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Department of Biomedical Sciences, Iowa State University, Ames, IA 50011-1250, USA
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and C fibre monosynaptic pathways, whereas presynaptic GluR5-KARs play a limited role in inhibiting the C fibre-activated pathway. The results obtained support the hypothesis that KARs are involved in bi-directional regulation of excitatory synaptic transmission in the spinal cord SG region, and that these actions may be of critical importance for nociception and the clinical treatment of pain.
(Received 3 November 2003;
accepted after revision 5 January 2004;
first published online 14 January 2004)
Corresponding author M. Randi: Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA. Email: mrandic{at}iastate.edu
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, 1996). The primary function of neurones in the SG is to integrate noxious afferent information arriving at this region via high-threshold A and C fibres. The SG neurones function as excitatory and inhibitory interneurones and regulate the output of projection neurones in other laminae of the DH (Willis & Coggeshall, 1991). The SG neurones express AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) (AMPARs and NMDARs, respectively) to mediate fast excitatory synaptic transmission. Recently, a series of studies with pharmacological tools (for review, see Chittajallu et al. 1999; Lerma et al. 2001) suggests functions for the kainate receptor (KAR), another type of iGluR, as a mediator and modulator of excitatory synaptic transmission, or a modulator of inhibitory transmission, in the spinal cord SG neurones (Li et al. 1999; Kerchner et al. 2001a,b). KARs are composed of homomeric and heteromeric configurations of five cloned subunits: GluR5–7, KA1 and KA2 (Herb et al. 1992; Chittajallu et al. 1999). In primary sensory neurones, there is evidence suggesting the existence of apparent homomeric GluR5 receptors or heteromeric assemblies of GluR5 and KA2 subunits on the dorsal root ganglion (DRG) and trigeminal ganglion neurones (Bettler et al. 1990; Sommer et al. 1992; Partin et al. 1993; Sahara et al. 1997; Swanson et al. 1998; Wilding & Huettner, 2001). On the other hand, in the spinal DH, KARs are expressed in both myelinated and unmyelinated primary afferent terminals (Partin et al. 1993; Sato et al. 1993; Petralia et al. 1994; Hwang et al. 2001), where they regulate glutamate release (Kerchner et al. 2001b). Besides being presynaptic, they may also be expressed on the postsynaptic membrane of SG neurones (Tölle et al. 1993; Petralia et al. 1994; Yung, 1998; Li et al. 1999; Hwang et al. 2001; Kerchner et al. 2001a,b; Dai et al. 2002; Guo et al. 2002).
The development of KAR subunit-deficient mice (Mülle et al. 1998, 2000; Contractor et al. 2003) has revealed important and distinct roles for each KAR subunit in synaptic transmission and plasticity in the brain (Bureau et al. 1999; Chergui et al. 2000; Contractor et al. 2000, 2001, 2003), and spinal cord (Kerchner et al. 2002). Recent electrophysiological and pharmacological findings in the rat spinal cord DH indicated that KARs regulate synaptic transmission at excitatory, as well as inhibitory, synapses (Kerchner et al. 2001a,b; Huettner et al. 2002). However, it is unclear which KAR subunits underlie the regulation of primary afferent A fibre and C fibre-evoked monosynaptic glutamatergic transmission in the substantia gelatinosa region, both under normal conditions and in the absence of GABAA and glycine receptor-mediated inhibition.
We have briefly reported that the exogenous activation of KA receptors with a high concentration of KA (3 µM) depresses AMPA receptor-mediated excitatory synaptic transmission in all cells examined in the SG region in slices obtained from adult mice (Gerber et al. 1999; Youn et al. 2002). In addition, a recent study showed that KA and ATPA suppress NMDAR-mediated EPSCs in rat DH neurones evoked by the stimulation of synaptically coupled DRG cells in DRG–DH neurone cocultures (Kerchner et al. 2001b). Based on the selectivity of ATPA for the GluR5 subunit (Clarke et al. 1997; Hoo et al. 1999), this effect was postulated to arise from the activation of presynaptic GluR5-KARs because the GluR5 subunit is expressed at a high level only on small-diameter DRG neurones (giving rise to C fibres), but not on the DH neurones (Bettler et al. 1990; Partin et al. 1993; Sato et al. 1993; Tölle et al. 1993; Swanson et al. 1998; Hoo et al. 1999; Hwang et al. 2001; Kerchner et al. 2001b; Wilding & Huettner, 2001). However, the pharmacology of ATPA is not definitive in this regard. It can activate some heteromeric receptors that do not contain the GluR5 subunit (Paternain et al. 2000). Therefore, the contribution of other subtypes of KAR, besides GluR5, to the AMPAR or NMDAR-mediated spinal sensory transmission and plasticity, has yet to be more directly demonstrated.
In the present study, we used gene-targeted mice lacking GluR5 or GluR6 KAR subunits to identify the subunits comprising KARs in the adult mouse SG region. We have investigated the effect of KAR activation on glutamatergic excitatory postsynaptic transmission at A and/or C primary afferent fibre–SG synapses. We report here that KARs, in the absence of GABAergic and glycinergic inhibition, biphasically and dose-dependently modulate the excitatory transmission. In addition, the GluR6 subunit is critically involved in inhibiting the excitatory transmission at both primary afferent A and C fibre monosynaptic pathways, whereas GluR5 plays a role in inhibiting the C fibre-activated pathway. Part of this work has been reported as an abstract form (Gerber et al. 1999; Youn et al. 2002).
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All experiments were approved by the Iowa State University Animal Care and Use Committee and were consistent with the ethical guidelines of the National Institutes of Health and of the International Association for the Study of Pain. Moreover, all efforts were made to minimize the number of animals used, and their suffering. Transverse spinal cord slices were obtained from 50- to 138-day-old mice, of either sex, and with the following genotypes: wild-type, GluR5 mutant (-/-), GluR6-/-, and GluR5-/-/GluR6-/-/KA2-/-. The genetic background of the wild-type and GluR5-/- mice was isogenic 129SvEv, whereas GluR6-/- and GluR5-/-/GluR6-/-/KA2-/- had a hybrid 129SvEv/C57BL/6 background. Developmental compensation could be a concern in knockout experiments; however, we feel this is unlikely because levels of mRNA expression of other KAR subunits were unchanged in GluR6-/- (Mülle et al. 1998) or GluR5-/- (Mülle et al. 2000) mice. Furthermore, functional replacement of whole-cell KAR currents does not occur in CA3 pyramidal neurones (Mülle et al. 1998), cerebellar Purkinje neurones (Brickley et al. 1999), or DRG neurones (Mülle et al. 2000). Each knockout mouse used in this study was genotyped by Southern blot analysis of tail DNA. Under deep isoflurane anaesthesia, the lumbosacral (L1–S1) part of spinal cord with long (8–15 mm) dorsal roots was removed from a mouse, and the mouse was killed by cervical dislocation. Transverse spinal cord slices were made from L4–L5 spinal segments using a Vibratome (Lancer, Bridgetown, MO, USA), as previously described (Randiet al. 1993). Several transverse slices (400–500 µm thick) were cut from L4–L5 spinal segments with attached dorsal root in an oxygenated (95% O2, 5% CO2) Krebs-bicarbonate solution (4°C) on a vibratome, and placed in a holding chamber (36 ± 1°C) to recover for at least 1 h. Slices were cut and maintained in a medium containing (mM): NaCl, 124; KCl, 5; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 10; pH 7.4, and was equilibrated with 95% O2, 5% CO2. A single slice was then transferred into a recording chamber where it was submerged beneath an oxygenated superfusing medium (flow rate of about 3 ml min-1, 34–35°C) of a similar composition to the incubation solution except for 128 mM NaCl and 1.9 mM KCl.
Dorsal root stimulation and intracellular recording
Intracellular recordings with sharp microelectrodes were made from substantia gelatinosa (SG, lamina II) neurones. When viewed under a dissecting microscope at a magnification of x 10–40 with transmitted illumination, the SG was distinguishable as a translucent band in the superficial DH, although it was difficult to discern with certainty the border between laminae I and II. Under visual control, a single fibreglass (no. 6010; o.d and i.d., 1.0 and 0.58 mm, respectively; AM Systems, Carlsborg, WA, USA) microelectrode filled with 4M potassium acetate (pH 7.2) (DC resistance: 140–220 M
) was placed in the SG, and neurones were impaled by oscillating the capacity compensation circuit of a high-input impedance bridge amplifier (Axoclamp 2A, Axon Instruments, Foster City, CA, USA). A DC pen-recorder was used to record membrane potentials continuously, and a Digidata 1200 system with pCLAMP (version 6) software (Axon Instruments) for data acquisition and analysis. Most recordings were obtained from neurones with a stable resting membrane potential (more negative than -60 mV) and with overshooting action potentials. The protocol for assessing the effects of KAR agonists and antagonists on synaptic responses was as follows. Monosynaptic and polysynaptic excitatory postsynaptic potentials (EPSPs) in SG neurones were evoked by orthodromic electrical stimulation of primary afferent fibres in the lumbar dorsal root using a bipolar platinum wire electrode or glass suction electrode (with the cathode internal). Single shocks at a fixed suprathreshold strength (0.01-0.5 ms pulses, 2–35 V), repeated at 2 min intervals, were given for at least 10 min before, during (2 min), and for a 20–30 min period after bath administrations of chemicals. This frequency of stimulation was chosen for sampling data because it did not result in response facilitation or depression. A stimulus intensity that yielded a 5–15 mV EPSP was chosen to standardize the baseline synaptic strength across slices, and it was below threshold for eliciting an action potential in most of the slices chosen for study. The classification of EPSPs in relation to the primary afferents activated was done solely on the basis of conduction velocity, which was calculated either by measuring the distance between the stimulating electrode and the recording site on the dorsal root and dividing by the conduction latencies of action potentials recorded, or from the latency of evoked EPSPs and the distance from the stimulating electrode to the recording site. Primary afferents conducting at velocities above 15 m s-1 were classified as Aß (Park et al. 1999), whereas those conducting between 1.5 and 15 m s-1 were classified as A, and those conducting below 1.5 m s-1 as C fibres. The minimum stimulus intensities and durations used to activate A and C fibres were 3 V/0.1 ms and 5 V/0.5 ms, respectively. Stimulation of the dorsal root led to the generation of an EPSP. With a small stimulus strength this EPSP was graded in amplitude, and had a fixed latency and monophasic decay. As the stimulus strength was increased, however, a later slow polysynaptic component(s) was apparent. Identification of the A fibre-evoked EPSPs as monosynaptic EPSPs was based on their short and constant latencies and absence of failures with repetitive stimulation at a frequency of 10 Hz (Fig. 1A; Randiet al. 1993). The shapes and amplitudes of monosynaptic EPSPs were similar in different trials when the dorsal root was stimulated at a constant intensity. C fibre-activated responses with long latencies could not easily be distinguished by high frequency stimulation as ‘monosynaptic’ or ‘polysynaptic’ because they frequently had failures with constant latencies during repetitive 10 Hz stimulation. Therefore, only EPSPs showing no failures at low frequency (
1 Hz) stimulation, which are presumably monosynaptic C fibre-evoked EPSPs (Ataka et al. 2000), were analysed. Input resistance was measured by passing a hyperpolarizing current pulse of 0.05 nA across the cell membrane and measuring the voltage deflection produced. The current values were of sufficient duration (200–300 ms) to fully charge the membrane capacitance. Bridge balance was monitored throughout the experiments and corrected when necessary.
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Whole-cell patch-clamp recordings from SG neurones were performed using the ‘blind method’ (Blanton et al. 1989). Patch-pipettes (7–10 M) contained (mM): potassium gluconate, 135; KCl, 5; EGTA, 5; Hepes, 5; CaCl2, 0.5; MgCl2, 2; adjusted to pH 7.2 with KOH, and a final osmolality of 290–295 mosmol kg-1. Neurones were voltage clamped at -70 mV using the Axopatch 200B amplifier (Axon Instruments), and access resistance was monitored and neurones discarded if this parameter changed by more than 20%. Dorsal root stimulation-evoked excitatory postsynaptic currents (EPSCs) were recorded in SG neurones at a holding potential of -70 mV. Spontaneous miniature EPSCs (mEPSCs) were recorded in the presence of tetrodotoxin (TTX, 1 µM), bicuculline (10 µM), strychnine (1 µM), and D-AP5 (50 µM, in some experiments) to block voltage-dependent Na+ channels and action potential-dependent release events, GABAA, glycine and NMDA receptors, respectively. Application of 50 µM GYKI 53655, a specific AMPA receptor antagonist, or 10-20 µM CNQX, a non-NMDA receptor antagonist, eliminated mEPSCs, confirming that the postsynaptic events resulted from the release of glutamate and the predominant activation of AMPARs. The mEPSCs were sampled at 10 kHz (Digidata 1200), low-pass filtered at 2 kHz, and stored onto a Gateway EV700 computer programmed with pCLAMP (version 8, Axon Instruments) software. Analysis of mEPSCs was performed by the use of the Mini Analysis Program (Synaptosoft, Leonia, NJ, USA). All records were fitted manually by screening through and picking events from the digitized data. The whole data file was fitted to check for stability of the recordings. Two to three minute stretches of data before and during KA application (or 10–20 min after washout of KA) were used for mEPSC frequency and amplitude analysis.
Chemicals
Chemicals used and their sources were as follows: (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propionic acid (ATPA), (–)-bicuculline methiodide, and strychnine hydrochloride from Sigma (St Louis, MO, USA); 2S-3[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP 55845 A), a gift from Novartis Pharma AG Research, Basel, Switzerland; 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655), a gift from Dr Antal Simay (IVAX Drug Research Institute, Budapest, Hungary); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5), 2S,3S,4R-carboxy-4-(1-methylethenyl)-3-pyrro-lidineacetic acid (kainic acid) (S)-
-methyl-4-carboxyphenyl glycine (MCPG), and 2,3-dioxo-6-nitro-1,2,3,4 tetrahydrobenzo-[f]quinoxaline-7-sulphanamide (NBQX), 2S-4R diastereomer of 4-methylglutamate (SYM 2081), all obtained from Tocris Cookson (Bristol, UK); (3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY 382884) was a gift of Lilly Research Laboratories, Eli Lilly and Co. (Indianapolis, IN, USA). All solutions were freshly prepared every day from stock solutions that were stored at -20°C. The solution entered the recording chamber within 30 s of changing solutions, with complete exchange occurring within 2 min
Data analysis
Data are presented as the mean ±S.E.M. Parameters were compared by the use of Student's unpaired t test or the Wilcoxon signed rank test. P < 0.05 was considered significant.
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203.4 ± 32.8 versus 184.2 ± 28.1 versus 201.5 ± 50.0), threshold to evoke A fibre-evoked monosynaptic EPSP ((V) 5.4 ± 0.7 at 0.1 ms versus 5.2 ± 0.6 versus 5.6 ± 0.4), and conduction velocity for A and C afferent fibres, respectively ((m s-1) A fibre: 3.1 ± 0.2 versus 3.0 ± 0.3 versus 3.3 ± 0.4; C fibre: 0.6 ± 0.1 versus 0.6 ± 0.1 versus 0.7 ± 0.2). Single shock electrical stimulation of the primary afferent A and/or C fibres in a L4 or L5 dorsal root elicited monosynaptic and/or polysynaptic EPSPs in SG neurones that were completely blocked by 50–100 µM GYKI 53655 or LY 300164, AMPAR-selective antagonists, and 50 µMD-AP5 in a reversible manner, suggesting that they were primarily mediated by the AMPA/NMDA subtypes of glutamate receptor (Randiet al. 1993). Activation of kainate receptors bi-directionally modulates primary afferent neurotransmission
Superfusion of slices with KA (0.01–10 µM, 2 min) depressed the peak amplitude of primary afferent-evoked EPSPs in a dose-dependent manner (Fig. 1B and C). In particular, at a concentration of 3 µM KA, the depression was consistently produced (Fig. 1B and D; 58.0 ± 3.9% of control, n= 19 neurones/18 mice; P < 0.001); depression typically peaked at 3–5 min, and persisted at least for more than 20 min (11 out of 19 neurones; the rest recovered) after the application of KA was terminated. Therefore, the calculation for the effect of KA on EPSP amplitude was made at the interval of 3–5 min after the start of KA application. The depressant effect of KA was associated with a slow and reversible membrane depolarization in 14 out of 19 SG neurones (5.6 ± 0.7 mV, n= 14). The depolarization typically recovered within 12 min whereas the depressant effect of KA remained even after the recovery of depolarization. Moreover, 3 µM KA could also depress evoked EPSCs under whole-cell voltage-clamp recording conditions (holding potential, -70 mV) at the time (3–5 min) when the peak depolarization in current-clamp conditions might occur (62.7 ± 4.3%, n= 3, P < 0.01; data not shown). Therefore, the membrane depolarization did not seem to entirely contribute, via a decrease in the driving force of synaptic potentials, to the KA-induced depression of EPSPs. The depressant effect of 3 µM KA was partially but significantly reduced by 10 µM LY 382884, a selective GluR5 subunit antagonist (Bleakman et al. 1996; O'Neill et al. 1998; Bortolotto et al. 1999), and was almost completely prevented in the presence of 3 µM SYM 2081, the agent that rapidly desensitizes KAR (Fig. 1D). These results further indicate that AMPAR is unlikely to be responsible for the depression of EPSPs in the SG region.
To test the possibility that KA-induced depression is caused by a change in the strength of synaptic inhibition (GABAergic and glycinergic), we established a dose–response curve for the effects of KA on primary afferent-evoked EPSPs, in the presence of 5 µM bicuculline and 2 µM strychnine (Fig. 1E). Interestingly, the effect of KA in these conditions was biphasic, which was not obvious in the normal Krebs solution, and was characterized by facilitation at a low concentration (30 nM) and depression at a higher concentration (3 µM). The magnitude of the average depression in the presence of bicuculline and strychnine (25.3 ± 4.3%, n= 16 neurones/10 mice; P < 0.01) was significantly smaller than that in normal Krebs solution (42%). However, the depression remained under conditions with further blockade of GABAB and/or group I/II metabotropic glutamate receptors (data not shown), indicating that the effect is a result of direct activation of KARs rather than the indirect or secondary action of other modulators released through interneuronal activity in the SG region (Kerchner et al. 2001a).
GluR5 and GluR6 subunits differentially contribute to KA-induced modulation in A and C fibre synaptic transmissions
To determine the identity of the subunit(s) comprising KARs responsible for depression of excitatory synaptic transmission in the SG, we compared the effect of KA on the peak amplitude of primary afferent A and/or C fibre-evoked monosynaptic EPSPs in spinal cord slices obtained from mice lacking GluR5 or GluR6 subunits. In a normal Krebs medium, bath application of 3 µM KA for 2 min reduced the peak amplitude of primary afferent A fibre-evoked monosynaptic EPSPs to a similar degree in wild-type mice and mice in which the GluR5 or GluR6 subunits had been disrupted (Figs 2A and B, and 4). When bicuculline and strychnine were included in the perfusion solution, the magnitude of KA-induced depression was still similar in GluR5-/- mice, if compared with wild-type mice, whereas, in GluR6-/- mice, the depressant effect was completely blocked or even opposite in the direction of change (Figs 2C and 4). On the other hand, the KA-induced depression of C fibre-evoked monosynaptic EPSPs in normal Krebs solution was significantly but partially reduced both in GluR5-/- or GluR6-/- mice (Figs 3A and B, and 4). However, in the presence of bicuculline and strychnine, the remaining depression was completely disappeared only in GluR6-/- mice (Figs 3C and 4). Taken together, these data suggest that in the absence of synaptic inhibition mediated by GABAA and glycine receptors, the GluR6 subunit is critically involved in inhibiting transmission at both primary afferent A and C fibre monosynaptic pathways in the SG region, whereas GluR5 plays a role partially in inhibiting the C fibre-activated pathways.
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To test for the presence of the functional presynaptic KARs at adult mouse spinal SG excitatory synapses and for their possible role in the KAR-mediated depression of evoked EPSPs, we recorded action potential-independent mEPSCs by using whole-cell voltage-clamp recordings. mEPSCs were visualized as inward currents at a holding potential of -70 mV with an averaged background frequency of 10.8 ± 5.6 s-1 (n= 10 neurones from 7 mice). Upon application of KA (3 µM, 2 min), the frequency of mEPSCs during the first 120 s of exposure decreased to 64.8 ± 4.1% of the control value (n= 10, P < 0.01; Fig. 5), suggesting that KA acts at a presynaptic locus. However, no significant change in mEPSC amplitude was observed during KA exposure (Fig. 5).
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In addition, a change in the whole-cell holding current during KA application occurred in the SG neurones from wild-type (-18.3 ± 6.5 pA) and GluR5-/- mice (-24.9 ± 6.1 pA), but not from GluR6-/- mice (-0.7 ± 1.7 pA, P < 0.05 versus+/+; Fig. 6A), which was also observed as depolarization during conventional intracellular recording (Fig. 6B). These data suggest that the GluR6 subunit localizes both pre- and postsynaptically at excitatory synapses onto SG neurones, whereas the GluR5 subunit preferentially localizes at presynaptic terminals (Fig. 8).
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In the presence of bicuculline and strychnine, the facilitation of EPSPs by a low concentration of KA (30 nM; Fig. 1E) was long-lasting in the spinal cord SG region (134.7 ± 6.7% of control at 13–19 min from the start of KA application, P < 0.01 versus baseline; Fig. 7A). The long-lasting potentiation by KA was reliably induced in all slices (n= 5 neurones/5 mice) and NMDAR dependent (Fig. 7A). However, the long-lasting potentiation was variable in GluR5-/- mice, i.e. a short- or long-lasting potentiation occurred in six out of nine neurones with no change in the rest. The facilitation of EPSPs was not seen in the GluR6-/- mice. As a result (Fig. 7B), the mean EPSP amplitude at 13–19 min from the start of KA application was 108.6 ± 8.4% of control in GluR5-/- (n= 9 neurones from 8 mice, P < 0.05 versus+/+) and 97.2 ± 4.4% in GluR6-/- mice (n= 7 neurones from 5 mice, P < 0.01 versus+/+). In addition to the KA-induced potentiation of synaptic transmission, we also found that another KAR agonist, ATPA (1–3 µM), classified as a selective GluR5 subunit agonist, can cause potentiation of EPSPs in approximately half (7/13) of SG neurones in wild-type mice (Fig. 7C and D) under normal condition or conditions with blockade of inhibitory transmission. The ATPA-induced potentiation appears to be absent in GluR5-/- and triple GluR5/GluR6/KA2-/- mice, but not in GluR6 (Fig. 7D). Taken together, these results indicate that weak activation of KARs, possibly involving a limited number of KARs due to agonist concentration or selectivity, may potentiate synaptic transmission.
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In the present study, we found that in the presence of synaptic inhibition mediated by GABAA and glycine receptors, KA strongly suppressed A and C fibre-evoked monosynaptic EPSPs in neurones from wild-type mice. In contrast, the KA depressant effect on EPSP amplitudes recorded from SG neurones in slices from GluR5- or GluR6-deficient mice was significantly reduced to a similar extent, but not eliminated, suggesting that both GluR5 and GluR6 receptor mechanisms account for, at least a part of, the KA depressant effect. The exact mechanism involved in this partial reduction of KA depressant action on the AMPAR-mediated EPSPs recorded under control conditions in the SG neurones following GluR5 and GluR6 deletions, is unclear. However, our data are at least consistent with the present evidence suggesting that GluR5 and GluR6 can exist as homomeric (Bettler et al. 1990; Sommer et al. 1992; Sato et al. 1993; Petralia et al. 1994; Swanson et al. 1996, 1998; Wilding & Huettner, 2001) and/or heteromeric receptor combinations (Bettler et al. 1990; Partin et al. 1993; Sahara et al. 1997) at primary afferent neurones. Recent experiments performed in mouse DRG–spinal neurone cocultures (Kerchner et al. 2002) showed that the depressant action of KA on NMDA receptor-mediated EPSCs was partially reduced by the deletion of either the GluR5 or GluR6 KAR subunit. To support this result, they showed that in mixed cocultures of GluR5-/- DRG and GluR6-/- DH neurones, KA or ATPA had no significant effect on NMDA EPSCs. Our result of the partial reduction of KA depressant action by GluR5 or GluR6 deletion in Krebs solution may be explained by their findings. However, the complete blockade of the KA depressant action by the knock-out of the GluR6 subunit in the absence of inhibitory transmission emphasizes the prominent role of the GluR6 subunit in eliminating the KAR modulation of both A and C primary afferent-evoked monosynaptic excitatory transmission. We are not sure why this discrepancy exists between our results and those of Kerchner and coworkers. However, it is possible that the subunit expression of KAR in early postnatal cultures may differ from that in the acutely prepared adult slices. Moreover, the present study focuses on the KA effects on the primary afferent A and/or C fibre-evoked monosynaptic EPSPs whose amplitudes are mostly mediated by AMPARs, whereas Kerchner et al. (2002) analysed the depressant action of KA on the NMDA receptor-mediated EPSCs evoked by extracellular stimulation directed at DRG cell bodies in culture.
The prominent role of the GluR6 subunit in inhibiting the transmission of primary afferents could be mediated by presynaptic KARs present on both A and C primary afferent fibres (Agrawal & Evans, 1986) and boutons (Hwang et al. 2001). Lee et al. (2001) provided evidence that KARs are present on C fibre-type DRG neurones projecting to lamina II of spinal DH. This report may explain part of our results which show that the GluR5 subunit plays a significant role only in C fibre-mediated excitatory transmission. However, evidence in favour of heterogeneity of primary afferents that express GluR5/6/7 subtypes has been provided by immunostaining of both myelinated and unmyelinated afferents (Hwang et al. 2001), supporting our result that GluR6 KARs are expressed on both A- and C-fibre terminals. In view of the blockade of KA-induced whole-cell currents in cultured DH neurones (Kerchner et al. 2002) and slices (Fig. 6A) from GluR6-/- mice, it is apparent that postsynaptic GluR6 subunits, together with presynaptic GluR6 subunits, may have the major functional role in the regulation of transmission at both primary afferent A and C fibre synapses. This conclusion, however, does not completely agree with some of the present anatomical data demonstrating regional expression of individual subunits in the spinal DH (Tölle et al. 1993; Stegenga & Kalb, 2001; Dai et al. 2002; but see also Guo et al. 2002; Kerchner et al. 2002), requiring further studies with more sensitive methods.
Activation of presynaptic kainate receptors decreases glutamate release
KARs are expressed on DRG cell bodies (Huettner, 1990), on peripheral fibres and axon terminals (Ault & Hildebrand, 1993), and preferentially on central terminals of sensory neurones, on which they could act as true autoreceptors (Agrawal & Evans, 1986; Hwang et al. 2001; Kerchner et al. 2001b; Lee et al. 2002). Previous work has demonstrated strong depolarizing responses when KA was applied to dorsal roots (Agrawal & Evans, 1986). To provide further evidence for the functional KARs on the adult mouse primary afferent neurones, and to determine their subunit composition, we have examined the effects of KAR activation on the frequency and amplitude of mEPSCs recorded from the SG neurones in slices of wild-type and GluR5- or GluR6-deficient mice. We found that in a subset of the mouse SG neurones, mEPSC frequencies were reversibly decreased after activation of KARs; this decrease arose from the activation of receptors incorporating both the GluR5 and GluR6 subunit. Although the excitatory synapses responsible for the mEPSCs are not identified (primary afferent versus local interneuronal synapses) with any degree of certainty (Lee et al. 1999), the observed decrease in frequency, and the dependence on GluR5 and GluR6 subunits supports the hypothesis that some, if not all, KA effects on mEPSC frequency occurred at primary afferent synapses, the result in agreement with presynaptic GluR5 and GluR6 localization in primary afferent terminals to the superficial laminae of the rat spinal cord (Hwang et al. 2001; Lee et al. 2002).
The mechanism by which KAR activation modulates excitatory synaptic transmission, at primary afferent synapses or other central synapses, remains unclear (Frerking & Nicoll, 2000). Studies of the effects of KAR agonists on glutamate release from synaptosomes have provided contradictory results (Zhou et al. 1995; Chittajallu et al. 1996; Perkinton & Sihra, 1999). One hypothesis, that KARs regulate glutamate release by a mechanism involving direct depolarization of axons or axon terminals, is supported by the finding that, in CA1 hippocampal neurones, KA induced a transient facilitation of evoked NMDAR-mediated EPSCs before a prolonged depression occurred (Chittajallu et al. 1996). More direct evidence that KARs mediate axonal depolarization comes from investigations of mossy fibre synapses demonstrating that presynaptic KAR-mediated depression of synaptic transmission was associated with increased mossy fibre excitability (Kamiya & Ozawa, 2000; Schmitz et al. 2000). This phenomenon was reproduced when synaptically released glutamate from mossy fibres, or associational x commissural fibres, was used instead of KA (Schmitz et al. 2000). A potential link between axonal depolarization and decrease of glutamate release has been proposed by Kamiya & Ozawa (1998, 2000), who showed reduced action potential-triggered Ca2+ influx into mossy fibre terminals with presynaptic KAR activation. The model proposed to explain these results is that KARs are present on the presynaptic terminal, which decrease release by a depolarization-induced inactivation of presynaptic calcium channels. Our data are at least consistent with such a model by providing direct evidence that presynaptic KAR activation by relatively high concentration (3 µM) of KA suppresses primary afferent transmission by strong depolarization of presynaptic fibres. GABA released by KAR activation from GABAergic interneurones may be additive to the KA-induced depolarization in axon terminals (Eccles et al. 1963; Kerchner et al. 2001a). In addition to the presynaptic depolarizing effect of KA, an antagonistic effect of KA on postsynaptic KARs which mediate spinal excitatory synaptic transmission (Li et al. 1999), may contribute the KA-induced depression of transmission because slow application of KA also desensitizes KARs (Lerma et al. 2001). However, this explanation can apply to only a small portion of C fibre-evoked EPSPs because postsynaptic KARs contribute only to synaptic currents evoked by high intensity stimulation and their portion in the entire synaptic current is relatively small compared to the AMPAR-mediated portion (Li et al. 1999). As an alternative to an ionotropic effect of KA on presynaptic sites, some evidence supports a possible G-protein-mediated action of presynaptic KAR stimulation at CA3–CA1 and DRG–spinal DH synapses (Frerking et al. 2001; Rozas et al. 2003).
Weak activation of kainate receptors potentiates excitatory synaptic transmission
Our finding showing a robust and long-lasting potentiation of excitatory synaptic transmission by a bath-applied low concentration of KA (30 nM, 2 min) has not been yet as described in the spinal cord SG region. In other brain areas, an excitatory effect of a low concentration of KA on evoked synaptic responses, which persisted only during its application, was recently discovered at the Schaffer collateral-commissural synapses (Chittajallu et al. 1996) and at mossy fibre synapses (Schmitz et al. 2001) in the hippocampus. On the other hand, Kerchner et al. (2001a) recently showed in the spinal DH that a low concentration (200 nM) of KA has the ability to enhance inhibitory synaptic responses induced by the stimulation of DH neurones. Although the effect occurred at inhibitory synapses under a condition of reduced neuronal excitability produced by raising the concentration of Ca2+ and Mg2+ from 2 mM to 6 mM, it provided a clue that the low concentration of KA may be able to potentiate synaptic strength in the spinal DH. A mechanism underlying the KA-induced long-lasting potentiation of excitatory transmission is still unknown. Our result, that the potentiation is an NMDAR-dependent and GluR5 and/or GluR6 KAR-mediated process, first provide some evidence for the possible mechanism. In addition, the findings that the 30 nM KA-induced potentiation reliably occurs in the presence of GABAA and glycine receptor antagonists, which reduce the amplitude of depolarization in primary afferent terminals, and can be mimicked by ATPA in a subpopulation of SG neurones, may fit to a recent proposal that weak presynaptic depolarization enhances transmitter release (Turecek & Trussell, 2001).
Significance
To summarize, in this study we show that KARs act via multiple and complex mechanisms to modulate excitatory synaptic transmission in the spinal cord substantia gelatinosa region. We have used gene-targeted mice lacking GluR5 and/or GluR6 KAR subunits to identify subunits involved in these effects. The results obtained support the hypothesis that KARs are involved in the bidirectional regulation of excitatory synaptic transmission in the substantia gelatinosa, and thus may be of critical importance in nociception and the clinical treatment of pain. Interestingly, recent behavioural studies have showed antinociceptive effects of specific KAR antagonists in models of persistent pain. Intravenous application of a mixed AMPA/GluR5 antagonist, LY 293558, in humans can prevent capsaicin-induced spontaneous pain and mechanical allodynia without any effect on brief pain sensations evoked in normal skin (Sang et al. 1998). In animal studies, LY 382884 reduced the formalin-induced secondary phase of nociceptive responses (Simmons et al. 1998). These reports indicate a functional role of KARs in the development, transmission and maintenance of nociception (Ruscheweyh & Sandkühler, 2002). Thus, our findings, which provide subunit profiles of KARs acting as excitatory or inhibitory regulators for the A or C fibre-mediated excitatory transmission on SG neurones, further suggest that the process of nociceptive transmission in the spinal SG neurones is finely tuned by the activation of KARs with different subunit compositions. Therefore, building up information on the synaptic or somatic location of KAR subunits and their physiological functions in the spinal cord DH will provide us with clues to develop more specific therapeutic strategies for the treatment of chronic pain.
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(P < 0.01) compared to GluR5-/- mice.
