Capsaicin, acid and heat-evoked currents in rat trigeminal ganglion neurons

Physiology & Behavior 69 (2000) 363–378

Capsaicin, acid and heat-evoked currents in rat trigeminal ganglion neurons: relationship to functional VR1 receptors L. Liu, S.A. Simon* Department of Anesthesiology, Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA Received 20 December 1999; accepted 31 December 1999

Abstract Activation of primary trigeminal (TG) neurons by protons, capsaicin, or heat can evoke a variety of sensations, including tingling, stinging, warmth, and burning. Capsaicin and acid are trigeminal stimulants that are important in gustatory physiology. These stimuli can activate H⫹-gated ion channels and heterologously expressed VR1 receptors (vanilloid receptor 1). We have obtained evidence by using electrophysiological and pharmacological measurements on TG neurons that these three stimuli can activate many receptors, and we have determined the extent they behave similarly to VR1 receptors and H⫹-gated channels from the DEGenerin/ENaC superfamily. Whole-cell recordings from rat TG neurons revealed that protons evoked transient (Tp), sustained (Sp), and biphasic (TSp) currents. Tp currents had reversal potentials (Vr) of 24–45 mV, a pH0.5 range from 5.5 to 6.5, and were inhibited by amiloride, suggesting the presence of functional H⫹-gated channels. Sp currents were inhibited by the VR1 antagonist capsazepine, had Vr’s ⵑ0 mV, and had pH0.5 ⫽ 6.4. Capsaicin also activated transient (Tc), sustained (Sc), and biphasic (TSc) currents. At pH 5.9, the sensitivity of the Sc currents increased by about a factor of 10, which may partially account for the synergistic responses of acid in foods containing capsaicin. Heating TG neurons evoked a thermally active, capsazepine-inhibitable current with threshold temperature of 43⬚C and Vr ⫽ 5 mV that is also present in neurons activated by and protons (Sp) and capsaicin (Sc). These data suggest that TG neurons have functional receptors that behave similarly to VR1. Activation of such receptors should result in a burning sensation, whereas activation of the transient and biphasic currents should result in other taste descriptors. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Pain; Taste; Vanilloid; Trigeminal; Acid

1. Introduction Mammals respond to mechanical, thermal nociceptive stimuli by moving away from them and to nociceptive chemical stimuli by diluting (salivating, tearing), or expelling them (coughing, sneezing). When irritating chemical stimuli are placed on the anterior tongue, they may activate two parallel but interacting pathways: the gustatory pathway, consisting of taste receptor cells in fungiform papillae and their associated chorda tympani fibers; and the trigeminal pathway, consisting of lingual epithelium and its associated nerve terminals that originate from the trigeminal ganglion (TG) [30]. Both systems are responsive to all three types of stimuli [60,61], although in regard to chemical stimuli, they evoke distinctly different sensations. For example, when acid interacts with the gustatory system, it produces a sour taste [63], whereas when it activates trigeminal neurons, it produces burning, tingling, or numbing sensations [14,58,64]. A burning sensation can also be produced * Corresponding author. Tel.: 919-684-4178; Fax: 919-684-4431. E-mail address: [email protected]

by high temperatures (ⵑ45⬚C) and by capsaicin, the primary pungent compound in hot pepper [7,29,55–57]. Both acid and capsaicin play important roles in sensory physiology: acid, because of its role in carbonated beverages, and capsaicin, as the principle pungent component of hot pepper. Because these three stimuli can all evoke burning sensations, it is important to determine whether they do so by activating the same receptors in the same neurons. Receptors for chemical, thermal and mechanical stimuli can be found on the terminals of several types of TG neurons, including polymodal nociceptors (PMNs), the most prevalent nociceptor that is activated by all three types of stimuli [78]. A major advance in elucidating the receptors for these stimuli involves the cloning and characterization of the vanilloid receptor 1 (VR1) that can be activated by capsaicin, acid, and heat and that has subunits in TG neurons [26,69]. VR1 subunits assemble to form an ion channel that is cation selective, has a reversal potential near 0 mV, and a rectifying current–voltage relationship [16,69]. VR1 receptors are characterized by their half-maximal concentrations for capsaicin (0.71 ␮M ⫽ ED50), acid (5.2 ⫽ pH0.5), and threshold temperature (Tth ⫽ 43⬚C) [16,69]. The responses

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to all three stimuli are inhibited to various extents by capsazepine (CPZ), a capsaicin analogue that inhibits responses to capsaicin in sensory neurons [9,41]. The reason that capsaicin is used clinically to relieve pain is because, upon repeated applications, VR1 receptors (and PMNs) desensitize [66]. VR1 receptors also becomes more sensitive to capsaicin when the pH is lowered [69]. Because VR1 receptors are extensively characterized, we can compare their parameters with those obtained from functional receptors in TG neurons. Our primary goal is to determine whether functional VR1-like receptors are present in TG neurons and then to relate their activation to the burning sensations evoked by these three stimuli. In this regard, there is physiological evidence that functional VR1-like receptors are present in PMNs from primary sensory neurons. Specifically, in PMNs having small soma diameters, it has been found that Tth ⫽ 44⬚C [7,45,49]. Moreover, subsets of cultured DRG neurons were found to be activated by capsaicin and temperature [31,32,49], and a different subset, by capsaicin and acid [35,36,47]. However, it is not clear whether the same receptor was activated. The reasons are that, unlike the case with VR1, there have been reports that some of the responses to acid and thermal stimuli are not inhibited by capsazepine [10,46,49,71] and also because there are many receptors for acid and heat [18,36,70,75]. To date, there have not been studies on TG neurons in which the responses to all three stimuli were tested. In this study, the responses of rat TG neurons to protons, capsaicin, and temperature are fully characterized and compared with those evoked in heterologously expressed VR1 receptors. We found that the sustained currents activated by capsaicin have marked similarities with the sustained currents activated by acid and heat. These currents can be evoked in the same neuron. Taken together, these data provide the best evidence for functional VR1–type receptors in rat TG neurons and that these receptors are part of the transduction machinery responsible for the burning taste sensations produced by these stimuli. 2. Materials and methods 2.1. Materials Salts were reagent grade. Unless otherwise specified, chemicals were obtained from Sigma Chemical Corp. (St. Louis, MO). Capsaicin and capsazepine (CPZ) were obtained from RBI (Natick, MA). 2.2. Methods 2.2.1. Reverse-transcription polymerase chain reaction Male and female adult rats (200–400 g) were anesthetized with 50 mg/kg sodium pentobarbital delivered i.p., whereupon their trigeminal ganglia (TG) were removed. Then they were killed with i.p injections of 150 mg/kg sodium pentobarbital. For each of these experiments, the TG

from a rat was excised, and the total RNA was isolated with the Trizol Reagent Kit at 100 mg TG/mL Trizol (Gibco BRL, Gaithersburg, MD). The same results were obtained using three different preparations. To eliminate residual DNA prior to testing, all RNA samples were treated with amplification grade DNase I (Gibco BRL). For all three preparations, the total RNA purification yielded an A260/ A280 ratio ⭓1.7. cDNA was synthesized using the 1st Strand cDNA Synthesis Kit (Boehringer Mannheim). The primers, synthesized by GIBCO BRL, for ASIC-␣, ASIC-␤, DRASIC, and VR1, respectively, were as follows: (658 bp) 5⬘-ATTGCTCTTCCCATCTCTAT-3⬘ and 5⬘-TTCAAGGCCCATACCTAAGT-3⬘ (346 bp) 5⬘-CCTTATGGGCAGTGGCCTTT-3 and 5⬘-TAGAGCAGCATGTCCTCCAG-3⬘ (502 bp) 5⬘-GCTATGCTGCGAAAGGACAC-3⬘ and 5⬘-TACGGTGGGAGGCAGAGAGT-3⬘ (342 bp) 5⬘-TGGAGTTCACTGAGAACTACG-3⬘ and 5⬘-CGTTGATGATACCCACATTGG-3⬘. Polymerase chain reaction (PCR) amplification was performed with a PCR Core Kit (Boehringer Mannheim). The reverse transcription (RT) and PCR-cycling profiles were performed with the Thermal Cycler 3200 (Perkin Elmer, Foster City, CA). The profile was one cycle at 94⬚C for 5 min, followed by 34 cycles of (94⬚C for 1 min (denaturation), 57⬚C for 1 min for annealing, 72⬚C for 1 min (extension and synthesis), and a final extension cycle for 7 min at 72⬚C. Care was taken to ensure that the number of cycles did not lead to a maximum in the amplification of the receptor subtypes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to verify the quality, and to determine the relative quantity of RNA. Each amplification reaction (20 ␮L) was separated by electrophoresis on a 2.4% agarose gel. The gels contained 0.6 mg/ mL ethidium bromide and were visualized with Foto/Analyst Image Analysis System (Fotodyny, Hartland, WI), and their optical density was measured using Image I (Universal Imaging Corporation, West Chester, PA.). Two negative controls were used in this experiment. One negative control was performed with all reactions except primers in every electrophoresis experiment, and the other negative control was performed before cDNA synthesis for every RNA pool to eliminate the possibility of DNA contamination. 2.2.2. Cell culture Rat trigeminal ganglion cells were excised from Sprague– Dawley rats (150–250g) anesthetized with sodium pentobarbital (50 mg/kg) [43]. The cells were washed several times in a cold (4⬚C) modified Hanks balanced salt solution (HBSS) adjusted to pH 7.4: NaCl (130 mM), KCl (5 mM), KH2PO4 (0.3 mM), NaHCO3 (4.0 mM), Na2HPO4 (0.3 mM), D-glucose (5.6 mM), and HEPES (10 mM). The cells then were incubated for 40 min at 37⬚C in HBSS containing 1 mg/mL collagenase (Type XI-S). They were then triturated with a flamed Pasteur pipette, incubated at 37⬚C for 8 min in the presence of 1 mg/mL DNAase I (Type IV), retrit-

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urated, and washed and centrifuged three times in F-14 medium (Gibco-BRL). They were then resuspended and placed in a Petri dish with F-14 containing 10% fetal calf serum and maintained in an incubator at 37⬚C for 12 to 24 h. At the beginning of each patch-clamp experiment, TG neurons were placed in a chamber containing Krebs–Henseleit (KH) buffer on an inverted microscope. The KH composition was NaCl (145 mM), KCl (5 mM), CaCl2 (2.0 mM), MgCl2 (1.0 mM), HEPES (10 mM), and D-glucose (10 mM) at pH 7.4. Experiments were performed at room temperature. 2.2.3. Whole-cell recordings Patch-clamp recordings were performed with an Axoclamp 1D patch-clamp amplifier (Axon Instruments, Foster City, CA). The output was digitized with a Digidata 1200 A/D converter (Axon) and was sampled every 5 ms. The electrode resistances were 2–5 M⍀. Series resistance was compensated at least 80%. In control experiments, the intracellular solution contained K or Cs aspartate (140 mM), CaCl2 (1.0 mM), MgCl2 (2.0 mM), BAPTA (1,2-bis(2-aminophenoyx)ethane-N⬘,N⬘,N⬘,N⬘-tetracetic acid) (10 mM), HEPES (10 mM), and K2-ATP (5 mM) adjusted to pH 7.3. The chamber containing the neurons was continuously perfused by solution flowing into the chamber. Extracellular solutions with different pH were buffered with HEPES (pKa ⫽ 7.4), MES (pKa ⫽ 5.9), and HOMOPIPES (pKa ⫽ 4.9). Liquid–junction potentials were measured to be less than 5 mV. These solutions were delivered to the cell with a multibarreled electrode (Adams and List Associated, Westbury, NY) placed 20 to 50 ␮m from a neuron. Markers associated with the opening or closing of the valves identified the onset and removal times of the stimuli. Unless otherwise specified, stimuli were applied for 30 s every 3 min. Acid (pH) dose–response curves were obtained in 0.5 pH unit steps with solutions buffered from pH 7.4 to 4.9. In a few early experiments in which capsaicin dose–response curves were obtained, capsaicin was introduced into the chamber as opposed to using a nearby pipette (see Fig. 9A). Under these conditions, the activation and deactivation kinetics are slower. To determine the inhibitory effects of CPZ, after application of a test stimulus, a cell was washed with KH buffer (pH 7.4) for a time sufficiently long for the current to return to baseline. It was then incubated with 10 ␮M CPZ in KH buffer (pH 7.4 or 5.9) for 2 to 3 min, whereupon the lower pH buffer containing 10 ␮M CPZ was applied for 30 s. To test for reversibility, after a 3-min wash, the acidic test buffer was reapplied. To determine whether the currents activated by protons and capsaicin in TG neurons have different reversal potentials,we used a method that simultaneously obtains current– voltage (I–V) curves and current–time plots [42]. To obtain I–V curves, the voltage was ramped ⫾ 80 mV every 100 ms before, during, and after application of either low pH buffers or capsaicin. The I–V plots presented usually corresponded to times at which the currents reached their peaks (e.g., Figs. 2 and 3). However, when multiple currents are activated, the I–V curves may contain contributions from

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both components. Under these conditions, the reversal potentials that are reported were obtained when one of the components clearly dominated (e.g., see arrows in Fig 3A). To diminish the activation of voltage-dependent sodium and L-type calcium channels, the extracellular solution included 3 ␮M TTX, 0.1 mM CdCl2 and 50 ␮M verapamil, respectively. After about 100 cycles, the voltage-dependent conductances were essentially eliminated, and the background current (BG) became relatively small (see Fig. 2A,B). To reduce the contributions of potassium channels to the currents, in the intracellular solution, Kasparatate was replaced by equimolar CsCl and CsF or, in some experiments, with Cs aspartate. For studying desensitization (or tachyphylaxis) produced by repeated applications of pH 5.9 buffer, or 1 ␮M capsaicin in pH 7.4 buffer or 1 ␮M capsaicin in pH 5.9 buffer, the stimuli were applied seven times for 30 s with either 30-s or 3-min interstimulus intervals. 2.2.4. Thermal studies For experiments in which thermal responsiveness was investigated, the temperature of the buffered extracellular solution was controlled with an in-line SH-27A solution heater and a TC-324B automatic temperature controller (Warner Instruments, Hamden, CT). The temperature of the perfusing solution was obtained by placing the recording thermistor immediately downstream of the neuron. The intracellular solutions contained Cs aspartate buffer, and the extracellular solution contained KH buffer (pH 7.4). To obtain a measure of the thermal sensitivity, Q10’s were calculated using the formula Q10 ⫽ exp (10 Ea/RT1T2), where R is the gas constant, T is the absolute temperature, and Ea, the activation energy which can be expressed ⫺Ea ⫽ 2.3 R log (I2/I1)/((1/T2 ⫺ 1/T1)), where I2 is the current at T2 and I1 is the current at T1. To calculate Ea, the currents were usually normalized to their maximal value. Current–voltage curves that were obtained during the heating and cooling cycles were generated as above. Experiments with capsazepine were performed by preincubating the cells for about 1 min at room temperature before heating the 10 ␮M CPZ–containing solution.

3. Results 3.1. Reverse-transcription polymerase chain reaction Figure 1A shows RT-PCR products obtained from TG ganglia. The products reveal the presence of subunits of the H⫹-gated channels ASIC-␣, ASIC-␤, and DRASIC, and VR1. Also seen are labels for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH (G)), a control without transcriptase (C), and molecular weight markers (marker). Figure 1B shows a histogram of the expression levels of VR1, ASIC-␣, ASIC-␤, and DRASIC subunits relative to GAPDH. The expression levels for

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L. Liu, S.A. Simon / Physiology & Behavior 69 (2000) 363–378 Table 1 Activation of trigeminal ganglion cells by pH 5.9 as a function of projected soma diameter and current density Projected Soma Diameter (␮m) Variable

⭐29

30–39 ⭓40 Total or mean

Number of cells tested 67 51 8 126 Number of cells responding 42 36 5 83 % Responding 62.7 70.6 62.5 65.9 Mean membrane capacitance (C), pF 21.2 46.3 93.0 30.7 Mean peak current (Ip), nA 5.1 5.5 3.1 5.1 Current density (Ip/C), pA/pF 224 126 32 182

Fig. 1. RT-PCR in rat trigeminal ganglion. (A) A gel showing bands for the H⫹-activated channel subunits ASIC-␣, ASIC-␤, and DRASIC; the vanilloid receptor, VR1; the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH-G); and molecular weight markers (marker). (B) Histogram of the expression levels of VR1, ASIC-␣, ASIC-␤, and DRASIC relative to GAPDH. Data represent the mean ⫾ SD of four experiments.

activated currents (Fig. 2A–2D and 3A); including two of which have not been previously reported (Figs 2D and 3A). The current–time patterns seen in the upper traces of Fig. 2 were obtained by applying a voltage ramp before, during, and after a 30-s application of acidic stimuli and by plotting the data at all times corresponding to ⫺60 mV (Figs. 2,3,5). The bottom four traces are their corresponding I–V (Fig. 2A,B) or ⌬I (I ⫺ IBG (background current)) –V traces (Fig. 2C,D) that were obtained at the times indicated by the arrows in the upper traces. Two representative traces of the background currents (labeled BG) are shown in Fig. 2A and B. It is evident that IBG is small relative to the evoked currents. An example of a transiently activated proton–gated current (Tp) is seen in Fig. 2B. Tp-type responses were found in 11% (9/83) of the responsive neurons. They have a peak current density of 166 ⫾ 103 pA/pF (mean ⫾ SD, n ⫽ 9), times to peak of 0.53 ⫾ 0.23 s, times to desensitization 50% of 0.49 ⫾

VR1, ASIC-␤, and DRASIC are essentially the same and are higher than for ASIC-␣. 3.2. Current–voltage relationships and currents that are activated by protons (H⫹) The responsiveness of TG neurons to acidic stimuli was tested by applying 30 s applications of pH 5.9–buffered solutions. This pH was chosen because the perception of pain occurs at pH ⬍ 6.2 [38] and because under most physiological or pathological conditions, the pH does not decrease much below this value [38]. Of the 126 neurons tested at pH 5.9, 83 (65.9%) evoked inward currents greater than 10 pA (Table 1). Neurons were considered to be unresponsive when the evoked currents were ⬍10 pA. We found that neurons with the smallest soma diameters had the highest current density and that the probability of a neuron being activated by pH 5.9 is independent of the soma diameter (range, 18–42 ␮m) (Table 1). In previous studies using cultured DRG and TG neurons, protons have been found to evoke three patterns of currents (transient, sustained, and transient and sustained) [5,11,21, 75,77,79]. In this (extended) investigation of TG neurons, we have observed five distinguishable patterns of proton-

Fig. 2. Proton-activated currents in rat trigeminal ganglion neurons. Representative responses evoked from four different neurons bathed in Krebs– Henseleit buffer (pH 7.4) with 3 ␮M TTX 0.1 mM CdCl2 and 50 ␮M verapamil. The upper traces were obtained from the generated family of current–voltage curves at ⫺60 mV taken before, during and after the application of pH 5.9 buffer. All the evoked currents were inward. The current– voltage curves in the lower traces were taken at the time indicated by the arrows in the upper trace. The responses included the following: (A) a transiently (Tp) activated current, (B) a sustained (Sp) current, (C) a biphasic current containing both transient and sustained components (TSp), and (D) a current consisting of two slowly activating components (DSp1 and DSp2). Note that the background current (BG), shown in the lower traces in Fig. 2A and B, whereas in Fig. 2C and D, it is subtracted from the evoked current and is denoted as ⌬I. Bar indicates duration of stimulus.

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Fig. 3. Current–time and voltage dependence of H⫹-gated currents in rat trigeminal ganglion neurons. The responses were evoked from different neurons originally bathed in Krebs–Henseleit buffer (pH 7.4) containing 3 ␮M TTX 0.1 MM CdCl2 and 50 ␮M verapamil. The upper traces were obtained from a family of current–voltage curves in 20 mV steps taken before, during and after the application of pH 5.9 buffer. The current–voltage curves on the right hand panels were taken at the time indicated by the arrows in the ⫺60 mV traces. (A) The responses of a TDSp-type current show that both the slow currents (“b” and “c”) have the same reversal potentials (⬵0 mV). Note also that the T component (“a”) is larger at ⫺60 mV and the S components (“b” and “c”) are larger at ⫹60 mV. The reversal potential of the T component was obtained at a time when the S-component contribution was small (unlabeled arrow in ⫺60 mV trace), that corresponds to the unlabeled arrow in ⌬I–V curve (32 mV). (B) The voltagedependence and current–voltage curves for a DSp-type current. ⌬I-V plots for components DSp1 and DSp2 are shown in the right hand panel. Note that the reversal potential for both components is near 0 mV. Bar indicates duration of stimulus.

0.21 s (n ⫽ 9), reversal potentials of 32 ⫾ 6 mV, and rectifying I–V curves (Fig. 2A). Tp-type currents were found in neurons with soma diameters ranging from 22 to ⭓40 ␮m. Application of pH 5.9 buffer also induced a sustained (Sptype) current that is a slowly activating and slowly desensitizing current (Fig. 2B). Sp-type currents were found in 30% (25/83) of the proton-responsive neurons. At the peak, the magnitude of the Sp-type current was 109 ⫾ 88 pA/pF (n ⫽ 25), and the times to peak ranged from 10 to ⬎30 s. Current– voltage curves of Sp-type currents had reversal potentials of 0.3 ⫾ 3 mV (n ⫽ 12) and outwardly rectifying current–voltage curves. Sp-type currents were found in neurons whose projected soma diameters ranged from 20 to 40 ␮m.

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A third type of proton-induced current, named TSp currents, appeared to be a combination of Tp- and Sp-type currents (Fig. 2C). They were found in 32% (27/83) of the proton-sensitive neurons. Although in the response shown in Fig. 2C the TSp1 component became completely desensitized before the TSp2 component activated, we present other examples where both components appear to be activated at the same time (see Figs. 4B, 5C). The ⌬I–V relationship of TSp currents (Fig. 2C) contained rapid (TSp1) and slow (TSp2) components having different reversal potentials. The TSp1 component had a reversal potential of 30 mV, and the TSp2 component had a reversal potential near 0 mV. A fourth type of proton-induced pattern was named the double slow, or DSp-type current; it consisted of two slowly activating currents (Fig. 2D). DSp-type currents were found in 17% (14/83) of the proton-sensitive neurons. The more rapidly activating component (DSp1) had times to peak that ranged between 4 and 10 s, and the more slowly activating component (DSp2) had peak times that ranged from 9 to ⬎30 s. The ⌬I–V plots for both components outwardly rectified, and both had reversal potentials near 0 mV (Figs. 2D and 3B). The reversal potentials for DSp1 and DSp2 were 1.0 ⫾ 4.2 mV and ⫺0.3 ⫾ 3. 1 mV (n ⫽ 8), respectively. The kinetics of the DSp-type currents do not markedly change with voltage (Fig. 3B). These data also indicate that the slower of the two components does not appear to be a different type of channel because it is seen at all voltages and has the same reversal potential as do DSp1-type currents. A fifth current pattern, named TDSp, consisted of a combination of Tp-and DS-type currents (Fig. 3A). TDSp cur-

Fig. 4. Current–time and current–voltage curves obtained at different pH’s. The current–time curves at ⫺60 mV (upper traces) were constructed from a family of I–V curves taken before, during, and after a 30-s application of buffers at pH 5.9. The cell was bathed in Krebs–Henseleit buffer (pH 7.4) plus 3 ␮M TTX 0.1 mM CdCl2 and 50 ␮M verapamil and was sequentially exposed to (A) pH 5.9 buffer, (B) pH 5.4 buffer 5.4, (C) pH 4.9 buffer, and (D) pH 5.9 buffer (for reversibility). The letters on the upper traces correspond to the times the ⌬I-V curves shown in the lower panels were obtained. Note that ⌬I ⫽ (I ⫺ IBG). The ⌬I–V curve taken at the time indicated by the unlabelled arrow in the pH 4.9 trace had a reversal potential of 43 mV (unlabelled arrow in lower panel). The “0” refers to the baseline current in nA. Conditions are the same as in Fig. 2. Bars indicate duration of stimulus.

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reversal potentials were 0.5 ⫾ 3.2 mV (range, ⫺4 mV to 6 mV) for the sustained components and 34 ⫾ 12 mV (mean ⫾ SD; range, 24–45 mV) for the transient components. In summary, we have identified several responses to acid stimuli. We have adapted the classification scheme (Tp, Sp, TSp1, etc.) merely to identify the proton-induced patterns. We acknowledge that not every Tp- or Sp-type of current may represent the same type of ion channel and that as the proton concentration increases, the categorization of the currents may also change as is seen in Fig. 4. 3.3. Does changing the pH change the reversal potential?

Fig. 5. Dose–response relationships for sustained and transient types of proton-gated currents. (A) Sp-type current. With the neuron bathed in KH (pH 7.4) buffer, the neuron was exposed to 30-s applications of buffers adjusted to pH’s 6.9, 6.4, 5.9, 5.4, and 4.9, respectively. Between applications, the neurons were washed 3 min with KH buffer (pH 7.4). The dotted lines indicate 0 nA. Note that after the application of pH 6.4 an outward current was evoked that nearly returned to baseline after wash. (B) Plot of the peak currents (Ip) normalized to the maximal current (Ipmax) versus the bulk pH for 6 experiments. The data were averaged and fit to the Hill equation (solid line—see Text). The fitting parameters were pH0.5 ⫽ 6. 4, and n ⫽ 1.54. (C) Continuous recordings obtained from a neuron held at ⫺60 mV and exposed to pH’s 6.9, 6.4, 5.9, 5.4, 4.9, and again to 5.4 (to show reversibility). Between applications the neuron was washed 3 min with KH buffer (pH 7.4). Note that at pH 4.9, the currents were smaller than at pH 5.4 (as measured from the baseline current just prior to the application), and the current would be classified as a TDSp-type current (see Fig. 2). We also note that after several applications, a leak current was generated. Bars indicate duration of the stimulus. (D) Plot of the normalized peak currents (Ip/Ipmax) versus pH for transient component of three currents having T-components. Two of the curves were “fit” to the Hill equation to obtain pH0.5, which were 5.88 and 5.57. The third curve (indicated by a dotted line to guide the reader) was used to obtain an apparent pH0.5 of 6.5 that is larger than the other two experiments.

rents were found in 10% (8/83) of the proton-responsive neurons. In this neuron, the peak times were 0.36, 6.2, and ⬎30 s (Fig. 3A). This experiment demonstrates that the more rapidly activating of the slowly activating peaks (labeled “b”) in the TDSp-type current (at 6.2 s) is not simply a less sensitive or desensitized Tp-type current but rather is an independent current with a different reversal potential. ⌬I–V relations for TDSp-type currents exhibit characteristics suggesting independent Tp and DSp pathways. That is, the slowly activating currents had outwardly rectifying ⌬I–V curves with reversal potentials near 0 mV, whereas the Tptype current had a reversal potential of about 38 mV (see unlabeled arrows in the ⫺60 mV traces and in the ⌬I–V curve). These data also show that the activation and desensitization kinetics of the TDSp-type currents are not markedly voltage-dependent (Fig. 3A). For the eight neurons that were identified as having TSp or TDSp-type currents, the

To test whether H⫹ is a major charge carrier and whether these currents are voltage or proton gated or both [22], current–voltage curves were obtained before, during (30-s application), and after application of increasing concentrations of acidic stimuli. We reasoned that if H⫹ is a major charge carrier, then as the proton concentration is increased 10fold, the reversal potential should move in a more positive direction. As before, we constructed the current–time plots (at ⫺60 mV, upper traces in Fig. 4A–4D) and ⌬I–V curves (lower traces) from the family of I–V curves. Fig. 5A shows that a Tp response was evoked at pH 5.9. At the time corresponding to the peak current (labeled a), the ⌬I–V had a reversal potential of 43 mV. After wash, the application of pH 5.4 buffer evoked a TSp-type current (Fig. 4B) whose corresponding ⌬I–V curves had reversal potentials of 35 mV and ⫺0.5 mV for the Tp and Sp components, respectively. At pH 4.9, the pattern resembled a TDSp-type response, although the magnitude of the Sp component was reduced compared with the slow currents at pH 5.4. At pH 4.9, the ⌬I–V curves rectified and had reversal potentials of 38 mV and 0.2 mV for the Tp- (arrow) and DSp-type currents, respectively. Reapplication of pH 5.9 buffer restored the Tp current to its initial value. We found that the reversal potentials of the Tp component and DSp components were independent of the extracellular pH, suggesting that under these conditions, protons are not major contributors to the current. Thus, the properties of these currents are inconsistent with them being voltagegated proton currents [22]. The current⫺time traces shown in Fig. 4 illustrate some of the problems with characterizing transient currents as just Tp types (as opposed to TSp or TDSp types). Fig. 5A provides an example of what appears to be a single Tp-type current that was activated at pH 5.9. However, at lower pH’s, TSp- and TDSp-type currents were activated. Consequently, the classification of currents into Tp type at one pH cannot be extended to all pH’s, even though the literature contains examples obtained from sensory neurons where this is the case [1,21]. 3.4. pH dose–response curves Lowering the pH, as occurs in the presence of CO2 or during inflammation, causes a burning sensation. That is,

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the more acidic the solution, the greater will be the neuronal response and the more pain that will be perceived [58]. For the purpose of comparing these responses to those obtained from in vivo recordings from TG neurons and from those obtained from VR1 receptors, we have measured the responses of TG neurons to increasing concentrations of protons. Fig. 5A shows an Sp-type current that was evoked from a neuron held at ⫺60 mV to which stimuli of monotonically decreasing pH were applied for 30 s every 3 min. In this neuron, the application of pH 6.9 buffer evoked a small, slowly activating inward current that did not reach its peak during the 30-s application time. Application of pH 6.4 buffer increased the magnitude of the inward current, as did decreasing the pH to 5.9. The maximum current was attained at pH 5.4 buffer because it decreased slightly at pH 4.9. The underlying reasons why the current decreased at pH 4.9 were not explored, although the blocking of the channels by protons, desensitization, or both are obvious possibilities. Because acidic stimuli can evoke kinetically complex patterns (e.g., Figs. 2,3,5), it is not surprising to find complexity in the dose–response curves. The concentration–response profile obtained from a neuron that exhibited TSp-type currents is seen in Fig. 6C. The application of pH 6.9 evoked a small Sp-type inward current, whereas pH 6.4 activated a large TSp type current, with the Tp component being much larger then the Sp component. At pH’s 5.9 and 5.4, both the Tp and Sp components markedly increased, but at pH 4.9, the amplitudes of both T and S components decreased. The currents partially increased upon returning to pH 5.4. Plots of the normalized peak current (Ip/Ipmax, or the current at 30 s) versus the bulk pH for Sp-type currents (n ⫽ 7) are shown in Fig. 6B. These data were fit to the Hill equation: Ip/Ipmax ⫽ ((1 ⫹ (pH /pH0•5)n)⫺1, where pH0•5 is the pH where the current reaches its half-maximal concentration (⫽ 6.4) and n, the Hill coefficient, ⫽ 1.6. For comparison, for VR1 (at room temperature) pH0•5 ⫽ 5.4 and n ⫽ 1. 6 [69]. We are aware that in these experiments, repeated applications may cause desensitization that may alter the parameters of the dose–response curve. However, we believe this disadvantage is outweighed by obtaining the entire dose–response profile in a single cell. For currents with transient components (we have not obtained a complete dose–response function only for Tp-type currents), a plot of the normalized peak current (Ip/Ipmax) versus the bulk pH for three complete experiments is shown in Fig. 5D. It is evident that there is a wide range in the apparent pH0.5 values (5.5 to 6.5), as well as a range in the steepness (cooperativity) of the response. In presenting these data, we have not corrected for tachyphylaxis or rundown (see Fig. 6). (The dotted line shown in Fig. 5D is only meant to guide the reader to illustrate the larger apparent pH0•5 value because we are aware that these data cannot, in actuality, be fit to the Hill equation). We have also found that the maximum amplitude of the peak currents evoked by acidic stimuli (at ⫺60 mV at pH 5.4) was not significantly larger

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Fig. 6. Tachyphylaxis experiments performed at pH 5.9. (A) Sp-type current. A current trace obtained from a neuron held at ⫺60 mV that was exposed to seven 30-s applications of pH 5.9 buffer interspersed by 2.5-h washes with pH 7.4 buffer. The magnitude of the currents remained relatively constant for the first five applications and then decreased for the last two applications. Note that after several repeated applications, a leak current developed. The dotted line indicates 0 nA. (B) TSp-type currents. A current trace obtained from a neuron held at ⫺60 mV that was exposed to seven 30-s applications of pH 5.9 buffer interspersed by 2.5-h washes with pH 7.4 buffer. The magnitude of the transient (T) component remained relatively constant for the first four applications and monotonically decreased for the last three applications. Note that the S component virtually disappeared by the seventh application. (C) Histogram showing the percentage of the peak current remaining relative to the first application (p remaining) for each of seven (S1–S7) experiments in which a slowly activating current was evoked. For each experiment the results of each of the seven applications of pH 5.9 are shown. The trace in 6A is represented by S1. Also shown are the results of three experiments in which a transient current was evoked (T1–T3). The trace in 6B is represented by T1. (D) A graph showing the mean ⫾ SD of the Ip remaining as a function of time for the S- and T-type currents.

(p ⬎ 0.11) for the Tp-type (⫺9.0 ⫾ 2.6 nA) than for the Sptype currents (⫺6.2 ⫾ 2.0 nA). 3.5. Desensitization (tachyphylaxis) of H⫹-gated currents Tachyphylaxis (sometimes called desensitization) is the diminution of a response to repeated applications of an agonist. In TG neurons held at ⫺60 mV, we measured the responses to 30-s applications of pH 5.9 buffer every 3 minutes. The responses of Sp-type currents to seven repeated applications of pH 5.9 are presented in Fig. 6 The first application produced a slowly activating, nondesensitizing inward current. Upon wash, a transient inward current was elicited that slowly returned to baseline (dotted lines). The second, third, and fourth applications evoked inward currents with about the same magnitude (the magnitude of the response was measured from the baseline current just prior to the application of the agonist to the peak current. In subsequent applications, the amplitude of the inward current

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3.6. Amiloride inhibits transient proton-gated currents

Fig. 7. Amiloride inhibits transient proton-gated currents. (A) The application of pH 4.9 evoked a transient current. This current was completely inhibited by 200 ␮M amiloride (middle trace) and, after a 3-min wash, was slightly reversible. (B) The application of pH 4.9 buffer evoked a sustained (Sp) current. In the presence of 200 ␮M amiloride at pH 4.9, the magnitude of the current increased. After wash with KH buffer, the reapplication of pH 4.9 buffer evoked a smaller current. Bars indicate duration of stimulus.

The effects of amiloride on the proton-gated currents were also investigated because ASIC-␣, ASIC-␤ and DRASIC subunits are all present in rat TG (Fig. 1), and because H⫹-gated channels containing MDEG/ENaC subunits are inhibited ⵑ80% by 200 ␮M amiloride [4,39,73–75]. We therefore tested which of the proton-gated currents would be inhibited by 200 ␮M amiloride. We found that in 70% (12/17) of the neurons tested, 200 ␮M amiloride in KH buffer (pH 7.4) did not evoke an inward current. We consequently used these neurons to directly test the effect of amiloride on the Tp-type (Fig. 7A) and Sp-type (Fig. 7B) currents. We found that 200 ␮M amiloride, reversibly inhibited the Tp-type current by 89.5 ⫾ 19.7 (n ⫽ 5). Amiloride produced a very different effect for the Sp-type currents. Fig. 7B shows an Sp-type current that was activated by pH 4.9 and whose magnitude at 30 s was 600 pA. In the presence of 200 ␮M amiloride, the application of pH 4.9 increased the inward current. After wash, the reapplication of pH 4.9 buffer produced a smaller inward current. On average, the S-type currents (which included two DS types) were increased by 54⫾ 69% (n ⫽ 9; range, 0 to 176%). 3.7. Effects of acid with capsaicin on TG neurons

monotonically decreased so that by the seventh application, the evoked current was 46% of the peak current of the initial application. The results of a tachyphylaxis experiment in which a TSp-type pattern was evoked is presented in Fig. 6B. In this experiment, the Sp component desensitized at a much faster rate than the Tp component. Indeed, by the seventh application, only a Tp-type component (of several nA) remained. In neuron T2, both the T and S components decreased in concert, such that at the seventh application they had decreased 82% and 75%, respectively, of their initial values (Fig. 6C). In neuron T3, the currents did not exhibit tachyphylaxis (Fig. 6C). Figure 6C shows histograms of the percentage of the peak current remaining (%Ip remaining) for the seven applications in each of the seven experiments (labeled S1 to S7) in which Sp-type patterns were elicited. The histograms for the three responses with transient currents (T1–T3) are also shown. Histogram S1 represents the trace in Fig. 6A, and histogram T1 represents the trace in Fig. 6B. These data show that the extent of tachyphylaxis was heterogeneous among neurons because the %Ip remaining after seven applications ranged from 60% for neuron S6 to 9% for neuron S7. The tachyphylaxis data, averaged for the Sp- and Tpcontaining currents, are shown in Fig. 6D. Here the %Ip remaining is plotted for each 3-min application and shows that on average, both Tp-and Sp-components decreased at about the same rate. For comparison, three applications of 10-mM acetic acid applied to TG polymodal nociceptors showed no change in the mean frequency of the response with a 5-min interstimulus interval [6].

The interaction of acid on responses to capsaicin has been shown to be synergistic. That is, the responses to capsaicin increase in the presence of acid [47,52]. Although

Fig. 8. Capsaicin-activated currents in rat trigeminal ganglion neurons. Representative responses evoked from four neurons originally bathed in Krebs– Henseleit buffer (pH 7.4) with 3 ␮M TTX, 0.1 mM CdCl2 and 50 ␮M verapamil. The upper traces are obtained from a family of current–voltage curves at ⫺60 mV taken before, during and after the application of 1 ␮M capsaicin. The current–voltage curves in the lower traces were taken at the time indicated by the arrows in the upper trace. The capsaicin-evoked responses included (A) a transiently activated (Tc) current, (B) a sustained (Sc) current, (C) a biphasic current containing both transient and sustained components (TSc), and (D) a current with two slowly activating components (DSc1 and DSc2). The unlabeled arrows in the upper traces in 8A and 8B represent the times an I–V curve was obtained that had reversal potentials corresponding to the unlabeled arrows in the lower traces. The lower traces show current– voltage plots ⌬I ⫽ (I ⫺ IBG) ⫺ V, where IBG is the background current. The dotted lines indicate 0 nA. Bar indicates duration of stimulus.

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acid has been shown to increase the dose–response relationship in VR1 receptors [69], this quantitative relationship has not been investigated in TG neurons. Here, we will first characterize the response of TG neurons to capsaicin and then show how the interaction of capsaicin with acid increases its sensitivity. Previous studies showed that 1 ␮M capsaicin activated currents in 60% of TG neurons and that capsaicin-induced currents were heterogeneous in regard to their rates of activation and desensitization and reversal potentials [3,42,53,65]. The data presented in Fig. 8A–D were obtained by the voltage–ramp method and are representative of the types of currents obtained (at ⫺60 mV) that are evoked by a 32-s application of 1 ␮M capsaicin at pH 7.4. Fig. 8A shows an example of a transiently activated inward current (named Tc). Tc-type currents have outwardly rectifying ⌬I–V curves (bottom curve) and reversal potentials of 17 ⫾ 7 mV (n ⫽ 9) and times to peak of 0.56 ⫾ 0.23 s (mean ⫾ SD; n ⫽ 22). Capsaicin also evokes slowly activating (Sc-type) and desensitizing currents such as seen in Fig. 8B. This current had a

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time to peak of 18.2 s, an outwardly rectifying ⌬I–V curve, and a reversal potential of ⫺2.4 mV. On average, the reversal potentials of Sc-type currents is 1.9 ⫾ 2.7 mV [3]. Capsaicin also activates TSc-type currents. The TSc-type current, shown in Fig. 8C, had peak times of 2.5 s and ⬎32 s. This TSc-type of response exhibited rectifying ⌬I-V curves and had reversal potentials of ⫺1.8 mV and 17.4 mV, for the S and T components, respectively. A fourth type of capsaicin-evoked pattern yielded two slowly activating currents (Fig. 8D). By analogy with the proton-gated currents, these currents were named DSc-type currents. The two components (DSc1 and DSc2) had peak times at 9 and ⬎32 s, rectifying ⌬I–V curves, and reversal potentials of 1.2 ⫾ 3 mV (n ⫽ 10) and 3 ⫾ 4 mV (n ⫽ 15), respectively. Figure 9A shows the current–capsaicin concentration traces obtained from a cell held at ⫺60mV. In this experiment, capsaicin was dissolved in KH (pH 7.4) buffer and was applied for 32 s every 5–6 min. For this cell, the threshold concentration was 0.1 ␮M, and the amplitude of the currents increased with increasing concentration until 2.5␮M, where it became saturated. In the presence of pH 5.9 buffer, TG neurons with Sc-type currents become more sensitive to capsaicin (Fig. 9B). For this cell, threshold was decreased less than 0.01␮M and the current saturated at about 0. 1␮M. The plot of the normalized peak current vs. capsaicin concentration is shown in Fig. 9C. In the plot, it is clear that the capsaicin dose–response curve shifts to lower concentrations at pH 5.9. The data were analyzed by fitting each of the dose–response curves to the Hill equation. This yields K1/2 ⫽ 1.19 ⫾ 0.28 ␮M (n ⫽ 7) for pH 7.4 and K1/2 ⫽ 0.05 ⫾ 0.027␮M (n ⫽ 7) for pH 5.9. These differences are significant (p ⬍ 0.01) using Student’s t-test. 3.8. Comparisons between Sc- and Sp-type currents TG neurons can be activated by both 1 ␮M capsaicin and protons (Fig. 14C). We found that 49% (32/65) of the neurons were activated by both stimuli, 18.5% (12/65) were activated only by capsaicin (Fig. 14B), and 10.7 % only by pH 5.9 (7/65). Because there are several types of currents that can be activated by protons and capsaicin (Figs. 2 and 8) we investigated whether the slowly activating Sc- and Sp-type currents activate the same receptor/channel by testing whether they are inhibited by capsazepine and determining the thermal sensitivity of TG neurons. 3.9. Capsazepine inhibits Sp-type currents

Fig. 9. Lowering the pH to pH 5.9 sensitizes the capsaicin dose–response curve. (A) Dose–response curve obtained for capsaicin that were obtained for capsaicin in KH buffer (pH 7.4). Between applications, the cell was washed for 5–6 min with KH buffer. (B) Dose–response curve obtained for capsaicin that were obtained for capsaicin in pH 5.9 buffer. Between applications, the cell was washed for 5–6 min with KH buffer. Bars indicate the duration of the stimulus and the arrows point to the inward currents. Holding potential ⫽ ⫺60 mV.

To test the possibility that the Sp- currents are in the family of vanilloid receptors, we examined whether 10 ␮M capsazepine (CPZ) would inhibit these currents. Although it has been shown that 10 ␮M CPZ partially inhibits the proton-activated currents evoked from the VR1 receptor [69], it remains controversial as to whether CPZ inhibits protongated currents or proton-gated responses in sensory neurons [10,23,40,46,49,71]. We found that 10 ␮M CPZ reversibly inhibited Sp- and (Fig. 10A), DSp- (Fig. 10B) type currents.

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Fig. 10. Capsazepine inhibits Sp-types of proton-gated currents. (A) An Sp-type of current was evoked by a 30-s application of pH 5. 9 buffer. After a 2 min wash with KH buffer (pH 7.4), the cell was incubated for 2 min with KH plus 10 ␮M capsazepine (CPZ) whereupon 10 ␮M CPZ in pH 5.9 buffer was applied for 30 s. Under these conditions, the protoninduced current was completely inhibited. After a 3-min wash, the current partially reversed (B) Application of pH 5.9 buffer evoked a DSp-type current. This current was markedly inhibited by 10 ␮M capsazepine. After a 3-min wash, pH 5.9 buffer could again evoke a large inward current. Bar indicates duration of stimulus. HP ⫽ ⫺60 mV.

The Sp-type current was completely inhibited, and the DSptype current was inhibited by 81%. On average, 10␮M CPZ inhibited 91 ⫾ 8% (n ⫽ 8) of peak amplitude of either Sp or DSp-type currents. In separate experiments, we found that 10 ␮M CPZ also inhibited the transient component about 30% (data not shown). 3.10 Thermal responses of TG neurons There have been several in vivo and in vitro investigations of the responses of primary afferent TG and DRG neurons to temperature [7,33,49,51,59]. However, there has not been a thorough investigation of the thermal responses of cultured TG neurons regarding the question of whether the response has similarities to those evoked by VR1. The application of temperature ramps (upper trace, Fig. 11A) to a subset of TG neurons held at ⫺60 mV induces an above-threshold temperature (Tth), a large temperature-sensitive inward current (lower trace) that is called Iheat (Fig. 11A,B). Upon cooling, the current is reversible. Fig 11B shows that Iheat increases slowly with increasing temperature until Tth ⫽ 45.2⬚C, whereupon small changes in temperature produced large changes in current (Fig. 11B). After normalizing the current (to its maximal value), the Q10’s of the low and high temperature regions (defined by Tth) were 3.3 and 84, respectively (Fig. 11C). For 23 neurons, the mean threshold temperature, Tth ⫽ 42.9 ⫾ 2.6 ⬚C (mean ⫾ sem), and Q10’s for the low and high temperature regimes were calculated to be 3.3⫾ 1.45, and 56.3 ⫾ 17.2, respectively. These values are in the range of those found in rat DRGs [49,72]. Currents that were classified as Iheat were observed in 60% (23/38) of the neurons. These neurons had

Fig. 11. Thermal responses of rat trigeminal ganglion neurons. (A) Plots of temperature (upper trace) and inward current (lower trace) versus time. Both heating and cooling cycles are shown. HP ⫽ ⫺60 mV. (B) Graph of current versus temperature (heating) for data in A. Currents with this characteristic are named Iheat. (C) Plot of log of the current normalized to its maximum current versus 1/ T (⬚K). Illustrated in this example are the threshold temperature (Tth ⫽ 46⬚C) and the Q10’s for the low- and hightemperature components.

a mean capacitance of 31.8 ⫾ 2.5 pF (n ⫽ 23). In addition, some of the neurons that exhibited DSc-type currents also exhibited two thermally induced peaks at 42.5 ⫾ 1.8 and 46.0 ⫾ 1.8 ⬚C (data not shown). In the subset of neurons in which Iheat-type currents were not evoked (in the range from 23–50⬚C), a variety of other thermally sensitive currents were identified (e.g., Fig. 14A). To test the hypothesis that Iheat arises from receptors containing VR1 subunits, we investigated whether it is inhibited by 10 ␮M CPZ. We chose this CPZ concentration to attain the maximal inhibition, especially given that the thermal responses evoked by VR1 [69] and the responses evoked from DRGs [32,49] are not completely inhibited by

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cantly different from the decrease in Iheat expected two applications (p ⬍ 0.05). To further characterize Iheat and also to test whether it arises from the activation of vanilloid receptors, we hypothesized that if it did, then its I–V curve should outwardly rectify and should have a reversal potential near 0 mV. Figure 13A shows temperature–time and current–time (at ⫺60 mV) plots. The current–time trace was constructed from a family of I–V curves and shows that the inward current remained unchanged until 25⬚C, whereupon it increased linearly until 40⬚C became independent of temperature until about 46⬚C (⫽ Tth), whereupon Iheat was activated. The ⌬I–V curves for T ⬍ Tth and for T ⬎ Tth (Fig. 13B) are similar in that they both outwardly rectify and have reversal potentials around 0 mV. The results of seven experiments revealed that Iheat had reversal potentials of 3.6 ⫾ 2.9 mV. These results are in good agreement with those reported in DRGs [32,64]. None of these TG neurons were found with a reversal potential between 20–30 mV, as would be expected if the T- or TS-type current evoked Iheat-type currents. Clearly, more experiments need to be conducted to make this point definitive.

Fig. 12. Capsazepine (CPZ) inhibits the thermally activated current (Iheat). (A) Currents evoked from neuron held at ⫺60 mV during two thermal cycles. Between applications the neuron was washed with KH buffer. Note that there is a small decrease in the amplitude of the second response. (B) A similar experiment performed in a different neuron. The major difference is that after the first cycle 10 ␮M CPZ was applied to the cell and after a 1-min incubation period, the second thermal cycle commenced. The CPZ-inhibitable current was comprised of two components. After treatment with CPZ, the thermal response was not reversible after a five minute wash. Bars indicate duration of stimulus.

this concentration of CPZ. Figure 12A shows two temperature–time (upper traces) and current–time traces, each interspersed with 3-min washes. Note that, relative to the initial response, the second thermal application produced a current whose peak was about 20% smaller. For the second thermal response, the peak current decreased 19.2 ⫾ 4.2% (n ⫽ 4). This control was necessary to test whether CPZ inhibits Iheat because of the partial inhibition of CPZ (see below) and also because of tachyphylaxis [64,72]. To test whether CPZ inhibits Iheat, a neuron was first heated to 48⬚C, and if Iheat was present, the neuron was incubated for at 23⬚C for 1 min in 10 ␮M CPZ in KH buffer, whereupon it was reheated to 48⬚C in the presence of 10 ␮M CPZ (Fig. 12B). In the control-heating cycle, the current slowly increased from 32⬚C to 42⬚C (⫽ Tth), where it increased abruptly with increasing temperature. The CPZ-insensitive current increased linearly from 25 to 29⬚C and did not markedly change temperature until 48⬚C, at which a small, slowly activating, inward current was observed. This latter current may represent the part of Iheat that was not inhibited by CPZ. On average, 42.0 ⫾ 12.2 % (n ⫽ 4) of the peak of Iheat was inhibited by 10 ␮⌴ CPZ. This percentage is signifi-

Fig. 13. Current–voltage curves of the thermally induced currents. (A) Temperature–time and current–time plots. In this experiment the current trace at ⫺60 mV was constructed from the family of current–voltage curves. From 25⬚C, the current increased linearly until 40⬚C and then remained constant until the threshold temperature for Iheat was reached. Upon cooling, the current reversed. (B) ⌬I-V plots taken at the times indicated by arrows in (A). Note that both the low temperature and high temperature plots have rectifying I–V curves and the same reversal potentials (5 mV).

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all three stimuli (Fig. 14C).These neurons were the most frequently observed, comprising 41% (13/32) of the neurons tested. We note that Iheat appeared in those neurons that exhibited Sp and Sc-type currents. 4. Discussion

Fig. 14. Responses of TG neurons to protons (pH 4.9), capsaicin (1 ␮M) and heat. This figure gives examples of neurons that (A) were not activated by protons or capsaicin and did not exhibit Iheat; (B) were not activated by protons but were activated by capsaicin and exhibited Iheat; and (C) were activated by all protons and by capsaicin and exhibited Iheat. Bars indicate duration of stimuli. HP ⫽ ⫺60 mV.

We next inquired whether all neurons that are activated by 1 ␮M capsaicin and protons (pH 4.9) exhibit Iheat-type currents. We hypothesized that if all three stimuli evoke S-type currents, this will provide additional evidence for the presence of functional VR1-type receptors in TG neurons. These proton and capsaicin concentrations were chosen because for VR1, 1 ␮M capsaicin is near the EC50 and pH 4.9 is below the pH0.5 ⫽ 5.4 [15,16,69]. Figure 14A provides an example of a neuron that is not activated by pH 4.9 or by 1 ␮M capsaicin yet elicits a small thermal response that is not Iheat. This response could be characterized by single Q10 ⫽ 4.1. We have also recorded two neurons that were not activated by pH 4.9 or 1 ␮M capsaicin but exhibited Iheat (data not shown), suggesting they could represent VRL-1 receptors [15]. Fig. 10B shows an example of a neuron that is not activated by pH 4.9, is activated by 1 ␮M capsaicin, and exhibits an Iheat-type current. These types of responses were observed in 19% (6/32) of the neurons tested. These cells had soma diameters of capacitance’s of 28.9 ⫾ 1.8 pF (31.6 ⫾ 6.6 ␮m ⫽ soma diameter). We have never observed a neuron that was activated by pH 4.9, was not not by 1 ␮M capsaicin, and had Iheat-type currents. Another category of neurons that we identified for the first time was activated by

When acid, capsaicin and heat are applied to lingual epithelium innervated by sensory neurons from the trigeminal ganglion, they may induce tingling, irritating, or burning sensations. Knowledge of the receptors that are activated by these stimuli is a first step in the understanding mechanisms involved in their gustatory physiology. The importance in gustation arises because capsaicin is eaten daily by over one third of the world’s population [62], and the interest in the responses to acid arises because of the gustatory responses of carbonated water [64] and its role in augmenting the taste of foods, including spices [46]. The responses to temperature are important because of its role in gustation and the burning sensation [57]. In this paper we report the results of our studies of the responses of cultured TG neurons to these stimuli. All three stimuli evoked a variety of currents, suggesting that TG ganglia express several subtypes of proton, capsaicin and thermal receptors. The slowly activating currents evoked by these three stimuli that have reversal potentials near 0 mV, and that are inhibited by CPZ, share many characteristics with VR1 receptors. It is highly probable that these receptors contribute to the burning sensation produced by these stimuli. Other gustatory sensations are likely to result from the activation of transient and biphasic currents. 4.1. Reverse-transcription polymerase chain reaction In intact rat TG, RT-PCR measurements revealed the presence of the mRNA for VR1, ASIC-␣, ASIC-␤, and DRASIC subunits (Fig. 1). With the exception of ASIC-␤, which has not been identified in TG, these data are largely confirmatory [16,26,39,73–75]. For the purposes of this discussion, these data reveal that rat TG contains at least four distinct subunits of channels that can be activated by protons. Immunocytochemical studies revealed that some of these subunits are co-localized in neurons with VR1 subunits [26,48], although extracellular recordings from TG neurons, calcium-imaging studies, and patch clamp recordings from cultured DRGs have shown that not every neuron activated by acid is activated by capsaicin [11,27,50,67]. These data, together with data showing that there are neurons with differential sensitivities to carbonic anhydrase [13,64], indicate the presence of several types of proton-activated receptors, some of which are present in capsaicin-sensitive neurons (Fig. 14C). Therefore, from an electrophysiological and pharmacological viewpoint, it is important to investigate the types of H⫹-activated receptors in capsaicin-sensitive neurons. 4.2. Proton-gated currents Psychophysical studies in which acid-induced irritation was reduced after capsaicin desensitization [25] suggest that

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they activate a common pathway but do not directly test whether they activate the same receptors. The burning sensation induced by acidic stimuli is thought to arise from the activation of sustained proton-gated channels (Sp) in nociceptors. However, there are many types of responses that have sustained currents (Figs. 2 and 3), and sustained currents can be generated with several types of subunits from H⫹-gated channels as well as from VR1 receptors [69,75]. In sensory and CNS neurons, there are numerous H⫹-gated, cation (usually Na⫹)-selective channels that belong to the DEGenerin (DEG)/ ENaC superfamily, e.g., DRASIC, ASIC-␣ [75], ASIC-␤ and -␥, DRASIC, MDEG1, MDEG1 with MDEG2 or with ASIC␣ and MNDG2 with DRASIC [36,75]. These channels exhibit diverse kinetics, sensitivities to acid, amiloride, and selectivities for cations [36,75]. In patch clamp studies with cultured dorsal root ganglion (DRG) or TG neurons, acidic stimuli have been shown to evoke transient (Tp), sustained (Sp), or both types (TSp) of currents, indicating that acidic stimuli can activate numerous receptors. [1,2,12,36,37,40,54,71]. We will explore which of these receptors exhibit characteristics that are similar to those evoked by VR1. Currents evoked by pH 5.9 buffer were found in 66% of the TG neurons (Table 1). This percentage is in reasonable agreement with other studies on DRG or TG neurons or both [11,71]. Moreover, proton-activated currents were found with equal probability in all sizes of neurons. In this regard, they differ from capsaicin-activated currents that are found with the highest probability in small-diameter neurons [66] and differ from neurons with Iheat-type currents that are found primarily in smaller neurons [64,72]. Thus, the probability of finding neurons that respond to both capsaicin and acid pH is greatest in neurons having the smallest soma diameter. We have also found that 59 % of the neurons exhibit both capsaicin sensitivity and Iheat (Fig. 14B,C). This is in reasonable agreement with results obtained using DRGs [31,72], which found that 70% of the small neurons were activated by both heat and capsaicin. We found that 41% of the neurons tested were activated by pH 4.9, 1 ␮M capsaicin, and exhibit Iheat (to 艑50⬚C). They were primarily in cells with soma diameters that averaged 30 ␮m. In this regard it was found that small and medium size neurons have threshold temperatures at 43⬚C (and presumably contain VR1 subunits [16,49], and larger neurons have threshold temperatures of 51⬚C and are mostly found in larger size neurons (and likely contain VRL-1 receptors [15]) 4.3. Types of proton-activated currents We have identified five kinetically distinct patterns of proton-activated currents (Figs. 2 and 3). These currents differ, to various extents, in their kinetics, proton sensitivity, reversal potentials, and pharmacology. 4.4. Tp-type currents The data obtained previously as well as in this study indicate the presence of several (independent) Tp types of channels in rat sensory DRG or TG neurons or both. The evi-

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dence includes the differential sensitivity to various antagonists [2,12,34,36,37,40,54,71], a relatively wide range of reversal potentials (24–45 mV, Figs. 2 and 3), and a wide range of pH0.5 values (Fig. 5D).The question of whether the Tp-current is related to the Tc-type current (Fig. 8B) cannot be definitively answered. We believe that it is unlikely because of their different reversal potentials and shapes of I–V curves (Figs. 2A and 8A). Also, Tp currents are inhibited by amiloride (Fig. 7), indicating that they share similarities with currents comprised of subunits of the DEG/ ENaC superfamily [75], rather than with the vanilloid family [66]. In this regard, Tp currents comprised of ASIC-␤ subunits are not inhibited by CPZ [19]. Nevertheless, because various subunits of H⫹-gated channels can combine to produce currents with very different kinetic properties [76] we can not determine the number of independent Tp-type currents that are present in rat TG neurons. Given that Tp and Tc currents are distinct, and distinct from Sp and Sctype currents it is likely that when activated they will produce different neuronal responses to each other and different responses than the slowly activating currents. 4.5. TSp-type currents TSp currents have been found in intact neurons and in channels expressed in oocytes and other cells (Figs. 2,3,5 and [8, 71,75]. Do TSp or TDSp-type currents with different reversal potentials represent independent transport pathways? Even though TSp1-and TSp2-components of TSp-type currents have different reversal potentials (Figs 2C and 3A), it cannot be concluded that they are separate entities because some protongated channels with DRASIC and MDEG2 subunits have been shown to exhibit biphasic currents with different reversal potentials [75]. Thus, only those TSp-type currents that can be separated by pharmacological manipulations can be considered to be comprised of two independent types of H⫹-gated transport pathways–that can co-exist in the same neuron. We have not identified a biphasic current having the same reversal potential for both the transient and sustained components, suggesting that H⫹-gated channels consisting only of DRASIC subunits are either rare, or absent, in TG [75]. 4.6. Slowly activating proton-gated currents (Sp and DSp) Slowly activating current types are believed to be involved in the burning response to acidic stimuli. Sp- and DSp-type currents constitute a classification of H⫹-gated receptors that can be distinguished from the Tp-types currents. The evidence is that they have significantly different reversal potentials (Figs. 2 and 3), that they can be completely inhibited by 10 ␮M CPZ (Fig. 10), and that they are not inhibited by 200 ␮M amiloride (Fig. 7). In addition, we have obtained data showing that Sp or DSp-type currents are not simply Tp-type currents that activated slowly at pH 5.9 because they remain distinctive at all voltages and have distinct reversal potentials (Fig. 3A). These data, taken as a whole, suggest that Sp- (or DSp-) and Tp-type currents are independent. We do not, how-

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ever, imply that Sp- and DSp-type (Fig. 2) currents are independent because it is possible that DSp-type currents may simply be a Sp-type current whose slower component (DSp2) becomes activated by intracellular modulators as a consequence of the activation of the DSp1 component. However, this point has not been rigorously investigated. From a physiological perspective, it is of interest that for both the transient and sustained proton-activated currents, we found that the inward current increased with decreasing pH to pH 5.5 but decreased at pH 4.9 (Figs. 5 and 6). This behavior may offer an explanation as to why the activity of C-fibers increases with decreasing pH until pH 5.2 and then decreases [35]. That is, at these lower pH’s, the depolarizing inward current may be reduced and hence a smaller number of action potentials should be evoked. Alternatively, voltage-dependent Na⫹-channels may be inhibited at these reduced pH’s [5,68]. Many characteristics of Sp-type currents are similar to those of Sc-type currents, and to currents evoked from VR1 receptors. Specifically, they all have reversal potentials around 0 mV, outwardly rectifying I-V curves (Figs. 2 and 8) that are inhibited by CPZ (Fig. 11), and can be found in the same neuron (Fig. 14C). Moreover, the dose–response curve of Sc currents is shifted to the left (Fig. 9) as it is for VR1 receptors [69], albeit further. From a physiological perspective, single-unit recordings of C fibers from cat TG neurons showed that neurons that were activated by acetic acid and thermal stimuli became much less responsive after pretreatment with capsaicin [6]. In other preparations, CPZ, ruthenium red, or both were shown to inhibit physiological responses to acid [23,24,44]. Although the above is strong evidence that Sp, Sc, and VR1 reflect the activation of the same receptor (or receptor family), it is not unequivocally so. For example, for Sp currents, pH0.5 ⫽ 6.4 (Fig. 5), for VR1 pH0.5 ⫽ 5.4 [69], and pH0.5 ⫽ 5.8 from 86Rb efflux measurements in neonatal rat DRGs, [12]. Thus, the acid sensitivity of DRGs [36] or TGs [47] is generally higher than it is for VR1. Also, not every neuron that is activated by capsaicin is activated by protons (Fig. 14B, [20,27]). This suggests that there may be subtypes of vanilloid receptors [15,28,66], or that these neurons may require even lower pHs to be activated (although pH 4.9 is quite low). There are numerous rationalizations for these differences, including the ages of the animals, days in culture, different cell types, and different subtypes or modifications of vanilloid receptors (Fig. 2; [28,65,66]). However, we believe that these data, in combination with results showing in DRGs the nearly identical NGF requirements for Sc- and Sp-type currents [11], provide strong evidence that Sp, Sc, and VR1 are closely related and that functional VR1 subunits are present in TG neurons. 4.7. Thermally activated currents Previous studies with DRGs have shown that neurons with thermally activated currents (Iheat), can also be acti-

vated by capsaicin [31,32,49], that CPZ partially inhibits [32] or does not inhibit [49] thermally induced currents, that Tth ⬵ 43–45⬚C [72,75], that for T ⬎ Tth the Q10’s are very large [72], suggesting that channel gating by thermal stimuli involves large conformational changes. The reversal potential for thermally activated currents was reported to be about 0–5 mV [72,75]. We obtained similar data in rat TG neurons (Figs. 11–14). All of these characteristics are similar, but not identical, to those found in VR1 [16,69]. The large range of Q10’s found in DRGs and in this study (Fig. 11), albeit by different heating rates, and the consistent changes in Q10 found in VR1, suggest that intact neurons have a variety of thermoreceptors [17,18,59,75]. We have also found, for the first time that a large percentage of neurons with Sp- and Sc- type currents also have Iheat-type currents (Fig. 14C). These data, showing that the same neuron can be activated by these three stimuli, represent the best evidence that Iheat, Sc and Sp⫺ activate the same receptor and that their characteristics are very similar, but not identical, to those obtained for VR1. We have also identified neurons that are activated by capsaicin and heat but not by protons (Fig. 14B) and neurons that are activated by heat but not by capsaicin. Neurons with these characteristics have been previously identified in DRG neurons [49,72]. Taken together, these data indicate the presence of a subset of neurons that contain subtypes of vanilloid receptors that differ from homomeric VR1 receptors. Finally, we identified two neurons that did not respond to protons or to capsaicin but did exhibit Iheat-type currents (Tth ⫽ 43⬚C and 45⬚C). Although it is difficult to draw any conclusions from two experiments, it is possible that this could represent other subtypes of vanilloid receptor (VRL-1) which are activated by heat but not by capsaicin or protons [15,68]. Finally, in TG, like in DRG and SCG neurons [17,49], there are thermally activated currents that are not Iheat (e.g. Fig. 14A). Although we have not extensively investigated these currents, they appear to be thermally sensitive over a 30–40⬚C range and likely represent different type of thermal receptors known to be present in sensory neurons [18,49, 55,56,70]. 4.8. Summary The responses elicited from TG neurons to protons, capsaicin, and heat demonstrated the presence of numerous receptors for these stimuli. We have obtained evidence for the presence of functional VR1-type subunits in TG neurons that respond similarly, but not identically, to homomeric VR1. We have also obtained evidence for functional receptors of the MDEG/ENaC superfamily that respond to acid. The recently cloned VR1 receptor that has been shown to be activated by protons, capsaicin, and heat has been proposed to act as a molecular integrator of nociceptive stimuli [69]. In mammals, the activation of slowly activating and desensitizing currents by any of these three stimuli in nociceptors should first result in a burning sensation. The augmentation of the capsaicin response at low pH (Fig. 9) should increase

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the responses to spices such as capsaicin, zingerone (horseradish), and piperine (black pepper). Activation of the transient currents will evoke different sensations (tingling, piercing). Thermoreceptors are necessary to sense the external temperature as well as changes in temperature. Electrophysiological recordings from sensory ganglia revealed the presence of cold fibers, warm fibers, and nociceptors [7,55,56], suggesting the presence of several different thermal receptors [18]. The thermal response in which the currents abruptly increase after 43⬚C (Iheat) that we have found in TG neurons correlates well with the change in the sensation of warmth to burning pain at about this temperature. It remains to determine the roles of the other receptors that were identified to understand their physiological roles. NOTE IN PROOF: A variety of responses of VR1 ⫺KO mice have recently been reported (Caterina et al, 2000). This study found that the burning taste sensation due to capsaicin is entirely due to VR1 subunits. Patch clamp studies of cultured DRG VR1 ⫺/⫺ neurons failed to evoke either transient or sustained capsaicin activated currents, and thermally activated currents with Tth ⫽ 43⬚C. Moreover, sustained currents activated by pH 5.0 were found in only 7% of the cells. These data confirm that the sustained currents evoked in TG neurons mostly arise from VR1 receptors. It is also clear from this elegant study that VR1 subunits are responsible for many of the currents seen in these studies. Nevertheless homomeric VR1 receptors can not completely explain all the results evoking sustained obtained in rat TG neurons, suggesting some small variants of VR1 are present. Caterina, M. J., Leffler, A., Malmberg, A. B., Martin, W. J., Trafton, J., Petersen-Zeitz, K. R., Koltzenberg, M., Basbaum, A. I., and Julius, D. (2000) Impaired nocicepton and pain sensation in mice lacking the capsaicin receptor. Science 288:306–313. Acknowledgments We thank Drs. Marga Oortgiesen and Allan McAlexander for useful discussions. This study was supported by NIH grant DC01065 and the Philip Morris Corporation. References [1] Akaike N, Krishtal OA, Maruama T. Proton-induced sodium current in frog isolated dorsal root ganglion cells. J Neurophysiol 1990;63:805–13. [2] Akaike N, Ueno S. Proton-induced currents in neuronal cells. Prog Neurobiol 1994;43:73–83. [3] Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 1996;379:257–62. [4] Bassilana F, Champigny G, Waldmann R, de Welle J, Heurteaux C, Lazdunski M. The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromeric H⫹-gated Na⫹ channel with novel properties. J Biol Chem 1997;272:28819–22. [5] Baumann TK, Burchiel KJ, Ingram SL, Martensen ME. Responses of adult dorsal root ganglion neurons in culture to capsaicin and low pH. Pain 1998;65:31–8.

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