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Journal of Neurophysiology logoLink to Journal of Neurophysiology
. 2010 Sep 22;104(6):3113–3123. doi: 10.1152/jn.00560.2010

Altered Purinergic Signaling in Colorectal Dorsal Root Ganglion Neurons Contributes to Colorectal Hypersensitivity

Masamichi Shinoda 1,3,, Jun-Ho La 1, Klaus Bielefeldt 2,3, G F Gebhart 1
PMCID: PMC3007642  PMID: 20861433

Abstract

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by pain and hypersensitivity in the relative absence of colon inflammation or structural changes. To assess the role of P2X receptors expressed in colorectal dorsal root ganglion (c-DRG) neurons and colon hypersensitivity, we studied excitability and purinergic signaling of retrogradely labeled mouse thoracolumbar (TL) and lumbosacral (LS) c-DRG neurons after intracolonic treatment with saline or zymosan (which reproduces 2 major features of IBS—persistent colorectal hypersensitivity without inflammation) using patch-clamp, immunohistochemical, and RT-PCR techniques. Although whole cell capacitances did not differ between LS and TL c-DRG neurons and were not changed after zymosan treatment, membrane excitability was increased in LS and TL c-DRG neurons from zymosan-treated mice. Purinergic agonist adenosine-5′-triphosphate (ATP) and α,β-methylene ATP [α,β-meATP] produced inward currents in TL c-DRG neurons were predominantly P2X3-like fast (∼70% of responsive neurons); P2X2/3-like slow currents were more common in LS c-DRG neurons (∼35% of responsive neurons). Transient currents were not produced by either agonist in c-DRG neurons from P2X3−/− mice. Neither total whole cell Kv current density nor the sustained or transient Kv components was changed in c-DRG neurons after zymosan treatment. The number of cells expressing P2X3 protein and its mRNA and the kinetic properties of ATP- and α,β-meATP-evoked currents in c-DRG neurons were not changed by zymosan treatment. However, the EC50 of α,β-meATP for the fast current decreased significantly in TL c-DRG neurons. These findings suggest that colorectal hypersensitivity produced by intracolonic zymosan increases excitability and enhances purinergic signaling in c-DRG neurons.

INTRODUCTION

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by abdominal pain, hypersensitivity, and changes in bowel activity in the relative absence of inflammation or other structural changes (Camilleri 2005; Gershon and Tack. 2007; Spiller 2007). Despite continuing investigation, the pathogenic mechanisms of IBS remain unclear (Drossman et al. 1997).

Several endogenous mediators have been implicated in gastrointestinal hypersensitivity in general and IBS in particular, including serotonin (Gershon and Tack. 2007; Mawe et al. 2006), proteases (Cenac et al. 2007; Coelho et al. 2002; Kirkup et al. 2003), and adenosine-5′-triphosphate (ATP). ATP has been of long-term interest as a signaling molecule in the gut (Burnstock 2001, 2008). ATP is released from a wide variety of cells (North 2002), including colonic epithelium in response to colorectal distension (Shinoda et al. 2009; Wynn et al. 2003). ATP and its metabolites can interact with a large number of ligand-gated ion channels (P2X receptors) and G-protein-coupled receptors of the P2Y family. P2X receptors are expressed on sensory neuron cell bodies in dorsal root ganglia (DRG) (Burnstock 2006), including visceral sensory DRG neurons (Dang et al. 2008; Wynn et al. 2003). ATP also contributes to colorectal mechanotransduction by activating pelvic nerve afferents (Wynn et al. 2003), a role that is enhanced after colon inflammation (Wynn et al. 2004). It also has been reported that P2X receptor antagonists reverse visceral hypersensitivity, further supporting the contribution of ATP to visceral sensory mechanisms (Xu et al. 2008). Finally, ATP is implicated in human colon pathology by increased expression of P2X3 receptors in inflammatory bowel disease (Yiangou et al. 2001).

We previously documented that colorectal mechanotransduction and zymosan-produced colon hypersensitivity, which develops and persists in the absence of a neutrophil-based colorectal inflammation, are attenuated and do not develop, respectively, in P2X3−/− mice (Shinoda et al. 2009). These outcomes further support a role for purinergic signaling in colorectal hypersensitivity. In the present study, we examined purinergic responses and P2X receptor expression in colorectal DRG (c-DRG) neurons after intracolonic saline or zymosan treatment (replicating the visceral hypersensitivity in IBS patients), hypothesizing that the robust colon hypersensitivity produced would be associated with increased cell excitability and enhanced purinergic sensitivity.

METHODS

Animals

Adult male C57BL/6 (Taconic, Germantown, NY) and congenic P2X3 knockout (backcrossed onto the C57BL/6 genetic background for ≥10 generations) mice (20–30 g) were used in these experiments. All procedures were approved by the Institutional Animal Care and Use Committee, University of Pittsburgh.

Cell labeling

Mice were anesthetized (2% isoflurane; Hospira, Lake Forest, IL), the colon exposed surgically (lower abdominal incision, ∼1 cm in length), and 50 mg/ml (in 100% DMSO) of 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate (DiI; Molecular Probes, Eugene, OR) was injected into one to three sites within the descending colon wall (∼1.5 cm from the anal verge) using a 30-gauge needle (∼6 μl per site). Visible leakage of DiI was removed with a cotton swab. The incision was closed (5–0 polyglactin sutures; Ethicon, Somerville, NJ), and mice were subsequently returned to their home cages until use 14–21 days later.

Intracolonic treatments

Mice were anesthetized with ketamine/xylazine (87.5/12.5 mg/kg ip) and 0.1 ml of saline or of a suspension of 30 mg/ml zymosan (derived from Saccharomyces cerevisiae; Sigma, St. Louis, MO) in saline was administered transanally via a 22-gauge, 24-mm-long stainless-steel feeding needle. Saline or zymosan was given daily for three consecutive days; lumbosacral (LS) and thoracolumbar (TL) DRG were harvested on day 4. This treatment protocol results in a robust and long-lasting colon hypersensitivity to balloon distension (Shinoda et al. 2009). We checked each mouse postmortem for leakage of DiI by examining the colon and surrounding tissues after removal of DRG. No labeling in any tissue other than the colon was noted.

Cell dissociation and culture

The mouse colon is innervated by pelvic and lumbar splanchnic nerves with cell bodies contained in LS and TL DRG, respectively. Because there are significant differences between the LS and TL innervations of pelvic organs (Brierley et al. 2005; Dang et al. 2008; Xu and Gebhart. 2008), c-DRG neurons from both innervations were studied. Mice were killed via CO2 inhalation and T11–-L1 or L6–S2 DRG removed rapidly and incubated at 37°C in 5% CO2 for 40 min in serum free, advanced Dulbecco's modified eagle medium (DMEM)/F12 containing 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA), collagenase (type 4; 2 mg/ml; Worthington Biochemical, Lakewood, NJ), and trypsin (1 mg/ml; Worthington). After washing three times with advanced DMEM/F12 containing 10% fetal bovine serum (Sigma; without enzymes), DRG were submerged in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin, 625 μM l-glucose, 4.5 g/l d-glucose, 110 mg/l sodium pyruvate and triturated gently to encourage cell dissociation. Neurons were plated on poly-d-lysine-coated coverslips (Becton Dickinson Labware, Bedford, MA) and incubated at 37°C in 5% CO2. Only c-DRG neurons (i.e., DiI-containing DRG neurons) were studied. All recordings were performed within 24 h after plating.

Solutions and electrophysiological recordings

Coverslips with cells were transferred to a recording chamber (1 ml) and superfused continuously (2 ml/min) with external solution containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with NaOH (305 mosM). Neurons that innervated the colon were identified by DiI content using a rhodamine filter. Fire-polished micropipettes with tip resistances of 2–4 MΩ were used for current- and voltage-clamp recordings. The uncompensated series resistance was generally <10 MΩ. The pipette was filled with an internal solution consisting of (in mM): 130 KCl, 0.2 CaCl2, 4 NaCl, 10 EGTA, 10 HEPES, 0.5 Na triphosphate, and 2 MgATP. The pH was adjusted to 7.25 using KOH (290 mosM). After establishing the whole cell configuration, the voltage was clamped at −70 mV using an Axopatch 200B amplifier, digitized at 1 kHz (Digidata 1350), and controlled by Clampex software (pClamp 10.0; all from Axon Instruments, Foster City, CA). Cell capacitance and resting membrane potential (RMP) were obtained from the Axopatch 200B amplifier. Recordings began 3 min after establishing whole cell configuration to ensure stable recording conditions. Only cells that had a RMP more negative than −40 mV and generated action potentials with a distinct overshoot >0 mV in response to depolarizing current injections were studied. Spontaneous activity was counted during a 1 min period before electrical or chemical stimulation. A series of 5-ms current pulses in 50-pA increments (1 s apart) was injected to determine action potential (AP) threshold, which was taken as the greatest membrane potential in the absence of an AP; the minimum current required to evoke an AP was considered rheobase. To examine firing patterns in c-DRG neurons, suprathreshold current (2 × rheobase) was injected for 500 ms and the number of APs counted. Drugs were applied using a fast-step SF-77B superfusion system (Warner Instruments, Hamden, CT) with a three-barrel pipette placed in close proximity (100 μm) to the cell, allowing complete solution exchange within 25 ms (Dang et al. 2005a). Purinergic agonists [ATP and α,β-methylene ATP (α,β-meATP); Sigma] were applied for 2 s, whereas antagonists were superfused for 30 s before the application of agonists. A washout period of 4 min was allowed between agonist applications. Drugs and chemicals were prepared fresh from stock solutions on the day of the experiment; experiments were performed at room temperature (21–23°C). We used current increases >20 pA or voltage changes >4 mV to identify responses to purinergic agonists because these levels exceeded baseline variability by a factor of two. To determine the kinetics of response onset, we measured the time from 10 to 80% of peak amplitude.

To isolate voltage-gated K+ (Kv) currents and minimize contamination by Na+ and Ca2+, NaCl was replaced with an equimolar amount of choline chloride, and CdCl2 (50 μM) was included in the external solution (pH was adjusted to 7.4 with Tris-base). Cells were held at −70 mV and subjected to either a −100 or −30 mV prepulse potential for 400 ms before outward currents were elicited by stepping the membrane potential from −60 to 40 mV for 400 ms in 10 mV increments. Inactivating Kv currents were isolated by subtracting the outward currents after −30 mV prepulse from those after −100 mV. The −30 mV prepulse for evoking the noninactivating currents was chosen based on the steady-state inactivation/availability curve of K+ currents in a pilot study. Series resistance was compensated by >80%, and leak currents were subtracted using the on-line P/4 protocol. Data were sampled at 10 kHz and low-pass filtered at 5 kHz.

Immunohistochemistry

Mice were anesthetized with ketamine/xylazine as in the preceding text and transcardially perfused with saline followed by a cold fixative containing 4% paraformaldehyde in 0.2% picric acid and 0.1 M phosphate buffer (pH 7.4). T11–L1 or L6–S2 DRG were removed rapidly and immersed in the same fixative for 4 h at 4°C. Postfixed DRG were cryoprotected for 12 h in 0.1 M phosphate buffered saline (PBS) containing 20% sucrose, then embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan) at −20°C and cut on a cryostat (10 μm). DRG sections were reacted with rabbit anti-P2X3 polyclonal antiserum (1:1000; Neuromics, Northfield, MN) overnight at 4°C. After rinsing with 0.01 M PBS, all samples were reacted with AlexaFluor488-conjugated goat anti-rabbit IgG (Invitrogen) at a dilution of 1:200 in 0.01 M PBS for 2 h at room temperature. After rinsing with 0.01 M PBS, sections were coverslipped in mounting medium (Dako, Carpinteria, CA) and examined under a fluorescence microscope. Using appropriate filters, double (DiI and AlexaFluor488)-labeled cells were identified and analyzed using ImageJ software (National Institutes of Health). Cells twofold or more intense than average background were considered positive for P2X3 immunoreactivity. No specific labeling was observed in the absence of primary antibody or in DRG from P2X3−/− mice. The percentage of P2X3 immunoreactive, DiI-labeled c-DRG neurons was calculated based on neuron counts in five randomly chosen sections separated by 100 μm to avoid double counting of cells. Histological analysis was performed blind with respect to treatment to colon.

Single-cell reverse transcription polymerase chain reaction (RT-PCR)

DiI-labeled c-DRG neurons (dissociated and cultured as in the preceding text) were collected with large-bore (∼50 μm) glass pipettes and expelled into microcentrifuge tubes containing reverse-transcriptase (RT) mix (Nealen et al. 2003). Cell lysis and RT reactions consisted of incubation at 65°C for 1.5 min, at room temperature for 2 min, at 37°C for 20 min after administrating 20 U SuperScript II (Invitrogen), and at 65°C for 10 min. For each experiment, negative controls were performed by omitting reverse transcriptase or using a cell-free bath aspirate as template. The first-strand complementary deoxyribonucleic acid (cDNA) from a c-DRG neuron was used as template in a polymerase chain reaction (PCR) containing 1xGoTaq reaction buffer (Promega, Madison, WI), 10 μM outer primers, 0.2 mM dNTPs, and 0.2 μl GoTaq DNA polymerase (Promega); primer sequences are listed in Table 1A. Reactions consisted of initialization at 95°C for 10 min, 35 cycles at 94°C/30 s, 52°C/30 s and 72°C/30 s before a final extension step at 72°C for 10 min. Each initial PCR product served as template in a subsequent PCR reaction using a nested primer pair. Each PCR product in a subsequent PCR reaction was electrophoresed on 2% agarose–ethidium bromide gels and photographed. Only neurons producing detectable amplification of a housekeeping gene (GAPDH) were analyzed further.

Table 1.

P2X3, P2X2, or both P2X3 and P2X2 mRNA expressed colon senory neurons

A. DNA primers used for PCR amplification
Target Gene/Predicted Size Outer Primers Nested Primers Genbank No.
P2X3 GCTCCGTAGAAGAAGATGGAGA TGTCCTAAGAGGATCCTGTACC NM_145526
251 bp, 141 bp CTGTGTGACCATGTTAGGGATG GGCATCTAGCACATAGAAGTGG
P2X2 CTCTTCAGTAACCATGTCCACG GAAGATAGGCATCTTGCTCTGG NM_153400
233 bp, 113bp CCGGAAGACAGCTCTAATTTGG GGGATCCTATGAGGAGTTCTGT
B. Colon sensory neurons expressing P2X3, P2X2 or both P2X3 and P2X2 mRNA
P2X3 Only, % Both P2X3 and P2X2, % P2X2 Only, % Neither P2X3 or P2X2, %
Saline
    LS 33/59; 55.9 20/59; 33.9 3/59; 5.1 3/59; 5.1
    TL 31/54; 57.4 20/54; 37.0 3/54; 5.6 3/54; 5.6
Zymosan
    LS 25/58; 43.1 29/58; 50.0 2/58; 3.4 2/58; 3.4
    TL 33/55; 60.0 18/55; 32.7 1/55; 1.8 3/55; 5.5

Sequence of P2X3, and P2X2 mRNA are indicated with 5′ at let. a, adenosine; c, cytosine; g, guanosine; t, thymidine; Outer primers were used for the first round of PCR; nested primers were used for the second round. Sequences used for primer design are indicated at right. bp, base pairs.

Colon sensory neurons expressing P2X3, P2X2 or both P2X3 and P2X2 mRNA are shown. The ratios of mRNA expression are calculated per total number of GAPDH mRNA-positive neurons in each group. LS, lumbosacral; TL, thoracolumbar.

Data analysis

Data are presented as means ± SE. The software packages pClamp 10.0 (Axon Instruments) and Prism 4 (Graph Pad, San Diego, CA) were used for data analysis. Dose–response curves were fit to sigmoidal functions using the following equation: Y = A/[1 + 10(logEC50 – X)B], where X is the logarithm of agonist concentration and Y is the response and starts at 0 and ends at A with a sigmoidal shape. B is the Hill slope. Desensitization kinetics were fitted with a standard exponential equation: Y = K0 + K1 × exp(−t/τ), where Y is the current amplitude at time t, K0 is the amplitude of the sustained component, and τ is the time constant. K0 and K1 represent the contribution to current amplitude from the fast and slow components of the current, respectively. For dichotomous variables, χ2 or unconditional exact tests were used. Statistical analyses were performed using Student's t-test and one- or two-way (repeated-measures) ANOVA where appropriate. Results were considered statistically significant when P ≤ 0.05.

RESULTS

Cell size

In previous studies, rat LS and TL colon (Gold and Traub 2004) and urinary bladder (Dang et al. 2005a; Yoshimura and De Groat. 1999) neurons were reported to differ in size and to increase after organ inflammation (Dang et al. 2008). Mean whole cell capacitances (as an index of cell size) did not significantly differ between mouse LS (33.5 ± 1.3 pF) and TL (32.96 ± 1.7 pF) c-DRG neurons and were not changed after zymosan treatment (Fig. 1).

Fig. 1.

Fig. 1.

Distribution of whole cell capacitance in thoracolumbar (TL) and lumbosacral (LS) colorectal dorsal root ganglion (c-DRG) neurons from saline- and zymosan-treated mice. c-DRG neurons from TL and LS DRG and from saline- and zymosan-treated mice exhibited the same distribution of capacitances.

c-DRG neuron excitability

ACTIVE AND PASSIVE MEMBRANE PROPERTIES.

Zymosan treatment was associated with a significantly depolarized RMP and enhanced membrane excitability in both LS and TL c-DRG neurons (Fig. 2). No c-DRG neurons from saline-treated mice exhibited spontaneous activity (LS: 0/35; TL: 0/37), but a significant proportion of LS neurons (6/37, 16%; unconditional exact test, P < 0.05) from zymosan-treated mice were spontaneously active (Table 2). Zymosan treatment did not affect action potential (AP) threshold, rheobase, number of APs evoked by suprathreshold current injection (2 X rheobase) or input resistance.

Fig. 2.

Fig. 2.

Passive and active properties of c-DRG neurons from saline- and zymosan-treated mice examined in current-clamp mode. Examples illustrate that neither LS (A, left) nor TL (F, left) neurons were spontaneously active in control mice, but ∼1/6 of LS neurons from zymosan-treated mice exhibited spontaneous activity (A, right). The excitability of LS and TL neurons as well as the number of TL neurons in which action potentials were produced by ATP (e.g., I, right; see also Table 1) was increased by zymosan treatment. Resting membrane potentials are indicated under each trace.

Table 2.

Passive and active electrical properties of LS and TL colon neurons from zymosan-treated and control (saline-treated) mice

Property Treatment TL LS
A. Current injection
Whole cell capacitance, pF Control 33.0 ± 1.7 (37) 33.5 ± 1.3 (35)
Zymosan 28.9 ± 1.2 (25) 34.8 ± 1.3 (37)
Series resistance, mohm Control 6.0 ± 0.3 (37) 6.5 ± 0.3 (35)
Zymosan 6.3 ± 0.5 (25) 6.3 ± 0.3 (37)
Resting membrane potential, mV Control *−63.5 ± 1.4 (37) −64.3 ± 1.4 (35)*
Zymosan *−58.4 ± 1.9 (25) −57.9 ± 1.6 (37)*
Spontaneous activity (frequency) Control 0/37 0/35*
Zymosan 1/25 6/37*
AP threshold, mV Control −27.6 ± 1.2 (31) −28.9 ± 1.2 (24)
Zymosan −29.8 ± 1.3 (16) −29.4 ± 1.4 (26)
Rheobase, pA Control 247.3 ± 13.0 (37) 227.1 ± 21.7 (24)
Zymosan 237.5 ± 23.5 (16) 211.5 ± 16.5 (26)
AP@2× rheobase (frequency) Control 2.8 ± 0.8 (31) 4.4 ± 0.9 (23)
Zymosan 4.8 ± 1.4 (16) 5.4 ± 1.1 (26)
Input resistance, mohm Control −32.4 ± 3.6 (32) −40.1 ± 3.3 (26)
Zymosan −28.8 ± 3.8 (16) −35.9 ± 3.7 (26)
B. ATP (30 μM)
Number of cells which induce AP, % Control *2.9 (1/34) 5.7 (2/35)
Zymosan *20 (4/20) 16.8 (5/30)
Number of cells depolarized, % Control 50 (17/34) 52.6 (20/38)
Zymosan 75 (15/20) 67.7 (21/31)
Depolazation, mV Control 16.8 ± 2.7 (17) 13.9 ± 1.5 (20)
Zymosan 16.9 ± 1.7 (15) 17.1 ± 1.8 (21)
C. α,β-meATP (30 μM)
Number of cells which induce AP, % Control 0 (0/32) 0 (0/31)
Zymosan 10 (2/20) * 0 (0/25)
Number of cells depolarized, % Control 50 (16/32) 25.8 (8/31)*
Zymosan 70 (14/20) * 60 (15/25)*
Depolazation, mV Control 15.7 ± 2.0 (16) 9.5 ± 1.5 (8)
Zymosan 17.2 ± 1.7 (15) 11.3 ± 1.5 (15)

Values are means ± SE, or percentages. Number of neurons are in parentheses.

*

P < 0.05. AP, action potential, ATP, adenosine-5′-triphosphate; α,β-meATP, α,β-m ethylene ATP.

Effect of purinergic agonists

In current clamp mode, ATP (30 μM) depolarized one-half of LS (20/38, 53%) and TL (17/34, 50%) c-DRG neurons from saline-treated mice, triggering APs in 6% of LS and 3% of TL neurons (Fig. 2, D and I; Table 2). Zymosan treatment did not change the magnitude of depolarization produced by ATP in either LS (21/31; 68%) or TL (15/20; 75%) c-DRG neurons compared with control (Table 2). Zymosan treatment did, however, significantly increase the proportion of TL c-DRG neurons in which APs were triggered by ATP (4/20, 20% vs. 1/34, 3% in control; unconditional exact test, P < 0.05).

α,β-meATP, a selective homomeric P2X3 and heteromeric P2X2/3 receptor agonist (North 2002), was applied to the same neurons previously challenged with ATP. The proportion of TL c-DRG neurons from saline-treated mice depolarized by α,β-meATP (30 μM) was significantly greater than the proportion of LS neurons (LS: 8/31, 26%. TL: 16/32, 50%. χ2 test, P < 0.05); α,β-meATP did not trigger APs in either LS or TL c-DRG neurons (Fig. 2, E and J, Table 2). In zymosan-treated mice, the proportion of LS c-DRG neurons depolarized by α,β-meATP was significantly increased (15/25, 60% vs. 8/31, 26% in control; χ2 test, P < 0.01). α,β-meATP (30 μM) also produced a greater depolarization in TL c-DRG neurons from control mice compared with LS counterparts (TL: 15.7 ± 2.0 mV; LS: 9.5 ± 1.5 mV. Student's t-test, P < 0.05; Table 2). While the fraction of responsive cells increased, the depolarization produced by α,β-meATP did not change after zymosan treatment in either TL or LS c-DRG neurons.

Whole cell Kv currents

Several currents may contribute to changes in neuron excitability (e.g., inward rectifying potassium currents, voltage-sensitive sodium or calcium currents). Accordingly, we examined whether Kv channels also were involved in the zymosan-produced increase in membrane excitability of c-DRG neurons. Total whole cell Kv current density was not changed in either TL or LS c-DRG neurons after zymosan treatment, and neither was the sustained nor transient Kv component (−30 mV prepulse; Fig. 3).

Fig. 3.

Fig. 3.

Whole cell Kv current in TL (A) and LS (B) colonic DRG neurons. Total whole cell Kv current (left), noninactivating (middle), and inactivating (right) Kv current by −30 mV prepulse were not changed after zymosan treatment.

Characterization of P2X receptor-mediated currents in c-DRG neurons

LS c-DRG NEURONS.

To evaluate functionally purinergic receptors on c-DRG neurons, we applied ATP (100 μM), which triggered inward currents in most LS neurons tested (saline-treated, 39/57, 68%; zymosan-treated, 28/39, 72%). Based on the kinetics of current decay, we differentiated fast, slow, and sustained currents in neurons from saline- and zymosan-treated mice (Fig. 4). In saline-treated mice, 14/39 (36%), 16/39 (41%), and 9/39 (23%) LS c-DRG neurons expressed fast, slow, and sustained current kinetics, respectively. Similar proportions of LS c-DRG neurons from zymosan-treated mice, 9/28 (32%), 9/28 (32%), and 8/28 (32%) expressed fast, slow, and sustained current kinetics, respectively. The 10–80% rise time and desensitizing time constants of fast and slow current kinetics generated by ATP did not differ between saline- and zymosan-treated mice in LS c-DRG neurons (Table 3).

Fig. 4.

Fig. 4.

ATP-activated inward currents in both LS and TL c-DRG neurons were characterized based on desensitization kinetics as fast, slow, and sustained.

Table 3.

Current kinetics for LS and TL colon sensory neurons from saline- and zymosan-treated mice

ATP (100 μM)
α,β-meATP (100 μM)
Current Treatment 10–80% Rise Time, ms tau. ms Current Density, pA/pF 10–80% Rise Time, ms tau. ms Current Density, pA/pF
LS
    Fast Saline 36.4 ± 6.5 96.8 ± 15.4 15.5 ± 3.1 27.3 ± 6.2 94.8 ± 20.6 21.9 ± 3.0
Zymosan 49.1 ± 9.9 127.7 ± 9.5 13.8 ± 3.2 30.1 ± 4.4 77.2 ± 10.5 22.0 ± 3.3
    Slow Saline 224.1 ± 29.4 N/A 18.7 ± 4.6 258.5 ± 11.0 N/A 16.3 ± 3.9
Zymosan 281.1 ± 36.3 N/A 20.1 ± 4.7 264.9 ± 22.8 N/A 17.3 ± 3.6
TL
    Fast Saline 53.5 ± 11.0 100.1 ± 15.7 17.3 ± 2.6 44.6 ± 7.2 84.1 ± 8.9 20.5 ± 2.9
Zymosan 68.8 ± 9.4 110.1 ± 9.7 17.4 ± 3.3 34.9 ± 4.6 83.4 ± 6.6 24.0 ± 2.0
    Slow Saline 220.6 ± 31.1 N/A 25.0 ± 4.0 358.0 ± 54.0 N/A 13.7 ± 8.6
Zymosan 224.6 ± 25.6 N/A 33.4 ± 10.4 349.1 ± 26.0 N/A 16.2 ± 3.4

We also examined the effects of α,β-meATP (100 μM), which produced inward currents in LS c-DRG neurons (saline-treated, 26/51, 51%; zymosan-treated, 33/63, 52%) (Fig. 5). Based on the current kinetics described in the preceding text, we identified fast (9/26, 36%) and slow (17/26, 65%) currents in LS c-DRG neurons from saline-treated mice and similar proportions of fast (15/33, 45%) and slow (18/33, 55%) currents in LS c-DRG neurons from zymosan-treated mice. The activation and desensitization kinetics were similar to those obtained in response to ATP and did not differ between neurons from saline- and zymosan-treated mice (Table 3).

Fig. 5.

Fig. 5.

α,β-Methylene adenosine-5′-triphosphate (α,β-meATP)-activated inward currents in both LS and TL c-DRG neurons were characterized based on desensitization kinetics as fast and slow. In both LS and TL c-DRG neurons, α,β-meATP produced currents with kinetics similar to that triggered by ATP.

TL c-DRG NEURONS.

Similar to LS neurons, ATP (100 μM) produced inward currents in most TL c-DRG neurons tested (saline-treated, 27/37, 73%; zymosan-treated, 41/57, 72%). In saline-treated mice, 18/27 (67%), 7/27 (26%), and 2/27 (7%) TL c-DRG neurons expressed fast, slow, and sustained current kinetics, respectively. The relative proportion of these kinetic properties did not differ in neurons from zymosan-treated mice [fast (31/41, 76%), slow (8/41, 20%), and sustained (2/41, 5%) currents; Fig. 4]. Similar to LS c-DRG neurons, the 10–80% rise time and desensitizing time constants of fast and slow current kinetics generated by ATP did not differ between saline- and zymosan-treated mice in TL neurons (Table 3).

A high proportion of TL c-DRG neurons tested (saline-treated, 38/46, 82%; zymosan-treated, 48/60, 80%) responded to α,β-meATP (Fig. 5). Like ATP, α,β-meATP triggered similar proportions of fast and slow currents in TL neurons from saline-treated (fast, 31/38, 82%; slow, 7/38, 18%) and zymosan-treated (fast, 37/48, 77%; slow, 11/48, 23%) mice. As described for LS c-DRG neurons, the activation and desensitization kinetics were similar to those obtained in response to ATP, 10–80% rise time, and desensitizing time of fast and slow current kinetics generated by α,β-meATP did not differ between saline- and zymosan-treated mice in TL neurons (Table 3).

Effects of P2X3 deletion and purinergic antagonists on P2X-dependent currents

To establish that the fast current triggered by ATP and α,β-meATP is dependent on P2X3 receptors, we examined c-DRG neurons from P2X3−/− mice. The genetic status of the mice was established by polymerase chain reaction for the P2X3 gene. In LS and TL c-DRG neurons from P2X3−/− mice, a transient current was not produced either by ATP or α,β-meATP (Fig. 6). However, we did observe a sustained current in response to ATP in 1/5 neurons (3 mice were used for harvesting cells; 1 cell from 1 mouse, 2 cells each from 2 mice). To further examine P2X-dependent currents, we applied the potent and selective homomeric P2X3 and heteromeric P2X2/3 receptor antagonist A-317491 (Jarvis et al. 2002) to c-DRG neurons from saline-treated mice. In the presence of A-317491 (10 μM), both ATP- and α,β-meATP-mediated fast and slow currents were reversibly attenuated (Fig. 7).

Fig. 6.

Fig. 6.

ATP- and α,β-meATP-activated fast and slow inward currents are absent in P2X3−/− mice; a sustained current, however, was present.

Fig. 7.

Fig. 7.

Antagonism of purinergic-activated fast and slow inward currents by co-application of the P2X3, 2/3 receptor antagonist, A-317491.

P2X receptor dose–response relationship

We next determined the dose-response relationship for both agonists by applying either ATP or α,β-meATP in increasing concentrations. In TL and LS c-DRG neurons, both ATP- and α,β-meATP-activated currents were concentration-dependent for both fast and slow currents (Figs. 8 and 9). The dose-response curves of fast but not slow currents induced by α,β-meATP shifted to the left in c-DRG neurons from zymosan-treated mice. The logEC50 for α,β-meATP to induce the fast current in LS c-DRG neurons from zymosan-treated mice (−5.30 ± 0.19) showed a tendency toward a decrease compared with that from saline-treated mice (−4.65 ± 0.45; Student's t-test, P = 0.18). When analyzed using two way ANOVA followed by the Holm-Sidak multiple comparison test, a significant increase in the fast current density was detected at 30 μM α,β-meATP in LS c-DRG neurons from zymosan-treated mice (P < 0.05). For fast currents in TL c-DRG neurons, logEC50 values for α,β-meATP (zymosan-treated; −5.37 ± 0.09. saline-treated; −4.65 ± 0.30. Student's t-test, P < 0.05) were significantly lower after zymosan treatment compared with saline treatment, and the current density at 10 μM α,β-meATP was significantly increased in TL c-DRG neurons from zymosan-treated mice (P < 0.05, 2 way ANOVA followed by the Holm-Sidak multiple comparison test).

Fig. 8.

Fig. 8.

Concentration–response relationship for ATP. Current responses were generated for kinetically distinct currents. Zymosan treatment did not affect the peak current of either the fast or slow component in TL or LS c-DRG neurons.

Fig. 9.

Fig. 9.

Concentration–response relationship for α,β-meATP. Current responses were generated for kinetically distinct currents. In TL c-DRG neurons, zymosan treatment significantly lowered the EC50 and increased the peak current of the fast component induced by 10 μM α,β-meATP. *P < 0.05.

P2X immunohistochemistry and mRNA expression in c-DRG neurons

DiI-positive c-DRG neurons co-labeled for P2X3 were present in both TL and LS DRG (Fig. 10). There was no significant difference in P2X3 immunoreactivity between LS (47/320; 15%) and TL (33/170; 19%) c-DRG neurons and zymosan treatment did not alter these proportions (LS: 52/286, 18%; TL: 39/155; 25%).

Fig. 10.

Fig. 10.

A: P2X3 immunoreactivity of neurons innervating the mouse colon (identified by content of the retrograde tracer, 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate). B: the number of P2X3 receptor-positive c-DRG neurons was unchanged by zymosan treatment.

To evaluate expression of P2X2 and P2X3 in LS and TL c-DRG neurons, we performed single-cell RT-PCR on DiI-labeled c-DRG neurons (Table 1A). Table 1B shows the percentage of c-DRG neurons expressing only P2X3, only P2X2 and both P2X3 and P2X2 transcripts. Transcripts for P2X2 and/or P2X3 were present in nearly all colon neurons examined with about half of the cells only containing P2X3 transcripts. While purinergic agonist triggered fast currents more frequently in TL than LS c-DRG neurons, the percentage of cells only expressing P2X3 did not differ between these two populations under control conditions and was not changed by zymosan treatment.

DISCUSSION

These findings add to a growing literature demonstrating the importance of peripheral sensitization and P2X receptors in visceral hypersensitivity. The novel aspect of the present report relates to the IBS-like colorectal insult produced by intracolonic instillation of zymosan, which is characterized by long-lasting colorectal hypersensitivity in the absence of colorectal inflammation assessed either by neutrophilic infiltration (myeloperoxidase assay) or histologically (Jones et al. 2005; Shinoda et al. 2009). Significantly, spontaneous action potential discharges of c-DRG neurons were increased as has been reported after significant insult and inflammation of visceral organs (Bielefeldt et al. 2002; Dang et al. 2004, 2005b; Yoshimura and De Groat. 1999). This enhanced excitability has been attributed to decreased transient outward potassium currents (Dang et al. 2004; Ibeakanma et al. 2009) and increases in voltage-gated sodium channel 1.8 protein expression (King et al. 2009) and its current density (Beyak et al. 2004; Bielefeldt et al. 2002), lowered input resistance, AP threshold (Moore et al. 2002), and rheobase (Ibeakanma et al. 2009) in DRG neurons innervating visceral organs.

In the present study, consistent with the absence of significant colonic insult, we did not see changes in neuron size or potassium currents that have previously been described in animal models of visceral hypersensitivity associated with inflammation (Dang et al. 2004; Yoshimura and De Groat. 1999). We found that zymosan had no effect on voltage-gated K+ currents, suggesting that voltage-gated K+ currents do not play a role in the mechanism(s) underlying zymosan-produced neuron excitability. Because of the present focus on purinergic receptors, we did not specifically investigate other currents that may contribute to changes in excitability, such as inward rectifying potassium currents or voltage-sensitive sodium or calcium currents. Zymosan treatment significantly increased the number of TL c-DRG neurons firing APs in response to ATP application. Because ATP-induced depolarization was not significantly changed by zymosan, the increase is likely the result of a decrease in RMP rather than a change in ATP current responses.

Recent studies have documented the importance of purinergic signaling in visceral sensory physiology. Colon distension increases ATP release into the colon lumen, which is further increased after induction of experimental colitis (Wynn et al. 2004). P2X3−/− mice have blunted basal responses to colorectal distension and do not develop hypersensitivity after zymosan treatment (Shinoda et al. 2009). The data presented here suggest that enhanced purinergic signaling contributes to the hypersensitivity, which has not previously been studied in c-DRG neurons.

Purinergic currents in c-DRG neurons

Using teased fiber recordings, we previously demonstrated significant differences between LS and TL colon sensory pathways (Brierley et al. 2004). Consistent with anatomic and functionally distinct roles, we document here differences in responses to purinergic agonists; most TL c-DRG neurons exhibited fast currents, whereas LS c-DRG neurons had similar proportions of fast and slow purinergic responses. The P2X purinergic identity of the currents was further confirmed by the absence of the fast current in c-DRG neurons from P2X3−/− mice and also by reversible inhibition of ATP- and α,β-meATP-mediated fast and slow currents by the P2X3-P2X2/3 antagonist A-317491. This suggests that the slow current is primarily due to activation of heteromeric P2X2/3 receptors, whereas the fast current is mediated by homomeric P2X3 receptors.

The single-cell PCR results for P2X3 and P2X2 transcripts revealed a greater frequency of expression in c-DRG neurons than percentages of neurons giving fast and slow responses to purinergic agonists, not surprisingly revealing greater sensitivity of the former. DiI-positive c-DRG neurons co-labeled for P2X3 were present in both TL and LS DRG. The proportions of co-labeled c-DRG neurons were less than reported by others (Brierley et al. 2005; Robinson et al. 2004). The discrepancy may be due to use of a different retrograde tracer (e.g., fast blue) or, more likely, due to the limited number of DiI injections made into the colon wall in the present experiments. In contrast, immunohisto-chemical evaluation of P2X3 in c-DRG neurons revealed less sensitivity than the functional whole cell results. The present electrophysiological results (purinergic responses in about 2/3 of the cells) are in line with prior publications (Chen and Gebhart. 2009; Dang et al. 2005a) suggesting that differences in the sensitivities of RNA-based or immunohistochemical assays are responsible for these discrepancies.

Purinergic responses and colorectal hypersensitivity

We noted a significant shift in the concentration-response relationship for the fast purinergic current; this may suggest a potential mechanism to explain why P2X3−/− mice do not develop zymosan-induced colon hypersensitivity (Shinoda et al. 2009). Although we did not detect any changes in kinetic properties of ATP (100 μM)- and α,β-meATP (100 μM)-evoked currents in TL or LS c-DRG neurons or the proportions or current density of P2X3-like fast current and slow P2X2/3-like currents after zymosan treatment, the EC50 for the α,β-meATP-produced fast current in TL c-DRG neurons was significantly lower. These findings differ from those produced by frank organ inflammation (e.g., cystitis) (Dang et al. 2008), which affect current kinetics and the relative contribution of fast and slow purinergic currents. Importantly, changes in excitability and purinergic responses were seen in TL and LS neurons innervating the colon, suggesting that both pelvic and splanchnic pathways participate in visceral hypersensitivity.

Although the mechanism(s) for the leftward shift of P2X3-agonists concentration-response curves in c-DRG neurons after zymosan treatment is unclear, it is noteworthy that P2X3 function can be upregulated by calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF), both of which are known to participate in visceral pain in IBS models (Barreau et al. 2008; Bourdu et al. 2005). CGRP stimulates trafficking of P2X3 receptors from intracellular stores to the cell membrane via protein-kinase-A- and protein-kinase-C-dependent mechanisms (Fabbretti et al. 2006), and NGF rescues P2X3 from an inhibitory control of conductance by C-terminal Src inhibitory kinase-dependent phosphorylation (D'Arco et al. 2009). It would be of interest to study whether these mechanisms underlie the increased P2X3-mediated responses in c-DRG neurons after zymosan treatment.

In summary, mild colonic insult enhanced membrane excitability and shifted the dose-response relationship to P2X3 agonists in TL c-DRG neurons, thereby contributing to the development of colon hypersensitivity. Responses to purinergic agonists differed between TL and LS c-DRG neurons, providing further evidence that these afferent pathways are indeed physiologically distinct. The properties of both TL and LS c-DRG neurons changed after zymosan treatment, arguing against a selective role of splanchnic pathways in the development of hypersensitivity during and after visceral insult. While we cannot exclude contribution of central P2X3 receptors as previously reported (Shinoda et al. 2009), the present results suggest that purinergic signaling contributes to peripheral sensitization. (Table 4)

Table 4.

EC50 of ATP and α,β-meATP-produced currents in LS and TL colon neurons fron saline- and zymosan-treated mice

ATP, μM
α,β-meATP, μM
Current Treatment logEC50 logEC50
LS
    Fast Saline −4.80 ± 1.31 (53) −4.65 ± 0.45 (41)]P=0.18
Zymosan −5.45 ± 0.28 (36) −5.30 ± 0.19 (42)
    Slow Saline −4.72 ± 0.32 (43) −4.29 ± 0.42 (54)
Zymosan −4.50 ± 1.26 (39) −4.19 ± 0.62 (55)
TL
    Fast Saline −4.47 ± 1.51 (95) −4.65 ± 0.30 (119)]*
Zymosan −5.19 ± 0.50 (90) −5.37 ± 0.09 (153)
    Slow Saline −4.72 ± 0.32 (30) −4.25 ± 0.34 (34)
Zymosan −4.50 ± 1.26 (32) −4.23 ± 0.42 (50)

Number of data points for a does-response curve construction are in parentheses.

GRANTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-19912. P2X3 knockout mice were graciously provided by Dr. Debra A. Cockayne, Neurobiology Unit, Roche Bioscience, Palo Alto, CA.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

We thank M. Gold and L. Fan for assistance with single cell RT-PCR and M. Burcham for preparation of the graphics.

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