Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability

Michael J. Beyak,1 Noor Ramji,1 Karmen M. Krol,2 Michael D. Kawaja,2 and Stephen J. Vanner1

1Gastrointestinal Diseases Research Unit and 2Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 5G2

Submitted 7 April 2004 ; accepted in final form 16 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.

tyrosine kinase A-positive neurons; sodium channels; nociception colon


ABDOMINAL PAIN RESULTING FROM colonic inflammation, such as inflammatory bowel disease, is a major cause of morbidity for affected patients. Voltage-gated Na+ channels (Nav) play a central role in neurotransmission of nociceptive signaling in dorsal root ganglia (DRG) sensory neurons (21). These channels are responsible for the fast upstroke of the action potential and set the threshold for action potential activation. Alterations in the properties of one or more of these Nav channels have been implicated in a variety of pathological pain states (16, 38, 45), including inflammation of the viscera (2, 39, 50, 52). There is growing evidence, however, that the ionic mechanisms underlying this pain may be specific to the pathophysiology of the pain state, e.g., inflammatory vs. neuropathic, and also the involved organ (17, 24).

Nav can be classified into two broad categories: TTX-sensitive (TTX-S) and resistant (TTX-R) on the basis of their relative sensitivity to the Na channel blocker TTX (19, 21). There are at least two TTX-R currents in adult DRG neurons, and these are preferentially found on the small nociceptive neurons. One is a slowly inactivating current (slow TTX-R INav), with slower kinetics of inactivation and faster recovery from inactivation (compared to the TTX-S INav). This slowly inactivating current is mediated by the Nav1.8 sensory nerve specific (SNS) channel (19) and plays an important role in action potential electrogenesis (32). The other is an ultraslow inactivating or persistent TTX-R current (persistent TTX-R INav), which is mediated by the NaV1.9 channel (8) and may contribute to the resting membrane potential. Together, this mixture of channels allows the neuron to have a specific repertoire of firing patterns (21, 32, 34, 44) and thus plays a critical role in neurotransmission in these sensory neurons. However, not all sensory DRG neurons express the same composition of channels.

Inflammation is known to modulate one or more of these TTX-R Nav channels, and these changes underlie increased excitability of nociceptive DRG neurons and associated hyperalgesia (2, 15, 39, 40, 52). Studies show a significant increase in the slow TTX-R INav (Nav1.8) density and changes in the steady-state availability curve. These changes appear to result from the acute actions of a number of inflammatory mediators, such as PGE2 (18), adenosine, 5-HT, ATP, as well as nerve growth factor (16, 27) and may persist during and possibly after inflammation as a result of increased expression of these channels (44, 48). Recently, increases in the persistent TTX-R INav (Nav1.9 channel) has also been implicated in the genesis of inflammatory pain (25), but its contribution to neuronal excitability in intestinal sensory DRG neurons is unknown.

Taken together, these studies show that multiple Nav channels are important in determining the excitability of DRG neurons and that inflammation can cause hyperalgesia by modulating one or more of these currents. To establish which currents are important in colitis, the aims of the present study was to perform an electrophysiological characterization of the INav in small sensory neurons innervating the mouse colon and to examine the effects of a model of chronic inflammation closely resembling human inflammatory bowel disease [trinitrobenzenesulfonic acid (TNBS) colitis] on sensory neuronal excitability and the properties of Nav channels. Neurons were further characterized by examining their immunohistochemical properties, because studies suggest important associations may exist between INav and phenotypic properties, but these have not been carefully studied in the intestine.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
CD-1 mice of either sex weighing 30–40 g were obtained from Charles River Laboratories (Montreal, PQ, Canada). Experimental protocols were approved by the Queen's University Animal Care Committee and conformed to the Guidelines of the Canadian Council of Animal Care.

TNBS Colitis and Acute Dissociation of Labeled Neurons

Animals were anesthetized with a combination of midazolam (1.25 mg/ml) and Hypnorm (0.315 mg/ml fentanyl and 10 mg/ml fluanisone) injected intraperitoneal (0.1 ml/20 g body wt). A midline laparotomy was performed, and the descending colon was carefully exposed. Under a dissecting microscope, a microliter syringe (Hamilton, Reno, NV) equipped with a 32-gauge needle was used to inject 1,1'-dioctadecyl-3-3-3'-3'-tetramethylindocarbocyanine (DiI) (5 mg/ml) or Fast Blue (17 mg/ml) into six to ten sites in the colon wall (volume = 1.0 µl/injection). To prevent leakage and labeling of adjacent structures, the needle was left in place for 30 s after each injection, and any leaked dye was removed with a cotton swab. Either 0.1 ml saline or TNBS (0.6 mg/ml in 25% EtOH) was injected into the colonic lumen. The abdomen was irrigated with copious amounts of warm saline and sutured closed. After surgery, animals were allowed to recover on a warming blanket and were given free access to food and water. After recovery from anesthesia, animals were monitored for signs of pain, feeding, and weight loss. Animals that displayed behavior consistent with ongoing pain or failure to thrive were killed.

Seven to ten days later, the mice were killed by isoflurane overdose, followed by exsanguination. The spinal column was removed and placed in ice-cold HBSS, and laminectomies were performed by using fine-bone rongeurs and DRG T9 to L1 were dissected out. These spinal levels were chosen, because it has been previously shown that sensory neurons from these regions are important in processing nociceptive stimuli arising from the colon (41, 42). Neurons were acutely dissociated as described previously (29, 39). Briefly, tissue was incubated in collagenase (1 mg/ml) and dispase (4 mg/ml) for 10 min, titurated with a fire-polished Pasteur pipette, and incubated again for an additional 5 min. Dissociated neurons were plated onto collagen-coated coverslips and stored in a humidified incubator at 37°C, under 95% air-5% CO2 until retrieval (4–24 h) for electrophysiological studies. The descending colon was removed for macroscopic scoring of damage and wet weight (dry/wet weight ratio). Macroscopic scoring of damage was accomplished as previously described (29), by assigning zero or one point for the absence or presence of hyperemia, erosion, petechial hemorrhage, and adhesions.

Electrophysiological Recordings

Whole cell current or voltage-clamp experiments were obtained 4–24 h after dissociation. Neurons adhering to the glass coverslip were placed in a RC-26 recording chamber (Warner Instruments, Hamden, CT), which was mounted on the stage of an inverted microscope (IX70; Olympus) fitted for both bright-field and fluorescence microscopy. With the use of the U-MWU2 filter (Olympus) for DiI and a U-MWIG2 filter for Fast Blue, labeled neurons were identified by their bright orange and blue fluorescence, respectively. Whole cell recordings were obtained from these neurons by using glass microelectrodes pulled with a P97 micropipette puller (Sutter CA), and fire polished by using a FP-830 fire polisher (Narishige). Final pipette resistance was between 2 and 5 M{Omega} for the current-clamp recordings. Larger pipettes were used (0.5–2 M{Omega}) to minimize voltage errors due to series resistance. Signals were amplified by using an Axopatch 200B amplifier and digitized with a Digidata 1322A A/D converter (Axon Instruments). Signals were low-pass filtered at 5 kHz, acquired at 20 kHz, and stored on disk.

Composition of the current-clamp solutions were (in mM): (Pipette) 140 KCl, 5 HEPES, 1 MgSO4, 1 EGTA, with pH adjusted to 7.2 using KOH (Bath), 140 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, 5 HEPES, and 5 D-glucose, pH adjusted by using NaOH. Cells were accepted for analysis if they had a stable resting membrane potential (mean = –52 mV) and displayed overshooting action potentials. Composition of the voltage-clamp solutions for isolating Na currents were (in mM): (Pipette) 110 CsCl, 1 MgCl2, 11 EGTA, 10 HEPES, 10 NaCl, pH adjusted to 7.3 with CsOH (Bath), 55 NaCl, 80 CholineCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 D-glucose, pH adjusted to 7.4 by using NaOH. CdCl (100 µM) was added to block calcium currents. Series resistance was compensated ≥85–90%, and the calculated junction potential was 4.9 mV. Recordings commenced 5 min after the establishment of whole cell access. Cells were excluded from analysis if uncompensated series resistance resulted in a maximum voltage error >5 mV or if the seal or access resistance were unstable. All experiments were performed at room temperature.

Recordings were analyzed by using the Clampfit 8.2 software (Axon Instruments). Linear leak subtraction was used for all experiments. Fitting of data was done with the least squares method using the fit function in Sigma Plot 8.0 (Jandel Scientific). Voltages of half activation (V50), time constants, and slope factors were obtained from means of the individual Boltzmann curve fits. Differences in categorical variables were analyzed for statistical significance using Fisher's exact test, and continuous variables were analyzed by using two-tailed unpaired Student's t-tests. Data are expressed as means ± SE. P < 0.05 was considered statistically significant. Dose response relationships were fitted with the Hill equation I/Imax = {[TTX]n/[TTX]n + (IC50)n}, where I/Imax is normalized current, IC50 is the concentration of TTX to obtain 50% of maximal inhibition, and n is the Hill coefficient.

Conductance was calculated as G = I/(VmVrev), where G is conductance, I is current, Vm is membrane potential, and Vrev is the reversal potential.

Activation and steady-state availability curves were fitted to a single Boltzmann function of the form: G/Gmax = 1/(1 + exp[V50Vm/k]), where G is conductance, Vm is membrane voltage, k is the slope factor, and Gmax is maximal conductance. Steady-state availability curves were fitted with a single Boltzmann function I/Imax = 1/(1 + exp[V50Vm/k]). In some cases, the G/V relationship seemed to be better fit by a double exponential; however, to facilitate comparison between groups, we used a single exponential function in all cases, as described by others (33). To determine the time course of inactivation, the decaying phase of the current evoked by a –10 mV pulse was fitted to a single decaying exponential function. The time course of recovery from inactivation was fitted to a single rising exponential function.

Drugs and Chemicals

Drugs were delivered to the cell under study using a fast flow solution switching system (VC6; Warner Instruments). All chemicals were obtained from Sigma (St. Louis, MO), with the exception of TTX (Calbiochem) and DiI (Molecular Probes, Eugene, OR).

Immunohistochemical Studies

Male CD1 mice (n = 10) were anesthetized with pentobarbital sodium (325 mg/kg ip) and killed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The thoracic DRGs from T9-T13 were dissected free, postfixed for a short period, and stored in a phosphate-buffered 30% sucrose solution. All ganglia were embedded in optimal cutting temperature by spinal segmental level and frozen in 2-methylbutane at –20°C. Fixed DRG sections were sectioned on a cryostat at 10-µm thickness, thawed, and mounted onto chrome alum-gelatin-coated slides, and stored at –20°C.

Tissue sections were incubated in 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories) diluted in 0.25% Triton X-100 in 0.1 M TBS for 1 h at room temperature. After a wash in TBS, the sections were incubated in rabbit anti-CGRP (1:500 dilution; Chemicon; Temecula, CA) or rabbit anti-tyrosine kinase A (trkA) (1:500 dilution; Chemicon), diluted in 3% NDS in 0.25% Triton X-100 in 0.1 M TBS for 72 h at room temperature. After washing in TBS, the sections were incubated for 2 h at room temperature in donkey anti-rabbit Texas Red-conjugated IgG, and diluted in 3% NDS in 0.25% Triton X-100 in 0.1 M TBS. After a final rinse, slides were coverslipped with Citifluor and viewed with a Zeiss fluorescence microscope.

Sections to be stained for Isolectin B4-FITC (IB4; Sigma) were thawed and incubated overnight at room temperature in a solution containing IB4 (1:20), diluted in 0.1 M PBS, to which an ion cocktail (1:10,000 dilution of 1 M CaCl2, 1 M MnCl2-4H2O, and 1 M MgCl2-6H20) was added. The tissue sections were rinsed and then placed on a coverslip, viewed, and immediately photographed with a Zeiss fluorescence microscope.

Photomicrographic images of immunostained DRG sections from all levels were obtained by using a Zeiss AxioCam high-resolution scanning digital camera mounted on a Zeiss microscope (Carl Zeiss Vision, München-Hallbergmoos, Germany). The Carl Zeiss software package AxioVision 3.0 was used to capture 48-bit RGB color images at 1,300 x 1,300 (scanning) pixels minimum. The raw image files (the intrinsic AxioVision file format) were optimized with regard to brightness, contrast, and color, and were then saved as uncompressed .tif files. Photomicrographic overlays and plates were then generated by using Adobe Photoshop 6.0 (also used to resize appropriately) and Corel Draw 8.0 (used to assemble and annotate images).

For quantitative analysis, multichannel overlays created by using Adobe Photoshop were used to count DRG neurons in all levels examined. First, the number of Fast Blue-labeled neurons was determined in images from DRG at a given level, and then the total number of neurons was counted in each field. For counting purposes, a neuron was defined as a cell having recognizable neuronal morphology and showing a clear nucleus. Several DRG fields were pooled by level, and each quantified field was separated by at least 40 µm to prevent double counting of neurons. The percentage of DRG neurons positively labeled by Fast Blue was then determined for each thoracic level. Images of DRG stained for CGRP, trkA, and IB4 were used to establish the extent of colocalization with Fast Blue. With the use of overlays having Fast Blue labeling in the blue channel and CGRP (or trkA) immunofluorescence in the red channel, or IB4 fluorescence in the green channel, the number of neurons having a clear nucleus and showing positive labeling for both Fast Blue and CGRP, trkA, and IB4 was counted. The percentage of cells colocalizing Fast Blue and each neurochemical marker was then calculated. Several fields at each level were used, no less than 40 µm apart for CGRP, trkA, and IB4. The data are expressed as mean ± SE percent and were tested for significance by using a one-way ANOVA with a post hoc Newman-Keuls test to examine differences among groups.

The long diameter of DRG neurons was measured by using Scion Image (Scion, Frederick, MD). With the use of the same fields used for neuron counting, the diameters of Fast Blue-labeled neurons was first measured, followed by the diameters of neurons colocalizing Fast Blue with either CGRP, trkA, or IB4. The data were pooled from all thoracic levels and expressed as mean ± SD, and were then tested for significance using a one-way ANOVA with a post hoc Newman-Keuls test to examine differences among groups.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Retrograde Labeling and Colocalization of CGRP, trkA, and IB4 in DRG Neurons Innervating the Colon

Injection of DiI or Fast Blue into the wall of the descending colon resulted in fluorescent labeling of a small proportion of acutely dissociated neurons in DRGs from the thoracolumbar region, predominantly in T9 to L1 (Fig. 1). Fluorescence labeling was not observed in cells obtained from animals in which the lumbar-splanchnic nerve was transected (n = 2) or in sham-injected animals (n = 6). Furthermore, cells dissociated from cervical DRG in labeled animals did not fluoresce (n = 3). Compared with DiI, Fast Blue provided brighter labeling. Quantitative assessments of the segmental distribution of Fast Blue-positive DRG neurons revealed an average percentage of cells ranging from 5 to 8% at each spinal segment. The mean diameter of labeled neurons was 23 µm and was normally distributed (Kolmogorov-Smirnov test). The vast majority of labeled neurons were <30 µm in diameter (see Fig. 1A).



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Fig. 1. A: immunohistochemical properties of small DRG neurons innervating the mouse colon. B: quantitative analyses of the distribution of Fast Blue (FB) neurons in dorsal root ganglia (DRG) from T9 to T13 in CD1 mice, 1 wk after tracer injection into the colon. The number of Fast Blue positive neurons was counted in several fields from each segmental level, as was the total number of neurons in that field. Results were pooled, and the data were expressed as an average %total DRG neurons showing Fast Blue labeling. Error bars represent means ± SE. C: representative photomicrographs of CGRP and tyrosine kinase A (trkA) immunostained DRG sections, colocalizing Fast Blue (stars; top), and DRG neurons colocalizing isolectin B4 (IB4) and Fast Blue (stars; bottom). D: quantitative analyses revealed the extent to which Fast Blue positive DRG neurons colocalize CGRP, trkA, and IB4. Significantly fewer Fast Blue neurons displayed IB4 fluorescence than did those that had either CGRP or trkA (P < 0.001; ANOVA). Scale bars = 50 µm.

 
Immunofluorescent staining for CGRP and trkA was localized predominantly to the neuronal cytoplasm and was distributed heterogenously throughout the DRG. Numerous CGRP (but not trkA) immunopositive fibers were also readily visible (Fig. 1C, top). IB4 staining of DRG neurons was also heterogeneously distributed throughout the DRG (Fig. 1C, bottom). In contrast to CGRP and trkA immunostaining, IB4 histofluorescence was present in the perinuclear cytoplasm of neurons and cell membrane and was additionally associated with numerous neuronal fibers.

Quantitative measurement of the colocalization of Fast Blue with the neurochemical markers demonstrated that 79.0 ± 5.22% of Fast Blue-labeled neurons exhibited CGRP immunofluorescence, 86.5 ± 4.53% of Fast Blue-labeled neurons also displayed fluorescence for trkA, and 13.0 ± 1.52% of Fast Blue neurons stained positively for IB4. Measurements of cell diameter of those neurons colocalizing Fast Blue and the three phenotypic markers revealed that most neurons had a small diameter; neurons colocalizing CGRP were 25.5 ± 5.7 µm, trkA were 25.9 ± 5.5 µm, and IB4 were 22.3 ± 5.1 µm in diameter.

INav in Colonic DRG Neurons

Whole cell recordings were successfully obtained from 188 labeled DRG neurons innervating the colon. Recordings were restricted to small cells (capacitance <40 pF) as our preliminary studies, as well as other studies (39, 50), have shown that these neurons have TTX-R action potentials and respond to capsaicin. These properties have been shown to be present in small diameter somatic and visceral unmyelinated afferents, a significant proportion of which are nociceptors (5). In current-clamp (n = 10) recordings, the mean resting membrane potential was –50.5 ± 2.3 mV and action potentials were resistant to TTX (1 µM; n = 7). In voltage clamp, two inactivating Na+ currents were identified on the basis of sensitivity to 1 µM TTX (Fig. 2). Currents were evoked by stepwise depolarizations between –80 and 40 mV from a holding potential of –100 mV, in both the absence and presence of TTX. Fast TTX-S INav was obtained by digital subtraction of slow TTX-R INav from the total. (Fig. 2). Total current was maximal at –10 mV, as were both the fast TTX-S and slow TTX-R INav. These currents reversed at ~50 mV, near the predicted reversal potential for Na+ in our solutions. Both slow TTX-R and fast TTX-S INav were seen in the vast majority of cells (88%). A small percentage, however, expressed only the slow TTX-R (7%) or fast TTX-S (5%) INav. Substitution of external Na+ with equimolar N-methyl-D-glucamine abolished these inward currents (n = 3).



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Fig. 2. Mouse colonic neurons exhibit TTX-resistance (TTX-R) and TTX-sensitive (TTX-S) voltage-gated Na+ channels currents (INav). A: representative whole cell current clamp recording demonstrating action potentials of small DRG neurons are TTX-R. B: representative traces of whole cell voltage-clamp recordings demonstrating fast TTX-S INav (left), slow TTX-R INav (middle), and persistent TTX-R INav (right). Inset shows tracing of persistent current obtained by using a long voltage pulse of 200 ms. Note, there is little inactivation of the persistent current over this time period. Fast and slow currents were generated by stepwise 30-ms voltage pulses between –80 and +40 mV from a holding potential of –100 mV. TTX-S Nav were obtained by subtracting TTX-R currents (recorded in the presence of 1 µM TTX) from the total. The persistent current was elicited by stepwise voltage pulses between –80 and –45 mV from a holding potential of –120 mV.

 
We also systematically investigated the persistent TTX-R INav (8) in colonic DRG neurons. With the use of a holding potential of –120 mV and 200-ms voltage pulses from –80 to –30 mV, we identified a persistent TTX-R INav in only 13% (n = 2/15) neurons examined (Fig. 2B). This current was maximal at approximately –50 mV with an extrapolated reversal potential near that for Na+ and showed little inactivation over the time of the test pulse (Fig. 2B, inset). The amplitude of this current was measured isochronally 200 ms after the onset of the test pulse to avoid contamination by the slow TTX-R INav. Prepulse inactivation and digital subtraction was not employed because preliminary experiments suggested the availability curves of these TTX-R INav overlap to some degree.

TTX Sensitivity of INav

To further characterize the slow TTX-R INav we constructed a TTX concentration-response curve (Fig. 3). Currents were evoked with a 30-ms pulse to –10 mV, fast TTX-S INav was eliminated by using 300 nM TTX, and subsequent peak currents were normalized to those obtained in 300 nM TTX. Cells were then exposed to various concentrations of TTX. This yielded an IC50 of 84 µM, similar to values reported previously (33). Slow TTX-R INav were not inhibited at concentrations ≤10 µM.



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Fig. 3. Concentration-response relationship of the slow TTX-R INav. TTX-S INav were blocked with 300 nM TTX and peak currents normalized to this value. Currents were generated from a holding voltage of –100 mV with voltage steps to –10 mV in increasing concentrations of TTX.

 
Voltage Dependence of Activation and Steady-State Availability of Inactivating TTX-S and TTX-R INav

The voltage dependence of activation of Na+ currents was examined by using 30-ms test potentials from –80 mV and +20 mV (Fig. 4). Conductance (G) was calculated by using the formula G = I(VmENa), where Vm is membrane potential, and ENa is equilibrium potential for Na. G/Gmax was plotted against membrane voltage, and individual conductance voltage relationships were fitted for each cell with a single Boltzmann function as described in MATERIALS AND METHODS. Voltage of half-activation (V50) and slope factors were determined from the mean of the individual curve fits. Slow TTX-R INav was isolated with 1 µM TTX, and the fast TTX-S INav was derived by digital subtraction. In control neurons, the V50 and k of the fast TTX-S INav were –35.6 ± 3.4 mV and 3.28 ± 0.69 (n = 10). The values for the slow TTX-R INav were V50 = –26.6 ± 3.9 mV and k = 5.8 ± 1.1 (n = 12) (Fig. 4A). The persistent TTX-R INav activated at more hyperpolarized potentials, with V50 = –47.8 ± 2.2 mV and k = 8.7 ± 1.1 (Fig. 4A).



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Fig. 4. A: conductance-voltage relationships for fast, slow, and persistent currents. Voltage dependence of activation was examined by using 30-ms test potentials given between –120 and +20 mV. Results were normalized and fitted by a single Boltzmann equation. Data for the persistent current are both from control and trinitrobenzenesulfonic acid (TNBS)-treated animals pooled together to obtain a sufficient number for cells (n = 6). B: steady-state inactivation curves for fast TTX-S INav and slow TTX-R INav. One-second conditioning pulses between –120 and +20 mV were used followed by a test pulse of –10 mV, 30 ms. Normalized current is plotted as a function of conditioning pulse voltage and fitted with a single Boltzmann function. The persistent TTX-R INav was seen too rarely to allow analysis of its inactivation properties.

 
Steady-state availability curves were constructed utilizing a two-pulse protocol. The first pulse was a 1-s conditioning pulse between –120 and +10 mV, followed by a test pulse of 0 mV. Normalized peak current was plotted against conditioning pulse voltage, and resultant curves were fitted with single Boltzmann function as described in MATERIALS AND METHODS. The V50 and slope factors (k) were obtained by averaging the individual curve fits (Table 1). The TTX-S INav were fit with a single Boltzmann function yielding V50 of –65 ± 5.7 and k = –3.9 ± 1.1. The slow TTX-R INav were also best fit with a single exponential, V50 = –47.7 ± 3.4 mV and k = –5.83 ± 0.32 (Fig. 4B).


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Table 1. Membrane properties of mouse colonic DRG neurons and the effect of TNBS colitis

 
Kinetics of Activation and Inactivation

The INav found in colonic DRG neurons could be distinguished on the basis of differences in their kinetics of activation and inactivation. The rate of activation of INav evoked by a 30 ms, –10 mV pulse was determined in both the presence and absence of 1 µM TTX. The time to peak of the slow TTX-R INav was 2.0 ms ± 1.1, and the fast TTX-S INav was 1.2 ± 0.34 ms. The inactivating phase of the current evoked by a –10 mV voltage step was fitted with a falling single exponential function. The inactivation time constant ({tau}) for the slow TTX-R INav was 3.21 ± 0.24 ms and for the fast TTX-S INav ({tau} = 0.355 ± 0.06 ms).

Kinetics of Recovery from Inactivation

The inactivating TTX-S and TTX-R currents also had differing repriming kinetics. The time course of recovery from inactivation was examined by using a two-pulse protocol. From a holding potential of –100 mV, two 30-ms, 10-mV pulses were given with an intervening step back to –100 mV. The interpulse interval was varied between 0 and 400 ms. Normalized current was plotted against time, and the resultant curves were fitted with single exponential function. The slow TTX-R INav recovered significantly more rapidly than the fast TTX-S INav ({tau} = 2.27 ± 0.37 vs. 25.3 ± 3.2 ms) (Fig. 5).



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Fig. 5. Time course of recovery for TTX-S (A)- and TTX-R (B)-inactivating INav. A 2-pulse protocol was employed. From a holding potential of –100, two 30-ms 10-mV pulses were given with an intervening step back to –100 mV. The interpulse interval was varied between 0 and 400 ms. Normalized current was plotted against time, and curves were fit with a single exponential function. Imax, maximum current.

 
Effect of TNBS Colitis on INav in Colonic DRG Neurons

Characteristics of TNBS colitis. Seven to ten days after instillation of TNBS in 25% EtOH into the colonic lumen, there was a marked degree of inflammation in all animals treated, compared with the control saline-treated group (Fig. 6). This was manifested by marked tissue thickening, hyperemia, petechial hemorrhage, and ulceration. The macroscopic pathological scoring (MATERIALS AND METHODS) was 0.64 ± 0.11 for control animals (n = 25) compared with 3.07 ± 0.21 (n = 25) (P < 0.001). This was associated with a large increase in both wet and dry weights (Fig. 6).



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Fig. 6. Assessment of inflammation induced by TNBS. A: TNBS treatment resulted in a significant increase in wet weight of 2-cm length of descending colon (n = 25). B: macroscopic damage was assessed on the basis of the presence/absence of erosions/ulceration, thickening, petechial hemorrhage, and adhesions with a score of 0 or 1 given for each. TNBS resulted in a significant increase in macroscopic damage score.

 
Effects of colitis on neuronal excitability. In current clamp, the resting membrane potential did not differ in neurons from TNBS animals compared with controls (–51.9 ± 3.0 mV, n = 16 vs. –50.5 ± 2.3 mV, n = 10, respectively) (Table 1). However, neurons from TNBS animals had a markedly reduced current threshold or rheobase (Fig. 7B). Furthermore, TNBS colitis resulted in a significant increase in the number of cells firing multiple action potentials in response to a two-times rheobase current injection (Fig. 7C). In addition, a subgroup of neurons showed firing of spontaneous action potentials (Fig. 7D), and the proportion of animals firing spontaneous action potentials was significantly greater in the TNBS group (37.5%) compared with control (10%) (Fig. 7E). Input resistance was estimated from the slope of the linear portion of the I-V relationship generated by hyperpolarizing test pulses between –70 and –10 pA. TNBS colitis resulted in a significant increase in input resistance (282.2 ± 89.0 vs. 892.8 ± 175.8 M{Omega}, P < 0.05) but had no effect on upstroke velocity, duration at half peak, amplitude, or afterhyperpolarization amplitude (Table 1).



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Fig. 7. Effect of TNBS colitis on neuronal excitability. A: representative traces showing effect of TNBS colitis on current threshold (rheobase) and firing pattern. Resting membrane potentials (–52 and –54 mV) were not significantly different between the 2 groups. B: summary of the effect of TNBS colitis on the rheobase. There was a marked reduction in rheobase in animals with TNBS colitis. C: firing pattern of the neurons. TNBS resulted in significantly more cells displaying repetitive firing (P < 0.05, Fisher's exact test). D: example of spontaneous firing of DRG neuron from a TNBS-treated animal. E: significantly more TNBS-treated cells displayed this type of spontaneous firing (P < 0.05, Fisher's exact test).

 
Effect of inflammation on INav density. Colitis resulted in a large increase in total INav density compared with control animals. The slow TTX-R INav component of this current was significantly increased (62%) (Fig. 8A), whereas the fast TTX-S INav density was not significantly altered (Fig. 8B). Only a small portion of cells exhibited the persistent TTX-R INav, and no obvious differences were observed in the number of cells exhibiting the current or the magnitude of the current between neurons from control and inflamed animals (Fig. 8, C and D).



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Fig. 8. Effect of TNBS colitis on peak INav density. A: mean TTX-R-inactivating INav density is significantly increased compared with controls at days 710. B: mean TTX-S INav density is not altered compared with controls. C: summary of the number of cells exhibiting the persistent TTX-R-inactivating INav in control and TNBS animals and the magnitude of the currents in those cells D: mean current density of persistent current in control (n = 2) and TNBS (n = 4) cells. Statistical tests were not applied due to the small numbers of cells exhibiting these currents.

 
Effect of inflammation on INav activation and inactivation. Inflammation did not have significant effects on the individual kinetics or the voltage dependence of activation and inactivation of the INav (Table 1). However, the small reduction in slope of the activation curve of the slow TTX-R INav combined with a left shift in the V50 lowered the threshold for activation of the slow TTX-R INav. Too few cells expressed the persistent TTX-R INav to allow analysis of the effects of inflammation on its properties.


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In the present experiments, DRG sensory neurons innervating the mouse colon were identified by using retrograde labeling techniques, and INav and neuronal excitability were characterized in animals with noninflamed or inflamed colons. Most retrogradely labeled cells were small, and the majority were CGRP positive, in agreement with a recently published report (35). In addition, we have also shown that a majority bear the high affinity NGF receptor, trkA. We focused on these small cells because they likely give rise to unmyelinated C fibers (2, 29, 39), a significant proportion of which transmit nociceptive stimuli (23, 28). The major finding was that these cells predominately express slow TTX-R INav and not persistent TTX-R INav and that colitis selectively increases the slow TTX-R INav. The percentage of cells expressing persistent TTX-R INav correlates closely with IB4 lectin binding.

Properties of INav in Small Mouse Colonic DRG Neurons

The fast TTX-S INav in unselected DRG neurons has been well characterized in a number of species including the mouse using electrophysiological and molecular techniques. These currents are mediated by the channels NaV1.7 and to a lesser extent NaV1.3 and 1.6. These channels have a relatively hyperpolarized threshold and steady-state availability curve. The kinetics of recovery of the TTX-S channels in DRG neurons tend to be slower than that of the TTX-R channels. The biophysical properties of the fast TTX-S INav in the mouse colonic neurons described in the present study (see Table 2) fit well with those reported for the NaV1.7 channel in the literature, suggesting important differences in the TTX-S channels do not exist in these neurons.


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Table 2. Biophysical properties of TTX-R and TTX-S INa in mouse colonic DRG neurons

 
The TTX-R Nav channels are unique in that they are relatively selectively expressed in primary sensory nerves (45). There are two major TTX-R channel {alpha}-subunits in DRG neurons that have been cloned and characterized (19). The first is Nav1.8, which predominates in smaller diameter, unmyelinated neurons. In the present study, the slow TTX-R INav had a TTX IC50 of 84 µM, which is similar to that reported for the Nav1.8 channel (~50–100 µM) (1, 33) and significantly higher than Nav1.5 (~1–2 µM) (33, 37). Compared with the properties of the fast TTX-S Nav channels, the Nav1.8 channels exhibits more positive thresholds for activation, inactivates more slowly, and recovers rapidly from inactivation. The slow TTX-R INav in the mouse colonic neurons exhibited similar properties (activation, inactivation kinetics, and TTX-sensitivity) to that described by others, except the activation and steady-state availability curves were more hyperpolarized. The explanation for these differences is unknown, but could reflect differences in regulatory signals such as cAMP (47) or in auxiliary {beta}-subunits (43). Given the importance of these currents in the genesis of the action potential (32), the low threshold for activating these currents may help to explain the relatively low threshold for action potential electrogenesis seen in these mouse neurons compared with our previous studies (39) and others (2, 52).

The second major TTX-R Nav channel in DRG cells, the Nav1.9 channel (8, 10, 19), has strikingly different properties. The current is a persistent or ultra slowly inactivating current and its voltage dependence of activation and inactivation are much more negative than Nav1.8. Although its action is incompletely understood, its distribution is also restricted to small unmyelinated DRG neurons (8), and it appears to play a role regulating neuronal excitability by setting the resting membrane potential closer to threshold (8). In contrast to studies of unidentified DRG neurons, which suggest this current is exhibited by over 40–60% of neurons (6), we found very few (<15%) mouse colonic DRG neurons that expressed a persistent TTX-R INav. Our findings are in keeping with recent data suggesting that Nav1.9 channels are not expressed homogenously, but rather are associated with specific sensory function or target (4). It has been suggested that binding of the isolectin IB4 may help to distinguish neurons that express Nav1.9 channels; a majority of IB4 positive cells exhibit Nav1.9 channels, but few IB4 negative cells do (12). IB4 positive DRG neurons also express the receptors for GDNF (31), on which Nav1.9 levels depend (12). Our studies have shown that only a small number (13%) of Fast Blue neurons from the colon were IB4 reactive, a similar proportion to labeled DRG bladder afferents (51) and similar to the proportion expressing the persistent TTX-R INav (Nav1.9) in the present study. Together these data support the contention that persistent TTX-R INav does not play a significant role in modulating excitability of the large majority of mouse DRG neurons innervating the colon and that neuronal phenotype varies depending on the innervation target, possibly determining the firing properties of the cell.

Effect of TNBS-Colitis on INav

TNBS in EtOH caused marked chronic inflammation at 7–10 days. EtOH alone causes only a mild, self-limited colitis, not chronic inflammation (30). Therefore, the changes seen are most likely due to the chronic colitis induced by TNBS/EtOH. The small DRG neurons innervating the inflamed colon were hyperexcitable, exhibiting a significant reduction in rheobase and increased repetitive firing. Furthermore, we observed that a significant proportion (38%) of neurons from inflamed animals displayed spontaneous activity. Together, these data suggest that inflammation significantly increases neurotransmission and could contribute significantly to the generation of hyperalgesia.

Our studies of the INav underlying electrogenesis of the action potential in these neurons demonstrated a significant increase in the slow TTX-R INav density. Although we did not find a significant difference in the voltage dependence of activation or inactivation, we did observe a trend toward a leftward shift in the activation curve and a reduction in the voltage dependence of activation (slope) that together lower the activation threshold. In contrast, we did not observe changes in the fast TTX-S INav density or its biophysical properties. The TTX-R NaV1.8 channel has been shown to be essential for spontaneous activity in a nerve injury model (36). The lowering of the threshold, increase in current density of the rapidly repriming slow TTX-R INav could support inflammation-induced repetitive and or spontaneous firing seen in our experiments. This selective increase in slow TTX-R INav is similar to that described in other visceral (2, 39, 52) and somatic organs (40) of differing species, enforcing the view that this is a common response to chronic inflammation.

Because some evidence suggests that inflammation increased cell size and that large neurons express less TTX-R current, it could be argued that the mean amplitude of the TTX-R current may have been artificially increased by sampling bias (i.e., cells that would have been at the upper limit of the <40 pF size range, would increase in size above 40 pF). However, we do not believe that this is the case for the following reasons. First, there was no increase in the mean capacitance of the cells recorded from. Second, the vast majority of the cells' size fell well below the <40 pF range, and therefore, a very large increase in size would have been required to shift a significant number out of this range. Finally, but most importantly, we failed to observe in this limited population a negative correlation between increasing cell size and slow TTX-R INav density or the ratio of slow TTX-R INav/Fast TTX INav density (R = 0.02, P < 0.05).

Several authors have suggested that Nav1.9 (NaN) currents may be involved in a variety of pain states (8, 25, 36, 49), and its selective localization in small DRG neurons supports a role in nociception (12). It may play a role in setting the membrane potential and/or action potential threshold in small DRG neurons (20). We compared the persistent TTX-R INav (Nav1.9) in control and inflamed animals. Although the numbers are relatively small, we found no obvious differences in either the numbers of neurons expressing the current or the magnitude of the currents. These data suggest that these currents are not important in the generation of the neuronal hyperexcitability observed with colitis. Because the slowly inactivating TTX-R channels are closed at resting membrane potentials, the absence of any change in resting membrane potential during colitis also supports the contention that these persistent TTX-R INav play little, if any, role in the increased excitability seen in neurons innervating the inflamed colon.

In summary, changes in INav are known to underlie the hyperalgesia observed in pathological pain states, but which channel(s) are involved appears to be specific for the pathophysiological process, (e.g., inflammatory vs. neuropathic) and the target organ (e.g., somatic vs. visceral). Neurons that innervated the inflamed mouse colon were hyperexcitable and exhibited a significant increase in the current density of the slow TTX-R INav (Nav1.8) but not the persistent TTX-R INav or the fast TTX-S INav. The increase in this rapidly repriming, low threshold current could decrease the threshold for action potential electrogenesis and support repetitive and/or spontaneous firing observed in inflammation (46). It will be important to determine whether this change in current density results from inflammatory mediators signaling through kinases to phosphorylate existing channels and/or whether increased Na+ channel expression occurs, possibly through altered gene expression (44, 46, 48). Several reports (3, 9, 11, 13, 22, 26) suggest that NGF may be an important mediator of inflammation-induced changes in ion channel expression. The presence of trkA receptors on these cells supports the view that NGF is one of the important inflammatory cytokines signaling to these neurons. Our finding that neuronal input resistance is significantly increased likely suggests important modulation of K+ channels also occurs, as we (29, 39) and others (7, 50) have previously found in other animals/organs. The relative roles played by the differing ion channels remain to be established. The use of gene deletion strategies in this mouse model of colitis will provide an additional strategy to address these issues.


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This study was supported by a grant from the Crohn's and Colitis Foundation of Canada (to S. Vanner). M. J. Beyak was the recipient of a Canadian Institute of Health Research/Canadian Association of Gastroenterology (CAG)/Astra-Zeneca Research Initiative award. K. M. Krol was the recipient of a doctoral research award from the Canadian Pain Society, and N. Ramji was the recipient of a Crohn’s and Colitis Foundation Canada/CAG summer research studentship.

D


    ACKNOWLEDGMENTS
 
The authors thank Iva Kotsatka, Margaret O'Reilly, and Verna Norkum for expert technical assistance

Portions of this work have appeared in abstract form.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Vanner, 166 Brock St. Hotel Dieu Hospital, Kingston, Ontario, Canada K7L 5G2 (E-mail: vanners{at}hdh.kari.net)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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