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GASTROENTEROLOGY 2004;127:166–178

planchnic and Pelvic Mechanosensory Afferents Signalifferent Qualities of Colonic Stimuli in Mice

TUART M. BRIERLEY,*,‡ R. CARTER W. JONES III,§,� GERALD F. GEBHART,§ and. ASHLEY BLACKSHAW*,‡,¶

Nerve-Gut Research Laboratory, Hanson Institute, Department of Gastroenterology, Hepatology & General Medicine, Royal Adelaideospital, Adelaide, South Australia, Australia; ‡Discipline of Physiology, School of Molecular & Biomedical Sciences, The University ofdelaide, Adelaide, South Australia, Australia; §Department of Pharmacology and �Medical Scientist Training Program, The University of Iowa,owa City, Iowa; and ¶Department of Medicine, The University of Adelaide, Adelaide, South Australia, Australia

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ackground & Aims: Mechanosensory information fromhe colon is conducted via lumbar splanchnic nervesLSN) and sacral pelvic nerves (PN) to the spinal cord.he precise nature of mechanosensory information en-oded by each pathway has remained elusive. Here, weharacterize and directly compare the properties ofechanosensitive primary afferents from these 2 path-ays. Methods: Using a novel in vitro mouse colon prep-ration, mechanosensitive primary afferents were re-orded from the LSN and PN and distinguished based onheir response to receptive field stimulation with 3 dis-inct mechanical stimuli: probing (70 mg–4 g), circulartretch (1–5 g), and mucosal stroking (10–1000 mg).esults: Five different classes of afferent were recorded

rom the LSN and PN. Three of these classes of afferentserosal, muscular, and mucosal) were conserved be-ween both pathways; however, their respective propor-ions, receptive field distributions, and response proper-ies differed greatly. In general, these 3 classes offferent recorded from the PN responded to lower stim-lation intensities, displayed greater response magni-udes, and adapted less completely to mechanical stim-lation compared with their LSN counterparts. Inddition, the LSN and PN each contain a specializedlass of afferent (mesenteric and muscular/mucosal),hich is unique to their respective pathway. Conclusions:he splanchnic and pelvic pathways contain distinct pop-lations of mechanosensitive afferents. These afferentsre capable of detecting an array of mechanical stimulind are individually tuned to detect the type, magnitude,nd duration of the stimulus. This knowledge contrib-tes to our understanding of the role that these 2 path-ays play in conveying mechanical information from theolon.

nhanced colonic mechanosensation is the hallmark ofthe functional bowel disease irritable bowel syn-

rome (IBS). Since its original description by Ritchie in973,1 increased perception of mechanical distention of

he distal colon/rectum has become the best characterizedlinical manifestation of IBS. This finding exists in theelative absence of overt colon pathology, suggestingaladaptive changes in the function of colonic mech-

nosensory pathways. Indeed, evidence from behavioralnd functional imaging studies of patients with IBSuggest that these changes occur at the level of therimary afferent neuron and/or spinal cord but not inigher cortical centers,2,3 thereby supporting the notionhat peripheral mechanosensation plays an important rolen the etiology of this disease.

Sensory information from the distal colon/rectum trav-ls to the central nervous system through 2 distinctnatomic pathways: the lumbar splanchnic nerves (LSN),hich terminate in the thoracolumbar spinal cord, and

he paired pelvic nerves (PN), which terminate in theumbosacral spinal cord. Mechanosensitive afferents haveeen identified in both of these nerve supplies using aombination of in vivo and in vitro electrophysiologicechniques in the cat4–6 and the rat.7–11 These studiesuggest important differences in the nature and sensitiv-ty of mechanosensory afferents in the LSN and PN, anding that would imply unique sensory signalling func-ions for these 2 nerve supplies. A meaningful functionalomparison of the 2 pathways is prevented, however, byhe diversity of species and experimental techniques thatave been used to characterize one or the other of theseathways individually, and, to date, no study that we areware of has compared the various classes of mechano-ensitive afferent in the pelvic and splanchnic pathwayssing the same technique in the same species. Theresent study had 2 purposes: (1) to address the lack of

Abbreviations used in this paper: LSN, lumbar splanchnic nerve; PN,elvic nerve.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2004.04.008

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July 2004 MECHANOSENSORY COLONIC AFFERENTS 167

undamental knowledge of afferent signalling from theistal colon by using an in vitro electrophysiologic ap-roach to directly compare the mechanosensitive affer-nts found in the LSN and PN; and (2) to document theechanosensory properties of colonic afferents in wild-

ype C57BL/6 mice, a mouse strain commonly used toenerate a growing library of transgenic mice, some ofhich have proven invaluable for elucidating mechano-

ransduction mechanisms in the skin.12,13

Our data indicate that mechanosensitive afferents existn both nerves that respond to mechanical stimulation ofhe colonic mucosa, muscle layer, serosa, and/or mesen-ery but that the splanchnic and pelvic pathways exhibitajor differences in the proportion of each class, the

ocation of their receptive fields, and their sensitivity toechanical stimulation. This study provides definitive

vidence that LSN and PN are distinct, not only ana-omically but also functionally and likely serve uniqueoles in the detection of mechanical stimuli in the distalolon.

Materials and MethodsAll experiments were performed in accordance with

he guidelines of the Animal Ethics Committees of the Insti-ute for Medical and Veterinary Science and the University ofdelaide, Adelaide, Australia, and the Institutional Animalare and Use Committee of The University of Iowa, Iowa City.

In Vitro Mouse Colonic Primary AfferentPreparations

Male and female mice (C57BL/6) 20–30 g were killedia CO2 inhalation and cervical dislocation. The colon (5–6m) and mesentery (containing the lumbar colonic nerves)ere removed intact, along with either the attached neurovas-

ular bundle containing the inferior mesenteric ganglion andSN or in separate preparations with the major pelvic ganglionnd PN. The tissue was transferred to ice-cold Krebs’ solution,nd, following further dissection, the distal colon was openedongitudinally along the antimesenteric border to orientateumbar colonic insertions to lie along the edge of the openreparation. The tissue was pinned flat, mucosal side up, in apecialized organ bath consisting of 2 adjacent compartmentsachined from clear acrylic (Danz Instrument Service, Ad-

laide, South Australia, Australia), the floors of which wereined with Sylgard (Dow Corning Corp., Midland, MI). TheN or neurovascular bundle containing the LSN was extended

rom the tissue compartment into the recording compartmenthere they were laid onto a mirror. A movable wall with a

mall “mouse hole” (to allow passage of the nerves) was low-red into position and the recording chamber filled witharaffin oil. The colonic compartment was superfused with a

odified Krebs’ solution (in mmol/l: 117.9 NaCl, 4.7 KCl, 25aHCO3, 1.3 NaH2PO4, 1.2 MgSO4(H2O)7, 2.5 CaCl2, 11.1

-glucose, 2 sodium butyrate, and 20 sodium acetate), bubbledith carbogen (95% O2/5% CO2) at a temperature of 34°C.ll preparations contained the L-type calcium channel antag-nist nifedipine (1 �mol/L) to suppress smooth muscle activitynd the prostaglandin synthesis inhibitor indomethacin (3mol/L) to suppress potential inhibitory actions of endogenousrostaglandins.10 Under a dissecting microscope, the LSNere dissected away from the neurovascular bundle and theerve sheath surrounding the LSN or PN peeled gently backxposing the nerve trunk. Using fine forceps, the nerve trunkas teased apart into 6–10 bundles, which were individuallylaced onto a platinum recording electrode. A platinum ref-rence electrode rested on the mirror in a small pool of Krebs’olution adjacent to the recording electrode.

Characterization of Lumbar Splanchnic andPelvic Afferent Properties

Receptive fields were identified by systematicallytroking the mucosal surface or the mesenteric attachmentith a brush of sufficient stiffness to activate all types ofechanosensitive afferent. Once identified, receptive fieldsere tested with 3 distinct mechanical stimuli to enable

lassification: probing with calibrated von Frey hairs (70, 160,00, 1000 mg, 2 g, and 4 g force; each force applied 3 timesor a period of 3 seconds), mucosal stroking with calibrated vonrey hairs (10, 200, 500, and 1000 mg force; each forcepplied 10 times), and circular stretch (1–5 g in 1-g incre-ents; each weight applied for a period of 1 minute, with a

-minute interval between each application). Stretch was ap-lied using a claw made from bent dissection pins attached tohe tissue adjacent to the afferent receptive field and connectedo a cantilever system via thread. Weights were applied to thepposite side of the cantilever system to initiate graded colonictretch. Only circular, and not longitudinal, stretch was testedn this study. Colonic afferents were characterized using thelassification system previously applied in the rat colon.7,10,11

Data Recording and Analysis

Electrical signals generated by nerve fibers placed onhe platinum recording electrode were fed into a differentialmplifier, filtered, sampled at a rate of 20 kHz using a 1401nterface (Cambridge Electronic Design, Cambridge, Unitedingdom) and stored on a PC. The amplified signal was alsosed for on-line audio monitoring. Action potentials werenalyzed off-line using the Spike 2 wave mark function andiscriminated as single units on the basis of distinguishableaveform, amplitude, and duration. A maximum of 2 activenits on each recorded strand was allowed to avoid errors iniscrimination. Data are expressed as mean � SEM (n � theumber of individual afferents). Adaptation profiles to probingere calculated as the mean number of spikes per 100 milli-

econd bin over the entire 3 seconds of a 1-g probing stimulus.

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168 BRIERLEY ET AL. GASTROENTEROLOGY Vol. 127, No. 1

daptation profiles to stretch were calculated as the meanumber of spikes per 10-second bin over the entire 60 secondsf a 3-g circular stretch. Curve slopes were derived fromegression analyses performed on stimulus-response functionsnd adaptation data to compare the gain and rates, respec-ively, of afferent responses. The limited number of stimulised at lower stimulus intensities prevented meaningful ex-rapolation of afferent thresholds to von Frey probing, mucosaltroking, and circular stretch. Extrapolation of threshold val-es for stimulation based on a linear regression analysis oftimulus-response functions was also not performed because ofhe nonlinear nature of the data within this stimulus range andonsensical (i.e., negative) x-intercepts that were obtainedrom this analysis. Therefore, to compare the sensitivity ofplanchnic and pelvic afferents with von Frey probing, theercentage of afferents that responded to each probe force wasalculated, based on the total number of fibers tested in eachfferent class (“percent responding”). Data were analyzed usingrism 4 software (GraphPad Software, San Diego, CA) and,hen appropriate, were analyzed using a 2-way analysis ofariance (ANOVA) or repeated measures, 2-way ANOVA toetermine significant differences between curves. Differencesere considered significant at a level of P � 0.05.

ResultsBasic Mechanosensory Properties

Four classes of splanchnic afferent fiber could beistinguished from one another by their responses toechanical stimuli (Figure 1A). These 4 fiber classesere termed mesenteric, serosal, muscular, and mucosal. All 4

lasses were responsive to von Frey probing. However, itas their location and response, or lack thereof, to thether types of mechanical stimuli (i.e., fine mucosaltroking and circular stretch) that clearly determinedheir class. Mesenteric afferents had receptive fields thatere located on the mesenteric attachment directly ad-

acent to the colon and responded in a graded manner torobing of their receptive fields with calibrated von Freyairs (Figure 1A, i). Because of their location on theesenteric attachment, these afferents were not testedith circular stretch or mucosal stroking. Serosal affer-

nts had receptive fields located in the colonic wall thatere reproducibly activated only by probing and did not

espond to colonic stretch or fine mucosal stroking with10-mg von Frey hair (Figure 1A, ii). Muscular afferentsad receptive fields in the colonic wall that were opti-ally activated by probing and maintained circular

tretch �2 mm but were not responsive to fine mucosaltroking (Figure 1A, iii). In response to circular stretch,uscular afferents responded with an excitation that

dapted completely during the stimulus (waning to pre-timulus levels within 10–20 seconds). Both serosal and

uscular afferents responded to mucosal stroking atigher stimulus intensities (500-1000 mg; data nothown), which we observed to distort the underlyinguscle and serosal tissue layers. However, mucosal re-

eptors were most sensitive to stroking of their receptiveelds with a brush or a 10-mg von Frey hair (Figure 1A,v). Their exact thresholds to mucosal stroking could note determined because smaller hairs would not penetratehe surface tension of the super fusate. These afferentsesponded in a graded manner to an ascending series oftroking stimuli (10–1000 mg; data shown for 10 mg)ut not to circular stretch.All classes of lumbar splanchnic afferent had small

�0.5 mm) punctate receptive fields from which re-ponses could be most readily evoked. The vast majorityf splanchnic afferents possessed a single mechanosensi-ive receptive field; only a small minority (15%) ofesenteric afferents possessed more than 1 receptive

eld, a property previously described for LSN mesentericfferents.7,14–17 Overall, the majority of mesenteric, se-osal, and muscular afferents and all mucosal afferentsecorded from the LSN were silent at rest. However, ainority of mesenteric (27%), serosal (37%), and mus-

ular (40%) afferents in this pathway did exhibit lowates of spontaneous activity (0.38 � 0.12 spikes/s,.48 � 0.09 spikes/s, 0.68 � 0.36 spikes/s, respectively)hat were not significantly different among fiber classesP � 0.05). The discharge rate of spontaneously activeSN afferents that we observed was similar to thosereviously reported.4,10

Pelvic afferents, similar to splanchnic afferents, coulde categorized into 4 different classes; 3 were also foundn the LSN (serosal, muscular, and mucosal), and 1 wasnique to the PN (muscular/mucosal). All PN afferentsesponded in a graded manner to probing of their recep-ive field but could be distinguished from one another byheir sensitivity to fine mucosal stroking and circumfer-ntial stretch. Unique to the PN were afferents thatesponded to both fine stroking and stretch, and thislass was therefore termed muscular/mucosal (Figure 1B, i).erosal, muscular, and mucosal afferents in the PN pos-essed the same general response properties as their re-pective counterparts in the LSN: Serosal afferents re-ponded only to probing of their receptive field (FigureB, ii), muscular afferents responded to stretch but notne stroking (Figure 1B, iii), and mucosal afferents re-ponded to fine stroking but not stretch (Figure 1B, iv).otably, mesenteric afferents, often recorded from the

planchnic nerve, were never found in the pelvic nerve.o pelvic afferents exhibited spontaneous activity, a trait

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igure 1. (A) Four types of lumbar splanchnic afferent fiber classified on the basis of their receptive field location and response to mechanical stimuli.i) Mesenteric afferents responded in a graded manner to an ascending series of probing stimuli (70 mg–4 g). (ii) Serosal afferents were activated onlyy probing their receptive field. (iii) Muscular afferents were activated by probing and maintained circular stretch but did not respond to fine mucosaltroking (10 mg). (iv) Mucosal afferents were activated by probing and fine mucosal stroking but did not respond to circular stretch. (B) Four types ofelvic afferent classified on the basis of their responses to mechanical stimuli. (i) Muscular/mucosal afferents were activated by probing, stretch, andne mucosal stroking (10 mg). (ii) Serosal afferents were activated by probing of their receptive field and did not respond to maintained circular stretchr fine mucosal stroking. (iii) Muscular afferents were activated by probing and circular stretch but did not respond to fine mucosal stroking. (iv) Mucosalfferents were activated by probing and fine mucosal stroking but not stretch. Upper traces show instantaneous firing frequency and lower traces show
aw electrophysiologic data. Horizontal bars indicate application of stimulus. Scale bars apply throughout except where indicated.

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imilar to that previously reported for mechanosensitiveelvic afferents in vivo6,18 and possessed single receptiveelds that were, in general, punctate like those found inhe LSN, although no formal analysis of their exactimensions was performed.

Topographic and Population Distribution ofAfferent Subtypes

Of the fibers recorded from the LSN, 50% hadeceptive fields in the mesenteric attachment (Figure 2And 2B). These were scattered along the mesentery,rimarily oral to the point at which the LSN and inferioresenteric artery branch out to form the lumbar colonic

igure 2. Distribution and proportions of afferent classes recordedrom the splanchnic and pelvic pathways. (A) Splanchnic receptiveelds were concentrated on or near the mesenteric attachment andere scattered down the entire length of the colon except for the

ectum and anal canal. (B) The majority of splanchnic afferents en-ountered were mesenteric (striped segment) and serosal afferentsgrey segment), with the remaining few composed of muscular (blackegment) and mucosal afferents (white segment). (C) Pelvic afferenteceptive fields were scattered across the entire width of the colonnd were generally clustered in the lower region of distal colon andectum. No receptive fields were found on the mesentery. (D) Theargest population of pelvic afferents encountered were serosal (greyegment), with similar proportions of muscular (black segment), mu-osal (white segment), and muscular/mucosal (checkered segment)fferents. IMG, inferior mesenteric ganglion; LSN, lumbar splanchnicerve; PN, pelvic nerve; MPG, major pelvic ganglion.

erves and lumbar colonic arteries. Commonly, mesen-eric afferent receptive fields were located on or near theserimary lumbar colonic blood vessels. Serosal afferentsere the second most abundant class of afferent fiber

36%; Figure 2A and 2B). Their receptive fields wereoncentrated near the mesenteric attachment throughouthe entire length of the colon except for the rectum andnal canal. Muscular and mucosal afferents were rela-ively scarce, consisting of only 10% and 4%, respec-ively, of the afferents recorded from the LSN (Figure 2And 2B). Muscular afferents were clustered in the loweregions of the distal colon, whereas mucosal afferentsere located more proximally. In general, the distribu-

ion of LSN afferent receptive fields located within theolonic wall (i.e., serosal, muscular, and mucosal) wasoncentrated near the mesenteric attachment, and noneere found more than 180 degrees around the colon from

he mesenteric attachment (Figure 2A), a pattern de-cribed previously in a similar in vitro preparation of theumbar splanchnic innervation of the rat colon.10,11

Compared with the LSN, the afferent population ofhe PN had a very different representation and receptiveeld distribution (Figures 2C and 2D). Receptive fieldsf pelvic afferents were distributed throughout the distalcentimeters of the colon, including the rectum and anal

anal, a region devoid of splanchnic receptive fields inhis study. Consequently, the majority of pelvic receptiveelds was situated distal to those of splanchnic afferents.lso, pelvic afferent receptive fields were found through-ut the circular axis of the colon, and, unlike those foundn the LSN, were not clustered near the mesentericttachment (Figure 2C). No obvious differences wereoted in the distribution of receptive fields among the 4lasses of PN afferents. Like the LSN, serosal afferentsere the most abundant class found in the PN (33%),ut considerably more afferents responsive to fine muco-al stroking and circular stretch were found in the PNhan the LSN, with mucosal, muscular, and muscular/ucosal afferents together comprising over two thirds

23%, 21%, and 23%, respectively) of all mechanosen-itive pelvic afferents (Figure 2D). As stated above, noesenteric afferents were found in the PN.

Dynamic Responsiveness to GradedProbing

All LSN afferents displayed graded responses ton ascending series of probing stimuli with similar stim-lus-response functions and adaptation profiles despiteajor differences in their receptive field location (Figure

A and 3B). No significant differences were detected inither the magnitude or the slope of stimulus-response

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unctions (Figure 3A) or adaptation curves (Figure 3B)mong the 4 classes of splanchnic afferents (P � 0.05 forll analyses). To compare the sensitivity of splanchnicfferents to probing, we plotted the percentage of affer-

igure 3. Mechanosensitivity, adaptation, and activation characteristairs. (A and D) Stimulus-response functions of splanchnic and pelvicraded responses to increasing probing stimuli (70 mg–4 g). Groupedifferent (P � 0.05). However, within each pathway, probing responlopes of stimulus-response functions significantly different (P � 0.0fferent responses during a 3-second application of a 1-g probe. The� 0.05). Among pelvic afferents, the adaptation profile of mucosa

fferents (E; P � 0.05). (C and F ) von Frey hair force required to activatfferents in the LSN were activated by probe intensities �1g, comparignificantly more sensitive to probing than all other LSN afferent clasprobing force of 2 g was required to activate all LSN afferents. (F )ost sensitive to probing among all pelvic afferents, with a significanfferent classes. In addition, more mucosal afferents were activatedrobing force of 1 g was required to activate 100% of PN afferents.

nts in each class that were activated by the range ofrobe forces used in this study (Figure 3C). All mucosalplanchnic afferents were activated by the lowest probingorce (70 mg). Significantly fewer serosal (21%; 4 of 19),

f splanchnic and pelvic afferents to graded stimulation with von Freyrents to probing. All afferent subtypes from both pathways displayedonses to probing of splanchnic and pelvic afferents were significantlyf individual afferent classes did not differ significantly nor were theall analyses). (B and E) Adaptation profiles of splanchnic and pelvictation curves of all splanchnic afferents displayed similar slopes (B;rents was significantly shallower than those of serosal or muscularanchnic and pelvic afferents. Significantly fewer serosal and muscularth similar afferents in the PN (P � 0.05). (C) Mucosal afferents werewith 100% recruited by the lowest probe intensity tested (P � 0.05).trast to splanchnic afferents, muscular/mucosal afferents were the

ifferent percentage responding curve compared with the other pelvicrobing intensities �1 g than were serosal afferents (P � 0.05). A

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uscular (20%; 1 of 5), and mesenteric (34%; 9 of 26)planchnic afferents were activated by this force (P �.05; Figure 3C). The percentage of these afferent classesctivated by ascending probing stimuli increased simi-arly until 100% were activated by 2-g and 4-g probingorce (P � 0.05; Figure 3C). Splanchnic afferents in the

esentery and serosa, despite originating from differentissue types, were notably similar in their responsiveness,daptation profiles, and activation thresholds to probingFigure 3A–C).

Pelvic serosal, muscular, mucosal, and muscular/mu-osal afferents all demonstrated graded responses to prob-ng that adapted throughout the stimulus (Figure 3Dnd 3E). No significant differences were detected amonghe 4 pelvic afferent classes, in terms of either the mag-itude or the slope of the probing stimulus-responseunctions (P � 0.05 for all analyses). However, amongelvic afferents, the adaptation profile of mucosal affer-nts was significantly shallower than those of serosal oruscular afferents (P � 0.05). All muscular/mucosal

fferents were activated by the lowest probing stimulus70 mg), whereas significantly fewer serosal, muscular,nd mucosal afferents (28%, 5 of 18; 42%, 5 of 12; and2%, 8 of 13, respectively) were activated by this probentensity (P � 0.05; Figure 3F). Probing with a 1-g vonrey hair activated 100% of all 4 classes of pelvic affer-nts.

The data described above on the dynamic mechanosen-ory properties of mouse splanchnic and pelvic afferentsonfirm that responses to von Frey probing discriminateoorly among afferent classes compared with stimuliirected at specific tissue layers (fine mucosal strokingnd circular stretch).

Comparison of Dynamic MechanosensoryProperties Between Splanchnic and PelvicAfferents

The most prevalent afferent classes encounteredn both the LSN and the PN were serosal and muscularfferents. A comparison of the dynamic properties ofhese afferents revealed that they differed considerablyetween the 2 pathways. Despite the fact that serosalfferents from both nerves displayed graded responseso increasing probing stimuli, splanchnic afferentsere significantly less responsive to stimulation thanelvic afferents (P � 0.001, Figure 4A). Splanchnicnd pelvic serosal afferents also displayed significantlyifferent adaptation profiles to probing (P � 0.001,igure 4B). Splanchnic serosal afferents adapted moreompletely to background activity levels than those inhe pelvic pathway, taking 0.3 seconds compared with

.4 seconds to adapt to 50% of initial response, re-pectively. Furthermore, splanchnic afferents adaptedo 25% of initial response in 1.1 seconds, whereaselvic afferents adapted only to 32% of initial re-ponse by the end of the 3-second stimulus. Althoughimilar proportions of splanchnic (21%, 4 of 19) andelvic (28%, 5 of 18) serosal afferents responded to theowest probing stimulus, pelvic afferents had a signif-cantly higher percent responding curve, with 100%ctivated by a 1-g probe compared with only 70% ofplanchnic serosal afferents (P � 0.05; compare Fig-res 3C and 3F). Therefore, we observed that splanch-ic serosal afferents required higher intensity probingtimuli to be activated, evoked smaller responses to aiven probing stimulus, and adapted more completelyo probing than pelvic serosal afferents.

A comparison of the functional properties of mus-ular afferents in the LSN and PN revealed differencesomparable with those observed between serosal affer-nts in the 2 pathways (Figure 4C–F). Splanchnicuscular afferents were less responsive to von Frey

robing than pelvic muscular afferents, with signifi-antly lower stimulus-response functions (P � 0.001,igure 4C) and percentage responding curves, with5% of splanchnic and 42% of pelvic afferents re-ponding to a 70-mg probe and 75% of splanchnic and00% of pelvic muscular afferents activated by a 1-grobe (P � 0.05, compare Figures 3C and 3F). Inontrast to serosal afferents, there were no significantifferences in the slopes of the adaptation profiles torobing stimuli when comparing splanchnic and pel-ic muscular afferents (P � 0.05). Overall, these datahow that pelvic muscular afferents evoked larger re-ponses to probing stimuli and were activated byower probing stimulus intensities than splanchnicuscular afferents. The functional differences between

planchnic and pelvic muscular afferents were alsoanifest in their response to stretch. Pelvic muscular

fferents were more responsive to circular stretch thanplanchnic afferents, with significantly higher stretchtimulus-response functions (P � 0.01, Figure 4E)nd stretch adaptation profiles (Figure 4F). Splanchnicuscular afferents adapted completely to background

evels within 20 seconds of the onset of the stretchtimulus (Figure 1A (iii) and Figure 4F), whereas mostelvic muscular afferents fired throughout the entireuration of the 1-minute stretch (Figure 1B (iii) andigure 4F). Thus, in response to stretch, pelvic mus-ular afferents evoked larger responses and adaptedess completely than splanchnic muscular afferents.

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igure 4. Comparison of serosal and muscular afferents between splanchnic and pelvic pathways. (A) Stimulus-response functions of splanchnicn � 19) and pelvic (n � 18) serosal afferents to probing. Serosal afferents from both pathways displayed graded responses to increasing probingtimuli (70 mg–4 g). However, pelvic serosal afferents were significantly more sensitive to probing, displaying larger stimulus-response functionsP � 0.001; 2-way ANOVA) and steeper slopes than splanchnic serosal afferents (P � 0.001; PN slope, 29.63 � 4.31 vs. LSN slope, 15.02 �.88). (B) Adaptation profiles of serosal afferents recorded from the PN (n � 18) and LSN (n � 19) to a 3-second, 1-g probing stimulus. The pelvicesponse was significantly larger throughout (P � 0.001, n � 18 vs. n � 19, respectively) and fitted a linear regression, whereas splanchnic dataas nonlinear. However, splanchnic serosal afferents demonstrated significantly faster adaptation over the first 0.5 seconds of the response

P � 0.01) and more complete adaptation than pelvic serosal afferents. (C) Stimulus-response functions of muscular afferents in the LSN (n �) and PN (n � 12) to graded probing stimuli. Muscular afferents from both pathways displayed graded responses to increasing probing stimuli70 mg–4 g). However, pelvic muscular afferents are more sensitive to probing, displaying significantly larger stimulus-response functions (P �.001) with steeper slopes than splanchnic muscular afferents (P � 0.001; PN slope, 30.89 � 5.5 vs. LSN slope, 9.01 � 3.3). (D) Adaptationrofiles of splanchnic (n � 5) and pelvic (n � 12) muscular afferents to a 3-second, 1-g probing stimulus. Pelvic muscular afferents respondedignificantly higher throughout the stimulus than splanchnic muscular afferents (P � 0.001); however, there was no difference in the slopes ofhe 2 curves (P � 0.05). (E) Circular stretch stimulus-response functions of splanchnic (n � 5) and pelvic (n � 12) muscular afferents. All pelvicnd splanchnic muscular afferents responded to the full range of stretch stimuli (1–5 g). However, pelvic muscular afferents were significantlyore responsive to stretch, displaying higher stimulus-response functions (P � 0.001) with steeper slopes than those of splanchnic muscularfferents (P � 0.001; PN slope, 0.30 � 0.03 vs. LSN slope, 0.11 � 0.02). (F ) Adaptation profiles of splanchnic and pelvic muscular afferentso a 1-minute, 3-g stretch. Pelvic muscular afferents displayed significantly more spikes per 10-second bin across the entire stimulus period thanplanchnic afferents (P � 0.001). Both splanchnic and pelvic muscular afferents adapted over the first 20 seconds of the stimulus. Althoughelvic afferents continued to discharge over the remainder of the stimulus, splanchnic afferents returned to their spontaneous level of firing 20econds after the stimulus onset. Thus, muscular afferents in the LSN adapted more completely to circular stretch than those in the PN. Note
hat pelvic afferents showed no spontaneous activity.

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Dynamic Properties of Pelvic NerveMuscular/Mucosal Afferents

The pelvic nerve contained afferent fibers withroperties not found in those recorded from the splanch-ic nerve. These fibers possessed properties of both mus-ular and mucosal afferents in that they were excited byircular stretch and fine mucosal stroking and were there-ore termed muscular/mucosal afferents. Figure 5A depicts

ucosal stroking stimulus-response functions for all 4fferent classes found in the PN. Muscular/mucosal af-erents were similar to mucosal afferents in their responseo stroking. Specifically, only mucosal and muscular/ucosal afferents generated responses to receptive field

troking with the lowest intensity filament (10 mg), andhese 2 classes of afferent had stroking stimulus-responseunctions significantly different from other PN afferentsP � 0.001, Figure 5A). Muscular/mucosal afferent re-ponses to circular stretch were more complex than theiresponses to stroking. Seven of 13 muscular/mucosalfferents exhibited stretch stimulus-response functionshat were similar to those of muscular afferents, whereashe remaining 6 muscular/mucosal afferents respondeduch more robustly, with significantly higher stimulus-

esponse functions compared with muscular afferentsP � 0.001, Figure 5B). Consequently, the former sub-lass of muscular/mucosal afferents was termed low re-ponders and the latter subclass termed high responders.his distinction was manifest as a significantly elevated

esponse to stretch during both the initial dynamichase, immediately following the application of stretch,nd the sustained tonic phase (P � 0.001, Figure 5C).

DiscussionThe present study defines the mechanosensory

roperties of spinal afferents from the mouse colon thatre found in the LSN and PN. We have identified 5ifferent classes of afferent fiber, each capable of detect-

igure 5. Comparison of dynamic properties of pelvic afferentlasses. (A) Mucosal stroking stimulus-response function of PN affer-nts. All 4 classes demonstrated graded responses to mucosal strok-ng (10–1000 mg). Notably, mucosal (n � 13) and muscular/mucosaln � 13) afferents were the only classes to respond to the 10-mglament and exhibited stimulus-response functions to mucosal strok-ng that were significantly higher than those of pelvic serosal (n � 18)nd muscular (n � 12) afferents (P � 0.001). (B) Circular stretchtimulus-response functions of muscular and muscular/mucosal pel-ic afferents. All muscular and muscular/mucosal afferents re-ponded to the full range of stretch stimuli (1–5 g). Two subclasses ofuscular/mucosal afferents could be distinguished, based on their

esponses to stretch: a high-responding population, with responsesignificantly different than muscular afferents (P � 0.001, n � 6) andlow-responding population, with responses similar to those of mus-

ular afferents (P � 0.05, n � 7). (C) Adaptation profiles of muscularnd muscular/mucosal pelvic afferents to a 1-minute, 3-g stretch.igh-responder muscular/mucosal afferents displayed significantlyore spikes per 10-second bin across the entire 1-minute stimuluseriod compared with both low-responder muscular/mucosal anduscular afferents (P � 0.001). No significant difference in the ratef adaptation, defined as the slope of the adaptation curve, wasbserved among pelvic muscular and muscular/mucosal afferentsP � 0.05).

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ng different types of mechanical stimuli. Three of theseber classes (serosal, muscular, and mucosal) were con-erved between both pathways but displayed signifi-antly different functional properties. Each pathway alsoontained a unique class of afferent fiber, mesentericfferents found in the LSN and muscular/mucosal affer-nts in the PN. Consequently, our data indicate thatoth nerves are critical in relaying mechanosensory in-ormation from the colon but that each contains afferentbers individually tuned to detect distinct types of me-hanical stimuli and respond with differing sensitivities.hese findings demonstrate the great detail in whichechanical events in the colonic environment are sig-

alled to the central nervous system. They lead us toonsider that the rich diversity in mechanosensory prop-rties of colonic afferents may be comparable with thosennervating the skin.19 Moreover, this investigation pro-ides the first information on colonic afferents in mice,stablishing a basis for studies of the role that specificene products play in colon sensory function.

Function of Afferent Classes Shared bySplanchnic and Pelvic Pathways

Based on their functional properties, serosal, mus-ular, and mucosal afferents found in the splanchnic andelvic pathways likely signal very different mechanicalvents. Serosal afferents were the most abundant popu-ation, accounting for approximately one third of allbers found in this study. They were activated optimallyy a perpendicular probing stimulus applied directly toheir receptive fields. Serosal afferents have been previ-usly described in the LSN of the rat10,11 and the cat.4,5

e found a similar distribution of splanchnic afferents inpproximately equal proportions as those found previ-usly in the LSN of the rat using an in vitro prepara-ion.10,11 However, the present study is the first to useraded stimuli to establish stimulus-response relation-hips, and we are, therefore, unable to qualitatively com-are functional differences with previous reports. Serosalfferents have not been encountered in prior studies ofechanosensitive PN afferents in rat8 or cat6 using in

ivo single-fiber recording techniques, probably becausefferents were identified in these studies based on theiresponse to distention without direct access to the loca-ion of their receptive fields. Indeed, these studies esti-ated that colon distention activated just 5%8 and 16%6

f all pelvic nerve afferents, making it likely that serosalfferents in these species were overlooked by the em-loyed mechanical stimulus.The function of serosal afferents is unclear. Because of

heir response profiles, neither pelvic nor splanchnic se-

osal afferents are likely to be activated by physiologicevels of distention or contraction in situ. Therefore, weropose that they signal transient, sharp pain at the onsetf spasm or distention because of rapid transit of contentsr experimental balloon inflation, during which acutentense mechanical stimulation might be achieved. Pel-ic serosal afferents differed from splanchnic afferents ineveral features: they were clustered more distally, exhib-ted less adaptation, and responded across a wider stim-lus range than splanchnic afferents. Consequently, pel-ic serosal afferents would be expected to generate a morentense and sustained afferent barrage in response tohese acute mechanical events when they occur in moreistal portions of the descending colon and rectum thanerosal afferents in the LSN would generate in response tohis type of event.

Muscular afferents accounted for 21% and 10% of allelvic and splanchnic afferents, respectively, recorded inhis study. For the LSN, the proportion of muscularfferents that we found is similar to previous reports ofuscular afferents in the LSN of rat using an in vitro

echnique;10,11 however, it is much lower than the per-entage of distention-sensitive afferents (presumablyquivalent to muscular afferents) among all mechanosen-itive afferents reported in the LSN of the cat in vivo.4 Its possible that the proportion of stretch-sensitive affer-nts was underestimated in the in vitro studies, includ-ng the current one, because only circular, and not lon-itudinal, stretch was used. Alternatively, the splanchnicnnervation of the cat may contain many more stretch-ensitive afferents than the rat or mouse. Because thistudy is the first report of muscular afferents in the pelvicerve using an in vitro technique in any species, it isifficult to compare with previous in vivo studies ofuscular afferents in the rat8 and the cat6 that used only

olon distention to identify mechanosensitive fibers.owever, stretch-sensitive afferents constitute over 50%

f the total mechanosensitive pelvic afferent populationn our hands (muscular and muscular/mucosal afferentsombined) and is similar to the predominance of disten-ion-sensitive afferents among afferents responding toechanical stimulation of the colon and anal canal de-

cribed in both rat8 and cat.6

Muscular afferents in the mouse LSN and PN wereocated primarily in the distal colon and activated byow-intensity circular stretch and direct probing of theireceptive fields. However, they differed in 3 criticalspects. First, splanchnic muscular afferents were lessikely to respond to probing at lower stimulus intensities�1 g) than pelvic muscular afferents (compare Figure

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C and 3F). Second, pelvic muscular afferents showedreater responses to both probing and stretch (Figure 4Cnd 4E) and did not adapt completely during stimuluspplication (Figure 4D and 4F) compared with splanch-ic muscular afferents. Third, stretch-sensitive pelvicfferents (including muscular/mucosal afferents, see be-ow), greatly outnumber stretch-sensitive splanchnic af-erents (compare Figure 2B and 2D). Taken together,hese differences indicate that the PN is better equippedo respond to stretch of the colonic wall and containsuscular afferents that respond more robustly to tonic

tretch than those found in the LSN. Also, their sus-ained response during maintained stretch suggests thatelvic muscular afferents are more likely to signal tonichanges in the caliber or wall tension of the distal colon,uch as during the presence of stool or gas. In contrast,he higher stimulus intensities required to activate andore completely adapting responses of splanchnic mus-

ular afferents, which occur in much lower proportions,ould be better tuned to signal the onset of higher

ntensity mechanical events, such as muscular contrac-ion or passage of material, which are of a more acuteature. This notion is supported by a study in ratsemonstrating that the pelvic nerve alone is sufficient toaintain behavioral responses to sustained colorectal dis-

ention20 and a discussion of human data proposing thatumbar splanchnic afferents are better able to conveyhasic rather than tonic changes in colonic pressure.2

owever, stretch-sensitive afferents in both pathwaysncode beyond the physiologic range of distention anday therefore transmit nociceptive information. Al-

hough the greater mechanosensitivity of pelvic muscularfferents relative to splanchnic muscular afferents haseen previously described in the cat,21,22 it was not untilhe current study that direct comparisons between the 2athways in the same setting have been performed toelineate clearly the different sensitivity of muscularfferents in the PN and LSN.

Afferents that respond to colonic stretch, applied ei-her directly in vitro or indirectly using colon distentionn vivo, have been identified and extensively character-zed previously.4–11,18,22,23 Muscular afferents, as a whole,re responsive to small changes in intraluminal pressure,espond to colonic stretch or distention with a linearelationship to wall tension, and encode these stimuliell into the noxious range. Additionally, 2 basic typesf distention-evoked afferent responses have been de-cribed: phasic and tonic.4–6,8,9,23 The roles of these 2ypes of afferent likely differ because phasic afferents arenly transiently excited during stimulus onset and offset,

hereas tonic afferents discharge throughout the stimu-us duration.6 Comparison of our results with thosereviously published are complicated by the heterogene-ty of species, techniques, and mechanical stimuli used inhese studies. However, the stretch-sensitive muscularfferents reported here closely resemble muscular affer-nts described in the LSN of the rat colon in vitro7,10,11

nd the distention-sensitive, low-threshold afferents de-cribed in vivo in the LSN of the cat4 and PN of theat4,5,23 and rat.8,9 All muscular and muscular/mucosalfferents that we recorded responded to the lowest in-ensity stretch (1 g), encoded stimuli well into the nox-ous, nonphysiologic range, and exhibited exclusivelyonic-type responses.

Mucosal afferents accounted for 23% of the pelvic and% of the splanchnic innervation in the present study.hey exhibited different distributions, with pelvic mu-osal afferents localized in the most distal region of theolon, whereas the few splanchnic mucosal afferents wereound more proximally. Although the rarity of splanch-ic mucosal afferents makes it difficult for direct com-arison with those in the PN, both display adaptingesponses to low-threshold mucosal stroking or probingcompare Figure 3B and 3E) and an insensitivity toircular stretch (compare Figure 1A, iv and 1B, iv). Ourata suggest that the signalling of fine mechanical stim-lation of the colonic mucosa occurs predominantly viahe pelvic pathway because nearly half of the pelvicfferent population (including muscular/mucosal affer-nts) was capable of detecting fine mucosal strokingFigure 2D). Colonic mucosal afferents have been char-cterized in vitro from the LSN10 and colonic hypogastricerves of the rat24 and in the PN in vivo with receptiveelds in the anal canal of the cat6,18 and perianal mucosaf the rat.8 However, the current study is the first toharacterize fully the mechanical response properties ofolorectal mucosal afferents in the LSN and PN of theouse and demonstrate a widespread distribution ofucosal afferents throughout the distal colon and not

estricted solely to the anal mucosa. In fact, we wereurprised to find so many afferents sensitive to fineucosal stimulation in such a large region of the distal

olon and rectum, considering the vagueness of colonicensations. We can only conclude that these and muscu-ar/mucosal afferents (see below) must be placed either torovide fine mucosal input to reflexes controlling motil-ty25–27 and/or to refine the quality of perceived stimuli.

hichever is the case, mechanical signals from the co-onic mucosa are carried predominantly by the PN and

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ay contribute to the large proportion of distention-nsensitive afferents reported previously.6,8

Mesenteric Afferents Specific to theSplanchnic Pathway

Mesenteric afferents are the largest fiber popula-ion recorded from the colon, accounting for 50% of theplanchnic innervation, and were specific to this path-ay. Mesenteric afferents displayed identical probing

esponse properties to splanchnic serosal afferents. Con-istent with other studies, mesenteric afferents in theresent study were located close to or on blood vessels orranching points of capillaries supplying the serosa andccasionally displayed multiple receptive fields, similaro mesenteric afferents previously reported in other spe-ies.4–8,10,14,17 Although not routinely tested in theresent study, it is clear that mesenteric afferents can bectivated briefly by intense mesenteric stretch and co-onic distention in addition to stimuli applied to theireceptive fields.4,10,17 It has been proposed that thesefferents may detect twisting and torsion of the colon andulsatile changes in blood pressure in mesenteric bloodessels, possibly critical during plasma extravasation re-ulting from colonic inflammation.28,29 The preponder-nce of mesenteric and serosal afferents in the LSN thatequired higher intensity mechanical stimulation for ac-ivation supports its role as a higher intensity nociceptiveathway than the PN.

Muscular/Mucosal Afferents Specific to thePelvic Pathway

Muscular/mucosal afferents accounted for 23% ofll pelvic afferents, yet they have not been reportedreviously in the colon. Similar afferents have, however,een reported in the vagal nerve supply to the ferretsophagus known as tension/mucosal receptors.30 Theresent study showed that pelvic muscular/mucosal af-erents were clustered in the distal colon (Figure 2C).hey displayed graded responses to mucosal stroking

hat were similar to pelvic mucosal afferents (Figure 5A)nd adapted only partially to circular stretch (Figure 5C).elvic muscular/mucosal afferents could be divided intodistinct populations, high responders and low respond-

rs, based on their stretch stimulus-response functionsFigure 5B): low-responding muscular/mucosal afferentsere comparable with muscular afferents, whereas theigh-responding population was the most sensitive of allfferents to circular stretch, generating larger responsesuring both the initial phase and throughout the entireuration of stretch (Figure 5C). The purpose of these 2istinct subpopulations of muscular/mucosal afferents

hat are unique to the pelvic pathway is unknown. Inddition, the anatomic structure of the receptive endingsf these afferents that confers on them the ability toetect both fine mucosal stimuli within the lumen andtretch of the colon wall is unknown; however, it haseen suggested that their endings might have a receptiveeld interposed in the muscularis mucosa and play apecialized role in the detection of rapidly moving bo-uses.30,31 Alternatively, muscular/mucosal afferents mayave 2 separate endings, one in the mucosa and anothern the muscle layers of the colon that are spatially indis-inguishable with the technique used here. Whicheveray be true, pelvic muscular/mucosal afferents are situ-

ted to detect both fine mucosal stroking and circulartretch, thereby enabling them to relay information on aiversity of mechanical events from the distal colon tohe spinal cord.

Functional Significance

Until now, the full complement of afferent fibersnvolved in detecting mechanical stimuli and the rolehat they potentially play in the physiology and patho-hysiology of the colon were unknown. The presenttudy permits, for the first time, a direct comparison ofhe functional properties of mechanosensitive afferentsrom the lumbar splanchnic and pelvic spinal innervationf the mouse colon. We conclude from these data thathese 2 pathways are equipped to detect multiple types ofechanical stimuli and are composed of 3 classes ofechanosensitive afferent common to both pathways but

ossessing distinct functional properties and a class offferent unique to each pathway. Afferents from bothathways are individually tuned to detect the type oftimulus and the magnitude and duration of stimulusntensity. Overall, the pelvic pathway contains a greaterumber of afferents tuned to detect circular stretch,ikely the primary mechanical stimulus generated byolorectal distention in natural or experimental situa-ions. They are, as a whole, more sensitive and responsiveo mechanical stimulation than the splanchnic pathway.herefore, our data indicate that the pelvic and lumbar

planchnic pathways constitute high- and low-gain path-ays, respectively, for the detection of mechanical stim-li in the distal colon. The sheer scale of mechanosensorynformation detected from the colon evident from thistudy is staggering and surely contributes to intrinsicnd extrinsic reflexes, as well as conscious innocuous andoxious perceptions from the colon. This study providesdetailed understanding of colonic mechanotransduction

hat is crucial to assessing the function of colon sensoryfferents under normal and diseased conditions. More-

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ver, the current study provides the basis for experimentssing transgenic mice on the very nature of mechano-ransduction, an area in which pharmacologic tools areimited and in which genetic approaches have provennvaluable.12,13 Such studies will be useful in revealingromising therapeutic molecular targets for the treat-ent of functional bowel disorders.

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E. Evidence for the hypersensitivity of lumbar splanchnic affer-ents in irritable bowel syndrome. Gastroenterology 1994;107:1686–1696.

3. Verne G, Himes N, Robinson M, Gopinath K, Briggs R, Crosson B,Price D. Central representation of visceral and cutaneous hyper-sensitivity in the irritable bowel syndrome. Pain 2003;103:99–110.

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7. Berthoud HR, Lynn PA, Blackshaw LA. Vagal and spinal mech-anosensors in the rat stomach and colon have multiple receptivefields. Am J Physiol Regul Integr Comp Physiol 2001;280:R1371–R1381.

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4. Brierley SM, Cooper N, Blackshaw LA. Pelvic colonic primaryafferents: mechanosensory and pharmacological subtypes. Gas-troenterology 2002;122:A527.

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Received November 5, 2003. Accepted April 1, 2004.Address requests for reprints to: Stuart M. Brierley, Nerve-Gut Re-

earch Laboratory, Level 1 Hanson Institute, Frome Road, Adelaide,outh Australia, Australia, 5000. e-mail: [email protected] or [email protected]; fax: (61) 8 8222 5934.Supported by an Australian Postgraduate Award (to S.M.B.), a Na-

ional Health and Medical Research Council of Australia Senior Re-earch Fellowship (to L.A.B.), NHMRC Australia grant number 104814to L.A.B.), and by National Institutes of Health Award NS 19912 (to.C.W.J. and G.F.G.).
S.M.B. and R.C.W.J. contributed equally to this work.

splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice

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