western blots. The
western blots were probed with the antibodies. As demon-
strated in figure 1 the western blots demonstrated that the
antibodies were highly selective for the appropriate NR1
splice variants.

Time Course of Carrageenan Induced Enhancement of
Nociception
Sixteen rats received injections of 6 mg of carrageenan
into the left hind paw and another sixteen rats received
vehicle injections. Eight of the carrageenan rats and eight
of the vehicle treated rats were then used for thermal noci-
ceptive testing and the remaining animals were used for
the mechanical nociception testing. The time course of the
thermal testing demonstrated that the limb withdrawal
latency was reduced by carrageenan injection within half
an hour (Figure 2A). This reduced withdrawal latency
reached a peak by 2 to 6 hours and recovery to baseline
levels was achieved within 24 hours (ANOVA, Dunnett's
test p < 0.05). Although there was a trend for greater ther-
mal hyperalgesia at 6 hours when compared to the 2 hour
time point this difference did not reach significance
(paired ttest, p = 0.12). In contrast the non-injected paws
demonstrated no significant changes in withdrawal
latency. No change in paw withdrawal latency was
observed in the vehicle injected animals (data not
shown).

In the mechanical allodynia assay the carrageenan
injected paws demonstrated significantly enhanced sensi-
tivity to mechanical stimuli within half an hour. The
enhanced sensitivity reached a peak within 6 hours. How-
ever, in contrast to the thermal assay the rats did not com-
pletely return to baseline levels for at least 2 days, but were
fully recovered by 5 days (Figure 2B). The non-injected
paws and vehicle treated animals (data not shown) dem-
onstrated no significant change in mechanical sensitivity
during the entire time course.

NRI Phosphorylation
To evaluate NR1 phosphorylation rats were injected with
either vehicle or 6 mg of carrageenan into the left hind
paw. The ipsilateral lumbar spinal cord tissue was har-
vested and NR1 subunits were precipitated with selective
antibodies for the N1 or C1 cassettes [18] or an antibody


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0 0.5 2 6 24 48 120
Time (Hrs)


SInflamed
Ol Non-Inflamed


tified by the anti-phospho antibodies, which was consist-
ent with NR1 [14].

Carrageenan injected into the left hind paw induced a sig-
nificant increase in serine phosphorylation of NR1 pro-
tein precipitated with the globally reactive NR1 antibody
(N = 6 rats/time point) and with protein precipitated with
the C1 cassette selective antibody (N = 8 rats/time point)
within 2 hours of the carrageenan injection (ANOVA fol-
lowed by Dunnett's test on raw data, p < 0.05) (Figure
3A). The phosphorylation of serine residues begins to
resolve within 6 hours of the treatment. Interestingly, NR1
protein precipitated with N1 cassette selective antibodies
did not demonstrate an increase in serine phosphoryla-
tion in response to carrageenan treatment (N = 8 rats per
time point, ANOVA followed by Dunnett's test on raw
data, p > 0.05).

To determine if the changes in phosphorylation were in
the dorsal horn the isolated spinal cord segments of 8 car-
rageenan injected rats, 8 saline injected rats and 8 non-
injected rats were further divided into dorsal and ventral
halves and analyzed for serine phosphorylation. Two
hours following the carrageenan injections the animals
demonstrated an increase in serine phosphorylation in
the dorsal horn, but not in the ventral horn (Figure 3B)
(ANOVA followed by Dunnett's test on raw data, p <
0.05).


I Phosphothreonine and phosphotyrosine antibodies dem-
IR I II I I I I I onstrated no changes in phosphorylation of C1 precipi-
0 0.5 2 6 24 48 120 tated NR1 (N = 9 rats/time point) or total NR1 protein (N
= 8 rats/time point) (Figure 4A and 4B) (ANOVA followed
Time (Hrs) by Dunnett's test on raw data, p < 0.05). The western blots


Figure 2
Time course of carrageenan induced inflammation on heat
hyperalgesia and mechanical allodynia. A. Paw withdrawal
latencies from a thermal stimulus before and following injec-
tion of 6 mg carrageenan into the plantar surface of the left
hind paw. Data are means SEM (N = 8 rats). Asterisks indi-
cate p < 0.05 ANOVA followed by Dunnett's test. B.
Thresholds for paw withdrawal from a mechanical stimulus
before and following the injection of 6 mg carrageenan into
the plantar surface of the left hind paw. Data are means
SEM (N = 8 rats). Asterisks indicate p < 0.05 ANOVA fol-
lowed by Dunnett's test.





that recognizes all 8 of the NR1 splice variants. The splice
variant selective antibodies were raised in house (Figure
1). The protein was then run on western blots and probed
with anti-phosphoserine, anti-phosphothreonine or anti-
phosphotyrosine antibodies. A band at ~118 kd was iden-


pluouc wul ule aiLu-Lp ospi oiulu os atuuou es wler
extremely faint (see Figure 4B) indicating that NR1 is not
phosphorylated on tyrosine residues to any significant
degree.

NR2 Phosphorylation
The NR2 subunits NR2A and NR2B were immunoprecip-
itated from rat spinal cord extracts as described in the
methods section, run on western blots and probed with
anti-phosphoserine, anti-phosphothreonine or anti-
phosphotyrosine antibodies. The anti-phospho antibod-
ies recognized proteins on the western blots with a molec-
ular weight of approximately 180 kd, which is consistent
with NR2A and NR2B [17].

An examination of phosphorylation of NR2A (N = 5 rats/
time point) and NR2B (N = 9 rats/time point)
demonstrated that there was no significant change in
phosphorylation on serine residues (Figure 5A) (ANOVA
on raw data, p > 0.05) after carrageenan treatment. Simi-
larly, no change in threonine or tyrosine phosphorylation


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2.5
2.. TotalNR1
*7 2 E( C1 Cassette
ScN1 Cassette
~1.5

S1 i

0.5

Co ntrol 2 hrs 6 hrs 24 hrs
Control 2 hrs 6 hrs 24 hrs


B




300

250

S200

150

S100

o 50

0


cY o
an dn am


SVentral Horn
SDorsal Horn


Control Saline Carrageenan


Figure 3
Serine phosphorylation of spinal cord NR I subunits following
carrageenan induced hind paw inflammation. A. Time course
of serine phosphorylation on NRI subunits immunoprecipi-
tated by antibodies that recognized all NRI subunits, N I
containing splice variants or CI containing splice variants.
The western blots at the top are representative experiments
where the immunoprecipitated protein was probed with
anti-phosphoserine antibodies. The bands migrated at
approximately I 18 kd. The graph summarizes the band den-
sity data from the western blots. Data are means SEM of
the normalized band density data (NRI: N = 6 rats per time
point, N I: N = 5 rats per time point, C I: N = 6 rats per time
point). Asterisks indicate p < 0.05 ANOVA followed by Dun-
nett's test on the raw data. B. Comparison of serine phos-
phorylation on NRI subunits from the dorsal and ventral
spinal cord four hours following the injection of either saline
or carrageenan (6 mg) into the left hind paws. At the top is a
representative western blot demonstrating the immunopre-
cipitated NRI after it was probed with anti-phosphoserine
antibodies. The graph is a summary of the normalized band
density data for control animals (N = 5), saline injected ani-
mals (N = 5) and carrageenan injected animals (N = 5). Data
are means SEM. Asterisks indicate p < 0.05 ANOVA fol-
lowed by Dunnett's test.


was detected on either NR2A (N = 6 rats/time point) or
NR2B (N = 5 rats/time point) (Figure 5B and 5C)
(ANOVA on raw data, p > 0.05).

NRI Protein
In western blots of spinal cord extracts the NR1 proteins
were probed with antibodies selective for the N1 (N = 8
rats/time point), C1 (N = 8 rats/time point), C2 (N = 4
rats/time point) or C2' (N = 4 rats/time point) cassettes
and all NR1 splice variants (N = 6 rats/time point).
Extracts were prepared from animals without carrageenan
injections, and 2, 6 and 24 hours following carrageenan
injection into the hind paw. All of the antibodies recog-
nized single bands on the westerns with a molecular
weight of approximately 118 kd. Lane loading was moni-
tored by probing for actin (data not shown). No change in
spinal cord NR1 protein or splice variant expression was
found following carrageenan inflammation (Figure 6)
(ANOVA on the raw data, p > 0.05).

NR2 Protein
To examine changes in NR2 protein spinal cord extracts
were collected at 2, 6, 24, 48 and 120 hours following car-
rageenan injections and probed on western blots with
antibodies to NR2A (N = 4 rats/time point) or NR2B (N =
4 rats/time point). Lane loading was monitored with anti-
bodies to actin. The western blots for NR2A indicated no
significant change in the expression of protein (ANOVA
on raw data, p > 0.05) (Figure 7). However, there was a
significant decrease in the expression of NR2B that was
observed 24 hours following the carrageenan injection
(ANOVA, Dunnett's test on raw data, p < 0.05). This
decrease in NR2B lasted throughout the remainder of the
week.

Discussion
The involvement of spinal cord N-methyl-D-aspartate
(NMDA) receptors in hyperalgesia and allodynia is well
documented in the literature [2-5,19-21]. However, the
mechanisms by which the receptors mediate the develop-
ment and maintenance of allodynia and hyperalgesia in a
chronic pain state are not well understood. Long lasting
changes in the NMDA receptors either through post-trans-
lational modifications or expression of different subunits
may lead to a chronic enhancement of nociception. In this
study we used a relatively long lasting nociceptive stimu-
lus to evaluate NR1 and NR2 subunit phosphorylation
and protein expression. We found that spinal cord NR1
subunits possessing the C1 cassette are phosphorylated
for a few hours following carrageenan injection (Figures 2
and 3). The NR1 phosphorylation was found only in the
dorsal horn indicating that the phosphorylation was asso-
ciated with nociception. Furthermore, the NR1 serine
phosphorylation paralleled thermal hyperalgesia with
recovery within 24 hours. The serine phosphorylation


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NR1 Threonine Phosphorylation C1 Threonine Phosphorylation
I$I Ia&J j 4 IH4

Ci C,^ ^ ^^B .


B.
NR1 Tyrosine Phosphorylation

NS ??
,&* .vir ,


C1 Tyrosine Phosphorylation


S.. .. ,.


a 2

% 1.5




| 0.5

0


Control 2 hrs 6 hrs 24 hrs


2 C1 Cassette
iC BTotal NR1

! 1.5




S0.5
a


Control 2 hrs


6 hrs 24 hrs


Figure 4
Phosphorylation of threonine and tyrosine residues on spinal cord NRI subunits. No phosphorylation of NRI threonine or
tyrosine residues was found in response to peripheral inflammation. A. Lumbar spinal cord NRI subunits were immunoprecip-
itated with an antibody that recognizes all NRI splice variants or an antibody that recognizes splice variants with the Cl cas-
sette before and after left hind paw inflammation with carrageenan. The protein was run on western blots and probed with an
anti-phosphothreonine antibody. The western blots at the top are representative experiments and the graph at the bottom
presents the mean SEM band density of all experiments (Total NRI; N = 8 rats per time point, C cassette; N = 9 rats per
time point). ANOVA p > 0.05. The molecular weight of the bands on the western blots was 118 kd. B. NRI subunit protein
was immunoprecipitated with an antibody that recognizes all NRI splice variants or an antibody that recognizes splice variants
with the C I cassette, run on western blots and probed with an anti-phosphotyrosine antibody. The western blots at the top
are representative experiments and the graph at the bottom presents the mean SEM band density of all experiments (Total
NRI; N = 4 rats per time point, C I cassette; N = 4 rats per time point). ANOVA p > 0.05.


peaked at 2 hours following the carrageenan injections
while the thermal hyperalgesia peaked in the range of 2 to
6 hours. This finding suggests that the NR1 serine phos-
phorylation may be a contributing factor to the thermal
hyperalgesia.

The inflammation did not produce phosphorylation of
series on NR2 subunits or phosphorylation of threonine
and tyrosine residues on either NR1 or NR2 subunits. The
NR1 phosphorylation data are consistent with the shorter
duration stimuli used by Zou et al. [11,13] and Brenner et
al. [14], and with our previous spinal cord injury study
[16]. However, we did not find that NR2B subunits were
phosphorylated on tyrosine residues as was reported by
Guo et al. [17]. The reason for this discrepancy is not
known, but it could be due to the differences in inflamma-
tion protocols. We used carrageenan to inflame the hind
paws while Guo et al. used Complete Freund's Adjuvant
(CFA) to induce inflammation.


In a previous study, Prybylowski and colleagues demon-
strated that NR1 subunits containing the C1 cassette in
the spinal cord of rats represented only about 5 percent of
the total NR1 protein [22], which suggests that C1 con-
taining NR1 subunits may not play a significant role in the
spinal cord. However, the group did not determine
whether these NR1 subunits were localized to any specific
area of the cord such as the dorsal horn or whether they
were distributed throughout the lamina. It is possible that
the C1 containing NR1 subunits are localized primarily in
the superficial lamina, thus enhancing their abundance
within neurons in the nociceptive pathways. From the
work of Brenner et al. [14] and Zou et al. [13] it is clear
that NR1 subunits are readily phosphorylated on series
890, 896 and 897 by serine/threonine kinases during
nociception, which is consistent with our findings. These
serine residues reside in the C1 cassette indicating that
NR1 subunits containing Cl are critical to nociceptive
processing in the spinal cord. Interestingly, Prybylowski et


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A.


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A B,
NR2A Serine Phosphorylation NR2B Serine Phosphorylation

Vic, f ^n


SNR2A
0 NR2B


S2


" 1.5


S1
N
.s

g 0.5


0


NR2A Threonine Phosphorylation NR2B Threonine Phosphorylation




2.5


* NR2A
SNR2B


S 2


1.5


1


' 0.5
Z


Control 2 hrs 6 hrs 24 hrs


Control 2 hrs 6 hrs 24 hrs


NR2A Tyrosine Phosphorylation NR2B Tyrosine Phosphorylation





2.5


2 NR2A
U NR2B

S1.5





, 0.5


0
ca T


Control 2 hrs


6 hrs 24 hrs


Figure 5
Phosphorylation of NR2A and NR2B subunits. NR2A and NR2B were immunoprecipitated from rat lumbar spinal before and
after carrageenan induced inflammation of the left hind paw. The protein was run on western blots and probed with anti-phos-
phoserine, anti-phosphothreonine or anti-phosphotyrosine antibodies. Single bands were identified on the western blots that
migrated at -180 kd. A. Phosphoserine on NR2A and NR2B subunits. The western blots at the top demonstrate representa-
tive experiments and the graph below presents the mean SEM band density for all experiments (NR2A N = 4 rats per time
point, NR2B N = 4 rats per time point). ANOVA p > 0.05. B. Phosphothreonine on NR2A and NR2B subunits. The western
blots at the top demonstrate representative experiments and the graph below presents the mean SEM band density for all
experiments (NR2A N = 4 rats per time point, NR2B N = 4 rats per time point). ANOVA p > 0.05. C. Phosphotyrosine on
NR2A and NR2B subunits. The western blots at the top demonstrate representative experiments and the graph below
presents the mean SEM band density for all experiments (NR2A N = 4 rats per time point, NR2B N = 4 rats per time point).
ANOVA p > 0.05.




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NR1 Protein


C1 Protein C2 Protein
m a -a MW M- ~ -
N1 Protein C2' Protein
m m 0 -a

C s5I0 Coo,


2

1.5


1
.N

S0.5
Z


* NR1
EC1


NR2B q.- IA% -18Okd


Actin


~43kd


1.2

1

M 0.8

0.6

S0.4

0.2

0


Control 2 hrs 6 hrs 24 hrs


Figure 6
Peripheral inflammation does not influence NRI protein
expression in the lumbar spinal cord. Spinal cords were har-
vested before and after carrageenan injection into the left
hind paw of rats, homogenized and run on western blots.
The western blots were probed with antibodies that recog-
nized all NRI splice variants (N = 6 rats per time point),
splice variants containing the N I cassette (N = 8 rats per
time point), splice variants with the Cl cassette (N = 8 rats
per time point), splice variants with the C2 cassette (N = 4
rats per time point) and splice variants with the C2' cassette
(N = 4 rats per time point). The western blots at the top are
representative experiments and the graph a summary of all
experiments. Bars are the mean SEM. ANOVA p > 0.05.







al. also demonstrated that the majority of the NR1 subu-
nits that contain N1 do not contain the C1 cassette [22].
This finding is consistent with our data demonstrating
that when N1 is used as the precipitating epitope we do
not find any injury induced phosphorylation of NR1 (Fig-
ure 3A) suggesting an absence of C1 in the precipitated
protein. The sum of the data therefore suggests that NR1
subunits containing the C1 cassette and lacking the N1
cassette represent a dynamic pool of NMDA receptors that
are rapidly recruited to the neuronal membrane following
a nociceptive stimulus.


Control 2hrs 6 hrs 24 hrs 48 hrs 120 hrs

Figure 7
Expression of spinal cord NR2A and NR2B protein following
peripheral inflammation. Rat lumbar spinal cords were har-
vested before and after carrageenan induced hind paw inflam-
mation. The tissue was homogenized and run on western
blots. The membranes were probed with antibodies that
were selective for either NR2A (N = 4 rats per time point)
or NR2B (N = 4 rats per time point). Single bands were iden-
tified at -180 kd. The western blots are representative for
NR2B and actin. Bars on the graph are means SEM of band
density data normalized to the control bands. Asterisks indi-
cate p < 0.05 ANOVA followed by Dunnett's test on the raw
band densities.





We further investigated the expression of NR1 splice vari-
ants, NR2A and NR2B protein. These data indicated no
significant changes in NR1 splice variants or NR2A pro-
tein (Figures 6 and 7). However, NR2B expression was
depressed by 24 hours following the carrageenan
inflammation and did not recover during the 5 day period
examined. The lack of change in NR1 subunits is consist-
ent with the findings ofZou et al. [11,13], Brenner et al.
[14] and Gaunitz et al. [23], but not with our spinal cord
injury study [16]. These findings suggest that NR1 protein
expression is not influenced by peripheral injuries, but is
altered by central nervous system injuries. NR2B protein
expression, in contrast, was suppressed by the peripheral
inflammation. Guo et al. [17] did not find a change in
NR2B protein following CFA induced peripheral


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40 We have not yet examined NR2C or NR2D to confirm this
hypothesis.

30 120 hrs Several investigators have demonstrated that pharmaco-
25 N logical agents that target NR2B subunits, such as Ifen-
S2 prodil, can be used to control pain [28-31]. The loss of
20 48hn NR2B subunits in the spinal cord as the nociceptive stim-
a 15 24hrs ulus progresses suggests that pharmacological agents tar-
10 o 6,rs getting NR2B may be less effective in chronic pain than in
\ acute pain. This idea is supported by the work of Nakazato
\ and colleagues and De Vry et al. who recently demon-
0 strated that intrathecally administered NR2B selective
0 0.2 0.4 0.6 0.8 1 agents are ineffective at blocking mechanical allodynia in
Normalized Band Density nerve constriction injury models [32,33]. These findings
contrast with data that demonstrate NR2B selective antag-
Figure 8 onists suppress allodynia in acute nociception models
Correlation between NR2B protein and recovery of mechan- [34] and that intrathecal siRNA knockdown of NR2B
ical allodynia following carrageenan induced hind paw inflam- formalin induced nociceptive behaviors [27
nation. Data from 6 hours to 120 hours from the NR2B suppresses formalin induced nociceptive behaviors [27].
mation. Data from 6 hours to 120 hours from the NR2B e R2
western blots (figure 7) and the mechanical allodynia data The efficacy of trathecal NR2B antagonists in acute
(figure 2) were plotted against each other. Deming linear models and the lack of effect in chronic injury models
regression indicates r2 = -0.93, p = 0.0363. support the concept that NR2B is down regulated in the
spinal cord as the injury progresses.

The temporal association of thermal hyperalgesia with
NR1 phosphorylation while recovery of mechanical allo-
dynia is correlated with the suppression of NR2B suggests
inflammation and Gaunitz et al. [23] found no change in that the two behavioral phenomena have distinct mecha-
NR2B mRNA 6 hours following peripheral formalin injec- nisms. Zou et al. and Brenner et al. have demonstrated
tion. However, Gaunitz et al. did find a modest increase in that NR1 phosphorylation is mediated by the serine/thre-
NR2A and NR2C mRNA. From our findings we may con- onine kinases protein kinase C (PKC) and protein kinase
elude that the NR2A mRNA increases may not translate A (PKA) [11,13,14]. Their findings suggest that phospho-
into increased levels of NR2A protein. We, however, did rylation of NR1 induces the migration of NMDA receptors
not examine NR2C protein, from the endoplasmic reticulum to synapses leading to
enhanced transmission of nociceptive information
The suppression ofNR2B following peripheral inflamma- [14,35,36]. It is possible that the phosphorylated
tion could have significant effects on nociception and the receptors remain in the synapses and remain functional
choice of treatments for chronic pain. NR2B subunits are following dephosphorylation. The dephosphorylation of
located primarily in laminas I and II of the dorsal horn the receptors may reduce stimulation of dorsal horn neu-
[24]. These subunits are involved in windup [25] and cen- rons produced by C-fibers transmitting thermal informa-
tral sensitization [26] suggesting a major role for NR2B tion. In contrast, the enhanced number of NMDA
subunits in the function of NMDA receptors mediating receptors remaining in synapses may still conduct a signif-
nociception. Furthermore, recent work by Tan and col- icant amount of information from A-delta fibers that
leagues [27] demonstrated that selective knockdown of transmit mechanical information. Only as the number of
NR2B in the dorsal horn using siRNA can suppress forma- NR2B subunits is reduced do the synapses return toward
lin induced nocifensive behaviors. Interestingly, our data normal function. Presumably, the loss of NR2B subunits
demonstrate a strong negative correlation between the would prevent the remaining NR1 subunits from forming
recovery period of mechanical allodynia and the level of functional receptors in much the same way as siRNA to
NR2B protein expression (Figure 8, Deming linear regres- NR2B was found to suppress nociceptive behaviors by
sion, r2 = -0.93, p = 0.0363). This might indicate that the selectively reducing the amount of NR2B protein [27].
suppression of NR2B is used to compensate for the Thus, thermal hyperalgesia may be mediated by the
enhanced nociceptive barrage. It is also possible; however, enhanced function of phosphorylated NMDA receptors,
that NR2B is replaced by either NR2C or NR2D during a while mechanical allodynia may simply require more
persistent nociceptive barrage. Thus, the NMDA receptors NMDA receptors in the synapse.
may still remain functional as the NR2B protein decreases.



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Alternatively, the phosphorylated NR1 subunits may be
inserted into C-fibers synapses while the NR2B subunits
are located primarily in synapses associated with A-delta
fibers. Thus, in the sensitized neurons suppression of the
NR1 phosphorylation results in reduction of thermal
hyperalgesia and a decrease in NR2B expression produces
an inhibition of mechanical allodynia. These hypotheses
are highly speculative at this time as the differences
observed between the two forms of stimuli may actually
be mediated by changes in the periphery rather than
centrally. However, it is intriguing to consider that ther-
mal hyperalgesia might be controlled via kinase inhibi-
tors, whereas established mechanical allodynia may
require selective NMDA receptor antagonists or suppres-
sion of NMDA receptor gene expression to be controlled.

In summary, our data indicate that peripheral inflamma-
tion with carrageenan results in a transient increase in
serine phosphorylation of spinal cord dorsal horn NR1
subunits and a long lasting decrease in the expression of
NR2B. These data indicate that NMDA receptors in the
spinal cord are highly dynamic and may represent a mov-
ing target for pharmacological control of chronic pain.

Methods
Male Sprague Dawley rats (200-300 g, Harlan Sprague
Dawley, Indianapolis, IN) were maintained on a 12 hour
light/dark cycle and fed standard rodent chow and water
ad libitum. All experiments were approved by the
University of Florida Institutional Animal Care and Use
Committee.

Hind Paw Inflammation
Inflammation was induced in the left hind paw of the rats
by subcutaneous injection of carrageenan (6 mg in 150 pl1
of saline) (Sigma, St. Louis, MO) into the plantar surface.
Control animals received only saline (150 ptl) injections.
Animals used for behavioral tests were tested prior to the
inflammation and at 0.5, 2, 6, 24, 48 and 120 hours fol-
lowing the injections. Rats used for western blots were
euthanized (CO2 inhalation) and the spinal cord tissue
was harvested at 2, 6 and 24 hours following the hind paw
injections. For the analysis of NR2 proteins tissue was har-
vested at 2, 6, 24, 48 and 120 hours following the hind
paw inflammation.

Thermal Nociception Assay
Thermal nociception was measured using the method of
Hargreaves et al. [37]. Briefly, the rats were placed on a
clear plastic surface and allowed 15 minutes to accommo-
date to the enclosure. An infrared light was directed onto
a hind paw's plantar surface approximately in the middle
of the foot. The latency for the animal to remove its foot
from the path of the light was used as the dependent
measure for thermal sensitivity. The light intensity was


initially adjusted to produce latencies of approximately 8
s and a cutoff time of 20 s was used to prevent injury to
the animals. Both hind paws were tested 3 times and the
average of the 3 tests was used as the paw withdrawal
latency for that time point. A rest period of at least 2 min-
utes was observed before the animals were retested to pre-
vent sensitization of the paws.

Mechanical Allodynia Assay
An electronic Von Frey device (Ugo Basile, Italy) was used
for analyzing mechanical sensitivity in the rats. The
animals were placed on a wire screen and allowed 15 min-
utes to become accustomed to the device. A small steel rod
(~0.5 mm in diameter) with a blunt end was pressed
against the plantar surface of the paw and a pressure gra-
dient was applied (0 50 g) over the course of 20 s. The
force at which the rat moved its paw was used as the
dependent measure of mechanical sensitivity. Both hind
paws were tested on each rat 3 times and the average of the
3 tests was used as the data point for that time period. A
rest period of at least 2 minutes was observed before the
animals were retested to prevent sensitization.

Immunoprecipitation and Western Blots
The rats were euthanized at the indicated times with CO2
and the spinal cords were removed by pressure ejection
with 5 mls ice cold phosphate buffered saline (pH 7.4).
The L2 to L5 region of the cord was isolated and divided
in half lengthwise. The side ipsilateral to the paw injection
was retained for the experiments. The tissue was immedi-
ately sonicated and boiled for 10 minutes in denaturing
buffer (10 mls/g tissue) containing 1% SDS, 10 mM Tris
pH 7.4 and 0.4 mM sodium ortho-vanadate. The solution
was then centrifuged at 25,000 g for 30 minutes and the
supernatant collected. A sample of the supernatant con-
taining 500 |tg of protein was diluted to 500 tl with
deionized water and mixed with 500 ptl 2 x immunopre-
cipitation buffer containing 2% triton X-100, 300 mM
NaC1, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.4
mM sodium ortho-vanadate, 0.4 mM PMSF and 1% NP-
40. The anti-NR1 or anti-NR2 antibodies (2-5 [tg) were
added to the solution and incubated over night at 40C.
The proteins were then precipitated with agarose beads
coupled to the appropriate anti-IgG antibody (Sigma, St.
Louis, MO). The beads were isolated and washed in
immunoprecipitation buffer. The beads were then sus-
pended in 30 ptl immunoprecipitation buffer and diluted
with 30 ptl 2 x Laemmli buffer (BioRad, Hercules, CA) and
boiled for 10 minutes. The samples were run on 3 20%
SDS-PAGE gels and semi-dry transferred to PVDF mem-
branes. Phospho-serine, -threonine and -tyrosine were
then examined using the appropriate phospho-selective
anti-bodies (Sigma, St. Louis, MO). The bands were
viewed using HRP chemiluminescence and film. Band
density was determined using Scion Image (Scion


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corporation, Gaithersburg, MD). To verify that the immu-
noprecipitations were quantitative samples of the super-
natant following the removal of the beads were run on
western blots and probed with the appropriate anti-NR1
or anti-NR2 antibodies. If the precipitated protein was
found in the supernatant the precipitation procedure was
repeated.

To analyze NR1 and NR2 protein a sample of the denatur-
ing buffer containing 20 |tg of protein was mixed with an
equal volume of 2 x Laemmli buffer, boiled (10 minutes)
and run as described above. The membranes were then
probed with the appropriate antibodies. Lane loading was
verified with anti-actin antibodies. The density of the actin
bands was determined. If the density of any band in the
gel diverged more than one standard deviation from the
mean of all the bands the gel was discarded and the exper-
iment repeated. All statistics were performed on the raw
band density, but were presented graphically as data
normalized to the non-inflamed control data to enhance
clarity.

Rabbit antibodies to the exon 5 region (N1), exon 21
region (Cl), exon 22 C-terminus (C2) and exon 22 minus
C-terminus (C2') cassettes [18] of the NR1 subunit were
raised at the National Institute for Dental and Craniofa-
cial Research, National Institutes of Health using the fol-
lowing peptide sequences (N1)
SKKRNYENLDQLSYDNKRGPC, (C1) DRKSGRAEPDP-
KKKATFRAC, (C2) PRRAIEREEGQLQLC and (C2')
QYHPTDITGPLNLSDPS. The antibodies were affinity
purified and tested for specificity using COS-7 cells trans-
fected with specific NR1 splice variants and western blots
(Figure 1). The secondary HRP coupled antibodies were
purchased from Sigma (ST. Louis, MO). The remainder of
the antibodies were purchased from Chemicon Interna-
tional (Temecula, CA), BD Biosciences (San Jose, CA) or
Santa Cruz Biotechnology (Santa Cruz, CA).

Data Analysis
Western blot band density was measured using Scion
Image (Scion Corp., Frederick, MD). All statistical analy-
ses were conducted on the raw data. Statistical analysis
consisted of ANOVA followed by Dunnett's tests or Dem-
ing linear regression using Prism statistical software
(Graphpad Software inc., San Diego, CA). Statistical sig-
nificance was assigned to p < 0.05.

Competing interests
The authors) declare that they have no competing
interests.

Authors' contributions
RMC designed the study, ran the behavioral experiments
and prepared the manuscript. FMP performed immuno-


precipitations and western blots. AYD performed
immunoprecipitations and western blots. MJI raised the
NR1 splice variant selective antibodies.

Acknowledgements
This work was supported by grants from the National Institute on Drug
Abuse DA 13166 (RMC), the McKnight Brain Institute Brain and Spinal Cord
Injury Research Trust Fund (RMC), and the National Institute of Dental and
Craniofacial Research intramural research program (MJI).

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