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1Department of Oral and Maxillofacial Surgery, McKnight Brain Institute and College of Dentistry and 2Department of Pathology, University of Florida, Gainesville, Florida 32610; and 3Cell Biology of Excitable Tissue Group, Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada
Submitted 26 December 2002; accepted in final form 10 February 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined 
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-methylene-ATP (meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 µM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 µM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 µM meATP could be largely inhibited by 10 µM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A-afferent fibers and play a role in modulating sensory transmission to lamina V neurons. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Jahr and Jessell 1983; Khakh et al. 2001; Krishtal et al. 1983; North 2002), are highly expressed on primary sensory neurons (Burgard et al. 1999; Cook et al. 1997; Guo et al. 1999; Li et al. 1999; Petruska et al. 2000a; Vulchanova et al. 1997; Xiang et al. 1998). Activation of P2X receptors at peripheral sensory nerve endings may initiate sensory impulses and may be associated with nociceptive and nonnociceptive sensory signals (Cook et al. 1997; Dowd et al. 1998; Sawynok and Reid 1997; Tsuda et al. 2000). At central terminals, P2X receptors play a role in modulating glutamate release from sensory terminals (Deuchars et al. 2001; Gu and MacDermott 1997; Li et al. 1998; Nakatsuka and Gu 2001). Activation of P2X receptors may also induce substance P release from primary afferent fibers (Nakatsuka et al. 2001).
Functional P2X receptors are formed by seven P2X receptor subunits (P2X1—P2X7) as homomers and heteromers (North 2002; North and Surprenant 2000). Six homomeric P2X receptors (P2X1—5 and P2X7) and at least four heteromeric P2X receptors (P2X2+3, P2X4+6, P2X1+5, and P2X2+6) have been shown to be functional P2X subtypes in heterologous expression system (Brown et al. 2002; Khakh et al. 2001). These P2X receptor subtypes can be classified into different groups based on their kinetics and pharmacological characteristics. Activation of P2X1 and P2X3 receptors produces fast currents manifested as rapid desensitization in the presence of agonists. Activation of the remaining eight subtypes yields slow currents manifested with weak or little desensitization. ,-methylene-ATP (meATP) can selectively activate P2X1- and P2X3-containing receptors at low concentrations (Khakh et al. 2001). However, at high concentrations, meATP can activate other P2X subtypes (e.g., P2X2 subtype), albeit with low efficacy (Spelta et al. 2002). Most subtypes of P2X receptors can be inhibited by suramin and iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). Advancements in determining possible subtypes of P2X receptors in native tissues have been impaired by the lack of highly subtype-selective antagonists. Recently, 2', 3'-O-(2,4,6-trinitrophenyl) adenosine triphosphate (TNP-ATP), an ATP analogue, has been shown to be a potent antagonist that selectively inhibits homomeric P2X1, P2X3, and heteromeric P2X2+3 receptors (Burgard et al. 2000; Khakh et al. 2001; Virginio et al. 1998). Therefore TNP-ATP is a much better discriminator of subtypes than meATP.
Most previous studies on sensory functions of P2X receptors have focused on homomeric P2X3 receptors and heteromeric P2X2+3 receptors. The predominant expression of P2X3 subunits on small-sized primary afferent neurons suggested their potential role in nociceptive signaling (Chen et al. 1995; Cockayne et al. 2000; Collo et al. 1996; Cook et al. 1997; Lewis et al. 1995; Souslova et al. 2000). Electrophysiological recordings from dorsal root ganglion (DRG) neurons showed three major phenotypes of currents, fast, slow, and mixed P2X currents (Burgard et al. 1999; Li et al. 1999; Petruska et al. 2000a; Ueno et al. 1999). It has been suggested that P2X3 receptors account for fast currents, P2X2+3 receptors account for slow currents, and the co-expression of P2X3 and P2X2+3 receptors account for the mixed currents (Burgard et al. 2000; Cook et al. 1997; Levis et al. 1995; Liu et al. 2001; Radford et al. 1997; Xu and Huang 2002). Little is known whether other P2X receptor subtypes may also contribute to P2X agonist-evoked currents in rat DRG neurons.
Using spinal-cord slice preparations, we have previously shown that A-afferent terminals innervating lamina V regions express meATP-sensitive P2X receptors (Nakatsuka and Gu 2001). These P2X receptors play a role in modulating sensory synaptic transmission and may be involved in sensory central sensitization in lamina V of the spinal cord. It has been widely believed that these P2X receptors are P2X2+3 receptors (North 2002) because of their sensitivity to meATP and their nondesensitizing property. However, we have recently found that the effects of meATP on afferent central terminals in lamina V are not blocked by the P2X2+3 antagonist TNP-ATP. Direct recordings from DRG neurons confirmed the presence of meATP-sensitive/TNP-ATP-resistant P2X receptor subtypes in some medium-sized primary afferent neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Principles of laboratory animal care (National Institutes of Health Publication No. 86-23, revised 1985) were followed in all the experiments described in the present study. Spinal-cord slice preparation and patch-clamp recordings are described in detail in a previous study (Nakatsuka and Gu 2001). In brief, transverse spinal cord slices (500-µm-thick) were prepared from L5 spinal cords of Sprague-Dawley rats at the postnatal age of 14–21 days. The slices were superfused with Krebs solution (22°C) at the flow rate of 10 ml/min. The Krebs solution contained (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose; the solution was saturated with 95% O2-5% CO2 and had pH of 7.3. Whole cell patch-clamp recordings were made from DH neurons in lamina V with microelectrodes filled with a solution containing (in mM): 135 K+-gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, and 5 HEPES, pH 7.3. The electrode resistance was 
5M
after filling the electrode solution. Signals were amplified with Axopatch 200B (Axon Instruments, Union City, CA), filtered at 2 kHz, and sampled at 5 kHz using pCLAMP 7 (Axon Instruments). Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded at holding potential of —60 mV in the presence of 20 µM bicuculline and 2 µM strychnine. In some experiments, 0.5 µM tetrodotoxin (TTX) was included in the bath solution during the recordings of sEPSCs, i.e., miniature EPSCs (mEPSCs). To record stimulation-evoked EPSCs (eEPSCs) from lamina V, stimuli (50 µA, 0.1 ms) were applied to a dorsal root with a suction electrode (Nakatsuka and Gu 2001). Monosynaptic connection was judged by constant latency of eEPSCs when multiple stimuli were applied. The latency of eEPSCs and the length of dorsal roots were used for conduction velocity calculation. For testing the effects of meATP, meATP (10 or 100 µM) was applied through bath solution. When TNP-ATP was used as an antagonist, 1 µM TNP-ATP was first preapplied for 5 min, and then meATP was co-applied.
Patch-clamp recordings from dissociated DRG neurons
Adult male rats (Sprague-Dawley, 100–250 g) were deeply anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL). DRGs were rapidly dissected out and incubated with dispase II at 5 mg/ml (Boehringer Mannheim, Germany) and type I collagenase at 2 mg/ml (Sigma, St. Louis, MO) in 2 ml S-MEM medium (GIBCO Invitrogen, Carlsbad, CA) at 37°C for 60 min. After a rinse, DRGs were triturated to dissociate the neurons in minimum essential medium (MEM), GIBCO Invitrogen Corporation). The dissociated cells were plated on coverslips precoated with poly-D-lysine (Sigma); the coverslips were in 35-mm petri dishes containing 2 ml MEM medium. Cells were maintained in a CO2 incubator (5% CO2) and used between 24 and 72 h after plating.
Coverslips with DRG neurons were mounted in a 0.5-ml recording chamber and placed on the stage of an Olympus IX70 microscope. Cells were continuously perfused with bath solution (22°C) flowing at 1 ml/min. The bath solution contained (in mM) 150 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4, osmolarity adjusted to 320 mosM with sucrose. Recording signals were amplified with Axopatch 200B (Axon Instruments), filtered at 2 kHz, and sampled at 5 kHz using pCLAMP 6 (Axon Instruments). Recordings were made from cells with diameters in a range of 15–50 µm. Each cell size was determined by calculating the average of the shortest and the longest diameters. We regarded neurons with mean diameters of 15 < diameters 
30 µm as small-sized neurons, 30 < diameters 50 µm as medium-sized neurons, and >50 µm as large-sized neurons. Cells were voltage-clamped at —70 mV in whole cell configuration. The recording electrode internal solution contained (in mM) 110 Cs2SO4, 2 MgCl2, 0.5 CaCl2, 5 TEA-Cl, 5 EGTA, and 5 HEPES, pH 7.3. The junction potential was —9 mV and not adjusted. Recording electrode resistance was 5 M and the access resistance <25 M. The duration of agonist applications (meATP or capsaicin) was 2 s. These agonists were rapidly applied to neurons through a glass tube (500 µm in diameter) positioned 1.0 mm away from the recorded cells. The gravity-driven solution flow was electronically controlled by solenoid valves and triggered from a computer. The drug-on time was <100 ms. The interval between agonist applications was 
5 min. To examine the inhibitory effect of TNP-ATP or PPADS on meATP-evoked currents, TNP-ATP (0.1, 1, and 10 µM) or PPADS (10 µM) was first preapplied for 2 min, and then meATP (10 or 100 µM) was co-applied with TNP-ATP or PPADS. Capsaicin sensitivity of DRG neurons was tested with 1 µM capsaicin. To examine the effect of ivermectin (IVM) on meATP-evoked currents, IVM (3 µM) was first preapplied for 3 min, and then meATP was co-applied for 2 s with IVM.
Drugs
meATP (10 or 100 µM), TNP-ATP (0.1, 1, or 10 µM), PPADS (10 µ), capsaicin (1 µM), IVM (3 µM), bicuculline (20 µM), strychnine (2 µM), and TTX (0.5 µM) were used in this study. meATP, PPADS, IVM, capsaicin, bicuculline, and strychnine were purchased from Sigma; TNP-ATP was from Molecular Probes (Eugene, OR); TTX was from Tocris Cookson (St. Louis, MO).
Data analysis
Synaptic events including sEPSCs and mEPSCs were analyzed using Mini Analysis Program (Jaejin software, Anderson Place, GA) with criteria being the same as previously described (Gu and MacDermott 1997). In analyzing the change of EPSC frequency after bath application of meATP, the time course of EPSC frequency before and after meATP was first constructed with time bin of 10 s. Then the average response in continuous three bins (30 s) around the peak was used to calculate the changes in reference to the control. Data recorded from DRG neurons were analyzed using Clampfit 8.2 (Axon Instruments). The amplitudes of fast currents and fast-current components in the mixed currents were measured at their peaks. The amplitudes of slow currents and slow components (steady-state phases) in the mixed currents were measured at the end of 2-s meATP application. All data are presented as means ± SE. Paired Student's t-tests were used for statistical comparison, and significance was considered at the level of the P < 0.05.
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RESULTS |
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meATP-induced changes of excitatory synaptic responses recorded from dorsal horn lamina V neurons in the spinal cord slice preparations
Patch-clamp recordings were performed in lamina V neurons in the spinal-cord slice preparations. Monosynaptic A-afferent EPSCs were evoked by dorsal root stimulation (Fig. 1A, left) and conduction velocity was 2.6 ± 0.3 m/s. A-afferent-evoked EPSCs became failed 5 min after the continuous application of meATP at concentrations of 10 µM (Fig. 1A, middle, n = 3) or higher (100 µM, n = 3, not shown). Recovery from failure was observed after washout of meATP (Fig. 1A, right). The failure of A-afferent EPSCs was accompanied by the increased sEPSC frequency in the same neurons (Fig. 1B). mEPSC frequency was also increased in lamina V neurons after bath application of 100 µM meATP (Fig. 1C, left) (also see Nakatsuka and Gu 2001) or 10 µM meATP (see Nakatsuka et al. 2003), consistent with the expression of meATP-sensitive P2X receptors at afferent central terminals in lamina V. However, in the presence of 1 µM TNP-ATP, meATP still induced a large increase of mEPSC frequency (Fig. 1C, right). The increases of mEPSC frequency were 514 ± 95% of control with 100 µM meATP alone, and the increases were 478 ± 111% of control in the presence of 1 µM TNP-ATP (n = 5). These results confirmed that A-afferent fibers innervating lamina V indeed express meATP-sensitive P2X receptors as shown in our previous study (Nakatsuka and Gu 2001).
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meATP-evoked currents in DRG neurons
To provide direct evidence that meATP-sensitive/TNP-ATP-resistant P2X receptors are expressed on primary afferent fibers, we characterized meATP-evoked currents and TNP-ATP sensitivity in the somata of the dissociated DRG neurons in the following experiments (Fig. 2 to Fig. 8). Small (15–30 µm)- to medium-sized (30–50 µm) DRG neurons were abundant; many of them had smooth membrane surfaces and good seals could be formed with patch electrodes for recordings (Fig. 2A). Large-sized (>50 µm, not shown) DRG neurons were relatively less abundant and were not recorded in the present study because most large DRG neurons had no response to P2X agonists (Petruska et al. 2000a). A total of 238 DRG neurons, 53 small-sized neurons and 185 medium-sized neurons, were recorded to have inward currents evoked by 100 µM meATP. meATP-evoked currents from these DRG neurons could be classified into three major phenotypes (Fig. 2, B—D) based on their current kinetics (see Petruska et al. 2000a), specifically, fast currents (Fig. 2B), mixed currents (Fig. 2C), and slow currents (Fig. 2D).
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Fast currents showed rapid desensitization during 2-s applications of 100 µM meATP (Fig. 2B). These were observed in 37 neurons that had mean diameter of 25.6 ± 1.2 µm (Fig. 2E). The fast currents could further be divided into two subphenotypes, single- and double-peak fast currents (Fig. 2B). It is unlikely that second peak was due to chloride ion efflux because electrode solution contained low concentration of chloride ions. Of 37 cells showing fast currents, 29 cells had single-peak and 8 cells had double-peak currents.
Mixed currents (Fig. 2C) had an initial fast desensitizing phase (fast component) followed by a steady state phase (slow component). Double-peak fast components were also observed in some cells. Mixed currents were observed in 132 neurons. The mean diameter of these cells was 37.2 ± 0.7 µm (Fig. 2E).
Slow currents show weak or no desensitization during 2-s meATP applications (Fig. 2D). These were observed in 69 neurons. The mean diameter of these cells was 38.8 ± 0.8 µm (Fig. 2E). Despite equivalent rates of drug application in each experiment, slow currents appeared to have a large variation in current rising phases. As indicated in Fig. 2D, some slow currents had rapid rising phases and others had slow rising phases.
The relationship between current phenotypes and cell sizes was further analyzed. Although fast currents were mainly expressed on small-sized DRG neurons, small-sized neurons also expressed mixed and slow currents. In 53 small-sized DRG neurons recorded (Fig. 2F, left), 31 neurons (59%) showed fast currents, 17 cells (32%) showed mixed currents, and 5 cells (9%) showed slow currents. Medium-sized neurons also had three major phenotypes of currents, but mixed currents predominated (Fig. 2F, right). In 185 medium-sized DRG neurons recorded, 6 of them (3%) had fast currents, 115 neurons (62%) had mixed currents, and 64 neurons (35%) had slow currents.
TNP-ATP sensitivity of meATP-evoked fast, mixed, and slow currents in DRG neurons
Fast currents evoked by 100 µM meATP were completely inhibited by 1 µM TNP-ATP (Fig. 3). Both single- and double-peak fast currents were sensitive to TNP-ATP. In the presence of 1 µM TNP-ATP, the amplitude of residual currents was 0.7 ± 0.3% (n = 28) of controls. The inhibitory effect was reversible after a washout of TNP-ATP.
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The inhibitory effects of TNP-ATP on mixed currents that were evoked by 100 µM meATP were variable (Fig. 4). In a total of 77 neurons with mixed currents, the fast components were substantially inhibited by 1 µM TNP-ATP in all neurons. The amplitude of fast components was only 5.0 ± 1.1% of controls in the presence of 1 µM TNP-ATP (n = 77). On the other hand, slow components in the mixed currents showed variable sensitivity to 1 µM TNP-ATP, ranging from complete (Fig. 4A) to little inhibition (Fig. 4B). Overall, 1 µM TNP-ATP reduced slow components to 58.0 ± 4.2% of controls (n = 77). Even at 10 µM TNP-ATP, slow components in some cells were only partially inhibited (Fig. 4C).
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The inhibitory effects of TNP-ATP on slow currents evoked by 100 µM meATP were also variable (Fig. 5). Slow currents in some cells were completely blocked by 1 µM TNP-ATP (n = 8, Fig. 5A) and in other cells were less sensitive or insensitive to 1 µM TNP-ATP (n = 24, Fig. 5B). Overall, 1 µM TNP-ATP inhibited slow currents to 40.9 ± 7.1% of control (n = 32).
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Effects of TNP-ATP on meATP-evoked currents were also examined at different TNP-ATP concentrations (Fig. 6A). TNP-ATP was tested at concentrations of 0.1, 1, and 10 µM. Fast currents and fast components in the mixed currents were inhibited to 2.7 ± 1.4 (n = 14), 3.8 ± 0.9 (n = 105), and 0.9 ± 0.4% (n = 7) of controls in the presence of 0.1, 1, and 10 µM TNP-ATP, respectively. On the other hand, slow currents and slow components in the mixed currents were 72.9 ± 10.0% of control in the presence of 0.1 µM TNP-ATP (n = 19), 54.0 ± 3.5% of control in the presence of 1 µM TNP-ATP (n = 109), and 27.9 ± 9.5% of control in the presence of 10 µM TNP-ATP (n = 10).
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To determine cell distribution at different degree of inhibition, the effects of 1 µM TNP-ATP on slow component in the mixed currents and slow currents were further analyzed (Fig. 6B). Results were pooled together from 77 neurons with mixed currents (Fig. 4) and 32 neurons with slow currents (Fig. 5). The inhibition by 1 µM TNP-ATP was highly variable among these cells (Fig. 6B), ranging from nearly complete inhibition (<10% of control) to little inhibition (>90% of control). If we arbitrarily term meATP-evoked currents as TNP-ATP-resistant when the currents were >80% of control in the presence of 1 µM TNP-ATP, 25% of DRG neurons expressed TNP-ATP-resistant P2X receptors (Fig. 6B).
Capsaicin sensitivity of the DRG neurons that have TNP-ATP-resistant currents
We further examined whether DRG neurons expressing TNP-ATP-resistant P2X receptors were also capsaicin-insensitive because our previous studies indicated that meATP-sensitive afferent central terminals innervating lamina V neurons are capsaicin-insensitive (Nakatsuka and Gu 2001; Nakatsuka et al. 2002). Of those neurons (in Fig. 6B) displaying <20% inhibition by 1 µM TNP-ATP, eight cells were tested for capsaicin sensitivity. In these eight cells, five of them had mixed currents (Fig. 7A) and three had slow currents (Fig. 7B). Capsaicin (1 µM) did not evoke significant inward currents in any of these eight cells (Fig. 7). However, in cells expressing fast P2X currents, 1 µM capsaicin could evoke large inward currents (data not shown, also see Petruska et al. 2000a).
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Effects of IVM and PPADS on TNP-ATP-resistant P2X currents
In seven neurons for which 100 µM meATP-evoked currents were resistant to 1 µM TNP-ATP, we tested if P2X receptors on these cells could be modulated by IVM (Khahk et al. 1999). After a 3-min treatment of these cells with 3 µM IVM, 100 µM meATP (co-applied with 3 µM IVM) was applied for 2 s. The meATP-evoked currents were 81.6 ± 15.0 pA after IVM treatment, which was not significantly different from meATP-evoked currents in normal bath (94.1 ± 18.3 pA, n = 7, Fig. 8A).
TNP-ATP resistant currents were evoked by meATP at 100 µM in the preceding experiments. To determine if lower concentration of meATP could also evoke TNP-ATP-resistant currents in DRG neurons, meATP at concentration of 10 µM was tested. In this set of experiments, medium-sized DRG neurons (41.9 ± 0.6 µm, n = 45) were chosen, and TNP-ATP (0.1 µM) was first preapplied for 2 min. meATP (10 µM) was then co-applied with TNP-ATP for 2 s. Of 45 cells tested, 16 of them showed slow currents (33.4 ± 4.4 pA). Of these 16 cells, 8 of them were further examined to determine if the TNP-ATP-resistant currents could be inhibited by PPADS (Fig. 8B). TNP-ATP-resistant currents evoked by 10 µM meATP were significantly inhibited to 22.7 ± 8.0% of control, from 31.0 ± 16.2 pA in the absence of PPADS (control) to 10.9 ± 12.6 pA in the presence of 10 µM PPADS (n = 8, P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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meATP-induced changes of sensory synaptic transmission from A-afferent fibers to lamina V neurons (Nakatsuka and Gu 2001). In addition to their central role in modulating synaptic transmission to lamina V regions, TNP-ATP-resistant P2X ionic current may be also involved in sensory signaling at peripheral terminals of A-afferent fibers.
We have previously shown that low concentrations of meATP (1 µM) potentiate A-fiber-evoked EPSCs recorded in lamina V (Nakatsuka and Gu 2001). However, failure in A-fiber-evoked EPSCs has been observed with high concentrations of meATP (10 µM) in the present study. The underlying mechanism for the failure of the evoked EPSCs should be due to conduction block after the opening of P2X receptor channels on A-fiber terminals. It has been known for many years that strong activation of ligand-gated ion channels on afferent central terminals produces primary afferent depolarization (PAD), which leads to conduction block and presynaptic inhibition (Rudomin and Schmidt 1999). Failure of A-fiber-evoked EPSCs by meATP in our study confirms the presence of meATP-sensitive P2X receptors on A-afferent fibers innervating lamina V although meATP-sensitive P2X receptors were also found to be expressed on DH inhibitory interneurons (Jang et al. 2001). Our results also suggest that the meATP-sensitive P2X receptors are most likely to be nondesensitizing (or weakly desensitizing) P2X receptor subtype.
Great attention has been given to P2X2+3 receptors for their sensory functions due to its nondesensitization property (Lewis et al. 1995; North 2002). It has been widely believed by many research groups that P2X2+3 receptors mediate the modulation of synaptic transmission from A-afferent terminals to lamina V neurons (North 2002). This speculation, however, is likely to be incorrect because P2X3 subunits are mainly located on VR1-expressing afferent terminals (Guo et al. 1999) while meATP-sensitive afferent terminals to lamina V neurons are capsaicin-insensitive (Nakatsuka and Gu 2001; Nakatsuka et al. 2002). The lack of inhibition by 1 µM TNP-ATP on meATP-mediated synaptic responses in lamina V (Fig. 1) (also see Nakatsuka et al. 2003) also does not support the involvement of P2X2+3 receptors because P2X2+3 receptors are highly sensitive to TNP-ATP. The lack of TNP-ATP inhibition in the lamina V recordings was unlikely due to the potential poor delivery of intact TNP-ATP to the recorded cells (Lewis et al. 1998) because TNP-ATP (1 µM) could completely abolish meATP-induced increases of mEPSC frequency when recordings were made in lamina II (Nakatsuka et al. 2003).
When directly examined from the somata of primary afferent fibers, multiple phenotypes of meATP-evoked currents were identified. In addition to the three major phenotypes (fast, mixed, and slow P2X currents) described previously (Grubb and Evans 1999; Li et al. 1999; Ueno et al. 1999), we have also found that some subphenotypes of P2X currents were present in each major P2X phenotypes in DRG neurons. Fast P2X currents in primary sensory neurons are thought to be mediated by homomeric P2X3 receptors (Cook et al. 1997). Consistently, we found that fast currents in DRG neurons were highly sensitive to the subtype-selective P2X receptor antagonist TNP-ATP (Virginio et al. 1998). We have found double-peak fast currents in some small-sized DRG neurons, which may be due to the co-expression of two subtypes of fast P2X receptors (Petruska et al. 2000b). Because of their rapid desensitization and high sensitivity to TNP-ATP, P2X receptors that show fast currents are unlikely to be involved in meATP-induced long-lasting increases of mEPSC frequency recorded in lamina V neurons.
Mixed currents and slow currents have been demonstrated previously (Burgard et al. 2000; Grubb and Evans 1999; Li et al. 1999; Ueno et al. 1999) and were reported to be sensitive to TNP-ATP (Burgard et al. 2000; Xu and Huang 2002). We have also found that TNP-ATP (1 µM) completely inhibited mixed currents and slow currents in some DRG neurons. These results are consistent with the idea that P2X2+3 subtypes are expressed on DRG neurons. However, we have found that slow currents and slow components of the mixed currents in 25% of DRG neurons had low sensitivity to TNP-ATP block. These results indicate that in addition to P2X2+3 receptors, other P2X receptors may also mediate meATP-evoked slow currents and slow component in the mixed currents in some DRG neurons. The presence of meATP-sensitive/TNP-ATP-resistant ionic currents on the somata of primary afferent neurons is consistent with the TNP-ATP-resistant responses in lamina V recordings in the spinal-cord slice preparation. TNP-ATP resistant currents were not reported previously, probably because cells with these P2X receptors became less abundant after dissociation procedures. Alternatively, the meATP-sensitive/TNP-ATP-resistant P2X ionic current may be neglected previously because their current amplitude was usually much smaller than meATP-sensitive/TNP-ATP-sensitive slow currents in DRG neurons (Fig. 5).
The meATP-sensitive/TNP-ATP-resistant currents were unlikely mediated by P2X4, P2X6, and P2X7 receptors because these three subtypes are insensitive to PPADS (see Khakh et al. 2001). The lack of effect by IVM also excludes P2X4 subtype. P2X2, P2X5, and P2X2+6 receptors were unlikely to be involved because these subtypes are not sensitive to meATP (Khakh et al. 2001). The use of 100 µM meATP might compromise its subtype selectivity. However, meATP at 10 µM also induced meATP-sensitive/TNP-ATP-resistant currents. P2X1, P2X3, and P2X1+2 receptors (Brown et al. 2002) could be excluded because they showed rapid desensitization; both P2X1 receptors and P2X3 receptors are highly sensitive to the block by TNP-ATP.
P2X4+6 and P2X1+5 receptors were sensitive to meATP with weak desensitization (Haines et al. 1999; Le et al. 1998; Torres et al. 1998). P2X1+5 receptors were found to have low sensitivity to TNP-ATP (Haines et al. 1999). TNP-ATP at 1 µM did not significantly inhibit ATP-evoked currents on Xenopus oocytes co-injected with P2X4 and P2X6 subunits (data not shown), raising a possibility that P2X4+6 receptors were not sensitive to TNP-ATP. Moreover, both of these two subtypes were sensitive to the block by low concentrations of PPADS (Khakh et al. 2001). Thus of the 11 functional P2X subtypes identified so far, only P2X4+6 and P2X1+5 receptors could not be excluded based on the pharmacological and electrophysiological profiles. However, other possibilities remain since unidentified heteromeric P2X receptors may be present (North 2002) in native cells.
We have shown that slow currents and slow components of mixed currents have a broad range of sensitivity to 1 µM TNP-ATP. This could be due to the co-expression of TNP-ATP-resistant P2X receptors and TNP-ATP-sensitive P2X receptors in the same cells in different abundance (Spelta et al. 2002; Thomas et al. 1998; Zhong et al. 2000). Another possibility is the presence of many subtypes of nondesensitizing P2X receptors in different sensory neurons. The current development of subtype-selective P2X receptor antagonists will greatly help us to identify P2X receptor subtypes expressed on different functional groups of sensory neurons and to understand the roles of these P2X receptor subtypes in modulating sensory transmission.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by a Canadian Institute of Health Research Grant MOP-14718 to P. éguéla and by a National Institute of Neurological Disorders and Stroke Grant NS-38254 to J. G. Gu.
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FOOTNOTES |
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Address for reprint requests: J. G. Gu, Dept. of Oral and Maxillofacial Surgery, McKnight Brain Institute and College of Dentistry, University of Florida, Box 100416, Gainesville, FL 32610 (E-mail: jgu{at}dental.ufl.edu).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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