European Journal of Pain 14 (2010) 49.e1–49.e11

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European Journal of Pain

journal homepage: www.EuropeanJournalPain.com

Gap junctions in dorsal root ganglia: Possible contribution to visceral pain

Tian-Ying Huang 1, Vitali Belzer, Menachem Hanani *

Hebrew University-Hadassah Medical School, Mount Scopus, Jerusalem 91240, Israel

a r t i c l e i n f o

Article history:Received 13 September 2008Received in revised form 12 February 2009Accepted 16 February 2009Available online 3 April 2009

Keywords:Gap junctionsSatellite glial cellsDorsal root gangliaNeuronsVisceral painColon

1090-3801/$36.00 � 2009 European Federation of Intdoi:10.1016/j.ejpain.2009.02.005

* Corresponding author. Tel.: +972 2 5844721; fax:E-mail address: hananim@cc.huji.ac.il (M. Hanani)

1 Deceased.

a b s t r a c t

Peripheral injuries can lead to sensitization of neurons in dorsal root ganglia (DRGs), which can contrib-ute to chronic pain. The neurons are sensitized by a combination of physiological and biochemicalchanges, whose full details are still obscure. Another cellular element in DRGs are satellite glial cells(SGCs), which surround the neurons, but little is known about their role in nociception. We investigatedthe contribution of SGCs to neuronal sensitization in isolated S1 DRGs from a mouse model of colonicinflammation induced by local application of dinitrosulfonate benzoate (DNBS). Retrograde labelingwas used to identify DRG neurons projecting to the colon. Cell-to-cell coupling was determined by intra-cellular dye injection, and the electrical properties of the neurons were studied with intracellular elec-trodes. Pain behavior was assessed with von-Frey hairs. The dye injections showed that 10–12 daysafter DNBS application there was a 6.6-fold increase in gap junction–mediated coupling between SGCssurrounding adjacent neurons, and this occurred preferentially (another 2-fold increase) near neuronsthat project to the colon. Neuron–neuron coupling incidence increased from 0.7% to 12.1% by colonicinflammation. Inflammation led to an augmented neuronal excitability, and to a reduced pain threshold.Gap junction blockers abolished the inflammation-induced changes in SGCs and neurons, and signifi-cantly reversed the pain behavior. We propose that inflammation induces augmented cell coupling inDRGs that contributes to neuronal hyperexcitability, which in turn leads to visceral pain. Gap junctionblockers may have potential as analgesic drugs.

� 2009 European Federation of International Association for the Study of Pain Chapters. Published byElsevier Ltd. All rights reserved.

1. Introduction

The mechanisms underlying chronic pain are far from clear,but it is known that sensitization of neurons in sensory gangliais a contributing factor (Zhang et al., 1997; Devor, 2006).Sensory neurons are individually surrounded by satellite glialcells (SGCs; Pannese, 1981), whose functions are only recentlybeginning to be unraveled (Hanani, 2005; Ohara et al., 2008).We found that gap junction–mediated coupling among SGCs isenhanced after nerve section or inflammation and proposed thatthis can contribute to chronic pain (Cherkas et al., 2004; Dublinand Hanani, 2007; Hanani et al., 2002). Recent work on a ratpain model provided molecular, pharmacological and behavioralevidence supporting this idea (Ohara et al., 2008; Vit et al.,2006).

Glial cells in the central nervous system (CNS) contribute tochronic pain (McMahon et al., 2005; Watkins and Maier, 2002;

ernational Association for the Stud

+972 2 5823515..

Scholz and Woolf, 2007). There is evidence that astrocytes (Wies-eler-Frank et al., 2005) and microglia (Farber and Kettenmann,2005) can release cytokines and also carry receptors for inflam-matory agents. It appears that activation of P2X4 nucleotidereceptors in spinal microglia is required for allodynia after nerveinjury (Tsuda et al., 2003). In contrast to the progress made onCNS glia, little is known on the role of SGCs in pain, but on thebasis of the presence of receptors for bradykinin (Heblich et al.,2001), endothelin (Pomonis et al., 2001), and ATP (Weick et al.,2003) on SGCs, it was suggested that SGCs have a role in nocicep-tion (Hanani, 2005). Recent work supported this view by showingthat SGCs release the cytokines interleukin-1b (IL-1b; Takedaet al, 2007) and tumor necrosis factor a (Zhang et al., 2007). Assensory neurons display IL-1b receptors, this cytokine can servefor SGC–neurons communication, which will be enhanced follow-ing inflammation.

The mechanisms of visceral pain have received relatively littleattention, but progress in this area has taken place in recent years(Bielefeldt and Gebhart, 2006). It was reported that inflammationin the stomach (Bielefeldt et al., 2002a; Bielefeldt et al., 2002b),intestine (Moore et al., 2002; Beyak et al., 2004), and bladder

y of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

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(Yoshimura and de Groat, 1999) increased the excitability of DRGneurons innervating these organs. These studies were done on iso-lated or cultured DRG neurons, and did not address the possiblerole of SGCs or gap junctions in pain. We have demonstrated thatpartial colonic obstruction in mice, which is associated with colo-nic inflammation, increased SGC coupling and neuronal excitabil-ity, and also produced hyperalgesia (Huang and Hanani, 2005).We proposed that augmented glial coupling contributes to chronicpain, as suggested for spinal cord astrocytes (Spataro et al., 2004).In this study we induced colonic inflammation in mice by localapplication of dinitrobenzene sulfonate (DNBS), and examinedthe resulting changes in sensory neurons and SGCs in DRGs in or-der to further understand the interactions between these cell typesand their potential contribution to visceral pain.

2. Methods

2.1. Induction of colonic inflammation

The experiments were done on Balb/c mice 2–4 months old ofeither sex, weighing 22–25 g. The experimental protocol was ap-proved by the Animal Care and Use Committee of the Hebrew Uni-versity-Hadassah Medical School, and adheres to the guidelines ofthe International Association for the Study of Pain. Colonic inflam-mation was induced by instilling DNBS (Sigma, St. Louis, MO, USA;50 ll, 100 mg/ml in 50% ethanol) into the distal colon via anal tube(Blau et al., 2000); the tip of the tube was located 1.2–1.5 cm fromthe anal verge. The animals were anesthetized with chloral hydrate(300 mg/kg, 2% in saline). Ten to twelve days later the animalswere sacrificed with CO2, and S1 DRGs were removed from bothsides. Control animals were instilled with saline or 50% ethanolin saline; no difference was found between these two controltypes.

2.2. Retrograde labeling

To identify DRG neurons that project into the distal colon we la-beled the neurons retrogradely with the florescent tracer 1,10-dioc-tadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DiI,Molecular Probes, Eugene, OR, USA; 5% in methanol, 10 ll) (Suet al., 1999). Mice were anesthetized as described above. The distalcolon was externalized through a low abdominal midline incisiononto a mat of cotton soaked with saline, and the colon wall was in-jected with DiI. Several injections were made circumferentially inabout 1 cm long segment of the colon, 1.5 cm proximal to the anus.The incision was sutured in two layers, and 4 days later the ani-mals were treated with DNBS or saline (controls); 10-12 days laterS1 DRGs were removed and studied in vitro.

2.3. Intracellular labeling and recording

Ganglia were fixed to the bottom of a silicon rubber-coateddish using fine pins. The dish was placed on the stage of an up-right microscope (Axioskop FS, Zeiss, Jena, Germany), equippedwith fluorescent illumination and a digital camera (Pixera 120 e,Pixera Corp. Los Gatos, USA) connected to a personal computer.The dish was superfused with Krebs solution, which contained(in mM): 118 NaCl, 4.7 KCl, 14.4 NaHCO3, 1.2 MgSO4, 1.2 NaH2-

PO4, 2.5 CaCl2, and 11.5 glucose; pH 7.3. Individual neurons andSGCs in DRGs were injected with the fluorescent dye Lucifer yel-low (LY, Sigma, St. Louis, MO, USA), 3% in 0.5 M LiCl solution fromsharp glass microelectrodes, connected to a preamplifier (NeuroData Instrument Corp., New York, NY, USA). The dye was passedby hyperpolarizing current pulses, 100 ms in duration; 0.5 nA inamplitude at 10 Hz for 3–5 min (Huang et al., 2005). Single SGCs

or neurons were injected with LY under visual inspection to allowcell’s identification (neuron or glia). At the end of the injection ofeach cell, we counted the number of cells that were labeled as aresult of dye passage from the injected cell (dye-coupled cells).The coupling incidence was calculated as the ratio between thetotal number of injected cells of each type to the number ofdye-coupled ones. For example, if 50 neurons were injected andfive of these showed coupling to other neurons, the incidenceneuron–neuron coupling was 10%. We also determined the aver-age number of cells coupled to the injected one by calculating theratio between the total number of cells coupled to the injectedones and the total number of injected cells. For example, if 50SGCs were injected, resulting in dye coupling to 100 SGCs, theaverage number of coupled SGCs was 2.0.

The fluorescent molecule dextran–rhodamine (Sigma, molecu-lar weight 10,000), 3% in 1 M KCl was injected in the same manner.During and after the injections living labeled cells were imaged.After the experiments DRGs were fixed overnight at 4 �C in 4%paraformaldehyde in phosphate buffered saline (PBS), washed withPBS and mounted in Gel/Mount (Biomeda. Foster City, CA, USA).Cells labeled with LY were imaged with a confocal microscope(Biorad, Macclesfield, UK).

After identifying DRG neurons that project into the colon by DiIretrograde labeling, labeled neurons or SGCs near (<50 lm) theseneurons were injected with LY. After LY injection the microscopicfield was photographed with filters for FITC (for LY) and with filtersfor TRITC (for DiI); the two images were then merged to determinethe spatial relationship between DiI-labeled neurons and dye-in-jected cells. The number of cells coupled to the LY-injected cellwas counted 4–5 min after the injection by changing microscopicfocus level.

For intracellular electrical recordings, DRGs were bathed inKrebs solution at 31–32 �C. This temperature was used rather than37 �C to allow better cell viability. Sharp glass microelectrodeswere filled with 2 M KCl, with 80–120 MX tip resistance. Trans-membrane currents were passed through the recording electrodeusing the bridge circuit of a preamplifier. The threshold for firingan action potential was determined by measuring the minimaldepolarizing current (100 ms duration) that elicited a single aspike. Electrophysiological data were recorded with VCR using aNeuro-corder (Neuro Data Instrument Corp., model DR 390), andwere analyzed later using pCLAMP 9 (Axon Instruments, FosterCity, CA, USA).

All neurons reported here had a stable resting membrane po-tential more negative than �35 mV and an overshooting spike.Neurons displaying subthreshold membrane potential oscillations(Amir et al., 1999), were defined as those showing membrane po-tential fluctuation of at least 1 mV in amplitude. Membrane poten-tials were sampled for 2 s at 2 kHz for spectral analysis (powerspectrum) with the fast Fourier transform module of pCLAMP 9.

2.4. Assessment of visceral pain

Von-Frey hairs (Stoelting, Wood Dale, IL, USA) were used tomeasure withdrawal responses to mechanical stimulation of thelow abdominal skin. These responses are attributed to pain re-ferred from the colon (Laird et al., 2001). Appearance of one ofthe following behaviors on application of a hair was consideredas a withdrawal response: sharp abdominal retraction, immediatelicking, scratching of the stimulation site, or jumping. Hairs cali-brated for forces of 0.5, 1.0, 2.0, and 4.0 g were applied 10 timeseach at 5 s intervals in ascending order (Laird et al., 2001).Care was taken not to stimulate the same point on the skin insuccession. The threshold of withdrawal response (pain threshold)was defined as the minimum force eliciting two subsequentresponses.

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2.5. Administration of gap junction blockers

In the experiments of intracellular dye injection and recording,three gap junction blockers (all from Sigma): carbenoxolone(50 lM) (Spray et al., 2002), meclofenamic acid (100 lM) (Harkset al., 2001), and palmitoleic acid (30 lM) (Burt et al., 1991) wereadded separately into the bathing solution. In the experiments de-signed to observe the changes in the pain threshold, each of the gapjunction blockers was injected intraperitoneally; carbenoxolone(100 mg/kg), meclofenamic acid (10 mg/kg), and palmitoleic acid(25 mg/kg). Pain behavior was tested 1 h after the injection.

2.6. Statistical analysis

Values are expressed as mean ± S.D. Fisher’s exact test, Dunn’sMultiple Comparisons test, Mann–Whitney test and one-way ANO-VA were used for comparison. p < 0.05 is considered as statisticallysignificant. The statistics on the mean number of SGCs coupled tothe LY-injected cell excluded the cases without dye coupling.

3. Results

3.1. Dye coupling between SGCs

Previous investigations showed that the extrinsic sensoryinnervation of the mouse colon derives from DRGs L1 and S1 (Rob-

Fig. 1. Changes in dye coupling between satellite glial cells (SGCs) of mouse S1 DRG 10coupling between SGCs in live tissues. (a) A Lucifer yellow (LY)-injected SGC is coupled tsurrounded by a large number of SGCs. (b) Dye coupling between SGCs around differenSGCs. Scale bars, 20 lm. (c–e) The histograms show the effect of gap junction blockers: ca(PA, 30 lM), on the augmented coupling among SGCs after inflammation. (c) Incidence ofSGCs around different neurons; N = 81 � 129 for each of the experimental conditions, p <gap junction blockers also reduced the mean number (± S.D.) of SGCs coupled to the LY-ininjected SGC, both within the same envelope and in envelopes of adjacent neurons. *p < 0for comparison.

inson et al., 2004; Huang and Hanani, 2005). Therefore our exper-iments were done on S1 ganglia.

When we randomly injected individual SGCs with LY wefound that in control ganglia, 19 of 77 (24.7%) LY-injected SGCswere coupled to other SGCs around a given neuron (Fig. 1a), andonly two (2.6%) of the injected cells were coupled to SGCs incontact with different neurons. At 10–12 days after treatmentwith DNBS, dye coupling incidence between SGCs around a givenneuron increased from 24.7% to 39.8% (n = 88, p < 0.05, Fisher’sexact test), and that between SGCs in contact with different neu-rons increased 6.6-fold (from 2.6% to 17.1%, p < 0.01), see exam-ple in Fig. 1b. The mean number of SGCs coupled to the injectedcells increased from 2.05 ± 0.35 to 3.22 ± 0.36 (n = 35, p < 0.05).Thus, inflammation led to a large augmentation of coupling be-tween SGCs.

We have shown previously (Hanani et al., 2002; Panneseet al., 2003) that the augmented dye coupling among SGCs aftersciatic nerve axotomy was associated with an increase in thenumber of gap junctions between SGCs. To test whether theDNBS-induced augmentation in dye coupling was also mediatedby gap junctions, we repeated the dye injections in the presenceof three different gap junction blockers: carbenoxolone (CBX,50 lM), meclofenamic acid (MFA, 100 lM) and palmitoleic acid(PA, 30 lM). Each substance was added separately to the bathingsolution during dye injection in ganglia from DNBS-treated mice.We found that each of these gap junction blockers significantlyreduced the coupling incidence among SGCs around a given

–12 days after induction of inflammation. The images show two examples of dyeo other SGCs only around the same neuron. This neuron is large, and is accordinglyt neurons, observed in DNBS-treated mouse. The asterisks indicate the LY-injectedrbenoxolone (CBX, 50 lM), meclofenamic acid (MFA, 100 lM) and palmitoleic acidcoupling between SGCs around the same neuron. (d) Incidence of coupling between

0.001 compared with results obtained after DNBS in the absence of blockers. (e) Thejected SGCs. This number represents the average number of SGCs coupled to a given.01, N = 17 � 29. Fisher’s exact test (c and d) and one way ANOVA test (e) were used

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neuron, the coupling among SGCs in contact with different neu-rons, and the mean number of SGCs coupled to the LY-injectedSGCs, see Fig. 1c and d. The effect on the coupling of SGCs envel-oping different neurons was the most striking and significant; itwas reduced from 17.1% to about 2% for all three substances(p < 0.001), see Fig. 1d. These findings indicate that theaugmented dye coupling between SGCs was mediated by gapjunctions.

We next tested whether the degree of SGC coupling is influ-enced by the proximity of SGCs to neurons projecting to the in-flamed colon. We identified neurons projecting to the colon byretrograde labeling with the fluorescent tracer DiI, and injectedLY into SGCs within 50 lm from the DiI-labeled neurons (Fig. S1,see the online version at doi:10.1016/j.ejpain.2009.02.005). Wefirst carried out a series of control experiments for the possible ef-fect of DiI itself on coupling. In these experiments DiI labeling wasperformed, but the animals were not treated with DNBS, and theresult was that DiI labeling itself did not alter coupling. However,after DNBS treatment dye coupling incidence between SGCs closeto DiI-labeled neurons rose from 26.5% (DiI without DNBS;n = 57) to 80.2% (n = 81, p < 0.01, Fisher’s exact test) around a givenneuron, and from 3.5% to 44.4% (p < 0.01) around different neurons.The mean number of SGCs coupled to the LY-injected SGCs in-creased from 2.13 ± 0.38 (n = 14) to 3.31 ± 0.32 (n = 65, p < 0.05,Mann–Whitney test), Fig. S1. Thus, after DNBS treatment LY injec-tion into SGCs near DiI-labeled neurons resulted in a 2-fold in-crease in dye coupling incidence compared with the randominjections (p < 0.0001, Fisher’s exact test).

We also asked whether there was any systemic effect due to co-lonic inflammation. This was tested by injecting LY into DRGs L4,5in mice after colonic DNBS application. We found that in treatedanimals coupling between SGCs surrounding different neurons inDRGs L4,5 was 7.6% (6/79), which is not statistically different fromthe value obtained in control mice (6.6%, 6/91).

3.2. Dye coupling between DRG neurons

In control S1 ganglia the incidence of neuron–neuron couplingwas extremely low (0.7%, 1/133, 6 mice). In contrast, 10–12 daysafter DNBS application, neuron–neuron coupling was present in12.1% of the cases (17/140, 6 mice), see Fig. 2. In most cases thedye passed to only one neuron. In a small number of cases addi-tional 1–2 neurons appeared to be stained, but the intensity ofthe labeling was very weak. Neuron-glia coupling was observedin only a small proportion in treated mice (2/140; 1.6%) and neverin control DRGs.

Fig. 2. Evidence for neuron–neuron dye coupling in DRG from a DNBS-treated mouse. (a)was injected into the descending colon. (b) The neuron shown in (a) was injected intrainjected neuron passed to a neighboring neuron (arrow). The results show that after DNBScale bar, 25 lm.

To test whether neuron–neuron coupling was mediated by gapjunctions and was not due to artifacts we injected neurons with alarge fluorescent tracer molecule, dextran–rhodamine, which doesnot cross gap junctions. We found that in all cases there was nocoupling when neurons from DNBS-treated mice were injectedwith this tracer (N = 37, 2 mice, p < 0.005 compared with LY injec-tion in DNBS-treated mice). Incubation of the tissues with carben-oxolone (50 lM) during LY injection greatly reduced couplingbetween neurons (1/51, 1.96%, p < 0.05). These experiments con-firmed that neuron–neuron coupling was mediated by gapjunctions.

To determine whether neuron–neuron coupling depended onneuronal projection into the colon we used DiI retrograde labelingas described above. Injection of LY into DiI-labeled neuronsshowed that 14/78 (17.9%, 10 mice) were coupled to other neu-rons; that is, nearly 50% more than with random injection, how-ever this difference was not statistically significant (p > 0.05).

When we injected LY into neurons in L4,5 DRGs from DNBS-treated mice we observed less than 1% incidence of neuron–neuroncoupling (1/102). This again indicated that neuron–neuron cou-pling was directly related to the DNBS application into the colon,into which some S1 neurons project.

3.3. Excitability of DRG neurons

To learn about electrophysiological changes in DRG neuronsafter DNBS treatment we carried out two types of intracellularrecording experiments. In the first, we recorded randomly fromDRG neurons, and in the second, from neurons projecting to thecolon (DiI-labeled). Type A neurons were defined by an action poen-tial duration of less than 10 ms, and the rest of the neurons wereclassified as C-type (Cabanes et al., 2002; Cherkas et al., 2004).

We determined the resting potential and threshold current forfiring an action potential by recording randomly from A-type andC-type neurons in control ganglia (Table S1, see the online versionat doi:10.1016/j.ejpain.2009.02.005). Ten to twelve days afterDNBS treatment the resting potential was 4–5 mV more positivein both A-type and C-type neurons, and the threshold current forfiring an action potential in A-type neurons and C-type cells waslower by 31.9% and 42.2%, respectively (for both, p < 0.05, Mann–Whitney test). Recordings of spontaneous activity revealed thatin control ganglia most (79.2%) of the neurons were electricallyquiescent (Fig. 3a), and the rest displayed spontaneous activity –subthreshold membrane potential oscillations (SPO, Fig. 3b andc) or spontaneous spikes (SPS, Fig. 3d and e). After DNBS treatmentthe proportion of neurons displaying SPO increased by 91.4% in

A DRG neuron (asterisk) was stained retrogradely by the red fluorescent dye DiI thatcellularly with the yellow fluorescent dye Lucifer yellow (LY). LY passed from theS injection DRG neurons that project to the colon can be coupled to other neurons.

Fig. 3. Changes in the electrophysiological properties of DRG S1 neurons 10–12 days after inducing colonic inflammation with DNBS. (a) Intracellular recording from a neuronin a ganglion from a control animal, showing an electrically quiescent cell. (b and c) Examples of membrane potential oscillations recorded from C- and A-type type neurons,respectively, after DNBS instillation. (d) Example of spontaneous action potentials (20 Hz) recorded from an A-type neuron in a ganglion after inflammation. (e) A portion of(d) displayed at faster speed to show individual spikes. (f) Spectral analysis (power spectrum) of spontaneous subthreshold membrane potential oscillations recorded from 28A-type cells from treated animals and nine cells in control animals. The power peaks were 35 Hz for neurons in the control animals, and 30 Hz in the treated mice. (g) Powerspectrum for 17 C-type cells in treated animals, and five cells in controls. The peak frequency was 30 Hz in the control mice and 28 Hz in the treated animals. The referencepower spectrum (without oscillations) in (f) and (g) was processed with 10 quiescent DRG neurons (5A- and 5C-type).

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A-type and by 139.7% in C-type cells, Table S1. Similar SPO havebeen described previously, and apparently underlie injury-inducedectopic firing (Amir et al., 1999; Liu et al., 2000). These results indi-cate that DNBS treatment led to increased excitability in S1 neurons.

We next asked whether the changes in neuronal excitability inDNBS-treated animals were restricted to the labeled colonic popu-lation. To address this question, we recorded from neurons thatproject into the colon, using retrograde labeling. In control animalsthe resting membrane potential, current threshold, and proportionof neurons displaying spontaneous activities for both A- and C-typeDiI-labeled cells were very similar to those of the randomly re-corded neurons, indicating that DiI labeling itself had no effecton neuronal behavior. After DNBS treatment the proportion ofDiI-labeled neurons displaying SPO was 44.4% in A-type and73.9% in C-type (Table S1). The proportion of neurons firing SPS in-creased to an even greater extent (Table S1). Thus, in DiI-labeledneurons spontaneous activity was significantly greater than in ran-domly impaled cells. The observations on SPO were quantified byspectral analysis. In control animals the oscillation frequency was35.3 ± 1.9 Hz in A-type, and 31.6 ± 1.8 Hz in C-type cells. AfterDNBS treatment the frequency was 31.9 ± 1.1 Hz in A-type, and28.4 ± 1.1 Hz in C-type neurons, see Fig. 3f and g. The amplitudeof oscillations recorded from DiI-labeled neurons was much moreprominent than that in controls (Fig. 3f and g).

3.4. Effect of gap junction blockers on neuronal excitability

As described above, DNBS treatment led to increased gap junc-tion–mediated SGC–SGC and neuron–neuron coupling, and also to

neuronal hyperexcitability. We hypothesized that this increasedcoupling contributed to the neuronal hyperexcitability. To testthis hypothesis we examined neuronal excitability after DNBStreatment when each of the three different gap junction blockerswas added separately to the bathing medium. In these experi-ments DRG neurons were impaled randomly. Each of the threegap junction blockers: CBX (50 lM), MFA (100 lM) and PA acid(30 lM), reversed to a large extent the augmented excitability ofDRG neurons after DNBS treatment (Fig. 4 and Table S2, see theonline version at doi:10.1016/j.ejpain.2009.02.005). The effectsof the gap junction blockers on the basic electrophysiologicalproperties of A-type and C-type neurons after DNBS treatmentare presented in Table S2. The results showed that gap junctionblockers suppressed neuronal hyperexcitability in DRG neuronsand support the idea that cell coupling plays a role in neuronalactivity.

There is evidence that CBX can either inhibit (Rouach et al.,2003) or increase (Jahromi et al., 2002) electrical excitability ofhippocampal and cortical neurons, independently of its actionson gap junctions. To find out whether CBX acted directly on targetsother than gap junctions we made electrical recordings from DRGneurons of untreated mice in the presence of CBX (50 lM). Afterapplying CBX the resting potential was 50.3 ± 1.3 mV (n = 33) forA-type cells, 51.4 ± 1.6 mV (n = 28) for C-type cells, and the currentthreshold for firing an action potential was 0.44 ± 0.04 nA and0.39 ± 0.03 nA for A-type and C-type neurons, respectively. Theproportion of neurons displaying spontaneous spikes was verysmall (3% for A-type; 0% for C-type), and the proportion of neuronsdisplaying subthreshold membrane potential oscillations was

Fig. 4. The effect of gap junction blockers on the electrophysiological properties of neurons in mouse S1 ganglia after colonic inflammation. Ten to twelve days after theinduction of inflammation, the proportion of the neurons with spontaneous action potentials (A) and spontaneous potential oscillations (B) increased, and also there was adecrease in the threshold for firing an action potential (C). The three gap junction blockers, CBX (50 lM), MFA (100 lM) and PA (30 lM), applied separately, reversed theneuronal hyperexcitability induced by the colonic inflammation, but statistical significance was reached only with CBX. All the comparisons are between data obtained for theDNBS-treated mice and data obtained for the other groups. *p < 0.05, **p < 0.01, Fisher’s exact test and one way ANOVA. The error bars indicate S.D.

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18.2% for A-type and 17.9% for C-type cells. These results showedthat the electrophysiological properties neurons in untreated micewere not affected by gap junction blockade.

3.5. Pain behavior

Referred pain responses of mice were assessed using von-Freyhairs applied to the abdominal wall (Laird et al., 2001). Pain thresh-old was decreased 12 days after DNBS treatment (Fig. 5). As gapjunction blockers largely reversed DNBS-induced neuronal hyper-excitability, we asked whether gap junction blockade could also re-verse the pain behavior in DNBS-treated mice. The three gapjunctions blockers were injected intraperitoneally to DNBS-treatedanimals (in separate experiments), 1 h before the measurements,at the following doses: CBX, 100 mg/kg; MFA, 10 mg/kg; and PA,25 mg/kg. These gap junction blockers partly, but significantly,reversed the sensitizing effect of DNBS as shown in Fig. 5. Theseresults indicate that gap junction blockers reduced the hyperalge-sia in the treated mice.

Fig. 5. The influence of gap junction blockers on mouse pain behavior after theinduction of colonic inflammation with DNBS. Withdrawal responses to mechanicalstimulation of the low abdominal skin with von-Frey hairs were measured. Thehistograms show that treatment with DNBS 10–12 days prior to the measurementsdecreased the threshold of withdrawal responses. Intraperitoneal injection of eachof the three gap junction blockers to DNBS-treated mice 1 h before the measure-ments significantly elevated the threshold, partly reversing the effect of DNBS. Thegap junction blockers were: carbenoxolone, CBX, 100 mg/kg; meclofenamic acid,MFA, 10 mg/kg; palmitoleic acid, PA, 25 mg/kg. All comparisons are with respect toDNBS. Dunn’s Multiple Comparisons test, * p < 0.01, N = 24 for Control and DNBS,N = 8 for each of the gap junction blockers.

4. Discussion

Neurons in sensory ganglia can be a source of ectopic electricalactivity, thus contributing to chronic pain (Devor, 2006). The mech-anisms underlying this activity are not entirely clear, and it is likelythat several changes combine to bring about abnormal firing. Ourfindings indicate that augmented coupling by gap junctions inDRG neurons and SGCs following colonic inflammation contributesto abnormal electrical activity in neurons, and hence to pain.

Electrical recordings from DRG neurons showed that after DNBStreatment the proportion of neurons with spontaneous activity in-creased from �20% to �40%, suggesting that at least 20% of neu-rons in S1 ganglia were affected by colonic inflammation. As onlyfew neurons in mouse S1 ganglia innervate the colon (�1%; Suet al., 1999), it is likely that neuronal hyperexcitability developednot only in colonic neurons but also in those that innervate othertargets and were not affected directly by colonic inflammation. Itfollows that hyperexcitability spreads among DRG neurons. Long-range spread of signals in sensory ganglia following damage is welldocumented (Thalakoti et al., 2007). There is evidence that chronicgastrointestinal pain (e.g. irritable bowel syndrome) is accompa-nied by somatic hyperalgesia in humans (Verne et al., 2003) andanimals (Bourdu et al., 2005), which was explained by a varietyof factors, including synaptic convergence in spinal dorsal hornneurons (Moshiree et al., 2006). The presence of coupling betweencolonic and non-colonic neurons, suggests that some convergenceoccurs at the DRG level.

Glial cells are essential for numerous functions of the nervoussystem under both normal and pathological conditions (Ketten-mann and Ransom, 2005). Abnormal glia–neuron interactions inthe spinal cord play a role in neuropathic pain (McMahon et al.,2005; Tsuda et al., 2003; Watkins and Maier, 2002; Woolf andScholz, 2007). Although peripheral injury causes striking changesin SGCs, the contribution of these cells to nociception has been lar-gely unexplored. In animal pain models SGCs proliferate, displayupregulation of GFAP and interleukin-1b (Li and Zhou, 2001; Pan-nese, 1981; Takeda et al., 2007; Woodham et al., 1989), consistentwith activation (gliosis). Following injury there is a great increasein gap junction–mediated coupling among SGCs (Hanani et al.,2002; Cherkas et al., 2004; Huang and Hanani, 2005), consistentwith an upregulation of the gap junction protein connexin43(Ohara et al., 2008). Our observation that DNBS induced an in-crease in coupling, together with these previous studies suggestthat augmented coupling is a part of glial activation following in-jury. The results support the hypothesis that this increase contrib-utes to post-inflammatory pain.

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In control ganglia coupling between neurons was rare, whereasin DNBS-treated mice 12.1% of the neurons were coupled to otherneurons. Little is known about coupling between primary sensoryneurons. In a study on a pain model in rats, no evidence for suchcoupling was found (Zuriel and Devor, 2001); however, the samplewas small. We used two methods to validate our findings: neuron–neuron coupling was largely blocked by CBX, and was completelyabsent when neurons were injected with a tracer that does notcross gap junctions. We conclude that neuron–neuron coupling ismediated by gap junctions. Recently we reported neuron–neuroncoupling in mouse DRGs associated with paw inflammation (Dub-lin and Hanani, 2007). Similarly, infraorbital nerve axotomy led toneuronal coupling in mouse trigeminal ganglia (unpublished). Itthus appears that such coupling may be a general response in sen-sory ganglia following peripheral injury. In earlier studies we didnot report neuron–neuron coupling because of its low incidenceand the possibility of an artifact. The control experiments men-tioned above rule out an artifact. It should be noted that neuronalcoupling appears relatively weak, as usually only a single neuronwas dye-coupled to the injected one, in contrast to the largernumber found for glia. Accordingly, the contribution of neuronalcoupling to the physiological changes found in the neurons follow-ing DNBS may be less important than that of SGCs.

To examine whether coupling influences neuronal excitability,we applied three different gap junction blockers during in vitroelectrical recordings – CBX, MFA and PA. These blockers, at con-centrations that completely blocked dye coupling, markedly re-versed DNBS-induced hyperexcitability. Currently no highlyspecific gap junction blockers are available (Spray et al., 2002),and it is possible that these substances acted via mechanismsnot related to gap junctions. Studies on CNS neurons showed thatCBX directly inhibited (Rouach et al., 2003), increased (Jahromiet al., 2002) neuronal excitability, or had no effect (Margineanuand Klitgaard, 2001). However, the blockers we selected arechemically very different, but share the ability to block gap junc-tions, and had similar effects on neuronal properties. Further-more, electrical recordings showed that CBX did not affectexcitability of neurons from control mice, where coupling islow. We therefore propose that enhanced cell coupling contrib-uted to neuronal hyperexcitability after DNBS treatment. It shouldbe added that the colonic inflammation selectively influencedganglia that innervate the colon, as evidenced by the absence ofaugmented coupling in DRGs L4,5 in DNBS-treated animals, againsuggesting that gap junctions in these ganglia were a major targetof the gap junction blockers. Previous studies have establishedthat gastrointestinal inflammation caused changes in ionic chan-nels in cultured DRG neurons, which could underlie neuronalhyperexcitability (Bielefeldt et al., 2002a; Bielefeldt et al.,2002b; Moore et al., 2002; Beyak et al., 2004). These results sug-gest that intrinsic neuronal changes contribute to visceral hyper-algesia, and are in apparent disagreement of ours. However, ourresults do not exclude other changes, and obviously, several cellu-lar events combine to bring about pain. It should be added thatcultured ganglia cannot be directly compared with intact ones,which are closer to the in vivo situation (see discussion by Lam-otte, 2007).

When each of the gap junction blockers was injected intraperi-toneally to mice it partly, but significantly, reversed DNBS-relatedhyperalgesia (Fig. 5). We therefore propose that formation and/oropening of gap junctions between DRG cells following DNBS con-tributes to visceral pain. Obviously, with systemic application wecannot be certain that the blockers acted on gap junctions in DRGs,but the in vitro experiments showed that gap junction blockers re-duced excitability of neurons from treated animals, suggesting thatgap junctions in DRGs were a likely target. The fact that CBX (Les-hchenko et al., 2006) and MFA (Pan et al., 2007) do not cross the

blood brain barrier, supports the idea that these blockers actedperipherally. That gap junction blockers have an analgesic actionis supported by a report that the gap junction blocker glycyrrheti-nic acid, which is chemically similar to CBX, has analgesic effects(Khaksa et al., 1996). Thus gap junction blockers might have a ther-apeutic potential as analgesic drugs. Recently it was reported thatCx43 RNA interference (RNAi) alters gap junction function in SGCs,and can reduce chronic pain a rat model (Ohara et al., 2008). Thiswork provided strong support for the hypothesis that gap junctionblockers can diminish chronic pain; and is consistent with the ideathat SGCs in sensory ganglia are a likely target of a peripheraladministration of carbenoxolone. Surprisingly, Ohara et al. (2008)also found that in naı̈ve animals, reducing gap junction expressionlowered pain threshold, indicating that gap junctions can contrib-ute to pain behavior in unexpected ways. Obviously, there is muchmore to learn about the contribution of gap junctions to pain undervarious physiological and pathological states.

How can coupling contribute to ectopic firing? A plausibleexplanation is that gap junctions augment communications be-tween DRG cells. A major mechanism by which glial cells transmitsignals is via intercellular Ca2+ waves (ICWs), which are mediatedby gap junctions and ATP (Scemes and Giaume, 2006). We demon-strated ICWs involving both SGCs and neurons in mouse trigeminalganglia (Suadicani et al., 2007). In some pain models neuronalsensitivity to ATP is enhanced (Zhou et al., 2007; Chen et al.,2005), and we have shown that inflammation increased SGC sensi-tivity to ATP (Hanani et al., 2007). Here we reported that inflamma-tion leads to augmented gap junction–mediated communication,and thus the two main factors controlling ICWs may be enhancedfollowing DNBS, promoting ICWs with the concomitant enhancedATP release. As ATP excites DRG neurons (Burnstock, 2007), theoverall effect will be neuronal excitation, consistent with our re-sults. This conclusion is in accord with reports on the contributionof ATP to neuropathic pain (Martucci et al., 2007; Khakh and North,2006). This explanation does not distinguish between neuronal andglial coupling, as both cell types will contribute to augmented ICWand to neuronal excitation.

Chronic visceral pain is a major clinical challenge, but its mech-anisms are largely obscure. Sensitization of DRG neurons after vis-ceral inflammation is well documented (Bielefeldt and Gebhart,2006) and is believed to play a role in pain. Alterations in ionicchannels in sensory neurons are likely to be a major factor in sen-sitization, but other mechanisms cannot be excluded. A cellularchange that may contribute to somatic pain is increased cell cou-pling via gap junctions (Dublin and Hanani, 2007; Spataro et al.,2004), but the role of gap junctions in visceral pain has received lit-tle attention. Our results indicate that augmented coupling is asso-ciated with visceral pain. Animal models of intestinal inflammationshowed that inflammation induced by trinitrobenzoate sulfonate(TNBS), which is even more potent than DNBS in inducing inflam-mation, decays in 7 days (Demedts et al., 2006). As we examinedDRGs 10–12 following DNBS, it is very likely that the colonicinflammation had completely healed, whereas the changes inDRG cells and visceral pain persisted. It can therefore be concludedthat colonic inflammation leads to long-term changes after theinflammation had resolved. Post-inflammatory pain is well recog-nized (Eijkelkamp et al., 2007), and may explain human disorderssuch as irritable bowel syndrome.

Acknowledgement

Supported by the Israel Science Foundation (Grant No. 577/03),by the US–Israel Binational Science Foundation (BSF, Grant No.2003262) and by the Hebrew University Center for Pain Research.We thank Dr. Pavel Cherkas for helpful comments on the manu-script and Mr. Pavel Dublin for technical assistance.

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Table S1Basic electrophysiological characteristics of DRG neurons 10-12 days after inducing colonic inflammation by DNBS.

Cells randomly recorded Cells projecting to the colon

Control Inflammation Control Inflammation

A-typen 43 70 16 18Rm (mV) 49.7 ± 8.5 45.1 ± 7.5 52.5 ± 6.4 49.7 ± 7.6*

APA (mV) 55.6 ± 11.2 54.9 ± 12.5 63.1 ± 9.6 60.8 ± 10.2APD (ms) 7.0 ± 1.3 7.1 ± 1.7 7.1 ± 1.2 7.3 ± 1.3Threshold (nA) 0.47 ± 0.2 0.32 ± 0.17* 0.63 ± 0.24 0.34 ± 0.21*

Ri (MX) 47.0 ± 5.9 43.2 ± 5.0* 42.3 ± 5.2 38.8 ± 5.1*

SPS 1 (2.3%) 6(8.6%) 0 2 (11.1%)SPO 9 (20.9%) 28(40.0%)* 2 (12.5%) 9 (50%)*

C-typeN 29 41 27 23Rm (mV) 51.6 ± 8.1 47.2 ± 6.4* 53.9 ± 8.8 48.9 ± 6.2*

APA (mV) 61.2 ± 2.4 57.2 ± 10.9 63.7 ± 9.4 60.9 ± 6.7APD (ms) 11.8 ± 0.5 11.5 ± 3.2 13.5 ± 3.1 13.4 ± 2.4Threshold (nA) 0.45 ± 0.04 0.26 ± 0.19* 0.43 ± 2.1 0.27 ± 0.24*

Ri (MX) 46.1 ± 1.0 42.8 ± 5.8* 44.4 ± 5.7 41.6 ± 5.3*

SPS 0 5 (12.2%)* 1 (3.7%) 6 (26.1%)*

SPO 5 (17.2%) 17 (41.5%)* 7 (25.9%) 16 (69.6%)*

*P < 0.05, as compared with control. RMP, resting membrane potential; APA, action potential amplitude; APD, duration of action potential; Ri, membrane input resistance; SPS,percentage of cells with spontaneous spikes; SPO, percentage of cells with spontaneous oscillations; ‘‘Threshold” indicates the minimal current required to elicit a singleaction potential. Fisher’s exact test and one way ANOVA test were used for comparisons.

Fig. S1. Inflammation-induced changes in dye coupling between SGCs in the vicinity of neurons that innervate the colon. (a) A micrograph showing two DiI-labeled neurons(marked N1,N2) that innervate the inflamed colon. (b) An SGC (asterisk) near N1 was injected with LY; this cell appears larger than its actual size because of light scatter. (c)Merged image obtained from (a) and (b) showing the relationship between DiI-labeled neurons and LY-injected SGC. Note that the LY-injected SGC is coupled to other SGCsthat ensheathe unlabeled (presumably non-colonic) neurons, and that the neurons were not labeled with LY. The images are of living cells during the injection experiment.Scale bar, 25 lm. The histograms show that glial cell coupling in the vicinity of the DiI-labeled neurons was much greater after inducing colonic inflammation. (d) Incidenceof coupling between SGCs around different neurons. (e) Incidence of coupling between SGCs around the same neurons. (f) The number (± S.E.M.) of SGCs coupled to the LY-injected SGCs. * p < 0.01, as compared with control. Fisher’s exact test (d and e) and Mann–Whitney test (f) were used for comparison.

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Table S2The effect of Gap junction blockers on neuronal excitability of DRG S1 neurons 10-12 days after inducing colonic inflammation by DNBS.

DNBS +CBX +MFA +PA

A-typen 70 48 51 57Rm (mV) 45.1 ± 7.5 49.2 ± 8.3* 48.9 ± 7.8* 48.8± 8.1*

APA (mV) 54.9 ± 12.5 56.1 ± 9.0 54.0 ± 10.0 55.6 ± 10.4APD (ms) 7.1 ± 1.7 6.9 ± 1.4 7.1 ± 1.4 6.9 ± 1.5Threshold (nA) 0.32 ± 0.17 0.44 ± 0.21* 0.420.21 0.40 ± 0.22Ri (MX) 43.2 ± 5.0 42.9 ± 6.9 43.0 ± 6.4 43.3 ± 8.1SPS 6(8.6%) 1 (2.1%)* 0* 1 (1.8%)*

SPO 28(40.0%) 8 (16.7%)* 9 (17.6%)* 9 (15.9%)*

C-typen 41 27 22 31Rm (mV) 47.2 ± 6.4 50.7 ± 7.3* 51.4 ± 8.4* 50.5± 6.1*

APA (mV) 57.2 ± 10.9 60.9 ± 10.9 61.4 ± 12.2 60.2 ± 8.9APD (ms) 11.5 ± 3.2 11.6 ± 2.1 11.9 ± 2.3 11.7 ± 2.2Threshold (nA) 0.26 ± 0.19 0.41 ± 0.21* 0.38 ± 6.1 0.39 ± 0.22Ri (MX) 42.8 ± 5.8 42.1 ± 1.2 42.5 ± 1.3 41.7 ± 6.1SPS 5 (12.2%) 0* 0* 0*

SPO 17 (41.5%) 5 (18.5%)* 5 (22.7%)* 6 (19.4%)*

The gap junction blockers used were: carbenoxolone, CBX, 50 lM; meclofenamic acid, MFA, 100 lM; palmitoleic acid, PA, 30 lM. * p < 0.05, as compared with the dataobtained from ganglia from DNBS-treated mice. RMP, resting membrane potential; APA, action potential amplitude; APD, duration of action potential;Ri, membrane inputresistance; SPS, percentage of cells with spontaneous spikes; SPO, percentage of cells with spontaneous oscillations; ”Threshold” indicates the minimal current required toelicit a single action potential. Fisher’s exact test and one way ANOVA test were used for comparisons.

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