Group Title: Molecular Pain 2006, 2:3
Title: Selective up-regulation of NMDA-NR1 receptor expression in myenteric plexus after TNBS induced colitis in rats
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Title: Selective up-regulation of NMDA-NR1 receptor expression in myenteric plexus after TNBS induced colitis in rats
Series Title: Molecular Pain 2006, 2:3
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Creator: Zhou QQ
Caudle RM
Price DD
Del Valle-Pinero AY
Verne GN
Publication Date: 38734
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

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Molecular Pain BioMedCentra


Research


Selective up-regulation of NMDA-NRI receptor expression in
myenteric plexus after TNBS induced colitis in rats
QiQi Zhoutl, Robert M Caudlet2,3, Donald D Pricet2,3, Arseima Y Del Valle-
Pinerot3 and G Nicholas Verne*t1,4


Address: 'Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA, 2Department of Oral and Maxillofacial
Surgery, University of Florida College of Dentistry, Gainesville, FL 32610, USA, 3Department of Neuroscience, University of Florida College of
Dentistry, Gainesville, FL 32610, USA and 4North Florida/South Georgia VA Health System, USA
Email: QiQi Zhou qzuou@yahoo.com; Robert M Caudle caudle@ufl.edu; Donald D Price dprice@dental.ufl.edu; Arseima Y Del Valle-
Pinero delvalle@ufl.edu; G Nicholas Verne* gnicholas.veme@med.va.gov
* Corresponding author tEqual contributors



Published: 7 January 2006 Received: 06 October 2005
Molecular Pain 2006, 2:3 doi:10.1186/1744-8069-2-3 Accepted: 17 January 2006
This article is available from: http://www.molecularpain.com/content/2/1/3
2006 Zhou 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: N-methyl-D-aspartic acid (NMDA) spinal cord receptors play an important role in
the development of hyperalgesia following inflammation. It is unclear, however, if changes in NMDA
subunit receptor gene expression in the colonic myenteric plexus are associated with colonic
inflammation. We investigated regulation of NMDA-NRI receptor gene expression in TNBS
induced colitis in rats. Male Sprague-Dawley rats (150 g-250 g) were treated with 20 mg
trinitrobenzene sulfonic acid (TNBS) diluted in 50% ethanol. The agents were delivered with a 24
gauge catheter inserted into the lumen of the colon. The animals were sacrificed at 2, 7, 14, 21, and
28 days after induction of the colitis, their descending colon was retrieved for reverse
transcription-polymerase chain reaction; a subset of animals' distal colon was used for two-
dimensional (2-D) western analysis and immunocytochemistry.
Results: NRI-exon 5 (NI) and NRI-exon 21 (CI) appeared 14, 21 and 28 days after TNBS
treatment. NR I pan mRNA was up-regulated at 14, 21, and 28 days. The NRI -exon 22 (C2) mRNA
did not show significant changes. Using 2-D western analysis, untreated control rats were found to
express only NRI001 whereas TNBS treated rats expressed NRI00l, NRIo0, and NRIIII.
Immunocytochemistry demonstrated NRI-NI and NRI-CI to be present in the myenteric plexus
of TNBS treated rats.
Conclusion: These results suggest a role for colonic myenteric plexus NMDA receptors in the
development of neuronal plasticity and visceral hypersensitivity in the colon. Up-regulation of
NMDA receptor subunits may reflect part of the basis for chronic visceral hypersensitivity in
conditions such as post-infectious irritable bowel syndrome.



Background ade research focusing primarily on alterations in the
Visceral pain is a common symptom involved in many peripheral and central nervous system has improved our
gastrointestinal disorders such as inflammatory bowel understanding of the pathophysiological mechanisms of
disease and irritable bowel syndrome. During the last dec- chronic visceral pain. These studies have demonstrated

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significant physiological changes following injury to the
viscera in the firing patterns of both primary afferent neu-
rons that transmit nociceptive information from the vis-
cera and in central neurons that process the nociceptive
information [1-7]. Furthermore, a number of receptors,
neurotransmitters, cytokines and second messenger sys-
tems in these neurons have been implicated in the
enhancement of visceral nociception [8-12]. Previous
research in the enteric nervous system has focused prima-
rily on altered motility. The potential role of altered
enteric nervous system function on visceral nociception
has not been fully explored. In this study, we examined
potential mechanisms of visceral pain produced by colitis.
We used an in vivo inflammatory model of TNBS colitis
which revealed N-Methyl-D-aspartate (NMDA) receptors
modulated neuronal plasticity.

In the spinal cord, NMDA receptors were found to play a
pivotal role in the development and maintenance of allo-
dynia and hyperalgesia in both visceral and somatic tissue
[13-18]. NMDA receptors integrate the activity of groups
of neurons and provide a mechanism to amplify nocicep-
tive signals. This process leads to central sensitization,
which is characterized by enlarged neuronal receptive
fields, allodynia and hyperalgesia [7,16,18-25]. Recent
work demonstrated the presence of NMDA receptors in
the enteric nervous system [22,26-29]. The role of these
receptors is currently not known, but it is likely that they
serve to integrate and amplify signals within the network,
possibly resulting in altered gut motility, secretion, and
enhanced nociception.

NMDA receptors are composed of at least two subunits,
NR1 and NR2 [30-32]. A third subunit of the NMDA
receptor, NR3, has also been described, but it is not
required for a functional receptor and its role is currently
unclear [33,34]. The NR1 subunit forms eight functional
splice variants based on the presence or absence of three
alternatively spliced exons, Exon 5 (N1), Exon 21 (C1)
and Exon 22 (C2) [35-39]. The presence of N1 enhances
the current flow through the NMDA receptors and pre-
vents glycine independent stimulation of the receptors by
spermine [40,41]. The C1 cassette contains four series
that are known phosphorylation sites and an ER retention
signal. Phosphorylation of the series blocks the ER reten-
tion signal and allows transport of the receptors to the
plasma membrane [42-44]. The presence of the C2 cas-
sette alters the C-terminus of the protein and changes the
targeting of the protein for different cell structures
[39,45]. Thus, the various splice variants of NR1 have dis-
tinct properties that significantly influence the function of
the fully formed receptor. In this current study, we exam-
ined the expression of the NR1 splice variants in the colon
of rats following TNBS treatment to determine if NMDA
receptor function was altered by an inflammatory injury.


We hypothesized that there may be enteric nervous sys-
tem sensitization mediated by increased expression of
NR1 splice variants following an inflammatory injury to
the gut by TNBS colitis. The resulting sensitization of the
colonic myenteric plexus would be similar to other
chronic disorders where peripheral sensitization is
present.

Materials and methods
Animal preparation
Male adult Sprague-Dawley rats weighing 150-250 g were
used in this experiment. The rats were housed in pairs
under constant temperature and humidity with 12-hour
light-dark cycles, and were given free access to food and
water. Administration of intracolonic trinitrobenzene sul-
phonic acid (TNBS) with 50% ethanol was used to pro-
duce colonic ulceration and inflammation [46]. Prior to
instillation of TNBS in the colon, the animals were anes-
thetized with an intraperitoneal injection of sodium
pentobarbital (50-90 mg/kg). Following this, 20 mg (per
rat) of trinitrobenzene sulfonic acid (TNBS) (1 M in
Water, Sigma Chemical Co.) diluted in 50% ethanol, was
instilled into the lumen of the colon via the anus (n = 58:
RT-PCR n = 3 per time point; 2-D western analysis n = 1
per treatment; immunohistocytochemistry n = 2 per treat-
ment; colorectal distension testing n = 8 per time point).
The agent was delivered in a volume of 0.3 ml/rat with a
24 gauge catheter. An equivalent volume of saline was
injected into control rats (n = 8: RT-PCR n = 3; colorectal
distension testing n = 5).

Evaluation of colonic inflammation
Immediately following the somatic and visceral pain test-
ing, all rats were euthanized using sodium pentobarbital
(120 mg/kg, ip). Following euthanasia, 3 cm of the
descending colon was removed and processed for histopa-
thology. The tissue was fixed in formalin and processed
using standard techniques for H & E staining. The severity
of the lesions in the colon and mucosa was graded using
a system previously described by Al Chaer et al [1]. The
grades of colitis included: mild (+1) infiltration of a lim-
ited number of neutrophils in the lamina propria with
minimal interstitial edema; moderate (+2) infiltration of
a moderate number of neutrophils in the lamina propria
with moderate interstitial edema; severe (+3) diffuse infil-
tration of neutrophils in the lamina propria with severe
interstitial edema.

Colorectal distension testing
A subset of treated and saline-treated rats underwent
colorectal distension. These rats were not used for any of
the molecular marker studies. A 3 cm long balloon was
used to perform colon distension. The balloon was lubri-
cated and placed into the rat's distal colon so that the tip
of the balloon was 1 cm from the anus. The rats were


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Table I: PCR Amplification Primers


Primer name

NRI Pan

NRIExon5 minus/
NRI Exon5 plus
NRI Exon 2148

NRI Exon 22

GAPDH


Sense/Antisense


Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense


Primer Sequences


5'-CCCTCAGACAAGTTCATCTACGC-3'
5'-AGGTTCTTCCTCCACACGTTCAC-3
5'-GCGAGTCTACAACTGGAACCAC-3'
5'-CTCGCTTGCAGAAAGGATGATG-3'
5'-TGTGTCCCTGTCCATACTCAAG-3'
5'-GTCGGGCTCTGCTCTACCAC-3'
5'-CATGGCAGGGGTCTTCATGCTG-3'
5'-GAACACAGCTGCAGCTGGCCCT-3'
5'-CCTTCATTGACCTCAACTACATGGTCTA-3'
5'-TAGCCCAGGATGCCCTTT-3'


allowed 10 minutes to acclimatize before behavioral test-
ing began. Using an automated distension device (G & J
Electronic Inc. Toronto, Canada) the rats received phasic
distension (0-80 mmHg in 5 mmHg ascending incre-
ments) of the colon until the first contraction of the testi-
cles, tail, or abdominal musculature occurred which was
indicative of the first nociceptive response as previously
described [47]. The colonic distensions were repeated 4
times and the mean pressures at the nociceptive threshold
were recorded for each rat.

Rats were euthanized with sodium pentobarbital (120
mg/kg, ip). Immediately following euthanasia the
descending colon (~2-3 cm) was removed at 2, 7, 14, 21,
and 28 days following TNBS treatment. Untreated rats (n
= 6) and saline rats (n = 3) were both used as controls. Tis-
sue was prepared for RNA and protein extraction for 2-D
western analysis, as well as immunocytochemistry.

All procedures were approved by the North Florida/South
Georgia Veterans Health System Institutional Animal Care
& Use Committee

Reverse transcription polymerase chain reaction
All primers of NR1 subunits for RT-PCR were synthesized
(GenoMechanix, Gainesville, FL). The NR1 receptor subu-
nit-specific primers were designed by targeting three alter-
natively spliced exons (Table 1) [48]. Glyceraldehyde
phosphate dehydrogenase (GAPDH) was used as an inter-
nal control since its expression is not regulated by inflam-
mation [49]. RNA was extracted using the RNeasy Mini Kit
(QIAGEN Inc). RT-PCR was carried out following the
manufacturer's instructions by using the RT-PCR Access
kit (Promega Corporation). The temperature cycle
(Eppendorf-Master Cycler Gradient from Brinkmann
Instruments Inc) followed the guidelines provided by
Promega Corporation. Reverse transcription (RT) period
was 48 C/45 min (RT time), 94 C/2 min (initial denatur-
ing of RT), 72C/1 min (extension of RT time). The tem-
perature cycle of PCR was 94 C/30 min denaturingg), 50-
62C/1 min (annealing), 72C/2 min and 72C/7 min


(extension). A total of 25-30 cycles were conducted. The
PCR product samples were loaded in parallel with a 100
bp DNA ladder (BIO RAD Inc) on 1.2 % ethidiumibro-
mde-stained agarose gel. The gels were imaged with the
AlphaEaseFC program using the FluorChem' 8900. Scion
Image program was used to analyze the data for statistical
analysis.

Two-dimensional polyacrylamide gel electrophoresis
Two-dimensional (2-D) electrophoresis was performed
according to the methods of O'Farrell [50] as follows
(Kendrick Labs): Isoelectric focusing was carried out in a
glass tube with an inner diameter of 2.0 mm pH 3.5-10
(Amersham Biosciences, Piscataway, NJ) for 9600 volt-
hrs. One jig of an IEF internal standard, tropomyosin, was
added to the sample. The protein migrates as a doublet
with lower polypeptide spot of MW 33,000 and pi 5.2.
Following equilibration for 10 minutes in 'O' buffer (10%
glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M
tris, pH 6.8), the tube gel was sealed to the top of a stack-
ing gel that overlaid a 10% acrylamide slab gel (0.75 mm
thick). SDS slab gel electrophoresis was carried out for
about 4 hrs atl2.5 mA/gel. After slab gel electrophoresis
the gel was placed in transfer buffer (12.5 mM Tris, pH
8.8, 8.6 mM Glycine, 10% MeOH) and transblotted onto
a PVDF membrane overnight at 200 mA and approxi-
mately 100 volts/two gels. The following proteins (Sigma
Chemical Co.) were added as molecular weight standards
to a well in the agarose that sealed the tube gel to the slab
gel: myosin (220,00), phosphorylase A (94,000), catalase
(60,000), actin (43,000) carbonic anhydrase (29,000)
and lysozyme (14,000). One membrane from each group
was used throughout the entire 2-D experiment. Mem-
branes from control rats and TNBS treated rats after 14
days were used. The western blots were viewed using
enhanced Chemiluminescent detection and radiographic
film. Primary antibodies were removed with western blot
stripping buffer (Pierce Co.) for multiple antibody prob-
ings. The efficiency of the stripping procedure was verified
by using the secondary antibody and re-exposing the
membrane to film.


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Product

563 bp

210 bp
273 bp
307 bp

362 bp

720 bp


Molecular Pain 2006, 2:3







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60-
S 0-
02
a-E
E E 30.
_ 20-
m 10-


N S 2d 7d 14d 21d 28d
Time

Figure I
Bar graph of colorectal distension in mmHg vs. days following
TNBS treatment. Normal control (N). Saline control (S). The
asterisks indicated Significant differences from respective
controls. One way ANOVA (p < 0.0001) with Dunnett's
multiple comparison test. ** p < 0.01. Values are expressed
as Means SEM.



Immunoflorescence
Rats were euthanized with sodium pentobarbital (120
mg/kg, ip) and their descending colon (~2-3 cm) was
removed at 14 days in both controls and those treated
with TNBS. The tissues were put into cold saline and fro-
zen in super-cooled isopentane. Five-micron-thick sec-
tions of descending colon were cut longitudinally in a
cryostat at -20C. Every fourth to fifth section was col-
lected and serially mounted on a glass slide and air-dried
for 1-3 hours. Tissues were placed in a blocking buffer
containing 3% Normal Goat Serum (NGS) or 2% of
Bovine Serum Albumin (BSA, Sigma) with PBS for 30-60
minutes, then incubated with anti-NRlpan antibody (BD
Biosciences), anti-NR1-C1 antibody (courtesy of Caudle
laboratory) and NR1-N1 antibodies (courtesy of Caudle
laboratory) in 3% of NGS/PBS overnight at 4 C. After 3-
4 washes the tissue was placed in PBS (15 minutes each)
and 60-minutes in a secondary IgG (Molecular Probes,
Inc.) with 3% goat serum in PBS. The NR1 pan was
probed by using Alex fluor 488 (Molecular Probes, Inc.)
with FITC broadband filter. The NR1-C1 and NR1-N1
were probed by using Alex fluor 594 (Molecular Probes,
Inc.) with Texas Red broadband filter (Zeiss Oberkochen,
Germany). The sections were washed with distilled water
before mounting the section. Double-labeled sections
were coded, viewed on an Axio-phot immuno-fluores-
cence microscope (Zeiss Oberkochen, Germany), and
photographs were made.

Data analysis
The RT-PCR data and the colorectal distension data were
analyzed using ANOVA followed by Dunnett's post-test
comparisons. GraphPad Prism software was used in all


.. .


Molecular Pain 2006, 2:3


data analysis. A p value of <0.05 was considered signifi-
cant.

Results
Histology
All rats treated with TNBS had severe (+3) colitis charac-
terized by diffuse infiltration of neutrophils in the lamina
propia with severe institial edema. The severe colitis was
present at all time points (2, 7, 14, 21, and 28 days) fol-
lowing TNBS treatment.

Colorectal distension testing
Hypersensitivity to colon distension was increased in
TNBS treated rats (n = 8 per time point) at 2, 7, 14, 21, and
28 days following TNBS administration compared to
saline treated rats (n = 5) and control rats (n = 5) One-way
analysis of variance indicated p < 0.0001 (Figure 1). Dun-
nett's post-test revealed that the p value was < 0.001 at 2,
7, 14, 21, and 28 days after TNBS treatment when com-
pared to saline controls.

RT-PCR
Primers of NR1 pan, NR1-Exon 21(C1), NR1-Exon 22
(C2) and NR1-Exon 5 (N1) were used to monitor NMDA
receptor subunit mRNA expression following TNBS coli-
tis.

The RT-PCR probe that recognized all splice variants (NR1
pan) demonstrated a significant increase in NR1 expres-
sion (ANOVA F6,14= 6.28, p = 0.0022) following TNBS
injection. Figure 2A demonstrates an increase in NR1
mRNA at 14 (p < 0.05), 21 (p < 0.05) and 28 days (p <
0.05).

The NR1-C1 splice variant mRNA was not detected in
untreated and saline control rats, or 2 and 7 days follow-
ing TNBS treatment. The NR1-C1 receptor mRNA
appeared at 14 (p < 0.01), 21 (p < 0.01) and 28 days (p <
0.01) following TNBS injection. ANOVA results showed
F6,14 = 11.46, p < 0.0001 (Fig 2B). The NR1-N1 minus
splice variant mRNA is not regulated by TNBS induced
colonic inflammation (ANOVA p = 0.99). The mRNA
expression was constant throughout the observation
period. However, NR1-N1 plus mRNA was up-regulated
(ANOVA F6,14 = 87.7, p < 0.0001) at 14 (p < 0.01), 21 (p
< 0.01) and 28 days (p < 0.01) following TNBS treatment
(Fig. 2C).

There was no apparent regulation of NR1 Exon 22 subunit
after TNBS treatment. The mRNA level was similar to
untreated and saline control rats (Fig. 2D).

Based on the RT-PCR results, we chose one time point,
which was 14 days after TNBS induced colitis, to examine
the functional protein expression during the colitis as well


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.-GAPDH 720bp
NRI 563bp

N S 2d 7d 14d 21d 28d
120-
I100



0-


N S 2d 7d 14d 21d 28d
Time


_f.APDH 720bp


.4_UAPUH (2Ubp
I I*IQI .cl307bp
N S 2d 7d 14d 21d 28d


2d
Ti


'd 14d 21d 28d
me


120
100
8 0
< 60-
S 40-
20-

N S


______ _._N 1


L 27 b 40 MD of&2WO0Wbp N


+273bp
-210bp


APDH 720bp
C2 362bp


N S 2d 7d 14d 21d 28d


N S 2d 7d 14d 21d 28d


Time lime


Time


Figure 2
(A-D) RT-PCR analysis of changes in NMDA NRI splice variants in rat descending colon following TNBS induced colitis. Exam-
ples of agarose gel electrophoresis of PCR products are shown on the top of each panel. The RT-PCR products with specific
size are indicated. The samples from naive animals are shown in "N", saline control animals are shown in "S". GAPDH PCR was
used as an internal Control. The bottom of each panel shows the summary of the effects on NMDA NRI receptor splice vari-
ant mRNA expression. Each value represents mean of three Individual experiments. The asterisks indicated significant differ-
ences from respective controls (One ANOVA with Dunnett's post-test comparison test); p < 0.05. Statistical comparisons
were made among all groups using raw data. The mRNA expression of NRI subunit normalized to GAPDH expression are
present as the Means SEM. A. Expression of NRI pan mRNA increase at day 14, 21 and 28 following colitis B. Expression of
NRI Cl cassette mRNA appeared at day 14, 21 and 28 after colitis C. Expression of NRI NI cassette of NI plus mRNA
appeared at day 14, 21 and 28 days after colitis; expression of NRI NI minus was not regulated by TNBS induced colitis D.
Expression of NRI C2 cassette mRNA was not regulated by TNBS induced colitis


as to detect location of the NMDA receptor in the
inflamed colon.


Two-dimensional (2-D) gel analysis
The eight splice variants of NR1 are predicted to have dif-
ferent isoelectric points and slightly different molecular
weights. We have adopted 2-D polyacryamide gel electro-
phoresis to detect the eight splice variants of the NR1 sub-
unit in the colon.


In the untreated control group, only one subunit of NR1
receptor was expressed NR,101 (Fig. 3A) [39]. However,
we found changes in protein profiles of NR1 subunits fol-
lowing TNBS treatment in rats (Fig. 3B). Among the eight
potential splice variants of NR1 protein 3 subunits are
observed following 14 days TNBS injection. They are NR1
001, NR1011, and NR1111 (Fig. 3B) [39].






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I--







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024.

C2-

N1

C,


Untreated






S-
4, .



e^


B. Treated


Figure 3
(A) 2-D western analysis of NRI splice variants in rat's mye-
nteric plexus. Fig. 3A: 2D gel analysis in normal control rat.
(B) 2-D gel analysis 14 days following TNBS injection. To
identify proteins with antibodies to the NRI-C2 plus, NRI-
C2 minus, NRI-CI and NRI-NI in descending colon two
separate membranes were used for all 4 experiments in the
treated and control animals. The membranes were stripped
of primary antibody between experiments. Label at the bot-
tom indicated the NRI splice variants.


Immunocytochemistry
Double-labeling fluorescent immunocytochemistry
showed that the NR1-C1 and NR1-N1 are not expressed in
the untreated control rat's colon (Fig. 4A and 4B). NR1
pan is present in enteric neurons in both untreated con-
trol rats and TNBS treated rats (see Fig 4A and 4B). Impor-
tantly, NR1-N1 appeared in TNBS induced colitis.
Double-labeling of fluorescent immunocytochemistry for
NR1 Pan and NR1-N1 indicated that they are located in
neurons in the myenteric plexus of colon (Fig 4A) at 14
days following TNBS treatment. NR1 pan with NR1-C1
double-labeling was also present in neurons in the mye-
nteric plexus (Fig. 4B) 14 days following TNBS treatment.
The NR1-N1 and NR1-C1 only appeared in the myentric
plexus (Fig. 4A and 4B).

Discussion
This study characterized changes in NR1 expression in the
colonic myenteric plexus in response to an inflammatory
injury to the colon. The changes in enteric NMDA recep-
tors is a novel finding in this study as previous studies
have described NMDA changes in the spinal cord in
response to a peripheral injury such as carrigenan or Com-
plete Freund's Adjuvant (CFA) injection of the paw
[20,51-53]. These changes in the NMDA receptor subunits
in response to inflammation may have profound implica-
tions and could be involved in the pathophysiology of
chronic visceral hypersensitivity seen in patients with
post-infectious IBS and other chronic visceral pain disor-
ders [54,55]. The current study examined the expression
of the NR1 splice variants in the colon of rats following


TNBS induced colitis. We found that protein expression of
NRl001, NR1011 and NR1111 appeared in TNBS treated rats
with active colitis. Untreated control rats only expressed
NRlo01 (Fig 3A). In addition, NR1-N1 and NR1-C1 pro-
tein expression was also present in the colonic myenteric
plexus in TNBS treated rats. Parallel to the protein expres-
sion, the mRNA of NR1-N1 plus and NR1-C1 were also
present at 14, 21 and 28 days after TNBS treatment.

The NR1 subunit forms eight functional splice variants
based on the presence or absence of three alternatively
spliced exons: Exon 5 (N1), Exon 21 (C1), and Exon 22
(C2). Splicing out the exon segment that encodes the C2
cassette removes the first stop codon, resulting in a new
open reading frame that encodes an unrelated sequence of
22 amino acids (C2 minus) before a second stop codon is
reached [35-39]. The NRlo01 has Exon 5 and Exon 21
spliced out, while the Exon 22 is spliced in; NR011 has the
Exon 5 (N terminal) spliced out, while the Exon 21 and
Exon 22 (C terminal) are spliced in; NR1 ,1, has all three
exons [39,45].

The functional properties of NMDA receptors depend on
the NR1 splice variant combination. NR1 receptors, lack-
ing the N-terminal exon, exhibited a high affinity for
NMDA and marked potentiation by spermine [45]. Pres-
ence of the N1 insert reduced the apparent affinity of
homomeric NR1 receptors for NMDA and almost abol-
ished potentiation by spermine at saturating glycine [45],
while splicing-in the N1 insert increased current ampli-
tude [36,41].

As demonstrated by Durand et al [45], NRlo01 did not
lead to generation of sufficiently large currents for analy-
sis, even if the amount of RNA injected was increased
from 10 to 50 ng per oocyte. In this study, we found
NR10o1 in the normal colon. Because Durand found that
NRlo01 does not generate significant current [45], NRlo01
may not be part of a functional receptor. We have shown
that NR1011 appeared only in rats following colonic
inflammation. This has very important implications for
visceral pain that NR1011 may play an important role in
the plasticity that occurs following transient inflamma-
tion. NR111, which has the N1 insert is also present in
inflamed colon. Splicing in the N1 insert increased cur-
rent amplitude [36,41]. Therefore NR111, may increase
the NMDA receptor activity in inflamed colon. In our
immunocytochemistry study, the NR1-N1 also was
expressed and presented on dendrites and cell membranes
in the myenteric plexus following TNBS treatment (see
arrow in Fig 4A). Expression of the N1 is associated with
large current amplitudes and an enhanced responsiveness
to PKC phosphorylation [39]. More importantly, NR1011
and NR1,11 could modulate increased visceral hypersensi-


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U ntreatedr


I IntrA tdr


TIMRQ TKRSP


Figure 4
(A) Photomicrographs illustrating NRI pan and NRI NI receptor double-labeling in descending colon. The upper panel is nor-
mal control rats. The lower panel is from inflamed rats. (B) Photomicrographs illustrates NRI pan and NRI Cl receptor dou-
ble-labeling in myenteric plexus of descending colon. The upper panel is normal control rats. The lower panel is from inflamed
rat.


tivity and alter colonic motility present in patients follow-
ing transient inflammatory injury to the colon.

Phosphorylation of NMDA receptors is thought to be an
important factor for cell modulation, regulation, and neu-
ronal plasticity to response to a variety of stimuli. It may
also play a critical role in long term potentiation (LTP)
underlying memory formation. A number of residues that
undergo phosphorylation are contained within a single
alternatively spliced exon in the C-terminal domain, the
C1 cassette [39,56]. Our immunocytochemistry revealed
that the NR1-C1 protein was expressed on dendrites in the
myenteric plexus (see arrow in Fig 4B) following TNBS
treatment. The NR1-C1 was not expressed in the sub-
mucosa. NR1-C1 contains an endoplasmic reticulum (ER)
retention signal suggesting that the presence of C1 may
alter translocation of NMDA receptors. The ER works as a
control center in coordinating the sequential assembly of
multi-subunit protein complexes within the ER and in
defining the number of receptors expressed at the plasma
membrane [57-61]. Scott et al [43] found that the ER reg-
ulates plasma membrane delivery of NMDA receptors. In
addition, the study indicated that ER retention signals in
the alternatively spliced C-terminal domain of the NR1
subunit control release of NR1 from ER and is regulated
by PKC phosphorylation [43]. We hypothesize that TNBS


induced colitis may produce assembled NR1 subunits and
transports them through the ER-Golgi secretion pathway
[62]. Phosphorylation blocks the NMDA receptor ER
retention signal leading to surface expression. Our find-
ings indicate that the activity of NMDA NR1 could be an
important factor in the neuroplasticity that occurs in the
colonic myenteric plexus following an inflammatory
stimulus.

As might be expected during an acute inflammatory
injury, there was visceral hypersensitivity present two days
after TNBS injection that persisted to 28 days. However,
the NMDA NR1 receptor up-regulation was present at 14-
28 days following TNBS injection. The delay in NR1
expression could suggest that NMDA receptor may be
involved in chronic nociception and may well persist fol-
lowing resolution of the colitis. Thus, it may take up to 14
days before more chronic changes occur, such as NMDA
NR1 receptor up-regulation. A study ofTNBS-induced col-
itis in rats [63] found hypersensitivity to visceral stimula-
tion at 2 days after TNBS treatment, yet hypersensitivity to
somatic stimuli was only present between 14 and 28 days.
Interestingly, this time period is the same as the one
wherein splice variants exhibited increased transcription
in the present study. Although NMDA NR1 receptor up-
regulation is not necessary for visceral hypersensitivity


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before 14 days, it may play a role in its maintenance at
later stages when somatic hypersensivity develops. NMDA
receptor upregulation is likely to be among one of multi-
ple factors involved in the expression of visceral hypersen-
sitivity.

Novel NR1 protein expression may be associated with per-
sistent increases in impulse activity originating from the
colon and rectum. This impulse activity could be gener-
ated as a result of increased NMDA receptor activity in the
myenteric plexus and/or terminals of primary afferent
neurons of the colon and rectum. The latter are known to
have NMDA receptors [22]. Increased activity in mye-
nteric neuronal NMDA receptors could lead to functional
changes that stimulate receptors in the rectum and colon
or increased NMDA receptors in primary afferent termi-
nals could directly lead to their increased impulse activity.
Regardless of the exact mechanism, increased impulse
activity in afferents innervating the colon and rectum may
then be transmitted to dorsal horn neurons of the spinal
cord. The chronicity of the tonic afferent input from the
viscera to the spinal cord may then lead to sensitization of
dorsal horn nociceptive neurons and would be associated
with visceral hypersensitivity. Somatic hypersensitivity
could develop later as a consequence of long term tonic
impulse activity and convergence of visceral and somatic
primary afferent impulse inputs onto the same dorsal
horn nociceptive neurons. In other words, somatic hyper-
sensitivity would develop over time as a result of increased
sensitization of somatovisceral convergent neurons

Spinal NMDA receptors are important in the induction
and maintenance of central sensitization, yet peripheral
NMDA receptors may also play an important role. Even
when the peripheral inflammatory injury to the colonic
myenteric plexus is healed, enduring neuroplastic changes
in enteric neurons of the colon/rectum, and/or primary
afferent terminals of the colon/rectum could lead to a
condition of increased rectal and colonic hypersensitivity,
as in irritable bowel syndrome. Similar to NMDA recep-
tors in other tissues, NMDA receptor expression in the
colonic myenteric plexus may be a major underlying fac-
tor in enhanced peripheral sensitivity in the rectum and
colon. This and other peripheral factors may operate in
concert with central sensitization mechanisms to produce
visceral hyperalgesia and secondary somatic hyperalgesia.

In our previous study as well as in this study, there was
persistent colitis up to 28 days following TNBS adminis-
tration [63]. These findings differ from Asfaha et al. [64]
in which the inflammation peaked at day 3 and resolved
at week 6. Our findings may be different for several rea-
sons. Asfaha et al. [64] used Wistar rats, whereas we used
Sprague-Dawley rats. Secondly, they examined different
time points which only included 3 days and then 6 weeks


postinflammaion. They did not examine any timepoints
inbetween to determine if there was persistent colitis (i.e.
7, 14, 21 and 28 days following TNBS injection). In addi-
tion, our study focused exclusively on neuronal plasticity
of NMDA receptor expression following colitis. Finally,
the intensity and duration of inflammation may very well
be strain related. The study by Wells et al. [65] indicated
that on days 2 and 4 post-TNBS, an overtly inflamed colon
was present, frequently with adhesions; but by days 16
and 36, the acute effects of inflammation had resolved,
but previously involved areas could be identified by mild
adhesions and bowel wall thickening by using independ-
ent criteria of weight loss, histological evaluation of trans-
mural inflammation and MPO (myeloperoxidase)
analysis. Microscopic evaluation ofTNBS-induced inflam-
mation resulted in a transient increase in damage score
with maximal inflammation present by days 4 post-TNBS
and full resolution by days 36. Our data showed colonic
inflammation present at day 2 through day 28 following
TNBS treatment, but somatic hypersensitivity appeared
from days 14 throughout to days 28. Several possibilities
for these differences exist including differing concentra-
tions of TNBS used and/or differing rat's age and/or
weight may be reasons that the colonic inflammation
healed early. Our data was most consistent with Morris'
group's findings [46]. Their data indicated that the ani-
mals that received varying doses of TNBS in 50% ethanol
developed areas of grossly visible bowel wall thickening,
inflammation, and ulcers. These inflammation and ulcers
were observed up to 8 weeks after administration of
TNBS/ethanol.

There is increasing evidence indicating an important role
for the NMDA receptor in mediating nociception in
colon. The up-regulation of NMDA receptors was also
shown in the spinal cord following colonic inflammation
[7,66] suggesting central sensitization. This central sensi-
tization may explain the somatic hypersensitivity our lab-
oratory has found in both animals and humans with IBS
[63,67,68].

Conclusion
In summary, our current study identified the inflamma-
tion-induced changes in expression of individual colonic-
NMDA receptor subunits. NR1 expression may play an
important role in mediating visceral hypersensitivity in
the TNBS colitis rat model. This could have important
implications in the treatment of patients with chronic vis-
ceral pain disorders as NMDA receptor antagonists specif-
ically targeting NR1 splice variants may be developed to
control visceral pain. Future studies are needed to deter-
mine if NR1 expression continues to be increased follow-
ing resolution of colitis.

Acknowledgements


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This study was supported by a Merit Review Award (PI: GN Verne) from
the Medical Research Service at the Department of Veterans Affairs and by
ROI Grant 1-ROI-NS053090-01 award (PI: GN Verne).

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736:7-15. scientist can read your work free of charge
67. Verne GN, Robinson ME, Price DD: Hypersensitivity to visceral dentist can read your work free of charge
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200 I, 93:7-14. disseminating the results of biomedical research in our lifetime.
68. Verne GN, Robinson ME, Vase L, Price DD: Reversal of visceral
and cutaneous hyperalgesia by local rectal anesthesia in irri- Sir Paul Nurse, Cancer Research UK
table bowel syndrome (IBS) patients. Poin 2003, 105:223-230. Your research papers will be:

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