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Proton Sensitivity Ca²⁺ Permeability and Molecular Basis of Acid-Sensing Ion Channels Expressed in Glabrous and Hairy Skin Afferents

¹Department of Oral Surgery and Diagnostic Sciences, Division of Neuroscience, J. H. Miller Health Center, University of Florida College of Dentistry and McKnight Brain Institute, ²Department of Physiological Sciences, College of Veterinary Medicine and University of Florida, McKnight Brain Institute, and ³Department of Neuroscience, College of Medicine and University of Florida, McKnight Brain Institute, Gainesville, Florida

Submitted 17 August 2005; accepted in final form 20 December 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)–like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration–response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH₅₀ = 6.54 and 6.75 vs. 6.20–6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1–0.3 vs. 0.8–1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca²⁺-permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca²⁺-permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca²⁺ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC–expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC–expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The dorsal root ganglia (DRG) contain the cell bodies of functionally distinct sensory afferents innervating highly diverse tissues. In dissociated DRG cells, nearly 70% will manifest some form of proton-activated currents; the form and kinetics of these currents vary substantially and their peculiar role in sensory function is uncertain (Bevan and Geppetti 1994; Bevan and Yeats 1991; Krishtal and Pidoplichko 1981). Some proton-sensitive afferents play a role in pain because proton reactivity is a common property of nociceptive populations, in vivo (Lindahl 1961; Steen and Reeh 1993; Steen et al. 1995b, 1996). Moreover, pain, allodynia, and hyperalgesia follow when acidic solutions are injected into skin, muscle or vascular tissues of human volunteers (Fransson and Espander-Jansson 1996; Hamamoto et al. 1998; Klement and Arndt 1991; Steen and Reeh 1993; Steen et al. 1995b).

There are multiple Na⁺-, Ca²⁺-, and K⁺-permeable channels that contribute to ionic fluxes that result from tissue acidity. Some proton-activated currents with slow activation and decay kinetics represent proton gating of the capsaicin receptor TRPV1 (Cortright and Szallasi 2004; Geppetti and Trevisani 2004). Other slowly activating and decaying currents represent proton gating of the K2p family of K⁺ leak channels: TASK-1, TASK-2, TASK-3, and TASK-4 (Baumann et al. 2004; Cooper et al. 2004; Patel and Honore 2001). In contrast to transient receptor potential (TRP) proteins, channels formed from TASK proteins close with increased acidity. Distinct fast activating and decaying proton-induced currents are manifested by the amiloride-sensitive, acid-sensing ion channels (ASICs). The relative importance of these proton-gated channels to inflammation and pain depends on their proton sensitivity, ion permeability, and differential distribution into superficial and deep nociceptors. As a family, ASICs seem particularly important to nociceptive processing: 1) they are widely expressed in DRG; 2) thresholds of activation can be near physiological pH; 3) their expression patterns are altered by inflammatory conditions; 4) some are Ca²⁺ permeable; and 5) they may play a role in mechanosensory transduction (Benos and Stanton 1999; Bianchi and Driscoll 2002; Krishtal 2003; Waldmann and Lazdunski 1998).

The molecular basis of channels with ASIC-like properties has been identified as deriving from proteins of the degenerin/epithelial sodium channel (Deg/ENaC) superfamily. In the last several years, multiple ASIC channel proteins have been cloned and extensively characterized in host cells. Six different proteins arise from four genes: ASIC1a (ASIC1, BNaC2; Waldmann et al. 1997a) and ASIC1b (ASIC; Chen et al. 1998) are spliced forms of the ASIC1 gene; ASIC2a (BNaC1 or MDEG1; Price et al. 1996; Waldmann et al. 1997a) and ASIC2b (MDEG2; Lingueglia et al. 1997) are spliced forms of the ASIC2 gene; ASIC3 (DRASIC; Waldmann et al. 1997b); and ASIC4 (SPASIC; Akopian et al. 2000). Except for ASIC2b and ASIC4, all subunits have the ability to form functional homomeric channels when expressed in Xenopus laevis oocytes or mammalian cells (Akopian et al. 2000; Hesselager et al. 2004; Lingueglia et al. 1997).

ASICs are expressed throughout the mammalian central and peripheral nervous system (Alvarez de la Rosa et al. 2002; Krishtal 2003; Waldmann and Lazdunski 1998; Waldmann et al. 1997b, 1999). Although early studies failed to identify the ASIC2a isoform in DRG (Waldmann and Lazdunski 1998), it was subsequently reported that all four ASIC proteins were expressed in DRG (Alvarez de la Rosa et al. 2002). Multiple ASIC subunits were shown to co-localize within the same DRG neuron. However, the specific distribution of ASIC subunits into functionally distinct afferent populations (such as nociceptive neurons) is unknown. Nor do we know how these heteromeric complexes differentially affect proton gating or reflect local function in native nociceptive neurons with distinct tissue distributions and histochemical phenotype.

When expressed in host cells, acid-sensing proteins are able to form into numerous heteromeric combinations that differ in kinetics, permeability, and proton sensitivity. Ca²⁺ permeability is uniquely important in nociceptive function. Although all channels formed from ASIC proteins are highly permeable to Na⁺, homomeric channels formed from complexes of ASIC1a can flux Ca²⁺ (pNa⁺/pCa²⁺ = 2.5; Chen et al. 1998; Chu et al. 2002; Waldmann et al. 1997a). Certain ASIC heteromers containing ASIC1a are not Ca²⁺ permeable, but testing of heteromeric combinations has not been exhaustive and can be difficult to interpret in host cells (Bassilana et al. 1997; Benson et al. 2002; Chen et al. 1998; Coscoy et al. 1999; Hesselager et al. 2004; Lingueglia et al. 1997) or in the absence of accessory proteins (Hruska-Hageman et al. 2002). The distribution of calcium-permeable ASIC1a is not known for nociceptive neurons of the DRG. Yet this distribution is likely to be of particular importance in the development of allodynia and hyperalgesia. Tissue acidity is a common sequela of inflammation. Because protons, unlike many other inflammatory mediators, are chronically present at inflamed sites, a conduit for Ca²⁺ could result by ASIC1a-expressing nociceptors. Calcium entry into nociceptors is linked to the activation of protein kinases and phospholipases with diverse proinflammatory actions on ion channels (Barber and Vasko 1996; Hou and Wang 2001; Jin et al. 2004; Lazar et al. 2004). Local peptide release by Ca²⁺-dependent mechanisms also plays a significant paracrine role in inflammation (Holzer 1988; Lam and Ferrell 1991; Lembeck and Holzer 1979). These peptidergic nociceptors are present in cutaneous, muscle, and visceral neurons, many of which would experience acidosis during ischemic or inflammatory conditions (Cervero 1994; Cervero and Laird 2004; Perry and Lawson 1998; Rau et al. 2005a). In our previous investigations, all subclassified nociceptors that expressed amiloride-sensitive ASIC-like channels were peptidergic (see following text). However, the molecular identity and the specific distribution of ASIC channels among peptidergic nociceptive populations that innervate skin, muscle, viscera, and vascular sites are not known.

We previously reported that small- and medium-sized DRG cells could be separated into nine subclasses by a current signature method (Petruska et al. 2000a, 2002). In these nine subclassified cells, types 3, 5, 6, 7, 8, and 9 manifested ASIC-like fast-decaying currents that varied in decay kinetics (Petruska et al. 2002). The IB4 negative (B4 isolectin of Griffonia simplicifolia), Nf-m positive, type 3 cells lacked capsaicin, adenosine triphosphate (ATP), or acetylcholine (ACh) sensitivity, lacked opiate receptors and manifested short afterhyperpolarizations consistent with nonnociceptive cells (Djouhri et al. 1998; Petruska et al. 2000a, 2002; Rau et al. 2005a,b). Cell types 5, 6, 7, 8, and 9 have properties consistent with nociceptors. All such cells expressed CGRP (types 6 and 9), coexpressed SP and CGRP (types 5, 7, and 8), expressed opiate receptors (types 5, 6, 7, 8, and 9), or are capsaicin sensitive (types 5, 7, 8, and 9; Petruska et al. 2000a, 2002; Rau et al. 2005b).

Although it is simple to conceptualize a role for ASICs as proton sensors in nociceptive neurons, the role of ASICs in nonnociceptive cells is less clear. We previously documented ASIC-like currents in a population that lacks all nociceptive markers. Other laboratories report fluorescent markers for ASIC2a in cutaneous low-threshold mechanosensory terminals. The latter include readily identifiable low-threshold mechanoreceptor structures such as Meissner corpuscles, Merkel, Penicillate, and Lancelot endings (Garcia-Anoveros et al. 2001; Price et al. 2000). Because many homomeric and heteromeric combinations of ASIC proteins manifest low proton sensitivity, it is possible that nonnociceptive neurons express isoforms whose sensitivity is shifted relative to nociceptors (Benson et al. 2002; Hesselager et al. 2004). If this were the case, the proton sensitivity of nonnociceptive cells could be outside the physiological range.

In the experiments described below, we used electrophysiological, pharmacological, immunocytochemical, and ratiometric methods to examine the molecular basis of proton sensitivity, decay kinetics, and calcium ion permeability in nociceptive and nonnociceptive subclassified DRG neurons expressing ASIC protein. Studies were complemented by tracings of peripheral terminals of cell classes from hairy and glabrous skin.

	METHODS

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Subjects

Adult male Sprague–Dawley rats (90–150 g) were used in all experiments. Animals were housed in American Association for Accreditation of Laboratory Animal Care–approved quarters. Procedures were reviewed and approved by the local Institutional Animal Care and Use Committee, and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Preparation of cells

Rats were preanesthetized with halothane. After decapitation, the spinal cord was rapidly removed, and all thoracic and lumbar dorsal root ganglia were dissected free from one side of the bisected vertebral column. Dissected ganglia were placed in a heated bath (35°C for 70 min) containing dispase II and collagenase (2 mg/ml; Sigma type 1). After wash and trituration, recovered cells were plated on 10 polylysine-coated, 35-mm petri dishes. In some instances glass-bottom dishes were used (MatTek). All recordings were made at room temperature within 10 h after plating. Cells were bathed continuously in a rat Tyrode's solution containing (in mM): 140 NaCl, 4 KCl, 2 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with NaOH. Only one cell was used from each dish. When applying acidic solutions with a pH <5.5, 2-[N-Morpholino]ethanesulfonic acid (MES) was substituted for HEPES.

Whole cell patch recording

Electrodes were prepared (2–4 M) from glass pipettes using a Brown and Flaming type horizontal puller (Sutter model P87). Whole cell recordings were made with an Axopatch 200B (Axon Instruments). Stimuli were controlled and records were captured with pClamp 8.2 software and Digidata 1322A (Axon Instruments). Series resistance (R_s) was compensated 40–60% with Axopatch 200B compensation circuitry. Whole cell resistance and capacitance were determined by Clampex 8.2 software utility. A liquid junction potential of about 4 mV was not corrected.

Cell classification protocols

Recordings were made exclusively from cells with diameters between 17 and 55 µm. Cell diameter was estimated from the average of the longest and shortest axis as measured through an eyepiece micrometer scale. Cells were classified as types 1–9 according to patterns of voltage-activated currents (current signatures) that were revealed by three classification protocols. Classification protocol 1 (CP1) was used to examine the patterns of hyperpolarization-activated currents. With CP1, currents were evoked by series of hyperpolarizing pulses presented from a V_h of –60 mV (10 mV per step to a final potential of –110 mV, 500-ms pulse, and 4-s interstimulus interval). Classification protocol 2 (CP2) was used to produce outward current patterns. From a V_h of –60 mV, a 500-ms conditioning pulse to –100 mV was followed by 200-ms depolarizing command steps (20 mV) to a final potential of +40 mV. Classification protocol 3 (CP3) was used to produce inward current patterns. With the cell held at –60 mV, a 500-ms conditioning pulse to –80 mV was followed by a series of depolarizing command steps (10-mV steps, 2.0-ms duration) to a final potential of +10 mV. Current signature patterns for the nine cell types were previously presented (Petruska et al. 2000a, 2002).

[Ca²⁺]_i measurement

A glass-bottom petri dish (MatTek Cultureware) was used for the measurement of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ca²⁺ currents through proton-gated channels were determined by loading the cells with the Ca²⁺ indicator dye fura-2 pentapotassium salt by the internal solution of the recording pipette (150 µM). At least 2 min were allowed for internal perfusion before cell classification and other experiments. For ratiometric recordings, cells were alternatively exposed at wavelengths of 340 and 380 nm (Bentham model FSM150Xe). A CCD camera (Cohu 4920) and videoimaging system were used to capture images selected for study (Intracellular Imaging). The fluorescence (510 nm) was measured and the ratio of that illuminated at 340 and 380 nm was calculated and referenced to a standard curve (Calcium Calibration Buffer Kit with Magnesium #2; Molecular Probes).

Afferent tracing

Under aseptic conditions, 35 young adult male rats (80–100 g) were anesthetized with a mixture of ketamine and xylazine (80 mg/kg ketamine; 10 mg/kg xylazine). The following signs were monitored during surgery: heart rate, respiratory rate, ventilatory status (end-expired pCO₂), and body temperature. Anesthetic depth was assessed by corneal, palpebral, and pinna reflexes. The animals were placed on a heating pad to maintain ideal body temperature (36–37°C). Intradermal injections of the fluorescent tracer FastDiI oil (1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; 25 mg FastDiI dissolved in 0.5 ml methanol; Molecular Probes) were then made with a 33-gauge needle coupled to a Hamilton microsyringe (16-ml volume per animal divided into eight injections per limb of 1 ml each). In 15 of the rats, these injections were made into the hairy skin overlying the gastrocnemius muscle. In the other 20 rats, these injections were made into the glabrous skin footpads or heel. Care was taken not to penetrate into subdermal tissues. After each injection, the needle was slowly removed and any leakage was controlled by cotton-tipped applicators. Rats were monitored daily and allowed to recover for 7 days. They were then killed for in vitro electrophysiological studies. Cells were plated in the usual manner but protected from ambient light. Dishes were mounted on a Nikon TE 2000 inverted microscope with an epifluorescence attachment. Tracer-labeled cells were viewed with the appropriate Vivid filter set (XF102, Omega Optical), and ultraviolet light exposure to all fields was about 1 min in duration. Only intensely fluorescent cells were considered positive and only one cell was recorded per dish. After a recording was completed, digital images of the brightfield and fluorescent fields of view were captured using a Dage MTI RC300 camera coupled to a PC running Scion Image 4.0.2. To assess the possible spread of DiI from injection sites, injected tissue and underlying muscle tissues were harvested before plating the DRG cells. The tissues were placed in vials containing 4% paraformaldehyde in phosphate-buffered saline (PBS) for a 24-h period. Subsequently, this fixative solution was replaced by 30% sucrose in PBS for cryoprotection. Once the tissue equilibrated it was embedded in TBS tissue freezing medium (Triangle Biomedical Sciences) and 10-mm sections were cut on a cryostat (HM 550; Microm). Sections were thaw-mounted onto slides and placed in a –20°C freezer until viewed under fluorescent microscopy. Cases in which DiI had leaked into underlying muscle tissue were not included (six cases).

Drugs and solutions

Plated cells were superfused in rat Tyrode's solution containing (in mM) 140 NaCl, 4 KCl, 2 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with NaOH. Test solutions were applied by a gravity-fed pipette positioned about 1 mm from the cell (sewer pipe). The recording electrodes contained (in mM): 120 KCl, 5 Na₂-ATP, 0.4 Na₂-GTP, 2.25 CaCl₂, 5 MgCl₂, 20 HEPES, and 5 EGTA, adjusted to pH 7.4 with KOH (osmolarity was approximately 300 mOsm). For ratiometric imaging experiments, EGTA was absent from the internal solution. Calcium-free solutions were prepared as a Tyrode's solution with Mg²⁺ substituted for Ca²⁺. For each experiment: capsazepine was prepared fresh from a 40 mM stock solution (in 100% DMSO) to a final concentration of 10 µM; ruthenium red was prepared from 25 mM stock solution to the final concentration of 10 µM; flurbiprofen solution (500 µM) was prepared from a 500 mM stock. Solutions of varying pH values were prepared from Tyrode's solution by addition of HCl or NaOH. Solutions lower than pH 5.5 were buffered with MES. All of the above drugs were purchased from Sigma Chemical. Psalmotoxin venom was purchased from Spider Pharm and diluted to a final concentration of 100 ng/ml. Fura 2-pentapotassium (Molecular Probes) was diluted with the internal solution from a 5 mM stock to a final concentration of 150 µM.

Immunohistochemistry

After recordings were completed, the electrode was removed from the cell surface. The recorded cell was sometimes photographed, but usually could be readily identified by etching the plastic petri dish below the field location. The bath solution was replaced with 4% paraformaldehyde (PFA) in PBS for 20–30 min and then replaced with a similar solution containing 0.4% Triton X-100. Fixed cells were kept refrigerated before the immunolabeling. For labeling studies, the cells were rinsed with PBS to remove residual PFA. Targeted cells were circled with a hydrophobic resin (PAP pen; The Binding Site); incubated with a solution of 2% Triton X-100 in PBS for 2 h; 1:30 normal goat serum in PBS with Triton X-100 for an additional 2 h; and incubated overnight by primary antisera for ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 (1:1,000 rabbit anti-ASIC; Chemicon International). Labeled cells were rinsed the next day and incubated with secondary antisera for 3 h (1:500 biotinylated goat anti-rabbit IgG; Jackson Immunoresearch Laboratories). This was followed with rinses and incubation with the avidin–biotin HRP complex (Vectastain Elite ABC reagent; Vectorlabs), followed with rinses and incubations with the tyramide signal amplification (TSA) fluorophore conjugate (Perkin Elmer) for 3.5 min. The cells were viewed with a Zeiss Axiophot microscope equipped with appropriate fluorescence filters (Omega Optical). The controls for possible independent binding of all secondary antisera and amplification systems were consistently negative.

Statistics

The peak of the rapidly decaying inward current was scored as the difference between the maximum inward deflection and the steady-state current that persisted in the presence of an acidic solution. The latter were relatively small (see Fig. 1). This scoring method was used to limit the contribution of residual TRPV1 and K2p currents to the peak amplitude responses. Proton-gated currents arising from TRPV1 can exceed 200 pA, but they were effectively blocked by capsazepine (Cooper et al. 2004; Petruska et al. 2000a). The small K2p currents diminish rapidly with increasing pH and were unlikely to contribute much error (Patel and Honore 2001).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Kinetic forms of acid-sensing ion channel (ASIC)–mediated currents in 8 subclasses of dorsal root ganglia (DRG) neurons. Types 3 and 15 are nonnociceptive. Types 8a and 8b are subphenotypes distinguished by ASIC current kinetics.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
In previous reports (Petruska et al. 2000a, 2002), we observed that a nonnociceptive type 3 cell and several nociceptive types (5, 6, 7, 8, and 9) expressed ASIC-like proton-gated currents. In the present report, we undertook detailed studies of the proton sensitivity, calcium permeability, and molecular basis for fast desensitizing proton-gated currents in nociceptive and nonnociceptive DRG cells. Investigations focused on classes of medium-sized capsaicin-sensitive (types 5, 8, and 9) and capsaicin-insensitive neurons (type 6; n = 98), as well as two small-diameter neuron types (17–30 µm, n = 17; types 3 and 7) with ASIC-like currents. Nociceptive cell classes were defined by sensitivity to capsaicin (types 5, 6, 7, 8, and 9) or algesics (type 6). Because we previously noted that multiple isoforms of proton-gated currents could be observed in the type 8 subclass, we formally separated this group into two subphenotypes defined by ASIC current kinetics (types 8a and 8b). We also included limited studies on large-diameter (>50 µM) capsaicin-insensitive neurons presumed to be nonnociceptive (n = 5). We classified these cells, using the protocols described in METHODS, and assigned them to a type 15 cell class. As in our prior report, all cells were classified according to patterns of the voltage-activated currents that were evoked by three classification protocols (Petruska et al. 2000a, 2002). Subphenotyped classes (types 8a and 8b) were separated by large distinctions in proton-evoked current kinetics (type 8a, fast kinetics; type 8b, slow kinetics).

Amplitude and kinetics of ASIC-like currents in nociceptive and nonnociceptive DRG cells

When acidic solutions were applied to subclassified cells, a fast desensitizing current consistent with ASIC proteins was observed in all cases examined (n = 119; Fig. 1). Fast components were accompanied by much smaller nondesensitizing currents that might have a distinct molecular basis (Baumann et al. 2004; Cooper et al. 2004). Both nociceptive and nonnociceptive cell classes expressed ASIC-like currents. We examined whether properties of these currents were distinct in afferent populations with distinct functional roles. As previously reported, decay kinetics of proton-activated currents (pH 5.0) varied between cell classes (Petruska et al. 2000a, 2002). In the present studies, we used pH 6.0 as a kinetics probe because this pH was unlikely to activate any TRPV1 proteins that were likely to be present in capsaicin-sensitive subclasses. Capsazepine (10 µm) was also present to ensure isolation from TRPV1 activity.

Currents with slow-decay kinetics ( > 1,000 ms) could be observed in types 3 and 5 and in certain subphenotypes of type 8 (type 8b). Only fast kinetic forms were observed in types 6, 7, and 9 and in a subphenotype of the class 8 neuron (type 8a; < 450 ms; see Table 1). There was little evidence that ASICs in nonnociceptive classes exhibited distinct decay kinetics. At pH 6.0, the decay of currents evoked from the nonnociceptive type 3 cell was significantly less than that of nociceptive type 5 cells (P < 0.02) but significantly greater than that of currents of nociceptive types 6, 7, 8a, and 9 (P < 0.00001). Kinetics in type 15 did not differ significantly from that of nociceptive types 6 and 9, but did differ from that of all other nociceptive and nonnociceptive groups. Clearly, decay kinetics did not predict nociceptive function (see Table 1), when such function was based on capsaicin sensitivity (types 5, 7, 8, and 9) or peptide expression (types 5, 6, 7, 8, and 9). The amplitude of peak currents (normalized for cell capacitance) also failed to distinguish nociceptive from nonnociceptive classes. Although the normalized peak current of type 3 cells was numerically largest in all types of cells we examined, it was significantly greater only than that of types 5 and 7 (P < 0.04). Current amplitudes of the large-diameter type 15 cell did not differ from capsaicin-sensitive or any other class of cell.

Potentially, the pH sensitivity of ASICs in nociceptive neurons could differ from that of nonnociceptive cell classes, and in some respect reflect differences in modality. To determine pH sensitivity, an ascending series of acidic solutions was presented to subclassified cells. Intervals of 2 min separated each test. Concentration–response curves (CRCs) were subsequently formed from normalized peak amplitudes (see METHODS). Regardless of modality, all subclassified cells had the same proton-gating threshold (pH 6.8); however, significant differences between nociceptors and nonnociceptive cells were apparent in several other respects (Fig. 2). Large-diameter, capsaicin-insensitive type 15 cells had the greatest sensitivity to acidic solutions (pH₅₀ = 6.75 ± 0.03; P < 0.001 vs. all cell classes). Nonnociceptive type 3 neurons exhibited similar proton potency to capsaicin-sensitive type 5. Both of these cell classes manifested significantly greater sensitivity to protons than that of other nociceptive cell classes (Fig. 2, Table 2; P < 0.02). Type 7 cells proved to be the least sensitive, whereas other subclassified nociceptive neurons had intermediate pH₅₀ values that were nearly identical (types 6, 8a, 8b, and 9).

View larger version (25K): [in this window] [in a new window]   FIG. 2. Concentration–response curves of 8 subclassified DRG neurons. Potency and reactive range of nonnociceptive classes (types 3 and 15) were shifted relative to nociceptive groups. Thresholds were similar in all cases.

To further clarify the role played by distinct nociceptive classes in the transduction of tissue pH, we complemented the above assessments with an examination of the proton reactivity of a capsaicin-sensitive nociceptive class that did not express ASIC-like currents, but did express capsaicin-sensitive TRPV1 proteins that are known to be gated by protons in expression systems (Caterina et al. 1997, 2000; Rau et al. 2003). Using methods identical to those above (no capsazepine present), we examined the concentration dependency of the type 2 cell for acidic solutions ranging form pH 7.0 to pH 3.5 (Fig. 3). As pH decreased, nondesensitizing currents could be observed as pH exceeded 6.0. Evoked currents continued to increase, reaching a stable peak at pH 4.0. Fitting of the normalized currents indicated a pH₅₀ of 4.98 (Hill slope of 2.03) that was well beyond the sensitivity and range of either nociceptive or nonnociceptive neurons that expressed ASIC-like currents.

View larger version (11K): [in this window] [in a new window]   FIG. 3. Concentration–response curves for the non-ASIC–expressing type 2 nociceptor. A: threshold and potency were substantially shifted relative to ASIC-expressing classes. B: capsazepine significantly blocks pH 5.0–evoked current in type 2 cells. Each bar represents consecutive exposures to pH 5.0 in the presence and absence of capsazepine.

Decay kinetics of ASIC-mediated currents are known to vary according to heteromeric composition. When expressed in human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cells, ASIC heteromers containing the Ca²⁺-permeable ASIC1a subunits have relatively slow-decay kinetics (Benson et al. 2002; Hesselager et al. 2004). We observed slow-decay kinetics in capsaicin-sensitive nociceptive classes that expressed substance P and CGRP (types 5 and 8; Petruska et al. 2002). We suspected that these nociceptors might express Ca²⁺-permeable ASIC1a. Accordingly, using ratiometric methods, we examined Ca²⁺ permeability in cell classes with slow kinetics (types 5 and 8b), and contrasted these findings with a related nociceptive class with relatively fast proton decay kinetics (type 8a).

Cell types 5 and 8 were patched and classified in the usual manner using pipettes containing fura-2 (150 µM). Proton-evoked currents were recorded simultaneously with Ca²⁺ fluorescence. Close superfusion was used to rapidly lower extracellular pH from 7.4 to 6.0. A rapid rise in [Ca²⁺]_i in cell types 5, 8a, and 8b accompanied both slow- and fast-decaying kinetic isoforms (Fig. 4). There were no differences in peak Ca²⁺ entry among nociceptive classes [155.2 ± 62.8 nM (n = 6); 525.3 ± 181.6 nM (n = 5); and 256.1 ± 97.0 nM (n = 7), respectively]. Substantial differences in integrated Ca²⁺ entry was apparent in cell classes with slowly decaying currents.

View larger version (34K): [in this window] [in a new window]   FIG. 4. Calcium permeability of ASIC-expressing nociceptors. Simultaneous electrical and ratiometric recordings were made from 3 nociceptive classes. A: increased fluorescence after application of pH 6.0 to a type 8 cell. B: summary graphs show Ca2+-mediated fluorescence in 3 nociceptive cells and the block of this fluorescence in 0 mM external Ca2+. C and D: representative traces of simultaneous electrical and ratiometric recordings from types 5, 8a, and 8b in 2 mM (left) and 0 mM Ca2+ (right).

Nonsteroidal antiinflammatory drugs (NSAIDs) are effective analgesics and retard eicosanoid-dependent inflammation and pain. It is reported that certain NSAIDs can also inhibit the activity and the inflammation-induced expression of ASICs in COS cells (Voilley et al. 2001). Specifically, flurbiprofen and ibuprofen were shown to be effective against currents evoked from ASIC1a homomeric channels. Accordingly, we examined the influence of flurbiprofen on cell types 5 and 8. Consistent with the presence of ASIC1a, proton-activated currents were significantly reduced by flurbiprofen (500 µM, 2-min application) to 38.1 ± 6.6% (n = 6), 54.2 ± 1.9% (n = 5), and 71.9 ± 2.8% (n = 6) in types 5, 8b, and 8a, respectively (Fig. 5). Control experiments consisting of repeated applications of antagonist-free pH 6.0 solutions at the same 2-min intervals gave little indication of tachyphylaxis using this procedure (94.3 ± 3.3, 88.0 ± 2.4, and 98.5 ± 2.8% residual current in types 5, 8b, and 8a, respectively; Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Pharmacology of subclassified cells is consistent with the presence of ASIC1a. A: flurbiprofen (500 µM) reversibly blocks pH 6.0–evoked currents in 3 representative cells expressing Ca2+-permeable ASICs. B: summary graph. No tachyphylaxis was observed with repeated exposure to pH 6.0 over the identical time course (see text). Two minutes separate each test. Numbers in parentheses represent total cases for each bar. *P < 0.05.

View larger version (16K): [in this window] [in a new window]   FIG. 6. ASIC1a-selective antagonist abrogates proton-evoked currents in subclassified nociceptive cells. A: representative traces illustrate block of pH 6.0–evoked current in types 5 and 8b by PcTX. B: summary graph. Currents are significantly reduced in types 5 and 8b only. No tachyphylaxis is observed with repeated exposure to pH 6.0 over the identical time course (see text). Two minutes separate each test. Numbers in parentheses represent total cases for each bar. *P < 0.05.

Physiological and pharmacological evidence indicated the presence of ASIC1a in types 5, 8a, and 8b. Using antibodies against ASIC1a protein, we labeled physiologically subclassified cells that exhibited ASIC-mediated currents. As expected, all three calcium-permeable, peptidergic nociceptive cell groups stained positively for ASIC1a (Fig. 7; Table 3). A variety of other ASIC proteins were also identified in these cell classes. Type 8a, a class manifesting fast kinetic proton-gated currents that were insensitive to psalmotoxin, exhibited ASIC3, ASIC1b, and ASIC1a immunoreactivity. Types 5 and 8b, exhibiting currents with slow kinetics, labeled positively for ASIC3, ASIC2a, ASIC1b, and ASIC2b in addition to ASIC1a. This staining pattern contrasted well with that observed in type 4 cells. The type 4 cell, a subclass that manifests only nondesensitizing proton-gated currents mediated by K2p channels (Cooper et al. 2004), was used as a control. Antibody concentrations, identical to those used for ASIC subunits in other classes, were used to stain type 4 cells for all five proteins. There was no indication of any ASIC protein immunoreactivity in type 4 cells (n = 32; Fig. 7).

View larger version (53K): [in this window] [in a new window]   FIG. 7. Differential distribution of ASIC protein immunoreactivity in nociceptive cell classes. Type 4 cells do not exhibit proton-evoked ASIC-like fast desensitizing currents (Cooper et al. 2004). Type 4 cells were negative for all ASIC antibodies examined ((T4; left column) when processed in the identical fashion as ASIC-expressing cell classes. Types 5, 8a, and 8b (T5, T8a, T8b) expressed multiple ASIC proteins. Arrows indicate the typed cell from each panel. Other positive and negative neurons can also be seen.

Although many subclassified nociceptors manifest ASIC-mediated currents, some do not (types 1, 2, and 4). It is likely that the specific role of ASIC proteins in nociceptor physiology is reflected by nociceptor innervation patterns in peripheral tissues. We previously reported that nociceptive type 2 (n = 14), type 4 (n = 7), type 6 (n = 3), type 8 (n = 2), and type 9 (n = 3) cells could be traced from hairy skin (Rau et al. 2005). Few ASIC-expressing nociceptors were identified in these limited tracing studies (types 8 and 9). To address this question more fully, we made multiple, 1-µl injections of Di-I into hairy and glabrous skin (27 rats; skin over gastrocnemius and hindfoot pad). After a period of 2 wk, cells were harvested from appropriate ganglia, processed as usual, and subclassified by standard recording methods. Capsaicin and proton sensitivity were consistent with previous reports (Petruska et al. 2000, 2002). Recording selectively from intensely fluorescent cells (n = 97; Fig. 8), we observed that the major, ASIC positive, subclassified nociceptive groups innervating hairy skin were the cell types 8 and 9 (n = 24 and 9). Twenty of 24 type 8 cells expressed ASIC-like currents, and 19 of 20 of these were the type 8a fast kinetic isoform. Twenty-two type 8a and seven of nine type 9 cells were capsaicin sensitive (1 µM; 65.0 ± 60.1 and 20.4 ± 3.6 pA/pF, respectively). Two other major nociceptive groups were also present in considerable numbers, including the type 2 cell (n = 25) and type 4 cell (n = 35; Fig. 8). All type 2 cells were capsaicin sensitive (132.2 ± 20.4 pA/pF), whereas all traced type 4 cells were capsaicin insensitive. Small numbers of capsaicin-sensitive type 1 cells (n = 4) were also identified in hairy skin tracings. Traced types 1, 2, and 4 did not express ASIC-like currents (see also Petruska et al. 2000; Rau et al. 2005a). These extensive tracing studies raised the number of identified nociceptors with hairy skin projections considerably (39, 42, 26, and 12 cases in types 2, 4, 8, and 9, respectively; Fig. 8).

View larger version (51K): [in this window] [in a new window]   FIG. 8. Subclassified DRG cell types traced from rat hairy and glabrous skin. Brightfield images (A–E), and corresponding fluorescent images (F–J), of recorded cells are shown. Examples of cell type 2 (A and F), 4 (B and G), type 8 (C and H), type 13 (D and I), and type 15 (E and J) are indicated by arrows. Type 13 and type 15 cell characteristics (K–O and P–T, respectively) are represented by current signatures for CP1 (K and P), CP2 (L and Q), and CP3 (M and R), evoked action potentials (N and S), and pH 5.0 response (O and T). U: summary graph illustrates differential distribution of subclassified cells into hairy and glabrous skin. Microscopy scale bar (J) indicates 50 mm for A–J. Horizontal scale bars indicate 100 ms (K, L, P, and Q), 4 ms (M and R), 10 ms (N and S), or 2,000 ms (O and T). Vertical scale bars indicate 500 pA (K and O), 10,000 pA (L and Q), 5,000 pA (M and R), 1,500 pA (P), or 3,500 pA (T).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Using a systematic method of classifying peripheral afferents, we contrasted the properties of ASIC channels expressed in six nociceptive and two nonnociceptive groups. We expanded on prior observations that kinetically diverse, amiloride-sensitive ASIC-like currents could be observed in distinct subclasses of DRG cells (Petruska et al. 2000a, 2002). Among the main findings: 1) activation thresholds of ASIC-like currents did not differ in nociceptive and nonnociceptive neuron subgroups; 2) proton potency of nonnociceptive classes was significantly greater than the potency of five nociceptive groups; 3) both nonnociceptive populations transduced proton concentration over a relatively narrow range compared with nociceptors; 4) peptidergic nociceptors expressed calcium-permeable ASICs with pharmacology and immunocytochemistry consistent with ASIC1a; 5) kinetics of proton-evoked currents varied with the pattern of ASIC protein expression; 6) only the type 8a nociceptor subclass could be traced from both glabrous and hairy skin; and 7) two novel cell classes were identified in glabrous skin that were not present in hairy skin.

High proton concentration is associated with nociceptor activation and pain (Belmonte et al. 1991; Issberner et al. 1996; Lindahl 1961; Steen and Reeh 1993; Steen et al. 1992, 1995a, 1996). Moderate shifts in acidity (0.8 pH units) are reported in the synovial fluid of knee joints of patients with rheumatoid arthritis (James et al. 1992; Simkin and Basset 1992; Stevens et al. 1991). After experimental ischemia, the pH of injured muscle drops 2.5 full pH units (Chambers et al. 1927; see also Caldwell et al. 1984). Shifts of 1–2 pH units can occur with brief experimental muscle ischemia (Jacobus et al. 1977; Poole-Wilson 1978; Victor et al. 1988). Clearly, ASIC expression could mediate ischemic pain that followed rheumatoid disease, injury, or exertion. It is curious that ASIC proteins are expressed in both nociceptive and nonnociceptive neurons (Garcia-Anoveros et al. 2001; Price et al. 2000). We have shown that both populations of afferents exhibit thresholds of pH 6.8; therefore both nociceptive and nonnociceptive cells containing ASIC protein would be vulnerable to activation with tissue shifts of only 0.6 pH units. Despite the possible presence of ASICs, Steen and colleagues were unable to activate nonnociceptive afferents with acidic solutions in vivo (pH 5.0–6.0; Steen et al. 1992). It is clear from our investigations that the physiology and perceptual correlates of nonnociceptive ASICs cannot be distinguished from those of nociceptive cells on the basis of threshold. These were identical in all DRG afferent groups. In fact, ASIC channels in nonnociceptive cells were actually more sensitive to suprathreshold tissue acidity than ASICs in nociceptors. Multiple indexes of acid sensitivity on nonnociceptive groups (potency, Hill coefficient, reactive range) exceeded that of nociceptive cells, both in an extensively characterized small-diameter nonnociceptive population (type 3) as well as in a new large-diameter cell type (>50 mm; type 15) that innervated glabrous tissues (see following text). The latter are likely to represent one of a family of low-threshold mechanoreceptors known to express ASIC2a (Garcia-Anaveros et al. 2001). Perhaps the inhibitory influence of protons on TTXs Na⁺ channels could play a role in suppressing discharge in nonnociceptive populations, although it cannot explain their presence in these afferents (Daumas and Andersen 1993; Mozhayeva et al. 1984).

It could be argued that the distribution of nonnociceptors and nociceptors along a gradient of acid sensitivity mimics their positions along gradients of mechanosensitivity. ASIC proteins might serve multiple sensory functions in sensory afferents. Many believe this includes mechanosensitivity. ASIC deletions were shown to disrupt, in a complex fashion, certain mechanosensory activity in skin, muscle, and viscera (Page et al. 2005; Price et al. 2000; Sluka et al. 2003); moreover, mechanogated currents of DRG neurons were reduced by the ASIC antagonist amiloride (Gschossmann et al. 2000; McCarter et al. 1999). It is especially interesting to us that this role may be distinct in cutaneous and deep tissues and specific to the particular protein disruption (Page et al. 2005; Sluka et al. 2003). It is important to note that ASICs do not confer mechanosensitivity to host cells, nor have all laboratories been able to demonstrate mechanosensory deficits with disruption of ASIC gene expression (Drew et al. 2004; Roza et al. 2004). Nevertheless, the proton binding by ASICs could represent allosteric modulation of channel whose main function is mechanotransduction. A similar relationship occurs between capsaicin or heat gating of TRP channels and extracellular acidity (Cortright and Szallasi 2004). The concept is attractive because it seems to provide an explanation for the presence of functional ASIC channels in nonnociceptive cells. Nevertheless, the role of ASICs in mechanosensitivity remains controversial and extensive studies will be necessary to finally resolve this important issue.

We previously reported that all nociceptive cell types with amiloride-sensitive ASIC currents expressed neuropeptides SP and/or CGRP (Petruska et al. 2000a,b, 2002). If their proton-gated channels were Ca²⁺ permeable, the consequence of their activation could include release of paracrine messengers with key roles in inflammation (Lam and Ferrell 1991; Lembeck and Holzer 1979). Detailed investigations, using ratiometric methods with simultaneous recording of proton-dependent currents, clearly demonstrated that all three nociceptive populations tested (types 5, 8a, and 8b) expressed Ca²⁺-permeable ASICs. Calcium permeability predicted the presence of ASIC1a because this is the only subunit of the ASIC family with the recognized capacity to confer Ca²⁺ permeability to channels assembled from ASIC proteins (Chen et al. 1998). Using immunocytochemical approaches, we were able to identify ASIC1a protein in all three Ca²⁺-permeable nociceptive classes. Functional ASIC1a was confirmed by inhibition of pH 6.0–evoked current by the ASIC1a-specific antagonist PcTX, as well as by inhibition by the ASIC1a antagonist flurbiprofen (Escoubas et al. 2000; Voilley et al. 2001). It was noteworthy that PcTX was ineffective in the one subclass with fast-decaying acid-evoked current (type 8a). This is not unexpected because fast-decaying ASIC currents, and specifically those incorporating ASIC3 and ASIC2a, have been shown to be resistant to the tarantula toxin (Escoubas et al. 2000). Our immunocytochemical labeling studies identified not only ASIC1a, but also ASIC1b and ASIC3 protein in type 8a. The presence of ASIC3 was consistent with a lack of PcTX sensitivity, as reported for heteromeric channels expressed in Xenopus. However, we found that ASIC3 was also present in types 8b and 5, which were significantly inhibited by PcTX. These cell classes also expressed ASIC2a protein. Heteromers of ASIC1a and ASIC2a have been shown to be insensitive to PcTX. The influence of PcTX on channels formed from multiple ASIC proteins has not been thoroughly investigated. When such channels are expressed in heterologous systems, the mechanism of its blocking action might be dependent on the stoichiometry of susceptible proteins. As yet, few of the many possible heteromeric combinations have been examined.

Native assemblies of ASIC proteins in DRG nociceptors differ in decay kinetics, but are generally similar in proton sensitivity (Petruska et al. 2000a, 2002). We observed that thresholds of pH 6.8 were universal, and potency hovered in a narrow range between pH 6.5 and pH 6.2. Immunocytochemical evidence suggested a molecular basis for these observed differences. When assembled in host cells, homomeric and heteromeric channels formed from ASIC proteins differ widely in their sensitivity to protons. Channels formed from ASIC3 were the most proton sensitive (pH₅₀ = 6.4), whereas functional channels formed from ASIC1a, ASIC1b, ASIC3, and ASIC2a were relatively resistant to proton gating (pH₅₀ = 6.3, 5.9, 4.9, 4.1, respectively). Homomeric ASIC2b or ASIC4 could not activated by pH 4.0 (Champagny et al. 1998; Chen et al. 1998; Lingueglia et al. 1997; Waldmann et al. 1997a,b; see also Benson et al. 2002; Hesselager et al. 2004; Neaga et al. 2005). When multiple ASIC proteins are expressed in host cells (CHO; Hesselager et al. 2004) the sensitivity to protons can shift substantially. Many combinations share sensitivity similar to those of DRG nociceptors: ASIC1a + ASIC1b (pH 6.0), ASIC1a + ASIC2b (pH 6.2), ASIC1a + ASIC3 (pH 6.3), ASIC1b + ASIC3 (pH 6.0), ASIC2b + ASIC3 (pH 6.5), and ASIC1a + 2b + 3 (pH 6.3). Many other combinations are reportedly relatively insensitive to pH.

It is noteworthy that substantial differences in sensitivity and other features are reported when ASIC proteins are coexpressed in CHO, COS7, or Xenopus (Babinski et al. 2000; Benson et al. 2002; Hesselager et al. 2004; Lingueglia et al. 1997). Decay kinetics of proton-activated currents varied substantially between subclassified sensory afferents (Petruska et al. 2000a, 2002). These variations could arise from distinct contributions of ASIC proteins. It is known that heteromeric combinations of ASIC1 or ASIC3 with ASIC2 can substantially alter current decay of proton-evoked currents in host cells (Bassilana et al. 1997; Chen et al. 1998; Coscoy et al. 1999; Lingueglia et al. 1997). We confirmed, by extensive immunocytochemical evidence, that nociceptive neurons with distinct proton-decay kinetics expressed distinct patterns of ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 protein. The presence of ASIC2a and ASIC2b in nociceptors with slow kinetic isoforms (types 5 and 8b) was consistent with the contribution of these protein subunits in functional channels examined in expression systems (Benson et al. 2002; Hesselager et al. 2004). The functional significance of kinetic diversity is unclear. Differential decay kinetics could reflect specific nociceptor adaptations devoted to dynamic proton sensing. That is, ASICs proteins might be important for the detection of pH change but make little contribution to encoding the absolute level of pH in the vicinity of an ending. Nociceptive populations innervating muscle, vessels, and viscera might have pH-sensing requirements that are distinct from those innervating superficial tissues.

Identifying the peripheral distribution of nociceptors with ASIC-mediated currents is key to understanding their functional significance in acute, inflammatory, and chronic pain conditions. We made extensive studies of this distribution in hairy and glabrous skin. In these regions, the main ASIC-expressing classes appeared to be type 8a (hairy and glabrous) and a novel type 13 cell (glabrous). We chose to examine cutaneous sites because they are used in many behavioral models of inflammatory and chronic pain conditions (Bennett and Xie 1988; Kim and Chung 1992; Seltzer et al. 1990). Tracings from hairy skin loci revealed the presence of substantial numbers of the ASIC-expressing type 8a and type 9 nociceptors but very minor or no representation of types 5, 6, 7, or 8b. Some subclassified cells were identified in such small numbers (types 1, 5, and 6) that we are unable to determine whether their identification indicates low-density representation or spread of dye label to subadjacent structures where they may be plentiful. Although we assessed dye spread in histological sections of peripheral tissue and eliminated cases contaminated by excessive leakage, we cannot rule out minor spread of dye into subcutaneous regions.

Populations of non-ASIC–expressing nociceptors were readily identified in large numbers in hairy skin. The latter confirms and extends limited observations in a previous report (Rau et al. 2005a). It is significant that nociceptive types 2 and 4, although plentiful in hairy skin, were absent in glabrous skin. We also identified a novel, ASIC-expressing, capsaicin-sensitive cell class with exclusive representation in glabrous skin. We label this population type 13 (Rau et al. 2005b). The physiological and immunohistochemical properties of this cell class, and other glabrous skin subclasses, will be fully presented in a separate report that contains a number of additional novel cell populations innervating glabrous skin.

Since the earliest electrophysiological studies of pain, investigators have been cognizant of diversity within the nociceptive population (Adrian 1931; Burgess and Perl 1967; Iggo 1960; Zotterman 1939). Springing from these reports—and reinforced in many subsequent studies—was the notion of a generic nociceptive class with widespread representation and a key role in inflammatory pain. This population was usually referred to as C polymodal nociceptors and associated with superficial tissues (C-PMN; Bessou and Perl 1969). Our extensive characterization of proton, capsaicin, cholinergic, and ATP-gated currents indicates a great diversity of nociceptors that differ substantially with respect to representation of heat- and ligand-gated currents (Cooper et al. 2004; Petruska et al. 2000a,b, 2002; Rau et al. 2005a,b). Further, DiI tracing of nociceptive processes strongly suggests that the diversity of nociceptor subpopulations extends to regional innervation patterns. Clearly, tracing studies indicate that the major nociceptor populations of hairy skin are largely distinct from those of glabrous skin. There is limited evidence of a generic C-PMN. If such a population exists, the leading candidate is the type 8a nociceptor. Otherwise, it is more nearly correct to recognize and be guided by the diversity of nociceptive populations. Rather than the presence of a generic C-PMN, our evidence strongly suggests that cutaneous tissues are selectively innervated by distinct nociceptor populations that are highly adapted to encode pain specific to local exigencies. Moreover, we could interpret the wide representation of the type 8a nociceptor as another highly tissue specific population innervating a widely dispersed tissue type (e.g., blood vessels). Peptidergic nociceptors, such as type 8a, are known to be associated with dermis, epidermis (Perry and Lawson 1998; Wallengren 1997), and deep cutaneous regions (Dux et al. 1999; Karanth et al. 1991; Lawson et al. 1997; Navarro et al. 1995; Petruska et al. 2002). Among the targets of deep cutaneous peptidergic nociceptors are blood vessels. Blood vessels are richly invested with axon and terminals expressing vasoactive peptides SP and CGRP (Dalsgaard et al. 1989; Wallengren et al. 1987). We have identified SP and CGRP in type 8 cells (Petruska et al. 2002). The presence of Ca²⁺-permeable ASIC channels, in a vascular nociceptor subpopulation, would be consistent with demonstrations of peptidergic release after injection of acidic solutions into vessels and associated with vascular pain in humans (Brazeau et al. 1998; Klement and Arndt 1991). Regardless of considerations of the microdistribution of ASIC-expressing cells, the discrete pattern of nociceptor innervation of glabrous and hairy skin was a striking outcome. Nociceptive populations with distinct properties were dedicated to particular skin regions. Accordingly, investigators using neuropathic or inflammation-based models to study nociceptor plasticity, in vitro, should be cautious when making recordings from randomly selected cells. Inferences taken from such experimental designs may not have general application.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The work was supported by National Institute of Neurological Disorders Grant NS-39874.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We gratefully acknowledge the technical assistance of D. Nene and R. Nene.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: B. Cooper, Department of Oral Surgery and Diagnostic Sciences, Division of Neuroscience, Box 100416, JHMHC, University of Florida College of Dentistry, Gainesville, FL 32610 (E-mail: bcooper{at}dental.ufl.edu)

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