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Peripheral inflammation selectively increases TRPV1 function in IB4-positive sensory neurons from adult mouse

Breese, Nicole M.; George, Annette C.; Pauers, Laura E.; Stucky, Cheryl L.*

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doi: 10.1016/j.pain.2005.02.010
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Abstract

1. Introduction

C-fiber nociceptors typically have small-diameter cell bodies and have been divided into two general classes. One group expresses TrkA receptors for nerve growth factor (NGF), depends on NGF for survival, and contains neuropeptides including calcitonin gene-related peptide and substance P. The other group expresses receptors for glial cell line-derived neurotrophic factor (GDNF), depends on GDNF for survival after birth, and expresses a surface carbohydrate group that binds isolectin B4 (IB4). Several studies report that IB4-positive neurons are poor in expression of neuropeptides and lack TrkA receptors for NGF (Averill et al., 1995; Bennett et al., 1996b; Molliver et al., 1995; Silverman and Kruger, 1990; Zwick et al., 2002), but others show significant overlap between IB4 binding and neuropeptide- or TrkA-expression (Kashiba et al., 2001; Wang et al., 1994). The central terminals of IB4-positive C-fibers terminate predominantly in inner lamina II whereas central terminals of IB4-negative C-fibers terminate in lamina I and outer lamina II (Gerke and Plenderleith, 2004; Molliver et al., 1995; Silverman and Kruger, 1990; Wang et al., 1994); although, not all studies agree with this anatomical distinction (Hantman et al., 2004; Woodbury et al., 2000).

The differences between the two classes of C-fiber neurons raise the question of whether they have different functional properties in conveying nociceptive information. Previously, we demonstrated that IB4-negative neurons from non-injured mice are highly responsive to capsaicin and protons whereas IB4-positive neurons are significantly less responsive (Dirajlal et al., 2003). Some authors have hypothesized that IB4-negative neurons specifically contribute to inflammatory pain and that IB4-positive neurons contribute to neuropathic pain (Mantyh and Hunt, 1998; Snider and McMahon, 1998). However, no study has used functional measures to determine whether peripheral inflammation selectively sensitizes IB4-negative C-fiber neurons compared to IB4-positive neurons.

The capsaicin receptor, TRPV1 (VR1), is an important transducer for heat stimuli in small- and medium-diameter nociceptors. TRPV1 is a member of the Transient Receptor Potential (TRP) family and constitutes a non-selective cation channel. Besides heat, TRPV1 can be activated or sensitized by other endogenous stimuli such as protons, N-arachidonoyl-dopamine, anandamide, and eicosanoids including leukotriene B4, 12-(S)-HPETE and 15-(S)-HPETE (Caterina et al., 1997; Huang et al., 2002; Hwang et al., 2000; Smart et al., 2000). Substantial evidence indicates that TRPV1 is a key mediator of inflammatory pain. Capsaicin sensitivity and TRPV1 expression both increase in peripheral neurons during experimental inflammation (Carlton and Coggeshall, 2001; Nicholas et al., 1999). Mice that lack TRPV1 do not develop heat hyperalgesia during inflammation (Caterina et al., 2000; Davis et al., 2000), which demonstrates that TRPV1 is critical for inflammatory heat hypersensitivity.

Here, we tested the hypothesis that peripheral inflammation selectively sensitizes IB4-negative but not IB4-positive C-fiber-type neurons to TRPV1 receptor stimuli. Using whole-cell patch clamp recordings of small-diameter (≤26μm) L4/L5 DRG neurons from adult mice, we determined the effect of CFA-induced inflammation on the responsiveness of IB4-positive and -negative neurons to the TRPV1 agonists, capsaicin and protons.

2. Methods

2.1. Animals

Experiments were performed in adult male C57BL/6 mice and adult male mice with a targeted deletion of the TRPV1 gene (Caterina et al., 2000). All animals were obtained from Jackson Laboratories (Bar Harbor, MN). Ages ranged from 10 to 16 weeks and body weight ranged from 20 to 27g. All procedures were approved by the Animal Care and Use Committee of the Medical College of Wisconsin and are in accordance with the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.

2.2. Induction and analysis of peripheral inflammation

Mice were anesthetized with isoflurane, and 50μl of straight complete Freund's adjuvant (CFA; 10μg/μl) was injected into the plantar surface of the left hind paw. Injection of this dose of CFA causes inflammation of the entire foot and hind leg of the mouse. Two days after CFA injection, the magnitude of inflammation was quantified by measuring the thickness and width of the hind paw with digital calipers and the mean of the two measurements was calculated. Thermal hyperalgesia was assessed by measuring withdrawal latencies to a radiant heat source (Paw Thermal Stimulator, University of California, San Diego, CA) applied to the medial plantar surface of the hind paw (Hargreaves et al., 1988). Mechanical sensitivity was determined by applying calibrated von Frey monofilaments (0.05–2.0g) to the medial plantar surface of the paw using the up and down method modified for mice (Chaplan et al., 1994). The 50% withdrawal threshold, which gives the force of the von Frey filament to which an animal responds in 50% of the presentations, was recorded. Testing was performed one time before CFA injection to obtain baseline values, and again 2 days after CFA injection. Control mice were either injected with saline or not injected and tested in parallel with the CFA-injected mice. Because there were no differences in the paw diameter, heat or mechanical behavior tests between the saline-injected and naïve controls, non-injected controls were used for the majority of data collected in this study. A total of 16 mice (8 CFA-injected; 8 control) were used for behavioral measures. A one-way ANOVA with Tukey's post hoc test (In Stat, GraphPad Software) was used for statistical measures. Error bars indicate±SEM.

2.3. Neuronal isolation

Two days after injection of CFA into the hind paw, mice were anesthetized with isoflurane and killed by cervical dislocation. The L4 and L5 dorsal root ganglia (DRG), which contain the majority of the somata of afferent fibers that innervate the hind limb, were removed. For CFA-injected mice, neurons were isolated from the L4 and L5 ganglia ipsilateral to the CFA-injection from two mice for each preparation. For control mice, neurons were isolated from the L4 and L5 DRG from both sides of one mouse. A total of 132 mice were used for electrophysiological recordings in this study. The ganglia were incubated with 1mg/ml collagenase IV (Sigma, St Louis, MO) and 0.05% trypsin (Sigma-Aldrich) for 40min each at 37°C and dissociated into single cells by passing though flame-constricted Pasteur pipettes of decreasing diameter. The cells were washed and resuspended in DMEM/Hams-F12 medium containing 10% heat-inactivated horse serum, 20mM glutamine, 0.8% glucose, 100 units penicillin and 100μg/ml streptomycin (Gibco, Invitrogen). Cells were plated onto glass coverslips coated with poly-L-lysine and maintained at 37°C, 5% CO2 for no more than 9h. No exogenous NGF was added to the medium, as NGF has been shown to alter the response properties of small-diameter neurons to capsaicin and heat within minutes (Galoyan et al., 2003; Shu and Mendell, 2001). Whole cell recordings from the cell soma were performed between 1.5 and 9h after isolation. Experiments performed within this acute time frame allow neurons to adhere to the coverslip but avoid many artifacts that occur in neurons cultured for longer time periods. The acute time frame also avoids the loss of capsaicin sensitivity that occurs in adult rat DRG neurons cultured without NGF (half-life of 3 days; Wach et al., 2003; Winter et al., 1988).

2.4. Electrophysiological recordings

Whole cell recordings were made from small-diameter (≤26μm) neurons using fire-polished glass electrodes (2–6MΩ resistance) pulled from borosilicate glass on a micropipette puller (P-87; Sutter Instruments). The recording chamber was continuously superfused with solution containing (in mM) 150 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 8 Glucose; pH brought to 7.4 with NaOH; osmolarity=320mOsm. Electrodes were filled with solution containing (in mM) 135 KCl, 10 NaCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 2.5 NaATP2; pH brought to 7.2 with KOH; osmolarity=290mOsm. All solutions were made fresh daily, filtered just before use and were used at room temperature (22–24°C). Neurons were viewed with a Nikon TE200 inverted microscope and soma size was estimated by calculating the mean of the longest and shortest diameters using a calibrated eyepiece reticle. Immediately, after each recording was complete, neurons were incubated with the plant lectin, IB4 conjugated to fluorescein isothiocyanate (IB4-FITC; 10μg/ml) for 10min and then rinsed for 2min. IB4-FITC staining was visualized with standard FITC filters (excitation 465–495nm; emission barrier filter 515–555nm). A neuron was considered IB4 positive if it had a continuous green ring around the perimeter at 10× magnification. The majority of IB4-positive neurons exhibited an intense green ring, but some neurons exhibited a thin yet continuous ring around the perimeter and these were also classified as IB4 positive.

For all chemical tests with capsaicin and protons, solutions were applied locally and rapidly (10s duration) to the neuron of interest using 28-gauge non-metallic needles (0.25mm ID; World Precision Instruments). The tip of each needle was placed 50μm from the cell soma using a manipulator (Narishige) and the gravity-fed solutions were controlled manually by switching one-way stopcock valves (Cole-Parmer). For experiments with capsaicin, a 100nM or 1μM concentration of capsaicin (in HEPES buffer) was prepared daily from a 10mM capsaicin stock solution dissolved in 1-methyl-2-pyrrolidinone (Sigma-Aldrich), a solvent that has no effect on the physiology of sensory neurons (Nicol et al., 1997). For stimulation with protons, the HEPES in the extracellular buffer was replaced with 2-[N-morpholino]ethanesulfonic acid (MES) and pH was brought to 5.0 with HCl. Because capsaicin and proton stimuli can interact even when applied to a neuron separately and with a wash between chemicals (Dirajlal et al., 2003), each neuron was tested with only one stimulus unless indicated otherwise, and only one neuron was tested per coverslip.

2.5. Data recording and analysis

Membrane voltage was clamped using an EPC-9 amplifier run by Pulse software (version 8.65, HEKA Electronic, Lambrecht, Germany). Data were sampled at 10kHz. Membrane potential was held at −70mV. Neurons were included in our analysis only if they formed seals greater than 1.5GΩ, had resting membrane potentials more negative than −45mV with no current applied, exhibited an action potential overshoot, and had an input resistance greater than 100MΩ in whole-cell configuration. The size of the cell capacitance transients was monitored throughout the recording and did not change by more than 10%. Pipette and cell capacitance were compensated using the computer-controlled circuitry. Series resistance was compensated at 70–80%. After establishing whole-cell configuration, the recording was switched to current-clamp mode and action potentials were generated by injecting ascending steps of current from 10 to 1000pA for 10ms each. The presence of an inflection on the falling phase of the somal action potential was determined using Igor software (version 4.01, WaveMetrics) to calculate the rate of change in voltage during the action potential (Dirajlal et al., 2003). All small-diameter (≤26μm) neurons included in this study had a clear inflection on the falling phase of the somal action potential. The recording mode was then switched to voltage-clamp to measure inward current evoked by chemical stimuli. The magnitude of inward current was determined using PulseFit software. Proton responses were classified as transient (rapidly inactivating within 1–2s) or sustained (slow inactivation >20s) as previously described (Dirajlal et al., 2003). To correct for differences in soma size, all inward current values are expressed as a function of cell capacitance (pA/pF). For statistical measures, groups were compared using Fisher's exact test or an unpaired two-tailed t-test using InStat. Error bars indicate±SEM.

2.6. Immunocytochemistry and image analysis

Lumbar 4 and 5 ganglia were taken from control mice and from mice 2 days after injection with CFA and were dissociated and plated as described above. A total of 17 mice were used for the immunohistochemical analysis in this study. Neurons were fixed with 4% paraformaldehyde (10min) 6–9h after plating. To reduce non-specific staining, neurons were incubated for 1h in 4% normal goat serum (Jackson Immunoresearch Laboratories) diluted in 1× PBS with 0.3% Triton X-100. Neurons were then incubated with a rabbit polyclonal anti-TRPV1 antibody (1:500; Oncogene Research) and IB4-biotin (10μg/ml) together for 24–48h, 4°C. Goat anti-rabbit Texas Red (1:1000; Jackson Immunoresearch) and Cy2-streptavidin (1:200; Jackson), incubated for 1h (RT), were used to visualize TRPV1 and IB4, respectively. Controls for each staining in which the TRPV1 primary antibody and biotinylated IB4 were omitted were performed in parallel. Images of random fields of neurons (4–8 per coverslip) were captured with a Spot II color camera (Diagnostics) and analyzed using Metamorph software (Universal Imaging). Neurons were measured for soma diameter and average brightness intensity of the soma. Neurons ≤26μm diameter were classified as positive for TRPV1 or IB4 if the average intensity of the soma was greater than twice the standard deviation of the average intensity values taken from six neurons that were clearly negative for TRPV1 or IB4 on the same coverslip. For statistical measures, groups were compared with a two-tailed unpaired t-test using InStat. Error bars indicate±SEM.

3. Results

3.1. CFA-induced inflammation, heat and mechanical sensitivity in mice

The CFA injection produced edema and erythema over the entire ipsilateral (left) foot and leg of the mouse. Two days after CFA injection, the ipsilateral hind paw was 40.9±0.01% larger than either the contralateral hind paw or the left hind paw of control, non-injected mice (Fig. 1(A)). CFA-injected mice exhibited significant behavioral hypersensitivity to both heat and mechanical stimuli on the side ipsilateral to the CFA injection when compared to either the contralateral side or to non-injected control mice (Fig. 1(B) and (C)).

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Fig. 1.:
CFA-injection induces inflammation, heat and mechanical sensitivity in mice. (A) Average diameter of the ipsilateral (injected) and contralateral hind paw of control mice and mice 2 days after injection with CFA (n=11 control; 82 inflamed). Symbol on graph is larger than error bars for inflamed data points. *** indicates that CFA-injected hind paws were significantly larger in diameter compared to the contralateral side and to non-injected controls (P<0.001; ANOVA, Tukey's post hoc test). (B) Latency of withdrawal from a radiant heat stimulus in control and CFA-injected mice (n=8 control; 8 inflamed). *** indicates that CFA-injected hind paws had significantly shorter thermal withdrawal latencies compared to the contralateral side and to the control group (P<0.001; ANOVA, Tukey's post hoc test). (C) Response threshold to von Frey monofilaments in control and CFA-injected mice (n=8 control; 8 inflamed). ** indicates that CFA-injected hind paws had reduced mechanical thresholds compared to the contralateral side and the control group (P<0.01; ANOVA, Tukey's post hoc test).

3.2. Inflammation increased the percentage of IB4-positive neurons from L4/L5 ganglia that responded to capsaicin but had no effect on IB4-negative neurons

To assess inflammation-induced changes in the responsiveness of small-diameter (≤26μm) IB4-positive and -negative neurons to TRPV1 stimuli, we used the specific TRPV1 agonist capsaicin (Caterina et al., 1997). Fig. 2(A) shows typical responses of an IB4-positive and -negative neuron to 1μM capsaicin. Because all responses to capsaicin in neurons from both control and inflamed mice were larger than 40pA, the criterion used for a capsaicin response was an inward current >40pA. In control, non-inflamed mice, 24% of the small-diameter IB4-positive neurons (mean diameter of neurons tested: 21.9±0.47μm) from the L4 and L5 ganglia responded to 1μM capsaicin with an inward current. Two days following induction of inflammation, the percentage of capsaicin-sensitive small-diameter IB4-positive neurons (mean 21.9±0.46μm) from the L4 and L5 ganglia increased 3.3-fold to 80% (Fig. 2(B)). IB4-positive neurons were specifically sensitized, as the percentage of capsaicin-sensitive small-diameter IB4-negative neurons (mean control: 20.8±0.57μm, mean CFA: 21.0±0.53μm) was unaltered by inflammation. Inflammation did not change the average magnitude of peak inward current evoked by capsaicin in either IB4-positive or IB4-negative neurons (Fig. 2(C)).

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Fig. 2.:
Inflammation sensitizes IB4-positive neurons to capsaicin. (A) Examples of whole cell voltage clamp recordings from an IB4-positive and -negative small-diameter neuron (≤26 μm) that responded to 1 μM capsaicin. (B) Percentage of IB4-positive and -negative neurons from the L4/L5 DRG of control or CFA-injected mice that responded to a 10 s exposure of 1 μM capsaicin. Capsaicin-evoked currents in all cells tested were >40 pA. *** indicates that significantly more IB4-positive neurons from inflamed mice responded to capsaicin compared to the control IB4-positive group (P<0.0001; Fisher's exact test). (C) Average magnitude of the peak inward current evoked by 1 μM capsaicin from control or CFA-injected mice in IB4-positive and -negative neurons. (D) Percentage of IB4-positive and -negative small-diameter neurons isolated from the L4/L5 DRG from control or CFA-injected mice that responded to 100 nM capsaicin. * indicates that significantly more IB4-positive neurons from inflamed mice responded to capsaicin compared to control IB4-positive neurons (P<0.05; Fisher's exact test). The percentage of IB4-negative neurons that responded to 100 nM capsaicin was unaltered by inflammation.

Because 1μM capsaicin activated nearly 70% of IB4-negative neurons from control, non-injured mice, this concentration of capsaicin may have been too high to observe an increase in response frequency following inflammation. Therefore, we tested a separate population of neurons with 100nM capsaicin (Fig. 2(D)). Inflammation did not alter the percentage of IB4-negative neurons that responded to a low concentration of capsaicin, as only 17% of IB4-negative neurons from CFA-injected mice responded to 100nM capsaicin with an inward current compared to 18% in non-injected control mice (P>1.0; Fisher's exact test). However, even with a small number of neurons tested, inflammation significantly increased the proportion of IB4-positive neurons (44%) that responded to 100nM capsaicin compared to 6% of neurons from control mice (P<0.05; Fisher's exact test). Together, these data indicate that peripheral inflammation selectively increased the proportion of IB4-positive small-diameter neurons that responded to capsaicin but had no effect on the capsaicin sensitivity of IB4-negative neurons.

3.3. IB4-positive neurons from TRPV1−/− mice with inflammation did not respond to capsaicin

The TRPV1 receptor is the only capsaicin-sensitive protein identified to date (Caterina et al., 1997). Furthermore, it has been shown that TRPV1−/− mice are completely insensitive to capsaicin (Caterina et al., 2000; Davis et al., 2000). To confirm that the inflammation-induced increase in capsaicin-responsiveness in IB4-positive neurons was due to enhanced expression or function of the TRPV1 receptor, we isolated L4 and L5 ganglia from TRPV1−/− mice 2 days following injection of CFA into the hind paw and tested the responsiveness of small-diameter neurons to capsaicin. None of the IB4-positive (n=23) or IB4-negative (n=30) neurons from CFA-injected TRPV1−/− mice responded to capsaicin. Likewise, none of the IB4-positive (n=29) or IB4-negative (n=24) neurons from control, non-inflamed TRPV1−/− mice responded to capsaicin. Thus, the increased capsaicin-responsiveness of IB4-positive neurons during peripheral inflammation was specifically mediated by the TRPV1 receptor.

3.4. Inflammation increased the percentage of IB4-positive neurons from L4/L5 ganglia that responded to protons

Because the concentration of protons is elevated during inflammation and protons are an endogenous stimulus for the TRPV1 receptor (Caterina et al., 2000; Tominaga et al., 1998), we examined the responsiveness of IB4-positive neurons to low pH. Two distinct profiles of inward current are evoked by pH 5.0 in mouse DRG neurons (Dirajlal et al., 2003). One is a transient current that is rapidly inactivated within 1–2s (Fig. 4(C) top) and the second is a sustained inward current that inactivates slowly (>20s) (Fig. 4(C) bottom). All pH 5.0-induced responses in IB4-positive neurons from both control mice and inflamed mice were sustained-only inward currents (Fig. 3(C)). Thus, as reported previously, IB4-positive neurons from mouse never exhibited a transient proton-evoked inward current (Dirajlal et al., 2003). Two days after injection of CFA in wild-type mice, the percentage of IB4-positive neurons from the L4/L5 ganglia that responded to pH 5.0 with a sustained inward current >40pA was significantly increased from 54 to 85% (Fig. 3(A)). There was no difference in the mean diameter of the IB4-positive neurons tested (control: 22.9±0.46μm; CFA: 22.9±0.43μm). Peripheral inflammation did not alter the average peak magnitude of the proton-evoked sustained inward current in IB4-positive neurons (Fig. 3(B)).

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Fig. 4.:
Inflammation decreases ASIC-like currents in IB4-negative neurons. (A) Percentage of IB4-negative neurons from the ipsilateral L4/L5 DRG that responded to pH 5.0 with a transient or sustained-only inward current in control and CFA-injected TRPV1+/+ mice. (B) Average magnitude of proton-evoked transient current (left) and sustained-only current (right) in IB4-negative neurons from control and CFA-injected TRPV1+/+ mice. * indicates that the average magnitude of the transient current was significantly decreased in neurons isolated from CFA-injected mice compared to control mice. (P<0.05; two-tailed unpaired t-test). (C) Examples of pH 5.0-evoked transient currents (top) and sustained-only currents (bottom) in IB4-negative neurons from control and CFA-injected TRPV1+/+ mice. (D) Percentage of IB4-negative neurons from control or CFA-injected TRPV1−/− mice that responded to pH 5.0. Compared to TRPV1+/+ mice, sustained-only pH currents are nearly gone in IB4-negative neurons from TRPV1−/− mice. (E) Average magnitude of proton-evoked transient current in IB4-negative neurons from control and CFA-injected TRPV1−/− mice. (F) Examples of pH 5.0-evoked transient currents in IB4-negative neurons from control and CFA-injected TRPV1−/− mice.
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Fig. 3.:
Inflammation sensitizes IB4-positive neurons to protons via TRPV1. (A) Percentage of IB4-positive small-diameter (≤26 μm) neurons from the L4/L5 DRG of TRPV1+/+ mice that responded to a 10 s exposure of pH 5.0 in control and CFA-injected mice. * indicates that significantly more IB4-positive neurons responded to pH 5.0 after inflammation compared to controls (P<0.05; Fisher's exact test). (B) Average magnitude of peak inward current in IB4-positive neurons that responded to pH 5.0 in control and CFA-injected TRPV1+/+ mice. (C) Examples of sustained inward current evoked by pH 5.0 in an IB4-positive neuron from a control and a CFA-injected TRPV1+/+ mouse. All proton-evoked currents in IB4-positive neurons from control or CFA-injected mice were sustained-only. (D) Percentage of IB4-positive small-diameter neurons from the L4/L5 DRG that responded to pH 5.0 in control and CFA-injected TRPV1−/− mice. (E) Magnitude of pH 5.0-evoked inward current in IB4-positive neurons from control and CFA-injected TRPV1−/− mice. (F) Examples of sustained inward current evoked by pH 5.0 in IB4-positive neurons from control or CFA-injected TRPV1−/− mice.

3.5. Sustained proton currents were present in IB4-positive neurons from TRPV1−/− mice and were unaltered by inflammation

To determine whether the inflammation-induced increase in proton-responsiveness of IB4-positive neurons was due to TRPV1, we recorded from L4/L5 neurons isolated from TRPV1−/− mice 2 days after CFA injection into the hind paw and tested responsiveness to pH 5.0. Interestingly, in spite of the absence of TRPV1, many IB4-positive neurons from TRPV1−/− mice exhibited a sustained-only proton-evoked current (Fig. 3(F)). In comparison to control data from TRPV1+/+ mice (Fig. 3(A) left), the same proportion of IB4-positive small-diameter neurons from non-inflamed TRPV1−/− mice responded to pH 5.0 with a sustained-only inward current (Fig. 3(D) left). The magnitude of proton responses in IB4-positive neurons from TRPV1−/− mice was not significantly different from those of TRPV1+/+ mice (Fig. 3(B) and (E)). IB4-positive neurons from inflamed TRPV1−/− mice were not increased in responsiveness to pH 5.0 when compared to TRPV1−/− controls (Fig. 3(D)). Similar to data from TRPV1+/+ mice, there was no difference in the magnitude of the proton-evoked sustained current in inflamed TRPV1−/− mice compared to TRPV1−/− controls (Fig. 3(E)). There was no difference in the sizes of IB4-positive neurons from TRPV1−/− mice tested (control: 20.9±0.6μm; CFA: 21.0±0.6μm). After IB4-positive neurons from TRPV1−/− mice (inflamed and control) were tested with pH 5.0, each neuron was tested with 1μM capsaicin and none exhibited any capsaicin-evoked current, thus confirming that all neurons from TRPV1−/− mice were indeed TRPV1 negative. These data indicate that the inflammation-induced increase in proton-responsiveness in IB4-positive neurons from wild-type mice is entirely due to increased TRPV1 function. Second, these data indicate that the sustained proton-evoked inward currents in mouse IB4-positive neurons are not mediated by TRPV1.

3.6. Inflammation decreased the magnitude of transient proton currents in IB4-negative neurons

Peripheral inflammation did not alter the percentage of IB4-negative neurons from wild-type mice that responded to protons as 73% (19/26) of IB4-negative neurons from CFA-injected mice responded to pH 5.0 with an inward current >40pA compared to 79% (19/24) from controls (Fig. 4(A)). There was no difference in the mean diameter of the IB4-negative neurons tested (control: 21.0±0.59μm; CFA: 21.7±0.48μm). In contrast to IB4-positive neurons which never exhibited transient proton-evoked currents, 47% of the IB4-negative neurons from control TRPV1+/+ mice that responded to pH 5.0 exhibited a transient proton-evoked current (Fig. 4(A) left). These transient proton currents were completely inhibited by the Acid-Sensing Ion Channel (ASIC) channel antagonist, amiloride as shown previously (Dirajlal et al., 2003). The percentage of IB4-negative neurons that exhibited a transient proton-evoked current was not significantly altered by inflammation as 32% of proton-sensitive IB4-negative neurons from CFA-injected mice exhibited a transient current (Fig. 4(A) right). However, peripheral inflammation significantly decreased the peak magnitude of the transient proton current by 3.9-fold (Fig. 4(B) left and (C) top). The remaining IB4-negative neurons from control mice (53%) and CFA-injected mice (68%) that responded to pH 5.0 exhibited sustained-only inward proton currents similar to those observed in IB4-positive neurons, and inflammation had no effect on the magnitude of the sustained currents (Fig. 4(B) right and (C) bottom). These data suggest that inflammation decreases the expression or function of ASIC-like proton channels in IB4-negative neurons.

3.7. Sustained proton currents were absent in IB4-negative neurons from TRPV1−/− mice

As expected from TRPV1+/+ data, inflammation had no effect on the percentage of IB4-negative neurons from TRPV1−/− mice that responded to pH 5.0 (Fig. 4(D)), and inflammation appeared to decrease the magnitude of the transient proton current in IB4-negative neurons (Fig. 4(E)).

Substantially fewer IB4-negative neurons from TRPV1−/− mice (control: 24%; CFA: 18%) responded to pH 5.0 compared to IB4-negative neurons from TRPV1+/+ mice (control: 79%; CFA: 73%; Fig. 4(A) and (D)). Neither the proportion nor the magnitude of the transient proton-evoked responses were significantly altered in IB4-negative neurons from TRPV1−/− mice compared to TRPV1+/+ mice (Fig. 4(D), (E) versus (A), (B)), suggesting that TRPV1 does not interact functionally with ASIC-family channels. However, the sustained-only proton responses in IB4-negative neurons from TRPV1−/− mice were virtually gone when compared to those in TRPV1+/+ mice. All IB4-negative neurons from TRPV1−/− mice were tested with 1μM capsaicin after pH 5.0 and none responded to capsaicin, confirming the absence of TRPV1. These data indicate that the sustained-only proton currents in IB4-negative neurons from wild-type mice are mediated by TRPV1 whereas sustained proton currents in IB4-positive neurons are independent of TRPV1.

3.8. IB4-positive neurons exhibit increased TRPV1-immunoreactivity after inflammation

To determine whether the increased percentage of IB4-positive neurons that responded to capsaicin is due to increased expression of TRPV1 protein in more IB4 neurons, we co-stained isolated L4 and L5 DRG neurons from CFA-injected and control wild-type mice with a TRPV1 antibody and IB4. Fig. 5 shows a confocal image of isolated L4/L5 DRG neurons from a control (A) and an inflamed (B) mouse. Overall, inflammation induced a 2.5-fold increase in the percentage of all small-diameter (≤26μm) neurons that were immunoreactive for TRPV1 (Table 1). More specifically, in control, non-inflamed mice, 4.5% of small-diameter IB4-positive neurons were immunoreactive for TRPV1, a finding that is highly consistent with other reports of TRPV1-immunoreactivity in IB4-positive neurons from mouse (Woodbury et al., 2004; Zwick et al., 2002). Two days after CFA-injection, the percentage of IB4-positive neurons that was immunoreactive for TRPV1 was increased over 3-fold to 15.5% (P<0.05, two-tailed unpaired t-test; Fig. 5 and Table 1). The percentage of IB4-positive neurons among total small-diameter (≤26μm) neurons was not significantly altered by inflammation (control: 35.0±4%; CFA: 45.0±6%; P>0.2; two-tailed unpaired t-test), indicating that the increased co-expression of TRPV1 and IB4 is not likely due to changes in IB4 binding. In addition, the percentage of IB4-positive neurons ≤26μm diameter isolated from the L4/L5 ganglia of control mice (35.0±4%) was not different than the percentage of IB4-positive neurons ≤26μm diameter in tissue sections of L4/L5 ganglia taken from adult non-inflamed C57BL/6 mice (38.2±0.8%; n=5 animals; Stucky, C.L., unpublished observations), indicating that in our laboratory, IB4 binding in acutely isolated neurons is representative of that in intact ganglia.

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Fig. 5.:
Inflammation increases TRPV1-immunoreactivity in IB4-positive neurons. Confocal images of acutely isolated DRG neurons from control (A) and CFA-injected (B) wild-type mice that were co-stained with IB4 and a TRPV1 antibody. Lumbar 4/5 DRG neurons were isolated from control, non-injected mice or mice 2 days after CFA injection and fixed 6–9 h later for staining. Merged confocal images show that few IB4-positive neurons from control mice are TRPV1-immunoreactive, but after CFA-induced peripheral inflammation, more IB4-positive neurons are immunoreactive for TRPV1. Although neurons from control and inflamed mice were fixed at the same time after isolation, neurons from CFA-injected mice typically exhibited more processes, less round somata and more clustering than controls.
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Table 1:
TRPV1-immunoreactivity in IB4-positive and -negative small-diameter neurons from control and CFA-injected mice

Consistent with our functional data, the percentage of IB4-negative small-diameter neurons that was immunoreactive for TRPV1 was not significantly altered by inflammation (Table 1; P>0.1; two-tailed unpaired t-test). These data confirm that the inflammation-induced increase in TRPV1 is specific to the IB4-positive population of small-diameter, putative C-fiber neurons and indicate that the increased TRPV1 function is at least partially due to increased expression of TRPV1 protein in IB4-positive neurons.

4. Discussion

Our study demonstrates that peripheral inflammation in mouse sensitizes IB4-positive, but not IB4-negative, small-diameter DRG neurons to TRPV1 receptor stimuli, capsaicin and protons. The enhanced responsiveness is specifically due to TRPV1, as IB4-positive neurons from inflamed TRPV1−/− mice are unresponsive to capsaicin and are not increased in responsiveness to protons. Immunocytochemical analysis indicates that the increased capsaicin sensitivity is due in part to novel TRPV1 expression in IB4-positive neurons. These data demonstrate that IB4-positive neurons are sensitized to TRPV1 ligands during inflammation and provide new evidence that IB4-positive C-fiber neurons may contribute to inflammatory hyperalgesia. Comparison of proton responses in TRPV1+/+ and TRPV1−/− neurons indicates that the sustained proton-evoked currents in IB4-positive neurons are independent of TRPV1 whereas the sustained-only proton currents in IB4-negative neurons are mediated by TRPV1.

4.1. Peripheral inflammation increases TRPV1 function and expression in IB4-positive neurons

Contrary to our hypothesis that IB4-negative small-diameter neurons would be sensitized by inflammation, we found that 3-fold more IB4-positive neurons responded to TRPV1 stimuli following inflammation but IB4-negative neurons were unaltered. In parallel, immunostaining showed that 3-fold more IB4-positive neurons express TRPV1 after inflammation. Our results agree with studies in rat showing that inflammation increases TRPV1-immunoreactivity in DRG somata, including IB4 binding neurons (Amaya et al., 2003, 2004; Ji et al., 2002), and indicate that in mouse, TRPV1 increases predominantly in IB4-positive neurons. Inflammation may also increase TRPV1 function in myelinated fibers (Amaya et al., 2003; Luo et al., 2004), but we did not investigate A-fibers here.

In agreement with other laboratories, we found that <5% of IB4-positive neurons from non-injured mice express TRPV1-immunoreactivity (Woodbury et al., 2004; Zwick et al., 2002). However, 25% of IB4-positive neurons responded to capsaicin. This discrepancy is most likely due to differential sensitivity in detection of immunofluorescence in fixed neurons compared to functional responses in live neurons. Although we used an anti-rat TRPV1 antibody, the discrepancy is not likely due to an inter-species antibody recognition problem because an anti-mouse TRPV1 antibody also labels only 2–5% of mouse IB4-positive neurons (Woodbury et al., 2004). Together, our data demonstrate that peripheral inflammation selectively increases TRPV1 function and expression in IB4-positive mouse neurons.

4.2. Physiological role of IB4-positive neurons in cutaneous inflammation

The majority of IB4-positive small-diameter neurons project to skin targets (Lu et al., 2001; Plenderleith and Snow, 1993; Wang et al., 1998), and electrophysiological recordings from identified cutaneous afferents show that most IB4 binding neurons from mouse and rat are nociceptors (Gerke and Plenderleith, 2001; Woodbury et al., 2004). In vivo deletion of IB4 binding neurons with IB4-saporin toxin demonstrates that IB4-positive neurons contribute to acute thermal and mechanical behavioral nociception in non-injured rats (Vulchanova et al., 2001) and participate in the early development of mechanical allodynia after spinal nerve injury (Tarpley et al., 2004). However, nothing is known about the physiological roles of IB4-positive neurons during persistent tissue inflammation. Our data suggest that enhanced responsiveness of IB4 binding neurons during cutaneous inflammation may contribute to inflammatory hyperalgesia. A 3-fold increase in the number of IB4-positive neurons that responds to TRPV1 stimuli including heat or endogenous TRPV1 inflammatory ligands could increase, by spatial summation, the amount of nociceptive information transmitted to the spinal dorsal horn and ultimately, contribute to inflammatory hyperalgesia. Behavioral assays and electrophysiological analyses in inflammatory animal models after deletion of IB4-positive neurons will further define the physiological role of these neurons during peripheral inflammation.

4.3. Mechanisms underlying the inflammation-induced increase in TRPV1 function in IB4-positive neurons

What mechanisms increase TRPV1 in IB4-positive neurons? A likely candidate is the neurotrophic factor GDNF. IB4-positive neurons express receptors for GDNF (GFRα1) and its relative neurturin (GFRα2) (Bennett et al., 1998; Molliver et al., 1997). GDNF increases the capsaicin sensitivity and TRPV1 expression in cultured DRG neurons (Bron et al., 2003; Ogun-Muyiwa et al., 1999). GDNF levels are increased in the DRG 2 days after CFA-induced inflammation in rat, and anti-GDNF antibody treatment prevents the increased TRPV1 expression in IB4-positive neurons and the inflammatory heat hypersensitivity (Amaya et al., 2004).

An alternative candidate is NGF. NGF levels increase in inflamed tissues (Aloe et al., 1992; Weskamp and Otten, 1987; Woolf et al., 1994) and NGF-sequestering antibodies prevent inflammatory heat hyperalgesia (Koltzenburg et al., 1999; Lewin et al., 1994). Inflammation-induced increases TRPV1 protein in DRG neurons have been shown to be NGF-dependent (Amaya et al., 2003; Carlton and Coggeshall, 2001; Ji et al., 2002). The conundrum is that some studies in rodents show that the NGF receptor, TrkA is not expressed by IB4-positive neurons (Averill et al., 1995; Molliver et al., 1995), but other studies report significant overlap between trkA and IB4 (Goodness et al., 1997; Kashiba et al., 2001; Orozco et al., 2001). In cultured adult mouse DRG neurons, NGF increases the heat-responsiveness of IB4-positive neurons more than that of IB4-negative neurons, indicating that some IB4-positive neurons are highly sensitive to NGF modulation (Stucky and Lewin, 1999). Furthermore, deletion of IB4-positive neurons in vivo with IB4-saporin toxin completely blocks NGF-induced thermal hyperalgesia, indicating that IB4-positive neurons are essential for development of NGF-mediated heat hypersensitivity (Tarpley et al., 2004).

Most IB4-positive neurons express trkA receptors until birth, but soon after lose trkA expression and NGF dependence (Bennett et al., 1996a; Molliver and Snider, 1997; Molliver et al., 1997). An intriguing though speculative possibility is that inflammation might induce re-expression of TrkA in adult IB4-positive neurons and confer NGF sensitivity to IB4-positive neurons in the setting of inflammation.

Surprisingly, although IB4-negative C-fiber neurons were hypothesized to contribute to inflammatory pain and they express trkA and respond to NGF (Mantyh and Hunt, 1998; Snider and McMahon, 1998), inflammation did not alter their TRPV1 function. The most parsimonious explanation is that TRPV1 and trkA function may already be maximal in IB4-negative C-fiber neurons in non-injured adult mice.

4.4. Inflammation decreases the transient proton currents in IB4-negative neurons

Transient proton-evoked currents in sensory neurons are mediated by members of the ASIC family including: ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 (Price et al., 1996; Waldmann and Lazdunski, 1998; Waldmann et al., 1997). The mRNA for all ASIC subtypes is found in rodent DRG neurons (Benson and Sutherland, 2001; Price et al., 2001), and mRNA levels for several ASIC isoforms increase substantially in rat DRG neurons 2 days after CFA-induced inflammation (Voilley et al., 2001). Furthermore, inflammatory mediators increase the ASIC-like transient proton currents in cultured rat neurons (Mamet et al., 2002). Therefore, we were surprised to find that CFA-induced inflammation in mouse decreased the magnitude of transient proton currents in IB4-negative neurons by 70%. This decrease in transient proton current may be due to altered stoichiometry of ASIC subunits within an ion channel complex. Different ASIC subunits co-localize to form heteromultimeric channels in DRG neurons (Benson et al., 2002). ASIC2b can suppress the function of ASIC1b by acting as a modulatory subunit (Hesselager et al., 2003; Lingueglia et al., 1997), and ASIC2b transcripts are increased 8-fold in small-diameter DRG neurons after peripheral inflammation in rat (Voilley et al., 2001). Other explanations for our apparently divergent results may be a species difference between mouse and rat, variation in the populations of neurons sampled, or a discrepancy between the milieu of true inflammation versus exogenous inflammatory chemicals.

4.5. TRPV1 does not mediate sustained proton currents in IB4-positive mouse neurons

Our finding that sustained proton currents in IB4-positive neurons from TRPV1−/− mice were present in normal proportions compared to those in TRPV1+/+ mice indicates that TRPV1 does not mediate the proton currents in IB4-positive mouse neurons. In contrast, the sustained-only proton currents in IB4-negative small-diameter neurons were virtually gone in TRPV1−/− mice and therefore, are mediated almost exclusively by TRPV1. Potential candidates for the non-TRPV1 sustained proton current in IB4-positive neurons include the acid-sensitive background K+ channels TASK-1, TASK-3, and Kir2.3 which are present in DRG neurons and are highly sensitive to changes in pH (Baumann et al., 2004; Cooper et al., 2004). During inflammation, proton-induced inhibition of these K+ channels may elevate the resting membrane potential, depolarize the neuron and thereby, increase the excitability of IB4-positive neurons.

Our data suggest that IB4-positive and -negative small-diameter mouse neurons express a relatively discrete composite of proton-sensitive ion channels: IB4-positive neurons express non-TRPV1 channels that convey sustained proton currents and lack ASIC-like transient proton channels. Conversely, IB4-negative neurons express both TRPV1 channels and ASIC-like transient proton-sensitive channels. During inflammation or tissue acidosis, these two classes of C-fiber neurons likely transmit distinct nociceptive information to the spinal dorsal horn.

Overall, our study provides new evidence that IB4-positive neurons are selectively sensitized to TRPV1 agonists during cutaneous inflammation. Their capacity to become sensitized highlights the IB4-positive subpopulation of unmyelinated neurons as a putative target for novel inflammatory therapeutics.

Acknowledgements

This work was supported by National Institute of Neurological Disorders and Stroke grant NS40538 to C.L.S. We thank Drs Quinn Hogan, Virginia Seybold and Michelle Mynleiff for critically reviewing the manuscript and Brett Schroeder for expert assistance with confocal imaging.

References

Aloe L, Tuveri MA, Levi-Montalcini R. Studies on carrageenan-induced arthritis in adult rats: presence of nerve growth factor and role of sympathetic innervation. Rheumatol Int. 1992;12:213-216.
Amaya F, Oh-hashi K, Naruse Y, Iijima N, Ueda M, Shimosato G, Tominaga M, Tanaka Y, Tanaka M. Local inflammation increases vanilloid receptor 1 expression in distinct subgroups of DRG neurons. Brain Res. 2003;963:190-196.
Amaya F, Shimosato G, Nagano M, Ueda M, Hashimoto S, Tanaka Y, Suzuki H, Tanaka M. NGF and GDNF differentially regulate TRPV1 expression that contributes to development of inflammatory thermal hyperalgesia. Eur J Neurosci. 2004;20:2303-2310.
Averill S, McMahon SB, Clary DO, Reichardt LF, Priestly JV. Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons. Eur J Neurosci. 1995;7:1484-1494.
Baumann TK, Chaudhary P, Martenson ME. Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain. Eur J Neurosci. 2004;19:1343-1351.
Bennett DL, Averill S, Clary DO, Priestly JV, McMahon SB. Postnatal changes in expression of the trkA high-affinity NGF receptor in primary sensory neurons. Eur J Neurosci. 1996a;8:2204-2208.
Bennett DL, Dmietrieva N, Priestly JV, Clary D, McMahon SM. TrkA, CGRP and IB4 expression in retrogradely labeled cutaneous and visceral primary sensory neurons in the rat. Neurosci Lett. 1996b;206:33-36.
Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestly JV. A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci. 1998;18:3059-3072.
Benson CJ, Sutherland SP. Toward an understanding of the molecules that sense myocardial ischemia. Ann NY Acad Sci. 2001;940:96-109.
Benson CJ, Xie J, Wemmie JA, Price MP, Henss JM, Welsh MJ, Snyder PM. Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons. Proc Natl Acad Sci USA. 2002;99:2338-2343.
Bron R, Klesse LJ, Shah K, Parada LF, Winter J. Activation of Ras is necessary and sufficient for upregulation of vanilloid receptor type 1 in sensory neurons by neurotrophic factors. Mol Cell Neurosci. 2003;22:118-132.
Carlton SM, Coggeshall RE. Peripheral capsaicin receptors increase in the inflamed rat hindpaw: a possible mechanism for peripheral sensitization. Neurosci Lett. 2001;310:53-56.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997;389:816-824.
Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Peterson-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288:306-313.
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Taksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55-63.
Cooper BY, Johnson RD, Rau KK. Characterization and function of TWIK-related acid-sensing K+ channels in a rat nociceptive cell. Neuroscience. 2004;129:209-224.
Davis JB, Gray J, Gunthrope MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC, Bingham S, Randall A, Sheardown SA. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature. 2000;405:183-187.
Dirajlal S, Pauers LE, Stucky CL. Differential response properties of IB(4)-positive and -negative unmyelinated sensory neurons to protons and capsaicin. J Neurophysiol. 2003;69:1071-1081.
Galoyan SM, Petruska JC, Mendell LM. Mechanisms of sensitization of the response of single dorsal root ganglion cells from adult rat to noxious heat. Eur J Neurosci. 2003;18:535-541.
Gerke MB, Plenderleith MB. Binding sites for the plant lectin Bandeiraea simplicifolia I-isolectin B4 are expressed by nociceptive primary sensory neurones. Brain Res. 2001;911:101-104.
Gerke MB, Plenderleith MB. Ultrastructural analysis of the central terminals of primary sensory neurones labeled by transganglionic transport of bandeiraea simplicifolia I-isolectin B4. Neuroscience. 2004;127:165-175.
Goodness TP, Albers KM, Davis FE, Davis BM. Overexpression of nerve growth factor in skin increases sensory neuron size and modulates Trk receptor expression. Eur J Neurosci. 1997;9:1574-1585.
Hantman AW, van den Pol AN, Perl ER. Morphological and physiological features of a set of spinal substantia gelatinosa neurons defined by green fluorescent protein expression. J Neurosci. 2004;28:836-842.
Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32:77-88.
Hesselager M, Timmermann DB, Ahring PK. pH Dependency and desensitization kinetics of heterologously expressed combinations of acid sensing ion channel subunits. J Biol Chem. 2003;270:11006-11015.
Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Petrocellis L, Fezza F, Tognetto M, Petros TJ, Krey JF, Chu CJ, Miller JD, Davies SN, Geppetti P, Walker JM, Di Marzo V. An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc Natl Acad Sci USA. 2002;99:8400-8405.
Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D, Oh U. Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci USA. 2000;97:6155-6160.
Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron. 2002;36:57-68.
Kashiba H, Uchida Y, Senba E. Difference in binding by isolectin B4 to trkA and c-ret mRNA-expressing neurons in rat sensory ganglia. Brain Res Mol Brain Res. 2001;95:18-26.
Koltzenburg M, Bennett DL, Shelton DL, McMahon SB. Neutralization of endogenous NGF prevents the sensitization of nociceptors supplying inflamed skin. Eur J Neurosci. 1999;11:1698-1704.
Lewin GR, Rueff A, Mendell LM. Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur J Neurosci. 1994;6:1903-1912.
Lingueglia E, de Weille JR, Bassilina F, Heureteaux C, Sakai H, Waldmann R, Lazdunski M. A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J Biol Chem. 1997;272:29778-29783.
Lu J, Zhou X-F, Rush RA. Small primary sensory neurons innervating epidermis and viscera display differential phenotype in the adult rat. Neurosci Res. 2001;41:355-363.
Luo H, Cheng J, Han JS, Wan Y. Change of vanilloid receptor 1 expression in dorsal root ganglion and spinal dorsal horn during inflammatory nociception induced by complete Freund's adjuvant in rats. NeuroReport. 2004;22:655-658.
Mamet J, Baron A, Lazdunski M, Voilley N. Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci. 2002;22:10662-10670.
Mantyh PW, Hunt SP. Hot peppers and pain. Neuron. 1998;21:644-645.
Molliver DC, Snider WD. Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons. J Comp Neurol. 1997;381:428-438.
Molliver DC, Radeke MJ, Feinstein SC, Snider WD. Presence or absence of TrkA protein distinguishes subsets of small sensory neurons with unique cytochemical characteristics and dorsal horn projections. J Comp Neurol. 1995;361:404-416.
Molliver DC, Wright DE, Leitner ML, Parsadanian AS, Doster K, Wen D, Yan Q, Snider WD. IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life. Neuron. 1997;19:849-861.
Nicholas RS, Winter J, Wren P, Bergman R, Woolf CJ. Peripheral inflammation increases the capsaicin sensitivity of dorsal root ganglion neurons in a nerve growth dependent manner. Neuroscience. 1999;91:1425-1433.
Nicol GD, Lopshire JC, Pafford CM. Tumor necrosis factor enhances the capsaicin sensitivity of rat sensory neurons. J Neurosci. 1997;17:975-982.
Ogun-Muyiwa P, Helliwell R, McIntyre P, Winter J. Glial cell line derived neurotrophic factor (GDNF) regulates VR1 and substance P in cultured sensory neurons. NeuroReport. 1999;10:2107-2111.
Orozco OE, Walus L, Sah DW, Pepinsky RB, Sanicola M. GFRalpha3 is expressed predominantly in nociceptive sensory neurons. Eur J Neurosci. 2001;13:2177-2182.
Plenderleith MB, Snow PJ. The plant lectin Bandeiraea simplicifolia I-B4 identifies a subpopulation of small-diameter primary sensory neurones which innervate the skin in the rat. Neurosci Lett. 1993;159:17-20.
Price MP, Snyder PM, Welsh MJ. Cloning and expression of a novel human brain Na+ channel. J Biol Chem. 1996;271:7879-7882.
Price MP, McIlwarth SL, Xie J, Cheng C, Qiao J, Tarr DE, Sluka KA, Brennan TJ, Lewin GR, Welsh MJ. The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice. Neuron. 2001;32:1071-1083.
Shu X, Mendell LM. Acute sensitization by NGF of the response of small-diameter sensory neurons to capsaicin. J Neurophysiol. 2001;86:2931-2938.
Silverman JD, Kruger L. Selective neuronal glycogonjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers. J Neurosci. 1990;23:789-801.
Smart D, Gunthrope MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB. The endogenous lipid anadamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol. 2000;129:227-230.
Snider WD, McMahon SB. Tackling pain at the source: new ideas about nociceptors. Neuron. 1998;20:629-632.
Stucky CL, Lewin GR. Isolectin B(4)-positive and -negative nociceptors are functionally distinct. J Neurosci. 1999;19:6497-6505.
Tarpley JW, Kohler MG, Martin WJ. The behavioral and neuroanatomical effects of IB4-saporin treatment in rat models of nociceptive and neuropathic pain. Brain Res. 2004;1029:65-76.
Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron. 1998;21:531-543.
Voilley N, de Weille J, Mamet J, Lazdunski M. Nonsteroidal anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci. 2001;21:8026-8033.
Vulchanova L, Olson TH, Stone LS, Riedl MS, Elde R, Honda CN. Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neuroscience. 2001;108:143-155.
Wach J, Marin-Burgin A, Klusch A, Forster C, Engert S, Schwab A, Petersen M. Low-threshold heat receptor in chick sensory neurons is upregulated independently of nerve growth factor after nerve injury. Neuroscience. 2003;117:513-519.
Waldmann R, Lazdunski M. H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol. 1998;8:418-424.
Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux C, Lazdunski M. Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J Biol Chem. 1997;72:20975-20978.
Wang H, Rivero-Melian C, Robertson G, Grant G. Transganglionic transport and binding of the isolectin B4 from Griffonia simplicifolia I in rat primary sensory neurons. Neuroscience. 1994;62:539-551.
Wang HF, Robertson B, Grant G. Anterograde transport of horseradish-peroxidase conjugated isolectin B4 from Griffonia simplicifolia I in spinal primary sensory neurons of the rat. Brain Res. 1998;811:34-39.
Weskamp G, Otten U. An enzyme-linked immunoassay for nerve growth factor (NGF): a tool for studying regulatory mechanisms involved in NGF production in brain and peripheral tissues. J Neurochem. 1987;48:1779-1786.
Winter J, Forbes CA, Sternberg J, Lindsay RM. Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin. Neuron. 1988;1:973-981.
Woodbury CJ, Ritter AM, Koerber HR. On the problem of lamination in the superficial dorsal horn of terminals: a reappraisal of the substantia gelatinosa in postnatal life. J Comp Neurol. 2000;417:88-102.
Woodbury CJ, Zwick M, Wang S, Lawson JJ, Caterina MJ, Koltzenburg M, Albers KM, Koerber HR, Davis BM. Nociceptors lacking TRPV1 and TRPV2 have normal heat responses. J Neurosci. 2004;24:6410-6415.
Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J. Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience. 1994;62:327-331.
Zwick M, Davis BM, Woodbury CJ, Burkett JN, Koerber HR, Simpson JF, Albers KM. Glial cell-line derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse. J Neurosci. 2002;22:4057-4065.
Keywords:

VR1; ASIC; Protons; Capsaicin; Nociceptor; C fiber

© 2005 Lippincott Williams & Wilkins, Inc.