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Changes of the excitability of rat trigeminal root ganglion neurons evoked by α2-adrenoreceptors
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Neuroscience Vol. 115, No. 3, pp. 731^741, 2002 H 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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CHANGES OF THE EXCITABILITY OF RAT TRIGEMINAL ROOT GANGLION NEURONS EVOKED BY K2 -ADRENORECEPTORS M. TAKEDA,a M. IKEDA,a T. TANIMOTO,a J. LIPSKIb and S. MATSUMOTOa a
Department of Physiology, School of Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan b
Department of Physiology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92-019, Auckland, New Zealand
Abstract9The aim of the study was to examine the e¡ects of K2 -adrenoreceptor agonists on the excitability of trigeminal root ganglion (TRG) neurons using the perforated patch-clamp technique, and to determine whether these neurons express mRNA for K2 -adrenoreceptors. In current-clamp mode, the resting membrane potential was 357.4 A 1.2 mV (n = 26). Most neurons (71%) were hyperpolarized by clonidine (5^50 WM) in a concentration-dependent manner. The response was associated with an increase of cell input resistance. In addition, clonidine reduced the repetitive ¢ring evoked by depolarizing current pulses. An K2 -adrenergic agonist, UK14,304, (10^20 WM) also hyperpolarized TRG neurons. The clonidine- and UK14,304-induced hyperpolarization was blocked by idazoxan (K2 -adrenoreceptor antagonist). In voltage-clamp, clonidine (1^50 WM) reversibly reduced the hyperpolarization- and time-dependent cationic current. The e¡ect was mimicked by UK14,304 (10^20 WM), and antagonized by idazoxan. Hyperpolarization-activated cationic current was blocked by extracellular Csþ (2 mM) or a speci¢c blocker, ZD7288 (20WM). Analysis of tail currents revealed that a reversal potential of the clonidine-sensitive component of hyperpolarization-activated cationic current was 346 mV. Single-cell reverse transcription-polymerase chain reaction analysis demonstrated the expression of mRNA for K2A - and K2C -adrenoreceptors. These results demonstrate that activation of K2 -adrenoreceptors can hyperpolarize TRG neurons, and that the inhibitory e¡ect is associated with inhibition of hyperpolarization-activated cationic current. Our results suggest that activation of K2 -adrenoreceptors in the absence of nerve injury may have an inhibitory e¡ect on nociceptive transmission in the trigeminal system at the level of both TRG neuronal cell bodies and primary a¡erent terminals. H 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: trigeminal root ganglion, perforated patch-clamp, reverse transcription-polymerase chain reaction, K2 -adrenoreceptors.
renergic sensitivity in nociceptive a¡erents is observed following many di¡erent neuropathic injuries, including the neuroma, a chronic nerve lesion, and the chronic constriction injury (CCI), there is increasing evidence to indicate that sympathetic^a¡erent interaction at the level of the DRG may not play a signi¢cant role in neuropathic pain (Devor et al., 1994; Xie et al., 1995; Ringkamp et al., 1999; Ha«bler et al., 2000). Less attention has been paid to injuries of trigeminal a¡erents than to those involving spinal nerves. There is some evidence that the anatomical and physiological consequences of trigeminal nerve crush or ligation di¡er from those seen after sciatic nerve injury. For example, sprouting of sympathetic ¢bers observed in the sensory ganglia associated with spinal nerve injury does not occur in the trigeminal root ganglion (TRG) (Bongenhielm et al., 1999; Benoliel et al., 2001), and the spontaneous activity in a¡erents ¢bers after trigeminal injury is signi¢cantly smaller than that seen after injury to the sciatic nerve (Tal and Devor, 1992). In£ammatory cytokines, such as interleukin (IL-6), and the nerve growth factor (NGF) may contribute to development of mechanical allodynia after trigeminal nerve injury, but release of these factors is not closely related
Injury of peripheral nerves can lead to neuropathic pain accompanied by hyperalgesia and/or allodynia (Bonica, 1979; Roberts, 1986; Chung et al., 1996; Anderson and Rao, 2001). Two forms of neuropathic pain have been identi¢ed: sympathetically-independent and sympathetically-maintained pain (Bonica, 1979; Roberts, 1986). With regard to the latter, a sympatho^sensory coupling has been found in dorsal root ganglion (DRG) neurons of the sciatic nerve-injured animals (McCormik and Pape, 1990; Chung et al., 1993; Devor et al., 1994), and it has been suggested that noradrenaline (NA) released from sympathetic nerve endings acts directly on K2 -adrenoreceptors expressed in these neurons (Maclachlan et al., 1993; Xie et al., 1995). Although ad-
*Corresponding author. Tel./fax: +81-3-3261-8740. E-mail address: [email protected] (M. Takeda). Abbreviations : CCI, chronic constriction injury; DRG, dorsal root ganglion; Ih , hyperpolarization-activated cationic current; IL-6, interleukin-6; NA, noradrenaline; NGF, nerve growth factor; RT-PCR, reverse transcription-polymerase chain reaction ; TRG, trigeminal root ganglion; VDCCs, voltage-dependent Ca2þ currents. 731
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to the onset or duration of pain after trigeminal injury (Anderson and Rao, 2001). Thus the question arises as to whether there are di¡erences between the trigeminal and sciatic sensory nerves in the response to activation of K2 -adrenoreceptors by NA, but there are no studies examining this possibility. Superfusion with NA hyperpolarizes dorsal horn neurons in a slice preparation obtained from the adult rat spinal cord, and the NA-induced hyperpolarization can be blocked by an K2 -adrenoreceptor antagonist, yohimbine (North and Yoshimura, 1984). This result indicates that NA can inhibit nociceptive input within the spinal cord. In addition, clonidine (an K2 -adrenoreceptor agonist) has an antinociceptive e¡ect (Davis et al., 1991; Glynn et al., 1992), and there is evidence that iontophoretic application of this agent inhibits N-methyl-Daspartic acid-evoked responses of nociceptive neurons in the trigeminal spinal nucleus caudalis (Zhang et al., 1998). The inhibition could be reversed by an K2 -adrenoreceptor antagonist, idazoxan, but not by an K1 -adrenoreceptor antagonist, prazosin (Zhang et al., 1998). However, these results were based on experiments, which did not discriminate between presynaptic and postsynaptic e¡ects, and the question arises which of two mechanisms is important for clonidine action. In addition, it has been reported that K2 -adrenoreceptor activation inhibits hyperpolarization- and time-dependent cationic current (Ih ) and modulates DRG neuronal activity (Yagi and Sumino, 1998). It has been demonstrated in a variety of neurons, and is thought to play an important role in modulating cell excitability, neuronal ¢ring and transmitter release (Mayer and Westbrook, 1983; McCormik and Pape, 1990; Scroggs et al., 1994; Villiere and Mclachlan, 1996; Parkis and Berger, 1997). Ligand binding studies have demonstrated the presence of K2 -adrenoreceptors on primary a¡erent neurons (Howe et al., 1987). There is also immunochemical evidence that more than 50% of cell bodies of DRG express K2A -adrenoreceptors. Gold et al. (1997) demonstrated that DRG neurons express mRNA for all three known K2 -adrenoreceptor subtypes: K2A , K2B and K2C , by using reverse transcription-polymerase chain reaction (RTPCR). An in situ hybridization study has demonstrated the presence of the corresponding mRNAs in normal rat DRG, and their up-regulation of K2A -message after peripheral nerve injury (Shi et al., 2000). The latter e¡ect suggests an involvement of K2A -adrenoreceptors in spinal (DRG) system in sympathetically-maintained pain. The aim of the present study was to examine the e¡ect of K2 -adrenoreceptor agonists on the excitability of TRG neurons using electrophysiological techniques. In addition, we investigated the expression of K2 -adrenoreceptor subtypes in these neurons with single-cell RT-PCR. EXPERIMENTAL PROCEDURES
Acute dissociation of TRG neurons Rat pups (P6-P17) were decapitated. Both TRGs were rapidly removed and immersed in oxygenated ice-cold PIPES bu¡er consisting of (in mM) 120 NaCl, 5 KCl, 0.1 CaCl2; 1 MgCl2; 20 PIPES (piperazine-N,NP-bis [2-ethanesulfonic acid]), 0.1
ascorbic acid and 25 glucose (pH 7.3). Each ganglion was cut into several smaller pieces which were preincubated in PIPES bu¡er for 30 min at room temperature and then incubated for 15 min (at 30‡C) with 20 U/ml papain (Worthington), 1.6 mM L-cysteine, 0.2 mM EDTA and 13.4 mM L-mercaptoethanol (Sigma, St. Louis, MO, USA) in a total volume of 5 ml. The incubation was followed by several washes and 30 min postincubation in the same bu¡er at room temperature. All incubations and washes were conducted in a custom-built chamber, with continuous gentle stirring and bubbling with 100% O2 . After post-incubation, the tissue was triturated with ¢re-polished Pasteur pipettes in the standard external solution containing (in mM): 155 NaCl, 3 KCl, 1 CaCl2 , 1 MgCl2 , 10 HEPES (N-2-hydroxyethylpiperazine-NP-2-ethanesulfonic acid) and 20 glucose (pH 7.3). Cells were plated at low density on poly-Llysine-coated coverslips placed in a recording chamber (RC-13, Warner Instr.; volume: 0.4 ml). The recording chamber was mounted on the stage of an inverted microscope (Nikon, TE300, Tokyo, Japan) equipped with phase-contrast, video camera and a micromanipulator (Narishige, MHW-3, Tokyo, Japan). The chamber was perfused under gravity with standard external solution at approximately 0.5 ml/ml. Whole-cell patch-clamp recording Recordings were conducted by using the ‘rapid’ perforatedpatch technique (Rae et al., 1991; Dean et al., 1997; Takeda et al., 2001). Fire-polished pipettes (2^5 M6) were ¢lled with a solution containing (in mM): 120 potassium methanesulphonate, 20 KCl, 7.5 HEPES and 2 EGTA, pH 7.3, with amphotericin B (200 Wg/ml). Current- and voltage-clamp recordings were conducted with an Axopatch 200B ampli¢er (Axon Instr., Foster City, CA, USA). A low-sodium solution (80 mM) was made by equimolar substitution of choline chloride for NaCl. A high-potassium solution (25 mM) was prepared by substituting 22 mM KCl for NaCl. Signals were low-pass ¢ltered at 1 or 5 kHz and digitized at 10 kHz. To monitor changes in cell membrane resistance during recordings in current-clamp, negative current pulses (5^30 pA, 250 ms, 0.2 Hz) were injected through the patch pipette. In the voltage-clamp mode, input conductance was measured by applying +20 mV voltage steps from the holding potentials of 360 mV (250 ms, 0.2 Hz). The step commands were within the linear portion of the I^V relationships established with depolarizing ramps (from 3140 mV to +60 mV, 300 ms). Ih was identi¢ed by its response to a speci¢c blocker ZD7288 (BoSmith et al., 1994; Saitow and Konishi, 2000). The reversal potential of this current was determined by tail-current analysis (Yagi and Sumino, 1998). All recordings were performed at room temperature. Data were stored on a computer disk for o¡-line analysis. Single-cell RT-PCR Single-cell RT-PCR was performed as described in a previous study (Takeda et al., 2001). Each dissociated neuron was aspirated into a pipette (tip diameter, 6^8 Wm) containing 1URT bu¡er (8.5 Wl). Care was taken to avoid aspiration of any cell debris. The contents of the pipette, transferred into a 0.5-Wl nonstick PCR tube, were freeze/thawed and vortexed to release RNA. Four to 6 Wl of cDNA was used for ¢rst-round ampli¢cation of 35 cycles in a ‘multiplex’ PCR reaction: one containing the external primers for glyceraldehyde phosphatase dehydrogenase (GAPDH) and the other containing the external primers for K2A -, K2B -, and K2C - adrenoreceptors, each at a 50-nM concentration. Primer sequences for GAPDH were as published previously (Comer et al., 1997). Second-round ampli¢cation (45 cycles) was performed separately for each gene with 3 Wl of the ¢rst-round product and 500 nM of the upstream (5P) and internal primer (‘semi-nested’ protocol). The internal primer sequences for K2A -, K2B -, and K2C -adrenoreceptors and conditions of PCR reactions have been published previously (Phillips and Lipski, 2000; Vidovic et al., 1994). PCR reactions were performed in a 25-Wl volume containing 0.625 U DNA polymerase, 1UPCR bu¡er, 0.25 mM dNTPs (Gibco, BRL), 200 nM of
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Fig. 1. E¡ects of clonidine and UK14,304 on the membrane potential and input resistance of acutely dissociated TRG neurons (current-clamp mode). (A) Neuron hyperpolarization and increase in input resistance after clonidine application (20 WM). The break in the trace is 3 min. (B) Dose^response relationship for clonidine application. (C) Neuron hyperpolarization and increase in input resistance after UK14,304 application (10 WM). (D) Clonidine-evoked e¡ects were abolished by idazoxan (1 WM). No obvious e¡ect of idazoxan application on the membrane potential observed. The break in the trace is 2 min. The dashed lines in A, C and D indicate the resting membrane potential.
each primer and 1.5^2.5 mM MgCl2 . Negative controls were conducted in the absence of both starting RNA and RT enzyme. Ampli¢cations were performed for 35 cycles in a PTC-1001 thermal cycler (MJ Research). The ¢nal cycle lasted for 5 min at 72‡. PCR fragments were run on a 3% agarose gel, stained with ethidium bromide and illuminated with UV light. The identity of individual PCR fragments, ampli¢ed with these primers from brainstem tissue was con¢rmed by sequencing.
(mean diameter 24.1 A 0.6 Wm, n = 74; range, 15^28 Wm) and had a bright appearance with a ‘halo’ around the cell body when viewed under phase contrast. Following perforation of the cell membrane with amphotericin B, the series resistance dropped to 6 20 M6 (17.9 A 0.8 M6; n = 74) within 5^10 min and remained stable for s 15 min. The cell capacitance was 18.4 A 0.9 pF (n = 68).
Application of drugs All drugs (stock solutions) were stored at 320‡C, and dissolved in standard external solution. Clonidine hydrochloride (1^50 WM, Sigma), UK14,304 (10^20 WM, Sigma), idazoxan hydrochloride (1^5 WM, Tocris, Cookson, Northpoint, UK), and ZD7288 (2^20 WM, Tocris) were added to the perfusate for a period of up to 90 s. Data analysis Data were stored on a computer disk for o¡-line analysis (pClamp 8.0). Values are expressed as means A S.E.M. Analysis of drug e¡ect was made with Student’s t-test. P 6 0.05 was considered signi¢cant.
RESULTS
Acutely isolated TRG neurons were spherical in shape
E¡ects of K2 -adrenoreceptor agonists on the membrane potential of TRG neurons The resting membrane potential recorded in the wholecell current-clamp mode was 357.4 A 1.2 mV (n = 26). Clonidine induced changes in the membrane potential in 12 of the 14 cells tested. Most neurons were hyperpolarized (10/14, 72%) as shown in Fig. 1A, but some were depolarized (2/14, 14.2%). The remaining two cells did not respond. The clonidine-evoked hyperpolarization was concentration-dependent (Fig. 1B). In neurons showing hyperpolarization (20 WM, clonidine), the cell input resistance increased by 33.9 A 4.9% (n = 5). The duration of hyperpolarization ranged from 5 to 18 min. A similar hyperpolarizing e¡ect (associated with input resistance increase was also observed after application
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Fig. 2. E¡ect of clonidine on ¢ring frequency of TRG neurons. (A) Application of 20 WM clonidine reduced the ¢ring in response to 60 pA depolarizing pulses and hyperpolarized cell membrane potential. (B) Mean reduction in number of spikes in response to 60 pA/250 ms pulses (n = 5). *P 6 0.01.
of an K2 -adrenoreceptor agonist, UK14,304 (in four out of ¢ve tested cells; Fig. 1C). The clonidine- and UK14,304-evoked membrane hyperpolarizations were abolished by an K2 -adrenoreceptor antagonist, idazoxan (1 WM;n = 3). No signi¢cant e¡ects of idazoxan application on its own on the membrane potential were observed (Fig. 1D).
E¡ect of clonidine on ¢ring of TRG neurons As illustrated in Fig. 2A, isolated TRG neurons ¢red repeatedly in response to depolarizing current pulses (300 ms, 60 pA). Application of 20 WM clonidine not only hyperpolarized the membrane potential, but also reduced the number of action potentials evoked by this depolar-
Fig. 3. Currents evoked by voltage steps in TRG neurons. (A) Example of Ih of small diameter neuron (diameter, 21 Wm) were evoked in response to 200 ms pulses (steps from 3120 mV to 340 mV, holding potential of 360 mV, 10 mV increments). Inhibitory e¡ect of Csþ on Ih . (B) Current^voltage relations for Ih recorded in the TRG neurons before and after application of Csþ (2 mM) (n = 5). Inset shows a hyperpolarizing voltage step which elicited an instantanenous inward current (Iinst ) followed by Ih .
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Fig. 4. E¡ect of K2 -adrenoreceptor agonists on Ih induced in TRG neurons by hyperpolarizing voltage steps. (A) Example of inhibitory e¡ect of clonidine (20 WM). (B) Current^voltage relationship for Ih recorded before and after clonidine application. (C) Concentration-dependent inhibition of Ih by clonidine (n = 5) (1 WM vs. 10, 20 and 50 WM, *P 6 0.01). (D) Example of inhibitory e¡ect of UK14,304 (20 WM). (E) Current^voltage relationship for Ih recorded in the TRG neurons before and after UK14,304 application. (F) A comparison of inhibition of Ih by clonidine and UK14,304.
ization. The inhibitory e¡ect on cell ¢ring was signi¢cant (Fig. 2B). Analysis of Ih in TRG neurons Under voltage-clamp, Ih was produced by steps
between 3120 and 340 mV from a holding potential of 360 mV (10 mV increments). The inward currents evoked by the hyperpolarizing commands consisted of two components: instantaneous inward current (Iinst ), and a slowly activating inward current. The Ih was determined by subtracting Iinst from the total current mea-
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Fig. 5. E¡ect of idazoxan on inhibition of Ih by clonidine. (A) Example of the antagonistic e¡ect of idazoxan. (B) Current^ voltage relationship for Ih recorded under control conditions, during clonidine application and during co-application of clonidine with idazoxan (n = 5).
Fig. 6. Reversal potential of clonidine-sensitive current. (A) In the voltage-clamp mode, the whole-cell current of small TRG neurons (diameter 24 Wm) were recorded in standard external solution in response to a twin pulse protocol (bottom) comprising a 200-ms hyperpolarizing prepulse (to 3120 mV from a holding potential of 360 mV), followed by test pulses to di¡erent voltage levels (from 370mV to 320 mV in 10 mV increments) before and after clonidine application. (B) Relationship between tail-current amplitude and test voltage steps in standard external solution and in low Naþ (80 mM) and high Kþ (20 mM) solutions (n = 5).
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Fig. 7. Comparison of inhibition of Ih by clonidine and a speci¢c blocker, ZD7288. (A) Example of original data. (B) Mean A S.E.M. of inhibitory e¡ects of both drugs (n = 5).
sured at the end of the pulses (Fig. 3). This current was observed in 40/48 neurons tested (83.3%). Its peak amplitude at 3120 mV was 291.2 A 20.5 pA (n = 35). Application of Csþ ions that are known to block Ih in di¡erent types of neurons (Mayer and Westbrook, 1983; Lucero and Pappone, 1990; Saitow and Konishi, 2000) abolished Ih in TRG neurons (Fig. 3A, B). E¡ects of K2 -adrenoreceptor agonists on Ih The e¡ect of K2 -adrenoreceptor agonists on Ih was studied in 40 neurons. Clonidine application (20 WM) reversibly reduced the currents within 5 min (Fig. 4A). The I^V relationships are shown in Fig. 4B. The inhibition was concentration-dependent (1^50 WM; Fig. 4C; n = 5) and the e¡ect was mimicked by application of UK14,304 (10^20 WM; Fig. 4D, E; n = 5). There was no signi¢cant di¡erence between the inhibition of Ih by clonidine and UK14,304 (Fig. 4F). As shown in Fig. 5A, B, clonidine induced inhibition of Ih , which was blocked by idazoxan (1 WM). The reversal potential of a clonidine-sensitive component of Ih was determined by tail-current analysis as described previously (Yagi and Sumino, 1998). The mean reversal potential of the clonidine-sensitive current was 346 mV (n = 4) in standard external solution ([Kþ ] = 3 mM, [Naþ ] = 155 mM; Fig. 6). In a low Naþ solution ([Kþ ] = 3 mM, [Naþ ] = 80 mM), the reversal potential shifted to 330 mV; in high Kþ solution ([Kþ ] = 20 mM, [Naþ ] = 155mM), it shifted 356 mV. These results indicate that the channels a¡ected by clonidine are permeable to both Naþ and Kþ ions.
Relationship between hyperpolarization of the membrane and inhibition of Ih evoked by K2 -adrenoreceptor agonist As illustrated in Fig. 7A, Ih was also inhibited by a speci¢c Ih blocker, ZD7288. To determine whether other ionic mechanisms were involved in the hyperpolarizing action of K2 -adrenoreceptor agonists, inhibition of Ih induced by clonidine (20 WM) was compared with that evoked by ZD7288 (20 WM). During voltage step to 3120 mV, the mean inhibition of Ih evoked by clonidine was similar to that observed after application of ZD7288 (n = 4; Fig. 7B). Expression of K2 -adrenoreceptors in the TRG neurons The expression of mRNAs for K2A -, K2B - and K2C adrenergic receptor subtypes was tested in dissociated TRG neurons by means of single-cell RT-PCR. Individual small-diameter TRG neurons (15^27 Wm in diameter, n = 9) were isolated, and their mRNA were reversely transcribed into cDNA. cDNA was ¢rst subject to PCR with primers speci¢c for GAPDH, a step necessary to con¢rm successful cell collection and RT reaction. Only cells that tested positive for GAPDH (n = 6) were further analyzed. Figure 8 shows the results for a neuron which was positive for K2A - and K2C - adrenergic receptor mRNA. K2A -adrenoreceptor mRNA was detected in two out of six TRG neurons tested, whereas K2C message was detected in one out of six neurons tested. K2B -adrenoreceptors were not detected in any of the neurons tested.
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nist, UK14,304 (Takeda et al., 2001), and was blocked by idazoxan, a speci¢c blocker of K2 -adrenoreceptors (Zhang et al., 1998). Although it has been reported that clonidine has a⁄nity for imidazoline receptors, resulting in membrane potential depolarization (Ernsberger et al., 1997), we found that in all neurons demonstrating repetitive ¢rings in response to depolarizing current pulses, clonidine application inhibited the ¢ring. These results suggest that the activation of K2 adrenoreceptors inhibits the excitability of TRG neurons. This conclusion extends previous observations that presynaptic K2 -adrenoreceptors are also located on primary a¡erent terminals in the trigeminal spinal nucleus, and mediate inhibition of glutamate release (Travagli and Wiliams, 1996). Our results indicate that modulation of nociceptive transmission in the trigeminal system by activation of K2 -adrenoreceptors can occur both at the level of TRG neuronal cell bodies and primary a¡erent terminals. Inhibition of Ih by K2 -adrenoreceptor agonist
Fig. 8. Example of mRNAs coding for K2A - and K2C -adrenoreceptor subtypes in the two acutely dissociated TRG neurons. Gel electrophoresis of PCR products obtained from TRG neurons (lanes 3 and 4) and brain stem tissue (positive control ; lane 1). Lane 2: Negative control (no reverse transcription) ; lane 3: PCR products obtained with primers for K2A -adrenoreceptor; lane 4: products obtained with primers for K2A - and K2C -adrenoreceptors. DISCUSSION
The present experiments provided evidence that activation of K2 -adrenoreceptors can hyperpolarize TRG neurons, and that the inhibitory e¡ect is associated with inhibition of Ih . In addition, we ¢rstly demonstrated that the expression of mRNA coding for K2 -adrenoreceptors in single TRG neurons. Our results suggest that activation of K2 -adrenoreceptors in the absence of nerve injury may have an inhibitory e¡ect on nociceptive transmission in the trigeminal system at the level of both TRG neuronal cell bodies and primary a¡erent terminals. Changes of the membrane potential and inhibition of ¢ring evoked by K2 -adrenoreceptor agonists Application of clonidine (10^50 WM) evoked a concentration-dependent hyperpolarization in the majority of TRG neurons tested and the response was associated with an increase in cell input resistance. However, in the some neurons, clonidine caused a depolarization or had no signi¢cant e¡ect on the membrane potential. The hyperpolarizing e¡ect of clonidine resembled that seen after application of a selective K2 -adrenoreceptor ago-
Application of clonidine inhibited a slowly activating cationic current (Ih ) evoked by membrane hyperpolarization and this inhibition was concentration-dependent (1^ 50 WM). Yagi and Sumino (1998) reported that EC50 of inhibition of Ih by clonidine in the DRG was about 2.2 WM. In the present study, on acutely dissociated TRG neurons, concentrations of K2 -adrenoreceptor agonist were higher than endogenous NA ( 6 1 WM). Thus, it is possible that clonidine-induced Ih inhibition may involve either a reduction of the number of functional receptors or, a non-speci¢c e¡ect of K2 -adrenoreceptors. We, however, found evidence that clonidine-induced inhibition of Ih was blocked by a speci¢c K2 -adrenoreceptor blocker, idazoxan. Similar concentrations used in the present study (10 WM) examining modulation of K2 -adrenoreceptor agonists have been used in the DRG neurons and hypoglossal motoneurons (Parkis and Berger, 1997; Xie et al., 1995; Homma et al., 1999). Therefore it is more likely that clonidine-induced inhibition of Ih may be mediated by the activation of K2 -adrenoreceptor e¡ect. The characteristics of clonidine-sensitive current were similar to those of the hyperpolarization-activated cationic currents described in previous studies in other neurons and non-neuronal cells (Mayer and Westbrook, 1983; McCormik and Pape, 1990; Villiere and Mclachlan, 1996). In this study, the identi¢cation of Ih was performed as follows: (1) The currents were blocked by Csþ application (the ability of this ion to block Ih in thalamic and DRG neurons was described previously (McCormik and Pape, 1990; Scroggs et al., 1994); (2) The reversal potential of the current was 346 mV, which resembled values reported previously, and suggests that the current was carried a mixed Naþ ^Kþ current (Mayer and Westbrook, 1983; Yagi and Sumino, 1998; Saitow and Konishi, 2000); (3) The magnitude of decrease of Ih after clonidine application was similar to that observed after application of the speci¢c blocker ZD7288 (McCormik and Pape, 1990; Saitow and Konishi, 2000). Our results show that the K2 -adrenore-
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ceptor agonists can reversibly inhibit Ih at least in neonatal TRG neurons. In current-clamp recordings, application of clonidine usually caused hyperpolarization of the membrane potential. We propose that this hyperpolarization occurs as a result of inhibition of Ih , because the reversal potential of Ih was more positive than the resting membrane potentials of TRG neurons. The fact that the holding potential was more negative than the reversal potential of Ih suggests that the Ih channel activated tonically at the holding level would produce a cation in£ux (Mayer and Westbrook, 1983; Scroggs et al., 1994). Therefore, Ih can be involved in both the setting of the level of the resting membrane potential as well as of the neuronal excitability. Functional signi¢cance of inhibition of TRG neuronal excitability by K2 -adrenoreceptors It has been reported that there is no sprouting of sympathetic nerve ¢bers in the trigeminal ganglion after peripheral trigeminal nerve injury (Bongenhielm et al., 1998; Benoliel et al., 2001) and that spontaneous activity observed in trigeminal a¡erents after trigeminal peripheral nerve injury is signi¢cantly smaller than that seen after injury to the sciatic nerve (Tal and Devor, 1992). Although the reason for this di¡erence is unclear, it is possible that it is due to some unique characteristics of the trigeminal nerve, For examples, this nerve carries almost equal numbers of myelinated and non-myelinated ¢bers, unlike many cutaneous nerves of spinal origin, which carry a considerably higher proportion of non-myelinated ¢bers (Matsuda et al., 1969; Fried and Hildebrand, 1982). The location of the nerve within a canal in the skeleton makes it more vulnerable to pressure after peripheral nerve injury (Bongenhielm et al., 1999). Finally, there is a di¡erence in embryonic origin; the sciatic nerve derives from the segmental neural crest, while many trigeminal a¡erents from the ectodermal placode (D’Amico-Martel and Norden, 1983). Chung et al. (1996) have demonstrated that sympathectomy performed at 4 days after L5^6 spinal nerve ligation reduces mechanical and cold allodynia and eliminates sympathetic sprouting. However other investigators were unable to reproduce this ¢nding (Ringkamp et al., 1999). Similarly, sympathectomy has no signi¢cant e¡ect on the early ectopic activity from neuromas of the ferret inferior trigeminal nerve (Bongenhielm et al., 1998). It is therefore possible that sympathetic sprouting is not required for the generation of ectopic activity in damaged trigeminal nerve. This idea is further supported by the ¢nding demonstrating that sprouting is not related to the sympathetic^sensory coupling in the DRG neurons: neurophysiological data (adrenergic sensitivity) are obtained 4^22 days after sciatic nerve lesion but sprouting is starting on the 20th day (Devor et al., 1994). In contrast, it has been reported that the increases in IL-6 and NGF may be related to the development of mechanical allodynia after trigeminal injury (Anderson and Rao, 2001). Thus, release of both IL-6 and NGF may be partly involved in the generation of ectopic dis-
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charges from injured trigeminal nerve. Because there is evidence that in the L5 spinal nerve injury model, sympathetic^sensory coupling does not occur during physiological activity in sympathetic neurons (Ha«bler et al., 2000), it is possible that sympathetic^sensory coupling may be generated indirectly via alternation of blood £ow in the TRG neurons. After peripheral nerve injury, population of neuronal cell bodies in DRG are known to generate spontaneous ectopic discharges (Chung et al., 1993; Maclachlan et al., 1993; Devor et al., 1994; Xie et al., 1995; Chung et al., 1996). Also there is a positive correlation between the ectopic discharges and allodynia-like behavior in spinal nerve-lesioned rats, and their activity occurred in A-¢bers, but not C-¢bers (Liu et al., 2000a,b). In contrast, ectopic discharges in most spontaneously ¢ring DRG neurons of both A- and C-¢bers were suppressed by sympathetic^a¡erent activity and this suppression predominant response in animals with long-standing nerve injury (Michaelis et al., 1996). In the rat DRG neurons, it is known that there is a positive correlation between the neuronal cell size and axonal conduction velocity both for AN- and for C-¢ber a¡erents (Harper and Lawson, 1985). The TRG neurons recorded in the present study are probably included both AN- and C-type neurons, because the cell body diameter of AN-type neurons overlaps the border between small and median diameter ( 6 30 Wm) acutely isolated DRG neurons (Harper and Lawson, 1985). Yagi and Sumino (1998) speculated that if the after-hyperpolarization following action potentials were su⁄ciently negative and lasted long enough to activate Ih in DRG neurons, then this current would contribute to facilitation of the DRG neuronal activity. In this study we found that K2 -adrenoreceptor agonists inhibited Ih and reduced the ¢ring rate of TRG neurons, suggesting that inhibition of Ih by K2 -adrenoreceptor activation may decrease the ¢ring rate of spontaneously activate TRG neurons. In contrast, it has been known that there is a di¡erence between intact and injured DRG neurons in respect to their sensitivity. For examples, NA inhibited voltage-dependent Ca2þ currents (VDCCs) in CCI rats, whereas it enhanced these currents in control rats (Homma et al., 1999). Moreover, K2 -adrenergic stimulation also inhibited outward Kþ currents in the CCI rats, while it had no e¡ects on the currents in control rats. The authors concluded that NA-induced inhibitory e¡ects on VDCCs and KCa channels in injured DRG neurons could lead to cell membrane depolarization, and increased frequency of action potentials (Homma et al., 1999). In the context of these ¢ndings, it is possible that K2 -adrenergic stimulus acts not only through modulation of Ih , but also on the VDCCs and KCa channels in injured TRG neurons. In the TRG neurons, Ih has been previously identi¢ed in large-, medium-, and small-diameter cell bodies, and its amplitude is cell size-dependent (Scroggs et al., 1994). Moreover, approximately 37^45% of small diameter (15^ 33 Wm) DRG neurons demonstrated Ih (Scroggs et al., 1994; Yagi and Sumino, 1998). In the present study, we found Ih in 83% of TRG neurons (15^28 Wm) tested. Although K2A - and K2C -adrenoreceptor mRNA expres-
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sion was identi¢ed in small population of TRG neurons, this is the ¢rst study to demonstrate expression of mRNA coding for K2 -adrenergic receptors in single TRG neurons. Because the trigeminal ganglion is nonhomogeneous and contains not only sensory neurons, but also glia and satellite cells (Matsuda et al., 1969), the fact that K2 -adrenoreceptor mRNA expression was found in the single TRG neurons leads us to suggest the possibility of neuronal modulation by K2 -adrenoreceptors. The previous study of in situ hybridization study has revealed that in normal rat DRG, the most common adrenergic receptor mRNA is for K2C -subtype (almost 80%), and that K2B -adrenergic receptor mRNA is only found in a small number of neuron pro¢les (Shi et al., 2000). In DRG neurons, K2A - adrenoreceptor mRNA increased to 45% after peripheral nerve injury, but K2C subtype showed a small decrease in expression (Shi et al., 2000). Thus, it appears that both K2A - and K2C -adrenergic receptors are closely related to the TRG neuronal function, including nociceptive transmission seen after trigeminal injury. In small DRG neurons in sciatic nerve-transected rats, there was a tendency to stronger inhibition of Ih by clonidine than in intact rats (Yagi and Sumino, 1998). Therefore, it is also possible that an increase in K2 -adrenoreceptor expression after trigeminal nerve injury may contribute to a stronger suppression of spontaneous activity in small TRG neurons. Intracellular recordings from DRG neurons have demonstrated that a large proportion of silent neurons is in£uenced by the spike activity of neighboring neurons
(Utzschneider et al., 1992). This suggests activity dependent cross-excitation in DRG neurons which could contribute to neuropathic sensory abnormalities triggered by nerve injury (Utzschneider et al., 1992). Amir and Devor (1996) reported repetitive ¢ring of DRG neurons induced by an increase in extracellular Kþ concentration and a non-synaptic release of neurotransmitters from adjacent neurons. In view of this observation, it is possible that certain substances accumulate in the extracellular space during stimulation of TRG neurons, di¡use toward neighboring neurons and modulate their activity. Actually, it has been recently reported that after nerve injury ongoing activity of DRG origins appears exclusively in small diameter myelinated muscle a¡erents, both axtomized and unlesioned, this ectopic activity is probably induced by paracrine signal released from DRG (Michaelis et al., 2000). Although we did not examine this mechanism in a neuropathic trigeminal pain model, our results suggest that inhibition of TRG excitability via K2 -adrenergic receptors may prevent ectopic discharges of injured TRG neurons as well as the increase of excitability of uninjured neighboring neurons. Further studies are needed to investigate such e¡ects. Acknowledgements,Thanks are due to Mrs. R. Dubey for technical help in preliminary experiments and Dr. J.K. Philips for advice regarding the RT-PCR experiments. This study was supported by a Grant from the Ministry of Education, Science and Culture of Japan (No. 13671953).
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Variation in Ih , Iir , and Ileak between acutely isolated adult dorsal root ganglion neurons of di¡erent size. J. Neurophysiol. 71, 271^279. Shi, T.J.S., Winzer-Serhan, U., Leslie, F., Ho«kfelt, T., 2000. Distribution and regulation of K2 adrenoreceptors in rat dorsal root ganglia. Pain 84, 319^330. Takeda, M., Philips, J.K., Dubey, R., Polson, J.W., Lipski, J., 2001. Modulation of Ach-induced currents in rat adrenal chroma⁄n cells by ligands of K2 -adrenergic and imidazoline receptors. Auton. Neurosci. 88, 151^159. Tal, M., Devor, M., 1992. Ectopic discharge in injuryed nerves : comparison of trigeminal and somatic a¡erents. Brain Res. 579, 148^151. Travagli, R.A., Wiliams, J.T., 1996. Endogenous monoamines inhibit glutamate transmission in the spinal trigeminal nucleus of the guinea-pig. J. Physiol. (Lond.) 491, 177^185. Utzschneider, D., Kocsis, J., Devor, M., 1992. Mutual excitation among dorsal root ganglion neurons in the rat. Neurosci. Lett. 146, 53^56. 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(Accepted 26 July 2002)
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Neuroscience Vol. 115, No. 3, pp. 731^741, 2002 H 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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CHANGES OF THE EXCITABILITY OF RAT TRIGEMINAL ROOT GANGLION NEURONS EVOKED BY K2 -ADRENORECEPTORS M. TAKEDA,a M. IKEDA,a T. TANIMOTO,a J. LIPSKIb and S. MATSUMOTOa a
Department of Physiology, School of Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan b
Department of Physiology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92-019, Auckland, New Zealand
Abstract9The aim of the study was to examine the e¡ects of K2 -adrenoreceptor agonists on the excitability of trigeminal root ganglion (TRG) neurons using the perforated patch-clamp technique, and to determine whether these neurons express mRNA for K2 -adrenoreceptors. In current-clamp mode, the resting membrane potential was 357.4 A 1.2 mV (n = 26). Most neurons (71%) were hyperpolarized by clonidine (5^50 WM) in a concentration-dependent manner. The response was associated with an increase of cell input resistance. In addition, clonidine reduced the repetitive ¢ring evoked by depolarizing current pulses. An K2 -adrenergic agonist, UK14,304, (10^20 WM) also hyperpolarized TRG neurons. The clonidine- and UK14,304-induced hyperpolarization was blocked by idazoxan (K2 -adrenoreceptor antagonist). In voltage-clamp, clonidine (1^50 WM) reversibly reduced the hyperpolarization- and time-dependent cationic current. The e¡ect was mimicked by UK14,304 (10^20 WM), and antagonized by idazoxan. Hyperpolarization-activated cationic current was blocked by extracellular Csþ (2 mM) or a speci¢c blocker, ZD7288 (20WM). Analysis of tail currents revealed that a reversal potential of the clonidine-sensitive component of hyperpolarization-activated cationic current was 346 mV. Single-cell reverse transcription-polymerase chain reaction analysis demonstrated the expression of mRNA for K2A - and K2C -adrenoreceptors. These results demonstrate that activation of K2 -adrenoreceptors can hyperpolarize TRG neurons, and that the inhibitory e¡ect is associated with inhibition of hyperpolarization-activated cationic current. Our results suggest that activation of K2 -adrenoreceptors in the absence of nerve injury may have an inhibitory e¡ect on nociceptive transmission in the trigeminal system at the level of both TRG neuronal cell bodies and primary a¡erent terminals. H 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: trigeminal root ganglion, perforated patch-clamp, reverse transcription-polymerase chain reaction, K2 -adrenoreceptors.
renergic sensitivity in nociceptive a¡erents is observed following many di¡erent neuropathic injuries, including the neuroma, a chronic nerve lesion, and the chronic constriction injury (CCI), there is increasing evidence to indicate that sympathetic^a¡erent interaction at the level of the DRG may not play a signi¢cant role in neuropathic pain (Devor et al., 1994; Xie et al., 1995; Ringkamp et al., 1999; Ha«bler et al., 2000). Less attention has been paid to injuries of trigeminal a¡erents than to those involving spinal nerves. There is some evidence that the anatomical and physiological consequences of trigeminal nerve crush or ligation di¡er from those seen after sciatic nerve injury. For example, sprouting of sympathetic ¢bers observed in the sensory ganglia associated with spinal nerve injury does not occur in the trigeminal root ganglion (TRG) (Bongenhielm et al., 1999; Benoliel et al., 2001), and the spontaneous activity in a¡erents ¢bers after trigeminal injury is signi¢cantly smaller than that seen after injury to the sciatic nerve (Tal and Devor, 1992). In£ammatory cytokines, such as interleukin (IL-6), and the nerve growth factor (NGF) may contribute to development of mechanical allodynia after trigeminal nerve injury, but release of these factors is not closely related
Injury of peripheral nerves can lead to neuropathic pain accompanied by hyperalgesia and/or allodynia (Bonica, 1979; Roberts, 1986; Chung et al., 1996; Anderson and Rao, 2001). Two forms of neuropathic pain have been identi¢ed: sympathetically-independent and sympathetically-maintained pain (Bonica, 1979; Roberts, 1986). With regard to the latter, a sympatho^sensory coupling has been found in dorsal root ganglion (DRG) neurons of the sciatic nerve-injured animals (McCormik and Pape, 1990; Chung et al., 1993; Devor et al., 1994), and it has been suggested that noradrenaline (NA) released from sympathetic nerve endings acts directly on K2 -adrenoreceptors expressed in these neurons (Maclachlan et al., 1993; Xie et al., 1995). Although ad-
*Corresponding author. Tel./fax: +81-3-3261-8740. E-mail address: [email protected] (M. Takeda). Abbreviations : CCI, chronic constriction injury; DRG, dorsal root ganglion; Ih , hyperpolarization-activated cationic current; IL-6, interleukin-6; NA, noradrenaline; NGF, nerve growth factor; RT-PCR, reverse transcription-polymerase chain reaction ; TRG, trigeminal root ganglion; VDCCs, voltage-dependent Ca2þ currents. 731
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to the onset or duration of pain after trigeminal injury (Anderson and Rao, 2001). Thus the question arises as to whether there are di¡erences between the trigeminal and sciatic sensory nerves in the response to activation of K2 -adrenoreceptors by NA, but there are no studies examining this possibility. Superfusion with NA hyperpolarizes dorsal horn neurons in a slice preparation obtained from the adult rat spinal cord, and the NA-induced hyperpolarization can be blocked by an K2 -adrenoreceptor antagonist, yohimbine (North and Yoshimura, 1984). This result indicates that NA can inhibit nociceptive input within the spinal cord. In addition, clonidine (an K2 -adrenoreceptor agonist) has an antinociceptive e¡ect (Davis et al., 1991; Glynn et al., 1992), and there is evidence that iontophoretic application of this agent inhibits N-methyl-Daspartic acid-evoked responses of nociceptive neurons in the trigeminal spinal nucleus caudalis (Zhang et al., 1998). The inhibition could be reversed by an K2 -adrenoreceptor antagonist, idazoxan, but not by an K1 -adrenoreceptor antagonist, prazosin (Zhang et al., 1998). However, these results were based on experiments, which did not discriminate between presynaptic and postsynaptic e¡ects, and the question arises which of two mechanisms is important for clonidine action. In addition, it has been reported that K2 -adrenoreceptor activation inhibits hyperpolarization- and time-dependent cationic current (Ih ) and modulates DRG neuronal activity (Yagi and Sumino, 1998). It has been demonstrated in a variety of neurons, and is thought to play an important role in modulating cell excitability, neuronal ¢ring and transmitter release (Mayer and Westbrook, 1983; McCormik and Pape, 1990; Scroggs et al., 1994; Villiere and Mclachlan, 1996; Parkis and Berger, 1997). Ligand binding studies have demonstrated the presence of K2 -adrenoreceptors on primary a¡erent neurons (Howe et al., 1987). There is also immunochemical evidence that more than 50% of cell bodies of DRG express K2A -adrenoreceptors. Gold et al. (1997) demonstrated that DRG neurons express mRNA for all three known K2 -adrenoreceptor subtypes: K2A , K2B and K2C , by using reverse transcription-polymerase chain reaction (RTPCR). An in situ hybridization study has demonstrated the presence of the corresponding mRNAs in normal rat DRG, and their up-regulation of K2A -message after peripheral nerve injury (Shi et al., 2000). The latter e¡ect suggests an involvement of K2A -adrenoreceptors in spinal (DRG) system in sympathetically-maintained pain. The aim of the present study was to examine the e¡ect of K2 -adrenoreceptor agonists on the excitability of TRG neurons using electrophysiological techniques. In addition, we investigated the expression of K2 -adrenoreceptor subtypes in these neurons with single-cell RT-PCR. EXPERIMENTAL PROCEDURES
Acute dissociation of TRG neurons Rat pups (P6-P17) were decapitated. Both TRGs were rapidly removed and immersed in oxygenated ice-cold PIPES bu¡er consisting of (in mM) 120 NaCl, 5 KCl, 0.1 CaCl2; 1 MgCl2; 20 PIPES (piperazine-N,NP-bis [2-ethanesulfonic acid]), 0.1
ascorbic acid and 25 glucose (pH 7.3). Each ganglion was cut into several smaller pieces which were preincubated in PIPES bu¡er for 30 min at room temperature and then incubated for 15 min (at 30‡C) with 20 U/ml papain (Worthington), 1.6 mM L-cysteine, 0.2 mM EDTA and 13.4 mM L-mercaptoethanol (Sigma, St. Louis, MO, USA) in a total volume of 5 ml. The incubation was followed by several washes and 30 min postincubation in the same bu¡er at room temperature. All incubations and washes were conducted in a custom-built chamber, with continuous gentle stirring and bubbling with 100% O2 . After post-incubation, the tissue was triturated with ¢re-polished Pasteur pipettes in the standard external solution containing (in mM): 155 NaCl, 3 KCl, 1 CaCl2 , 1 MgCl2 , 10 HEPES (N-2-hydroxyethylpiperazine-NP-2-ethanesulfonic acid) and 20 glucose (pH 7.3). Cells were plated at low density on poly-Llysine-coated coverslips placed in a recording chamber (RC-13, Warner Instr.; volume: 0.4 ml). The recording chamber was mounted on the stage of an inverted microscope (Nikon, TE300, Tokyo, Japan) equipped with phase-contrast, video camera and a micromanipulator (Narishige, MHW-3, Tokyo, Japan). The chamber was perfused under gravity with standard external solution at approximately 0.5 ml/ml. Whole-cell patch-clamp recording Recordings were conducted by using the ‘rapid’ perforatedpatch technique (Rae et al., 1991; Dean et al., 1997; Takeda et al., 2001). Fire-polished pipettes (2^5 M6) were ¢lled with a solution containing (in mM): 120 potassium methanesulphonate, 20 KCl, 7.5 HEPES and 2 EGTA, pH 7.3, with amphotericin B (200 Wg/ml). Current- and voltage-clamp recordings were conducted with an Axopatch 200B ampli¢er (Axon Instr., Foster City, CA, USA). A low-sodium solution (80 mM) was made by equimolar substitution of choline chloride for NaCl. A high-potassium solution (25 mM) was prepared by substituting 22 mM KCl for NaCl. Signals were low-pass ¢ltered at 1 or 5 kHz and digitized at 10 kHz. To monitor changes in cell membrane resistance during recordings in current-clamp, negative current pulses (5^30 pA, 250 ms, 0.2 Hz) were injected through the patch pipette. In the voltage-clamp mode, input conductance was measured by applying +20 mV voltage steps from the holding potentials of 360 mV (250 ms, 0.2 Hz). The step commands were within the linear portion of the I^V relationships established with depolarizing ramps (from 3140 mV to +60 mV, 300 ms). Ih was identi¢ed by its response to a speci¢c blocker ZD7288 (BoSmith et al., 1994; Saitow and Konishi, 2000). The reversal potential of this current was determined by tail-current analysis (Yagi and Sumino, 1998). All recordings were performed at room temperature. Data were stored on a computer disk for o¡-line analysis. Single-cell RT-PCR Single-cell RT-PCR was performed as described in a previous study (Takeda et al., 2001). Each dissociated neuron was aspirated into a pipette (tip diameter, 6^8 Wm) containing 1URT bu¡er (8.5 Wl). Care was taken to avoid aspiration of any cell debris. The contents of the pipette, transferred into a 0.5-Wl nonstick PCR tube, were freeze/thawed and vortexed to release RNA. Four to 6 Wl of cDNA was used for ¢rst-round ampli¢cation of 35 cycles in a ‘multiplex’ PCR reaction: one containing the external primers for glyceraldehyde phosphatase dehydrogenase (GAPDH) and the other containing the external primers for K2A -, K2B -, and K2C - adrenoreceptors, each at a 50-nM concentration. Primer sequences for GAPDH were as published previously (Comer et al., 1997). Second-round ampli¢cation (45 cycles) was performed separately for each gene with 3 Wl of the ¢rst-round product and 500 nM of the upstream (5P) and internal primer (‘semi-nested’ protocol). The internal primer sequences for K2A -, K2B -, and K2C -adrenoreceptors and conditions of PCR reactions have been published previously (Phillips and Lipski, 2000; Vidovic et al., 1994). PCR reactions were performed in a 25-Wl volume containing 0.625 U DNA polymerase, 1UPCR bu¡er, 0.25 mM dNTPs (Gibco, BRL), 200 nM of
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Fig. 1. E¡ects of clonidine and UK14,304 on the membrane potential and input resistance of acutely dissociated TRG neurons (current-clamp mode). (A) Neuron hyperpolarization and increase in input resistance after clonidine application (20 WM). The break in the trace is 3 min. (B) Dose^response relationship for clonidine application. (C) Neuron hyperpolarization and increase in input resistance after UK14,304 application (10 WM). (D) Clonidine-evoked e¡ects were abolished by idazoxan (1 WM). No obvious e¡ect of idazoxan application on the membrane potential observed. The break in the trace is 2 min. The dashed lines in A, C and D indicate the resting membrane potential.
each primer and 1.5^2.5 mM MgCl2 . Negative controls were conducted in the absence of both starting RNA and RT enzyme. Ampli¢cations were performed for 35 cycles in a PTC-1001 thermal cycler (MJ Research). The ¢nal cycle lasted for 5 min at 72‡. PCR fragments were run on a 3% agarose gel, stained with ethidium bromide and illuminated with UV light. The identity of individual PCR fragments, ampli¢ed with these primers from brainstem tissue was con¢rmed by sequencing.
(mean diameter 24.1 A 0.6 Wm, n = 74; range, 15^28 Wm) and had a bright appearance with a ‘halo’ around the cell body when viewed under phase contrast. Following perforation of the cell membrane with amphotericin B, the series resistance dropped to 6 20 M6 (17.9 A 0.8 M6; n = 74) within 5^10 min and remained stable for s 15 min. The cell capacitance was 18.4 A 0.9 pF (n = 68).
Application of drugs All drugs (stock solutions) were stored at 320‡C, and dissolved in standard external solution. Clonidine hydrochloride (1^50 WM, Sigma), UK14,304 (10^20 WM, Sigma), idazoxan hydrochloride (1^5 WM, Tocris, Cookson, Northpoint, UK), and ZD7288 (2^20 WM, Tocris) were added to the perfusate for a period of up to 90 s. Data analysis Data were stored on a computer disk for o¡-line analysis (pClamp 8.0). Values are expressed as means A S.E.M. Analysis of drug e¡ect was made with Student’s t-test. P 6 0.05 was considered signi¢cant.
RESULTS
Acutely isolated TRG neurons were spherical in shape
E¡ects of K2 -adrenoreceptor agonists on the membrane potential of TRG neurons The resting membrane potential recorded in the wholecell current-clamp mode was 357.4 A 1.2 mV (n = 26). Clonidine induced changes in the membrane potential in 12 of the 14 cells tested. Most neurons were hyperpolarized (10/14, 72%) as shown in Fig. 1A, but some were depolarized (2/14, 14.2%). The remaining two cells did not respond. The clonidine-evoked hyperpolarization was concentration-dependent (Fig. 1B). In neurons showing hyperpolarization (20 WM, clonidine), the cell input resistance increased by 33.9 A 4.9% (n = 5). The duration of hyperpolarization ranged from 5 to 18 min. A similar hyperpolarizing e¡ect (associated with input resistance increase was also observed after application
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Fig. 2. E¡ect of clonidine on ¢ring frequency of TRG neurons. (A) Application of 20 WM clonidine reduced the ¢ring in response to 60 pA depolarizing pulses and hyperpolarized cell membrane potential. (B) Mean reduction in number of spikes in response to 60 pA/250 ms pulses (n = 5). *P 6 0.01.
of an K2 -adrenoreceptor agonist, UK14,304 (in four out of ¢ve tested cells; Fig. 1C). The clonidine- and UK14,304-evoked membrane hyperpolarizations were abolished by an K2 -adrenoreceptor antagonist, idazoxan (1 WM;n = 3). No signi¢cant e¡ects of idazoxan application on its own on the membrane potential were observed (Fig. 1D).
E¡ect of clonidine on ¢ring of TRG neurons As illustrated in Fig. 2A, isolated TRG neurons ¢red repeatedly in response to depolarizing current pulses (300 ms, 60 pA). Application of 20 WM clonidine not only hyperpolarized the membrane potential, but also reduced the number of action potentials evoked by this depolar-
Fig. 3. Currents evoked by voltage steps in TRG neurons. (A) Example of Ih of small diameter neuron (diameter, 21 Wm) were evoked in response to 200 ms pulses (steps from 3120 mV to 340 mV, holding potential of 360 mV, 10 mV increments). Inhibitory e¡ect of Csþ on Ih . (B) Current^voltage relations for Ih recorded in the TRG neurons before and after application of Csþ (2 mM) (n = 5). Inset shows a hyperpolarizing voltage step which elicited an instantanenous inward current (Iinst ) followed by Ih .
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Fig. 4. E¡ect of K2 -adrenoreceptor agonists on Ih induced in TRG neurons by hyperpolarizing voltage steps. (A) Example of inhibitory e¡ect of clonidine (20 WM). (B) Current^voltage relationship for Ih recorded before and after clonidine application. (C) Concentration-dependent inhibition of Ih by clonidine (n = 5) (1 WM vs. 10, 20 and 50 WM, *P 6 0.01). (D) Example of inhibitory e¡ect of UK14,304 (20 WM). (E) Current^voltage relationship for Ih recorded in the TRG neurons before and after UK14,304 application. (F) A comparison of inhibition of Ih by clonidine and UK14,304.
ization. The inhibitory e¡ect on cell ¢ring was signi¢cant (Fig. 2B). Analysis of Ih in TRG neurons Under voltage-clamp, Ih was produced by steps
between 3120 and 340 mV from a holding potential of 360 mV (10 mV increments). The inward currents evoked by the hyperpolarizing commands consisted of two components: instantaneous inward current (Iinst ), and a slowly activating inward current. The Ih was determined by subtracting Iinst from the total current mea-
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Fig. 5. E¡ect of idazoxan on inhibition of Ih by clonidine. (A) Example of the antagonistic e¡ect of idazoxan. (B) Current^ voltage relationship for Ih recorded under control conditions, during clonidine application and during co-application of clonidine with idazoxan (n = 5).
Fig. 6. Reversal potential of clonidine-sensitive current. (A) In the voltage-clamp mode, the whole-cell current of small TRG neurons (diameter 24 Wm) were recorded in standard external solution in response to a twin pulse protocol (bottom) comprising a 200-ms hyperpolarizing prepulse (to 3120 mV from a holding potential of 360 mV), followed by test pulses to di¡erent voltage levels (from 370mV to 320 mV in 10 mV increments) before and after clonidine application. (B) Relationship between tail-current amplitude and test voltage steps in standard external solution and in low Naþ (80 mM) and high Kþ (20 mM) solutions (n = 5).
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Fig. 7. Comparison of inhibition of Ih by clonidine and a speci¢c blocker, ZD7288. (A) Example of original data. (B) Mean A S.E.M. of inhibitory e¡ects of both drugs (n = 5).
sured at the end of the pulses (Fig. 3). This current was observed in 40/48 neurons tested (83.3%). Its peak amplitude at 3120 mV was 291.2 A 20.5 pA (n = 35). Application of Csþ ions that are known to block Ih in di¡erent types of neurons (Mayer and Westbrook, 1983; Lucero and Pappone, 1990; Saitow and Konishi, 2000) abolished Ih in TRG neurons (Fig. 3A, B). E¡ects of K2 -adrenoreceptor agonists on Ih The e¡ect of K2 -adrenoreceptor agonists on Ih was studied in 40 neurons. Clonidine application (20 WM) reversibly reduced the currents within 5 min (Fig. 4A). The I^V relationships are shown in Fig. 4B. The inhibition was concentration-dependent (1^50 WM; Fig. 4C; n = 5) and the e¡ect was mimicked by application of UK14,304 (10^20 WM; Fig. 4D, E; n = 5). There was no signi¢cant di¡erence between the inhibition of Ih by clonidine and UK14,304 (Fig. 4F). As shown in Fig. 5A, B, clonidine induced inhibition of Ih , which was blocked by idazoxan (1 WM). The reversal potential of a clonidine-sensitive component of Ih was determined by tail-current analysis as described previously (Yagi and Sumino, 1998). The mean reversal potential of the clonidine-sensitive current was 346 mV (n = 4) in standard external solution ([Kþ ] = 3 mM, [Naþ ] = 155 mM; Fig. 6). In a low Naþ solution ([Kþ ] = 3 mM, [Naþ ] = 80 mM), the reversal potential shifted to 330 mV; in high Kþ solution ([Kþ ] = 20 mM, [Naþ ] = 155mM), it shifted 356 mV. These results indicate that the channels a¡ected by clonidine are permeable to both Naþ and Kþ ions.
Relationship between hyperpolarization of the membrane and inhibition of Ih evoked by K2 -adrenoreceptor agonist As illustrated in Fig. 7A, Ih was also inhibited by a speci¢c Ih blocker, ZD7288. To determine whether other ionic mechanisms were involved in the hyperpolarizing action of K2 -adrenoreceptor agonists, inhibition of Ih induced by clonidine (20 WM) was compared with that evoked by ZD7288 (20 WM). During voltage step to 3120 mV, the mean inhibition of Ih evoked by clonidine was similar to that observed after application of ZD7288 (n = 4; Fig. 7B). Expression of K2 -adrenoreceptors in the TRG neurons The expression of mRNAs for K2A -, K2B - and K2C adrenergic receptor subtypes was tested in dissociated TRG neurons by means of single-cell RT-PCR. Individual small-diameter TRG neurons (15^27 Wm in diameter, n = 9) were isolated, and their mRNA were reversely transcribed into cDNA. cDNA was ¢rst subject to PCR with primers speci¢c for GAPDH, a step necessary to con¢rm successful cell collection and RT reaction. Only cells that tested positive for GAPDH (n = 6) were further analyzed. Figure 8 shows the results for a neuron which was positive for K2A - and K2C - adrenergic receptor mRNA. K2A -adrenoreceptor mRNA was detected in two out of six TRG neurons tested, whereas K2C message was detected in one out of six neurons tested. K2B -adrenoreceptors were not detected in any of the neurons tested.
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nist, UK14,304 (Takeda et al., 2001), and was blocked by idazoxan, a speci¢c blocker of K2 -adrenoreceptors (Zhang et al., 1998). Although it has been reported that clonidine has a⁄nity for imidazoline receptors, resulting in membrane potential depolarization (Ernsberger et al., 1997), we found that in all neurons demonstrating repetitive ¢rings in response to depolarizing current pulses, clonidine application inhibited the ¢ring. These results suggest that the activation of K2 adrenoreceptors inhibits the excitability of TRG neurons. This conclusion extends previous observations that presynaptic K2 -adrenoreceptors are also located on primary a¡erent terminals in the trigeminal spinal nucleus, and mediate inhibition of glutamate release (Travagli and Wiliams, 1996). Our results indicate that modulation of nociceptive transmission in the trigeminal system by activation of K2 -adrenoreceptors can occur both at the level of TRG neuronal cell bodies and primary a¡erent terminals. Inhibition of Ih by K2 -adrenoreceptor agonist
Fig. 8. Example of mRNAs coding for K2A - and K2C -adrenoreceptor subtypes in the two acutely dissociated TRG neurons. Gel electrophoresis of PCR products obtained from TRG neurons (lanes 3 and 4) and brain stem tissue (positive control ; lane 1). Lane 2: Negative control (no reverse transcription) ; lane 3: PCR products obtained with primers for K2A -adrenoreceptor; lane 4: products obtained with primers for K2A - and K2C -adrenoreceptors. DISCUSSION
The present experiments provided evidence that activation of K2 -adrenoreceptors can hyperpolarize TRG neurons, and that the inhibitory e¡ect is associated with inhibition of Ih . In addition, we ¢rstly demonstrated that the expression of mRNA coding for K2 -adrenoreceptors in single TRG neurons. Our results suggest that activation of K2 -adrenoreceptors in the absence of nerve injury may have an inhibitory e¡ect on nociceptive transmission in the trigeminal system at the level of both TRG neuronal cell bodies and primary a¡erent terminals. Changes of the membrane potential and inhibition of ¢ring evoked by K2 -adrenoreceptor agonists Application of clonidine (10^50 WM) evoked a concentration-dependent hyperpolarization in the majority of TRG neurons tested and the response was associated with an increase in cell input resistance. However, in the some neurons, clonidine caused a depolarization or had no signi¢cant e¡ect on the membrane potential. The hyperpolarizing e¡ect of clonidine resembled that seen after application of a selective K2 -adrenoreceptor ago-
Application of clonidine inhibited a slowly activating cationic current (Ih ) evoked by membrane hyperpolarization and this inhibition was concentration-dependent (1^ 50 WM). Yagi and Sumino (1998) reported that EC50 of inhibition of Ih by clonidine in the DRG was about 2.2 WM. In the present study, on acutely dissociated TRG neurons, concentrations of K2 -adrenoreceptor agonist were higher than endogenous NA ( 6 1 WM). Thus, it is possible that clonidine-induced Ih inhibition may involve either a reduction of the number of functional receptors or, a non-speci¢c e¡ect of K2 -adrenoreceptors. We, however, found evidence that clonidine-induced inhibition of Ih was blocked by a speci¢c K2 -adrenoreceptor blocker, idazoxan. Similar concentrations used in the present study (10 WM) examining modulation of K2 -adrenoreceptor agonists have been used in the DRG neurons and hypoglossal motoneurons (Parkis and Berger, 1997; Xie et al., 1995; Homma et al., 1999). Therefore it is more likely that clonidine-induced inhibition of Ih may be mediated by the activation of K2 -adrenoreceptor e¡ect. The characteristics of clonidine-sensitive current were similar to those of the hyperpolarization-activated cationic currents described in previous studies in other neurons and non-neuronal cells (Mayer and Westbrook, 1983; McCormik and Pape, 1990; Villiere and Mclachlan, 1996). In this study, the identi¢cation of Ih was performed as follows: (1) The currents were blocked by Csþ application (the ability of this ion to block Ih in thalamic and DRG neurons was described previously (McCormik and Pape, 1990; Scroggs et al., 1994); (2) The reversal potential of the current was 346 mV, which resembled values reported previously, and suggests that the current was carried a mixed Naþ ^Kþ current (Mayer and Westbrook, 1983; Yagi and Sumino, 1998; Saitow and Konishi, 2000); (3) The magnitude of decrease of Ih after clonidine application was similar to that observed after application of the speci¢c blocker ZD7288 (McCormik and Pape, 1990; Saitow and Konishi, 2000). Our results show that the K2 -adrenore-
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ceptor agonists can reversibly inhibit Ih at least in neonatal TRG neurons. In current-clamp recordings, application of clonidine usually caused hyperpolarization of the membrane potential. We propose that this hyperpolarization occurs as a result of inhibition of Ih , because the reversal potential of Ih was more positive than the resting membrane potentials of TRG neurons. The fact that the holding potential was more negative than the reversal potential of Ih suggests that the Ih channel activated tonically at the holding level would produce a cation in£ux (Mayer and Westbrook, 1983; Scroggs et al., 1994). Therefore, Ih can be involved in both the setting of the level of the resting membrane potential as well as of the neuronal excitability. Functional signi¢cance of inhibition of TRG neuronal excitability by K2 -adrenoreceptors It has been reported that there is no sprouting of sympathetic nerve ¢bers in the trigeminal ganglion after peripheral trigeminal nerve injury (Bongenhielm et al., 1998; Benoliel et al., 2001) and that spontaneous activity observed in trigeminal a¡erents after trigeminal peripheral nerve injury is signi¢cantly smaller than that seen after injury to the sciatic nerve (Tal and Devor, 1992). Although the reason for this di¡erence is unclear, it is possible that it is due to some unique characteristics of the trigeminal nerve, For examples, this nerve carries almost equal numbers of myelinated and non-myelinated ¢bers, unlike many cutaneous nerves of spinal origin, which carry a considerably higher proportion of non-myelinated ¢bers (Matsuda et al., 1969; Fried and Hildebrand, 1982). The location of the nerve within a canal in the skeleton makes it more vulnerable to pressure after peripheral nerve injury (Bongenhielm et al., 1999). Finally, there is a di¡erence in embryonic origin; the sciatic nerve derives from the segmental neural crest, while many trigeminal a¡erents from the ectodermal placode (D’Amico-Martel and Norden, 1983). Chung et al. (1996) have demonstrated that sympathectomy performed at 4 days after L5^6 spinal nerve ligation reduces mechanical and cold allodynia and eliminates sympathetic sprouting. However other investigators were unable to reproduce this ¢nding (Ringkamp et al., 1999). Similarly, sympathectomy has no signi¢cant e¡ect on the early ectopic activity from neuromas of the ferret inferior trigeminal nerve (Bongenhielm et al., 1998). It is therefore possible that sympathetic sprouting is not required for the generation of ectopic activity in damaged trigeminal nerve. This idea is further supported by the ¢nding demonstrating that sprouting is not related to the sympathetic^sensory coupling in the DRG neurons: neurophysiological data (adrenergic sensitivity) are obtained 4^22 days after sciatic nerve lesion but sprouting is starting on the 20th day (Devor et al., 1994). In contrast, it has been reported that the increases in IL-6 and NGF may be related to the development of mechanical allodynia after trigeminal injury (Anderson and Rao, 2001). Thus, release of both IL-6 and NGF may be partly involved in the generation of ectopic dis-
739
charges from injured trigeminal nerve. Because there is evidence that in the L5 spinal nerve injury model, sympathetic^sensory coupling does not occur during physiological activity in sympathetic neurons (Ha«bler et al., 2000), it is possible that sympathetic^sensory coupling may be generated indirectly via alternation of blood £ow in the TRG neurons. After peripheral nerve injury, population of neuronal cell bodies in DRG are known to generate spontaneous ectopic discharges (Chung et al., 1993; Maclachlan et al., 1993; Devor et al., 1994; Xie et al., 1995; Chung et al., 1996). Also there is a positive correlation between the ectopic discharges and allodynia-like behavior in spinal nerve-lesioned rats, and their activity occurred in A-¢bers, but not C-¢bers (Liu et al., 2000a,b). In contrast, ectopic discharges in most spontaneously ¢ring DRG neurons of both A- and C-¢bers were suppressed by sympathetic^a¡erent activity and this suppression predominant response in animals with long-standing nerve injury (Michaelis et al., 1996). In the rat DRG neurons, it is known that there is a positive correlation between the neuronal cell size and axonal conduction velocity both for AN- and for C-¢ber a¡erents (Harper and Lawson, 1985). The TRG neurons recorded in the present study are probably included both AN- and C-type neurons, because the cell body diameter of AN-type neurons overlaps the border between small and median diameter ( 6 30 Wm) acutely isolated DRG neurons (Harper and Lawson, 1985). Yagi and Sumino (1998) speculated that if the after-hyperpolarization following action potentials were su⁄ciently negative and lasted long enough to activate Ih in DRG neurons, then this current would contribute to facilitation of the DRG neuronal activity. In this study we found that K2 -adrenoreceptor agonists inhibited Ih and reduced the ¢ring rate of TRG neurons, suggesting that inhibition of Ih by K2 -adrenoreceptor activation may decrease the ¢ring rate of spontaneously activate TRG neurons. In contrast, it has been known that there is a di¡erence between intact and injured DRG neurons in respect to their sensitivity. For examples, NA inhibited voltage-dependent Ca2þ currents (VDCCs) in CCI rats, whereas it enhanced these currents in control rats (Homma et al., 1999). Moreover, K2 -adrenergic stimulation also inhibited outward Kþ currents in the CCI rats, while it had no e¡ects on the currents in control rats. The authors concluded that NA-induced inhibitory e¡ects on VDCCs and KCa channels in injured DRG neurons could lead to cell membrane depolarization, and increased frequency of action potentials (Homma et al., 1999). In the context of these ¢ndings, it is possible that K2 -adrenergic stimulus acts not only through modulation of Ih , but also on the VDCCs and KCa channels in injured TRG neurons. In the TRG neurons, Ih has been previously identi¢ed in large-, medium-, and small-diameter cell bodies, and its amplitude is cell size-dependent (Scroggs et al., 1994). Moreover, approximately 37^45% of small diameter (15^ 33 Wm) DRG neurons demonstrated Ih (Scroggs et al., 1994; Yagi and Sumino, 1998). In the present study, we found Ih in 83% of TRG neurons (15^28 Wm) tested. Although K2A - and K2C -adrenoreceptor mRNA expres-
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sion was identi¢ed in small population of TRG neurons, this is the ¢rst study to demonstrate expression of mRNA coding for K2 -adrenergic receptors in single TRG neurons. Because the trigeminal ganglion is nonhomogeneous and contains not only sensory neurons, but also glia and satellite cells (Matsuda et al., 1969), the fact that K2 -adrenoreceptor mRNA expression was found in the single TRG neurons leads us to suggest the possibility of neuronal modulation by K2 -adrenoreceptors. The previous study of in situ hybridization study has revealed that in normal rat DRG, the most common adrenergic receptor mRNA is for K2C -subtype (almost 80%), and that K2B -adrenergic receptor mRNA is only found in a small number of neuron pro¢les (Shi et al., 2000). In DRG neurons, K2A - adrenoreceptor mRNA increased to 45% after peripheral nerve injury, but K2C subtype showed a small decrease in expression (Shi et al., 2000). Thus, it appears that both K2A - and K2C -adrenergic receptors are closely related to the TRG neuronal function, including nociceptive transmission seen after trigeminal injury. In small DRG neurons in sciatic nerve-transected rats, there was a tendency to stronger inhibition of Ih by clonidine than in intact rats (Yagi and Sumino, 1998). Therefore, it is also possible that an increase in K2 -adrenoreceptor expression after trigeminal nerve injury may contribute to a stronger suppression of spontaneous activity in small TRG neurons. Intracellular recordings from DRG neurons have demonstrated that a large proportion of silent neurons is in£uenced by the spike activity of neighboring neurons
(Utzschneider et al., 1992). This suggests activity dependent cross-excitation in DRG neurons which could contribute to neuropathic sensory abnormalities triggered by nerve injury (Utzschneider et al., 1992). Amir and Devor (1996) reported repetitive ¢ring of DRG neurons induced by an increase in extracellular Kþ concentration and a non-synaptic release of neurotransmitters from adjacent neurons. In view of this observation, it is possible that certain substances accumulate in the extracellular space during stimulation of TRG neurons, di¡use toward neighboring neurons and modulate their activity. Actually, it has been recently reported that after nerve injury ongoing activity of DRG origins appears exclusively in small diameter myelinated muscle a¡erents, both axtomized and unlesioned, this ectopic activity is probably induced by paracrine signal released from DRG (Michaelis et al., 2000). Although we did not examine this mechanism in a neuropathic trigeminal pain model, our results suggest that inhibition of TRG excitability via K2 -adrenergic receptors may prevent ectopic discharges of injured TRG neurons as well as the increase of excitability of uninjured neighboring neurons. Further studies are needed to investigate such e¡ects. Acknowledgements,Thanks are due to Mrs. R. Dubey for technical help in preliminary experiments and Dr. J.K. Philips for advice regarding the RT-PCR experiments. This study was supported by a Grant from the Ministry of Education, Science and Culture of Japan (No. 13671953).
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(Accepted 26 July 2002)
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