Activation of lamina I spinal cord neurons that express the substance P receptor in visceral nociception and hyperalgesia

ORIGINAL REPORTS Activation of Lamina I Spinal Cord Neurons That Express the Substance P Receptor in Visceral Nociception and Hyperalgesia Prisca Honoré,* Elizabeth H. Kamp,† Scott D. Rogers,* G.F. Gebhart,† and Patrick W. Mantyh* Abstract: Spinal lamina I neurons expressing the substance P receptor (SPR) have been shown to play a role in the transmission of somatic inflammatory and neuropathic pain. To evaluate their involvement in visceral nociception in both the noninflamed and inflamed colon, we examined the expression and ligand-induced internalization of the SPR in the rat spinal cord after distention of the noninflamed colon and in rats with inflammation induced by intracolonic instillation of zymosan (3 hours). In the noninflamed animal, acute noxious but not non-noxious colorectal distention induced SPR internalization in lamina I neurons at the thoracolumbar (T13) and lumbosacral (S1) spinal levels, whereas SPR internalization was not detected in lamina I neurons at spinal lumbar segment L4. Although zymosan-induced colorectal inflammation alone did not induce SPR internalization in lamina I neurons, there was an increased number of SPR-expressing lamina I neurons showing SPR internalization in segments T12 through S2 of the spinal cord after colorectal distention. These results show that acute noxious visceral stimuli induce activation of spinal lamina I neurons expressing the SPR and, that after visceral inflammation, there is a marked increase in both the number and rostrocaudal extent of lamina I SPR neurons activated in response to both normally non-noxious and noxious distention of the colon. © 2002 by the American Pain Society Key words: Colorectal distention, internalization, spinal cord, neurokinin-1 receptor, visceral pain, zymosan.

P

ain of visceral origin, such as that arising from intestinal obstruction, inflammatory bowel disease, irritable bowel syndrome, or ulcerative colitis, differs in many important ways from pain arising from cutaneous structures. Whereas cutaneous pain is well localized and typically produces rapid reflex motor

Received December 12, 2000; Revised March 21, 2001; Re-revised May 15, 2001; Accepted May 15, 2001. From the *Neurosystems Center, Department of Preventive Sciences, Psychiatry and Neuroscience, University of Minnesota, and VA Medical Center, Minneapolis, MN, and the †Department of Pharmacology, University of Iowa, Iowa City, IA. H.K. Proudfit served as Guest Editor for this report. Supported by the National Institute of Health NINDS 23970, NINDS 19912, NIHDA 11986, and T32 NIGMS 07069, a Department of Veterans Affairs Merit Review, and the Spinal Cord Society. Address reprint requests to Patrick W. Mantyh, PhD, Neurosystems Center, 18-208 Moos Tower, 515 Delaware St, Minneapolis, MN 55455. E-mail: [email protected], or to G.F. Gebhart, PhD, Department of Pharmacology, University of Iowa, Iowa City, IA 52242. E-mail: [email protected]. © 2002 by the American Pain Society 1526-5900/02/0301-0001 $35.00/0 doi:10.1054/jpai.2002.27001

responses, visceral pain is difficult to localize and treat, because it is diffuse in character, usually referred to superficial structures, and frequently refractory to therapies that are efficacious in relieving somatic pain. In general, gastrointestinal pathologic conditions are associated with diffuse tenderness of the gut and increased sensitivity to mechanical stimuli that may contribute to the characteristic symptoms of abdominal discomfort and pain.12,15 Afferent sensory fibers that innervate the descending colon and rectum travel in nerve bundles with the efferent fibers of both the sympathetic nervous system in the splanchnic nerve and the parasympathetic nervous system in the pelvic nerve. These visceral afferents originate in the T13-L2 and the L6-S2 dorsal root ganglia with central projections to the respective segments of the spinal cord. Within the spinal cord, visceral afferents project primarily to lamina I of the dorsal horn and to the deeper laminae V to VII and X.6,8,12,13 The highest concentration of substance P receptor (SPR) in the spinal cord is found in the regions in which

The Journal of Pain, Vol 3, No 1 (February), 2002: pp 3-11

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4 visceral afferents terminate (lamina I and lamina X).5,8 In recent studies it has been shown that after noxious but not non-noxious somatic stimulation there is release of substance P (SP) in the spinal cord (presumably being released primarily from SP-containing primary afferent fibers), which in turn induces SPR internalization in SPR-expressing lamina I spinal neurons. Under somatic inflammatory conditions, up-regulation of the SPR is observed in lamina I, and both non-noxious and noxious somatosensory stimulation induce SPR internalization in SPR-expressing lamina I neurons.1,16 In addition, under somatic inflammatory conditions, there seems to be an increased ventral diffusion of SP from primary afferents because SPR-expressing neurons located in laminae III and IV also show internalization of the SPR after noxious stimulation.1,16 These results suggest that after somatic inflammation, there is substantial neurochemical plasticity so that an increased number of SPRexpressing neurons now respond to normally non-noxious and noxious somatic stimulation. SPR-expressing neurons in the spinal cord appear to play a significant role in the ascending conduction of both somatic inflammatory and neuropathic pain. Thus, although lamina I neurons that express the SPR represent only 5% to 10% of the total number of lamina I neurons, nearly all lamina I neurons that express the SPR project to higher areas of the brain that are involved in nociceptive signaling.9,23,44 These lamina I SPR-expressing neurons receive substantial input from SP-releasing sensory Cfibers and terminate within the thalamus and parabrachial area of the brain.9,23,44 Neurons within this part of the parabrachial area respond to most types of noxious (but not non-noxious) stimulation, are intensity coded, and have large receptive fields that can include the whole or 1 side of the body.2,10 Ablation of these neurons with the neurotoxin SP-saporin results in a decrease in somatic inflammatory and neuropathic pain.34 In an effort to determine the potential involvement and neurochemical plasticity of SPR-expressing neurons in visceral nociception, we explored the expression and internalization of the SPR in the spinal dorsal horn in a model of acute and inflammatory visceral pain. A wellcharacterized visceral stimulus, colorectal distention (CRD),31 was used as an acute noxious stimulus in naïve rats as well as in a model of colonic inflammation associated with visceral hyperalgesia.7 The present results show that visceral nociception induces SP release and activation of SPR-expressing spinal lamina I neurons and that both the number and rostrocaudal extent of activated SPR-expressing neurons are significantly increased under inflammatory, hyperalgesic conditions.

Material and Methods Experimental Animals Experiments were performed on 24 adult male Sprague Dawley rats (Harlan, Indianapolis, IN), weighing 400 to 425 g. Rats were kept in a vivarium, main-

Substance P Receptors in Visceral Nociception tained at 22°C, with a 12-hour alternating light-dark cycle. Animals were housed singly, with food and water available ad libitum. All experiments were performed during the light cycle. All experiments were approved by the Institutional Animal Care and Use Review Committee of The University of Iowa.

Distention Method The noxious stimulus was distention of the descending colon and rectum as previously described.31 Briefly, a rat was allowed to crawl freely into a canvas garden glove. Then, a 7-cm long latex balloon was inserted into the descending colon and rectum. The balloon was positioned such that it was 1 cm into the rectum and the balloon catheter (Tygon tubing; Fisher Scientific, Chicago, IL) was connected to a pressure control device (Bioengineering, University of Iowa, Iowa City, IA) that regulated inflation of the balloon. The descending colon and rectum were distended once to 80 mm Hg for 20 seconds, and balloon pressure was monitored for the entire 20 seconds of distention. This distending pressure is considered noxious because it is reported to be painful in human studies and is aversive in rats. Either control animals received no visceral stimulus (handling only), or the distention balloon was inserted but was not inflated.

Induction of Visceral Hyperalgesia Colorectal inflammation was induced by intracolonic administration of zymosan. Coutinho et al7 have previously shown that intracolonic injection of zymosan induces visceral hyperalgesia observable at 1 hour that reaches a maximum at 3 hours. At 3 hours after zymosan injection, an increase in the magnitude of the response to normally nonpainful CRD (20 mm Hg) was observed, in addition to an increase in the response to noxious CRD (80 mm Hg) (the observed response was a recording of the contraction of the abdominal musculature during CRD in awake rats by using electrodes implanted in the external oblique abdominal musculature, just above the inguinal ligament). At 80 mm Hg, the response under zymosan inflammation was 2.5 times greater in comparison to the response observed under noninflamed conditions. Briefly, 1 mL of a solution of zymosan (25 mg/mL, freshly prepared in sterile saline; Sigma Chemical Company, St Louis, MO) was introduced into the colon with a 7-cm feeding needle (16-gauge) attached to a 1mL syringe, with the animal under brief halothane anesthesia. Rats were returned to their cages and allowed free access to food and water. Under noninflamed conditions, animals were perfused 15 minutes after insertion of balloon or colonic distention. Under inflammatory conditions, at 3 hours after zymosan injection, animals were perfused 15 minutes after insertion of balloon or colonic distention.

Experimental Groups In noninflamed rats, the effects of balloon insertion (n = 6) or noxious CRD (n = 6) were studied and contrasted

ORIGINAL REPORT/Honoré et al with a group of noninflamed rats that received no treatment (n = 3). Zymosan-treated rats were unstimulated (n = 3), submitted to balloon insertion (n = 3), or stimulated with noxious CRD (n = 3). Control animals were sham animals. For the noninflamed animals, controls for the balloon alone were naïve rats, and controls for the noxious distention were balloon alone. For the inflamed group, controls for the zymosan group were naïve rats; controls for the balloon alone in zymosan-treated animals were (1) balloon alone in noninflamed animals and (2) zymosan alone; controls for noxious stimulation in zymosan-treated animals were (1) noxious stimulation in noninflamed animals and (2) balloon alone in zymosan-treated animals.

Immunohistochemistry At the appropriate time (15 minutes after balloon insertion or CRD, 3 hours after intracolonic zymosan instillation, or 15 minutes after balloon insertion or CRD in zymosan-treated rats), rats were deeply anesthetized with halothane and perfused intracardially with phosphate-buffered saline (PBS) followed by a solution of 4% formaldehyde and 12.5% picric acid. The spinal cord was then removed, postfixed overnight, and cryoprotected in 30% sucrose. Serial frozen sections, 60-µm thick, were cut with a microtome and collected in PBS to be processed for immunohistochemistry as free floating sections as previously described.16 The tissue sections were incubated in a blocking solution of 1% normal goat serum in PBS with 0.3% TritonX100 and were then incubated overnight in the primary antiserum. The SPR was detected by a polyclonal rabbit anti-SPR antibody (1:5,000, raised in our laboratory). The antibody used in our study was raised against a 15–amino acid peptide sequence (SPR393-407) at the COOH-terminus of the rat SPR. The immunogen consisted of synthetic peptide conjugated to bovine thyroglobulin with the use of glutaraldehyde. The antiserum recognized a protein band of 80 to 90 kd on protein immunoblots of membranes prepared from cells transfected with the rat SPR. The incubated sections were washed in PBS and incubated in the secondary antibody solution (conjugated to Cy3, 1:600). Finally, the sections were washed, mounted on gelatincoated slides, air-dried, dehydrated via an alcohol gradient, cleared in xylene, and coverslipped. To confirm the specificity of the primary antibody, controls were performed; preabsorption with the corresponding synthetic peptide or omission of any stage in the protocol abolished the staining. Because staining intensity might vary between experiments, control sections were included in each run of staining (see protocol details16).

Quantification of Immunofluorescence Levels and SPR Internalization Sections from the thoracic, lumbar, and sacral spinal cord were analyzed by fluorescent and confocal micro-

5 scopy to characterize SPR immunofluorescence and SPR internalization, with an MRC-1024 Confocal Imaging System (Bio-Rad Microscience, Hercules, CA) and an Olympus BH-2 microscope (Olympus, Melville, NY) equipped for epifluorescence.16 In all cases, immunofluorescence level for SPR and the percentage of SPR immunoreactive (SPR-IR) neurons showing SPR internalization were determined. Analysis was performed in all laminae (I to X) of the dorsal horn of the spinal cord at thoracic T13 and sacral S1 levels, the main projections for the primary afferent fibers innervating the descending colon. In addition, SPR internalization was also evaluated at the lumbar level (L4) to study the rostrocaudal extent of the effects of noxious CRD. Sagittal sections were viewed through a 1-cm2 eyepiece grid divided into one hundred 1 mm × 1 mm units. In cell bodies that do not contain internalized SPR, SPR-IR is uniformly distributed on the cell surface; in contrast, in neurons that have internalized the SPR, the cytoplasm contained bright, immunofluorescent endosomes. An endosome was defined as an intense SPR-IR intracellular organelle between 0.1 and 0.7 µm in diameter that was clearly not part of the external plasma membrane. Unstimulated cells contained less than 5 endosomes per cell. In the present study, neurons containing 20 or more endosomes were considered to be internalized. Importantly, because neurons with less than 20 endosomes were not counted, it is possible that subtle changes in the magnitude of internalization were missed. For each spinal segment, 5 sagittal sections were viewed and the total number of SPR-positive neurons in lamina I in addition to the number of internalized SPR-positive neurons were counted. For the total number of SPR-positive neurons, results are given per section. The percentage of internalized-positive neurons was calculated by the formula ([Number of internalized lamina I SPR + neurons]/[Total number of lamina I SPR + neurons]) * 100. Immunofluorescence intensities were obtained with a confocal fluorescent imaging system and analyzed with NIH Image 1.7. These results were confirmed with a 12bit SPOT2 digital camera (Diagnostic Instruments Inc, Sterling Heights, MI) on an Olympus BX-60 fluorescence microscope with Image Pro Plus v.3.0 software (MediaCybernetics, Silver Spring, MD). The digital camera’s response was measured with 540/560 nm Inspeck fluorescent bead standards (Molecular Probes, Eugene, OR).

Statistical Tests Statistical analysis was performed with a 1-way analysis of variance. For multiple comparisons, the Fisher protected least significant difference post hoc test was used. A P value less than .05 was considered significant. In all of the cases, the investigator responsible for the collection of data and their analysis was blinded to the experimental situation of each animal.

6

Substance P Receptors in Visceral Nociception

Figure 1. Confocal images illustrating the distribution of SPR-IR in coronal sections of the spinal cord at the thoracic T13 (A1) and sacral S1 (B1) levels. Confocal image of preganglionic sympathetic neurons at the thoracic level T13 (A2) and lamina X neurons at the sacral level S1 (B2). The densest SPR staining is found in lamina I. In contrast, lamina II contains almost no SPR-IR neuronal cell bodies but is traversed by SPR-IR dendrites originating from SPR-IR neurons located in laminae III-V. SPR-IR is also observed in laminae V-VI and around the central canal. In addition, SPR-IR labeling is observed in a population of preganglionic sympathetic neurons. Images (A1, B1) were obtained from 60-µm thick tissue section acquired with a ×10 lens. Scale bar = 500 µm. Images (A2, B2), obtained from 60-µm thick tissue sections, were projected from 12 optical sections acquired at 0.8-µm intervals with a ×40 lens. Scale bar = 90 µm (A2) and 25 µm (B2).

Results Distribution of SPR Immunoreactivity in the Rat Thoracic and Sacral Spinal Cord Under normal or unstimulated experimental conditions, a distinct pattern of spinal SPR-IR is observed at

the spinal levels T13, L4, and S1. The densest SPR staining is found in lamina I (Fig 1A1 and B1), and SPR-IR covers almost the entire dendritic and somatic surface of each neuron that expresses the receptor (Fig 2A and C). In contrast, lamina II contains almost no SPR-IR cell bodies (Fig 1A1 and B1) but is traversed by SPR-IR

ORIGINAL REPORT/Honoré et al

7

Figure 2. Noxious CRD induces SPR internalization in lamina I SPR-expressing neurons at thoracic T13 and sacral S1 levels in rats with zymosan-induced visceral inflammation. Confocal images of lamina I SPR-IR neurons at the thoracic (A, B) and sacral (C, D) levels in noninflamed animals (A, C) and 15 minutes after noxious CRD (B, D) in zymosan-injected rats. These images, from 60-µm thick tissue sections, are projected from 25 optical sections acquired at 0.8-µm intervals with a ×60 lens. Scale bar = 25 µm.

dendrites originating from SPR-IR neurons located, in part, in laminae III-IV (Fig 1B1). SPR-IR is also observed in spinal neurons localized in laminae V and VI, around the central canal (lamina X, Fig 1B2), and in preganglionic neurons sending transversal processes toward the central canal (Fig 1A2). In all these regions most of the SPR-IR is associated with the plasma membrane, with few SPR-IR endosomes present in the cytoplasm. In the present study, SPR staining was evaluated in all spinal regions, but results are reported for lamina I because changes were only observed in this lamina.

Acute Noxious CRD Induces a Specific Pattern of SPR Internalization SPR internalization was not observed in lamina I SPRexpressing neurons in naïve rats and rats submitted to balloon insertion alone (Table 1). In contrast, after noxious CRD, significant SPR internalization was observed in SPR-IR lamina I neurons at both the thoracic level T13 and sacral level S1. At the thoracic level, noxious CRD induced 30% ± 2% internalization (P < .0001 compared with control or balloon insertion). At the sacral level, noxious CRD induced 23% ± 4% internalization (P < .0001 compared with control or balloon insertion). In

Noxious CRD-Evoked SPR Internalization in Lamina I Neurons Is Increased After Zymosan-Induced Colorectal Inflammation Table 1.

THORACIC T13 No. of SPR + neurons per section in lamina I in naïve rat Naïve rats Balloon alone Noxious CRD Zymosan alone Balloon in zymosan-treated rats Noxious CRD in zymosan-treated rats

6.2 ± 0.8 0% ± 0% 0% ± 0% 30% ± 2%*** 0% ± 0% 30% ± 4%*** 76% ± 1%***

LUMBAR L4 8.5 ± 0.7 0% ± 0% 0% ± 0% 0% ± 0% 0% ± 0% 17% ± 10%* 63% ± 4%***

SACRAL S1 6.4 ± 0.5 0% ± 0% 0% ± 0% 23% ± 4%*** 0% ± 0% 16% ± 4%** 69% ± 3%***

NOTE. Results are expressed as number of lamina I SPR-expressing neurons per section in lamina I in naïve rats and as mean percentages of SPRexpressing neurons showing SPR internalization at the thoracic T13, lumbar L4, and sacral S1 spinal levels in the various experimental groups. Note that zymosan treatment significantly enhanced the number of lamina I SPR-expressing neurons showing SPR internalization after normally non-noxious (balloon insertion alone) or noxious visceral stimulation. Significance compared to respective control group is *P < .05, **P < .01, ***P < .001 (analysis of variance and Fisher protected least significant difference test). In noninflamed rats, significance is expressed as compared with balloon insertion alone: in zymosan-treated animals, significance for the balloon insertion group is expressed compared with the effects of balloon insertion in noninflamed animals; and significance for noxious colorectal distension is expressed compared with the effects of the same stimulation in noninflamed animals.

8 contrast, noxious CRD produced no SPR internalization at the L4 lumbar level (Table 1). SPR internalization at the thoracic level was significantly higher than at the sacral level (P < .01). No SPR internalization was observed in SPR-expressing neurons in laminae III to X in spinal segments T13-S2 in naïve animals, in animals submitted to balloon insertion, or in noninflamed animals after noxious CRD.

Noxious CRD-Evoked SPR Internalization Is Increased in Zymosan-Induced Colorectal Inflammation Spinal SPR immunofluorescence levels remained unchanged in zymosan-treated rats relative to naïve rats. In addition, no alteration in the number of SPR-IR neurons in lamina I was observed at either of the spinal cord levels considered in the present study. Three hours after intracolonic zymosan (no visceral stimulus), there was no significant SPR internalization in lamina I neurons in spinal segments T13-S2 (Table 1). However, in animals with zymosan-induced inflammation of the colon, balloon insertion induced SPR internalization in lamina I neurons at spinal levels T13, L4, and S1 (Table 1). The magnitude of SPR internalization in these rats was significantly greater at the thoracic level T13 (30% ± 4%) than L4 (17% ± 10%) or S1 (16% ± 4%) (P < .01 for both comparisons). In addition, at T13, L4, and S1 levels, balloon insertion–induced SPR internalization was significantly greater in zymosantreated rats than in noninflamed rats (Table 1). Similarly, noxious CRD-induced SPR internalization was significantly enhanced in lamina I neurons at the T13, L4, and S1 spinal levels in zymosan-treated rats over balloon insertion alone (Fig 2B and D; P < .0001 for all 3 spinal levels). At spinal levels T13, L4, and S1, 76% ± 1%, 63% ± 4%, and 69% ± 3%, respectively, of SPR-IR lamina I neurons exhibited SPR internalization (Table 1). Also, noxious CRD-induced SPR internalization was significantly greater in zymosan-treated animals than in noninflamed animals (P < .0001 for all 3 spinal levels). No SPR internalization was observed in SPR-expressing neurons in laminae III to X in spinal segments T13-S2 in inflamed animals, in inflamed animals submitted to balloon insertion, or in inflamed animals after noxious CRD.

Discussion

Substance P Receptors in Visceral Nociception (L6-S2) dorsal root ganglia,6,8,12,13 which have previously been documented to contain SP.8,28 Internalization of the SPR was not observed in lamina I neurons located at the L4 spinal segment after acute, noxious CRD. Previous studies with other markers are in agreement with the present results. For example, Fos protein is expressed in L6-S2 spinal segments after noxious CRD45 or noxious urinary bladder distention3,4 consistent with the innervation of both pelvic organs by the pelvic nerve. In addition, it has been shown with fast dye or horseradish peroxidase that primary afferent fibers innervating the colon or rectum originate from the thoracolumbar (T13L2) and lumbosacral (L6-S2) dorsal root ganglia.20,21 Finally, electrophysiologic studies have shown that 35% of the pelvic nerve fibers recorded in the first sacral root responded to colonic distention38,40 and that spinal neurons in spinal laminae I and V at both the thoracolumbar (T13-L2) and lumbosacral (L6-S2) levels respond to noxious CRD in the rat.29,30,32 Acute release of SP in the spinal cord and subsequent internalization of the SPR after noxious CRD suggest that spinal SPRs are activated in some types of acute visceral pain. It has been shown that SPR antagonists inhibit nociceptive reflex responses to chemical stimulation of the gallbladder35 and to jejunal distention.26 In addition, SPR antagonists inhibit abdominal contractions induced by irritants such as phenylbenzoquinone or acetic acid,11,37 although not acetylcholine.39 In contrast, SPR antagonists were ineffective in attenuating nociceptive reflex responses to rectal17 or colonic distention.18 Interestingly, whether given systemically or intrathecally, antagonists at the SPR were ineffective when given alone,18 but a combination of SPR and neurokinin-2 receptor antagonists given into the intrathecal space significantly attenuated responses to noxious CRD in the rat.18 Recently, it has been reported that SPR knockout mice exhibit normal responses to CRD but are unable to encode the intensity of colonic distention and showed a profound deficit in their response to colonic instillation of capsaicin.22 In related experiments, SPR knockout mice revealed an impaired response to cyclophosphamide-induced experimental cystitis and had no acute reflex or primary hyperalgesia to intracolonic acetic acid injection.22 Together, these results suggest that the SPR is clearly one of at least several receptors in the spinal cord involved in signaling acute visceral pain.

SP Release in Acute Visceral Nociception

Neurochemical Plasticity in the Spinal Cord After Visceral Inflammation

The present results show that SP is released after an acute noxious visceral stimulus, in this case, CRD, complementing similar studies that have used noxious somatic stimuli.1,16 Importantly, SP release and subsequent SPR internalization were confined to anatomically appropriate spinal segments. The distal colon of the rat is innervated by afferent fibers whose cell bodies are located in thoracolumbar (T13-L2) and lumbosacral

A significant finding in the present study was that in the presence of zymosan-induced visceral inflammation, either normally non-noxious (balloon insertion) or noxious CRD induces a greater activation of lamina I neurons that express the SPR than is found in animals without visceral inflammation. Intracolonic instillation of zymosan induced exaggeration of visceromotor responses to CRD as early as 2 hours after instillation and

ORIGINAL REPORT/Honoré et al lasted for 36 to 48 hours7 (D. Beck and G. F. Gebhart, unpublished observations). In the periphery, recordings from pelvic nerve afferent fibers teased from the S1 dorsal rootlet reveal that sensitization of responses to CRD are apparent by 30 minutes after intracolonic instillation of zymosan.14 The visceral hyperalgesia produced in this model is evident across the dynamic range of distending stimuli (10 to 80 mm Hg) and reaches a maximum 3 hours after zymosan instillation into the colon. The present results suggest that recruitment of additional spinal lamina I neurons that express the SPR could contribute to exaggerated responses to both noxious and non-noxious intensities of CRD and also contribute to the diffuse character of visceral inflammatory pain because neurons outside the spinal segments that normally are activated in the noninflamed state are activated in the inflamed state. These observations correlate well with clinical studies that show that in patients with functional dyspepsia27 or irritable bowel syndrome,41 there is a significantly increased area of referred sensation, principally discomfort and pain, when compared with normal subjects. The absence of ongoing activation of SPR-expressing lamina I neurons in the absence of CRD in zymosantreated rats is consistent with behavioral observations. Zymosan-treated rats do not exhibit spontaneous pain behaviors or behavior suggestive of distress. Rather, they are typically quiet and exhibit reduced exploratory activity7 and thus appear to be attempting to minimize mechanically induced stimulation of sensitized primary afferent sensory fibers. Similarly, patients with inflammatory or functional bowel disorders typically do not have ongoing pain in the absence of movement but rather complain of discomfort and pain associated with ingestion, gas, and probing.42,43 Absence of ongoing SP activation of SPR-expressing lamina I neurons in the absence of CRD in zymosan-treated rats is consistent with the lack of increase in SPR immunoreactivity because it has been shown that up-regulation of the SPR is activity dependent.24,25 The increase in SPR messenger RNA observed in the spinal cord after peripheral inflammation has been reported to be blocked by morphine or SPR antagonists,24,25 which suggests that SP release and/or SPR activation is necessary for SPR upregulation. One major difference concerning the spinal cord in somatic versus visceral inflammation is the activation of SPR-expressing neurons in the deep laminae (III to X). After peripheral somatic inflammation induced by injection of carrageenan or complete Freund’s adjuvant into the hind paw, normally non-noxious or noxious stimulation of the inflamed hind paw induces internalization of the SPR in spinal neurons located in laminae I and III to V. In contrast, with visceral inflammation induced by intracolonic injection of zymosan, normally non-noxious or noxious mechanical stimulation of the colon only induces internalization of SPR-expressing neurons in lamina I, with SPR-expressing neurons lo-

9 cated in the deep spinal laminae (III to X) never showing internalization. This absence of SPR internalization in neurons located in the deep laminae, especially lamina X, was unexpected because it has been reported that 65% of visceral afferents that innervate the colon express SP and project primarily to lamina I of the dorsal horn and in deeper laminae V to VII and X.6,8,12,13

Referred Pain and the Recruitment of Lamina I SPR-Expressing Neurons in Inflammatory Visceral Pain One hypothesis as to the basis for referral of visceral sensations to somatic structures is convergence in the spinal cord of somatic and visceral inputs onto the same second order neurons.36 As indicated previously, spinal visceral afferent fibers terminate centrally in lamina I, deeper in the dorsal horn (lamina V and interomediolateral cell column) and in lamina X, a pattern of termination that overlaps significantly with the central terminations of somatic nociceptors.46 Consistent with this pattern of convergence, the principal conscious sensations that arise from the viscera are discomfort and pain. Stimuli that are adequate for the majority of visceral sensory receptors are rarely perceived (eg, mechanical activation of baroreceptors, chemical activation of gastric or hepatic receptors). For hollow organs, particularly those associated with the gastrointestinal tract, the adequate stimulus is a mechanical, distending stimulus such as used here. In the absence of organ inflammation, acute noxious distention of the colon produces central consequences readily observed in thoracolumbar and lumbosacral spinal cord. In electrophysiologic studies the convergent cutaneous receptive fields in naïve rats are anatomically appropriate to the spinal thoracolumbar or lumbosacral segments. 19,29,30 After experimental colonic irritation/inflammation, the sizes of convergent cutaneous receptive fields expand. In addition, responses of spinal neurons increase after colonic inflammation, and the stimulus-response function of these neurons shifts to the left33 as would be expected as visceral hyperalgesia develops. Thus, lower intensities of colonic distention such as that induced by balloon insertion alone resulted in SPR internalization in zymosantreated rats in all 3 spinal segments. In conclusion, the present data offer a unique view of the activation of a specific receptor in spinal cord neurons involved in the ascending conduction of visceral pain. In the noninflamed animal, SPR-expressing lamina I neurons are only activated after noxious stimulation. In contrast, in the presence of visceral inflammation, normally non-noxious and noxious CRD induces activation of these neurons as previously demonstrated for somatic inflammatory pain. However, in contrast to what has been shown under somatic inflammatory conditions, in visceral inflammation, SPR internalization is limited to lamina I neurons and does not diffuse to deeper laminae neurons.

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Substance P Receptors in Visceral Nociception

Nevertheless, in the presence of visceral inflammation, there is a significant recruitment of new populations of lamina I SPR-expressing neurons because lamina I neurons located in spinal segment T13 to S1 are now responding to visceral stimulation. This increase in both the number and rostrocaudal extent of lamina I SPR-expressing neurons activated could, in part, account for the extension of neuronal receptor field and the diffuse character of inflammatory visceral pain. Understanding the differences in terms of both

We would like to thank Ma’Ann C. Sabino for her helpful comments and discussion.

References

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neurotransmitters/receptors and population of neurons that are responsible for the ascending conduction of visceral versus somatic pain should aid in the development of new and more effective analgesics for visceral pain.

Acknowledgments

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