Background
Visceral pain is a common symptom involved in many gastrointestinal disorders such as inflammatory bowel disease and irritable bowel syndrome. During the last decade research focusing primarily on alterations in the peripheral and central nervous system has improved our understanding of the pathophysiological mechanisms of chronic visceral pain. These studies have demonstrated significant physiological changes following injury to the viscera in the firing patterns of both primary afferent neurons that transmit nociceptive information from the viscera and in central neurons that process the nociceptive information [
1‐
7]. Furthermore, a number of receptors, neurotransmitters, cytokines and second messenger systems in these neurons have been implicated in the enhancement of visceral nociception [
8‐
12]. Previous research in the enteric nervous system has focused primarily 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 allodynia 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 nociceptive 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 prevents glycine independent stimulation of the receptors by spermine [
40,
41]. The C1 cassette contains four serines that are known phosphorylation sites and an ER retention signal. Phosphorylation of the serines blocks the ER retention signal and allows transport of the receptors to the plasma membrane [
42‐
44]. The presence of the C2 cassette 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 distinct properties that significantly influence the function of the fully formed receptor. In this current study, we examined 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 system 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.
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 receptors 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 Complete 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 implications and could be involved in the pathophysiology of chronic visceral hypersensitivity seen in patients with post-infectious IBS and other chronic visceral pain disorders [
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 NR1
001, NR1
011 and NR1
111 appeared in TNBS treated rats with active colitis. Untreated control rats only expressed NR1
001 (Fig
3A). In addition, NR1-N1 and NR1-C1 protein expression was also present in the colonic myenteric plexus in TNBS treated rats. Parallel to the protein expression, 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 NR1
001 has Exon 5 and Exon 21 spliced out, while the Exon 22 is spliced in; NR1
011 has the Exon 5 (N terminal) spliced out, while the Exon 21 and Exon 22 (C terminal) are spliced in; NR1
111 has all three exons [
39,
45].
The functional properties of NMDA receptors depend on the NR1 splice variant combination. NR1 receptors, lacking the N-terminal exon, exhibited a high affinity for NMDA and marked potentiation by spermine [
45]. Presence of the N1 insert reduced the apparent affinity of homomeric NR1 receptors for NMDA and almost abolished potentiation by spermine at saturating glycine [
45], while splicing-in the N1 insert increased current amplitude [
36,
41].
As demonstrated by Durand et al [
45], NR1
001 did not lead to generation of sufficiently large currents for analysis, even if the amount of RNA injected was increased from 10 to 50 ng per oocyte. In this study, we found NR1
001 in the normal colon. Because Durand found that NR1
001 does not generate significant current [
45], NR1
001 may not be part of a functional receptor. We have shown that NR1
011 appeared only in rats following colonic inflammation. This has very important implications for visceral pain that NR1
011 may play an important role in the plasticity that occurs following transient inflammation. NR1
111 which has the N1 insert is also present in inflamed colon. Splicing in the N1 insert increased current amplitude [
36,
41]. Therefore NR1
111 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, NR1
011 and NR1
111 could modulate increased visceral hypersensitivity and alter colonic motility present in patients following transient inflammatory injury to the colon.
Phosphorylation of NMDA receptors is thought to be an important factor for cell modulation, regulation, and neuronal 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 regulates 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 findings 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 following 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 of TNBS-induced colitis in rats [
63] found hypersensitivity to visceral stimulation 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 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 multiple factors involved in the expression of visceral hypersensitivity.
Novel NR1 protein expression may be associated with persistent increases in impulse activity originating from the colon and rectum. This impulse activity could be generated 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 myenteric neuronal NMDA receptors could lead to functional changes that stimulate receptors in the rectum and colon or increased NMDA receptors in primary afferent terminals 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 hypersensitivity 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 receptors in other tissues, NMDA receptor expression in the colonic myenteric plexus may be a major underlying factor 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 administration [
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 reasons. 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 addition, 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 independent criteria of weight loss, histological evaluation of transmural inflammation and MPO (myeloperoxidase) analysis. Microscopic evaluation of TNBS-induced inflammation 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 concentrations 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 animals 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 sensitization may explain the somatic hypersensitivity our laboratory has found in both animals and humans with IBS [
63,
67,
68].