Br. J. Pharmacol. (1992), 107, 628-633
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Macmillan Press Ltd, 1992
The effect of centrally acting myorelaxants on NMDA receptor-mediated synaptic transmission in the immature rat spinal cord in vitro R.J. Siarey, S.K. Long & ',*R.H. Evans Department of CNS-Pharmacology, SOLVAY DUPHAR BV, P 0 Box 900, 1380 DA Weesp, The Netherlands and *The Department of Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 ITD 1 The effect of the myorelaxant drugs baclofen, diazepam and tizanidine have been compared on in vitro preparations of baby rat spinal cord and adult rat superior cervical ganglion. 2 Dorsal root-elicited long duration (time to half decay 9.71 ± 0.29 s.e.mean, n = 31) ipsilateral ventral root reflexes (DR-VRP), measured as integrated area, of immature rat spinal cord preparations were abolished by RS-2-amino-5-phosphonopentanoate (AP5) (EC50 8.13 ± 0.92 ILM, n = 3). The initial short latency component of DR-VRP was resistant to AP5. 3 Baclofen abolished both components of the DR-VRP. Respective EC50 values for the AP5-insensitive and AP5-sensitive components were 237 ± 68 nM (n ± 7) and 57 ± 10 nM (n = 7). These effects of baclofen were reversed by the GABAB antagonist, CGP35348. The apparent Kd values (16.7 ± 6.4 pLM, n = 3 and 14.3 ± 3.9 JtM, n = 6 respectively) for this reversal were not significantly different. 4 Tizanidine, clonidine and diazepam had no effect on the AP5-insensitive component of the DR-VRP. 5 The AP5-sensitive long duration component of the DR-VRP was depressed to respective maximal levels of 23.2 ± 1.4% (n = 7), 18.8 ± 3.8% (n = 4) and 47.6 ± 1.6% (n = 5) of control (100%) levels by tizanidine (EC50 135 ± 33 nM), clonidine (EC50 26.0 ± 2.2 nM) and diazepam (EC25 114 ± 12 nM, n = 4). The depressant effects of tizanidine and clonidine were reversed by idazoxan (1 1AM). Flumazenil (I tiM) failed to reverse the depressant effect of tizanidine. The depressant effect of diazepam was reversed by flumazenil (1 jLM) but not by idazoxan (1 JAM). Naloxone 1 jAM did not reverse the effects of either tizanidine or diazepam. 6 In the presence of tetrodotoxin (0.1 SAM) which abolished synaptic activity, clonidine, tizanidine or diazepam (10, 100 and 101JM respectively) produced no significant antagonism of NMDA-induced depolarizations recorded from ventral roots. 7 Control (100%) synaptic responses of rat superior cervical ganglion preparations were depressed respectively to near maximal levels of 60.0 ± 5.2% (n = 4) and 60.7 ± 5.6% (n = 5) by clonidine (0.5 JAM, EC25 15.3 ± 3.0 nM) and tizanidine (1 JAM, EC25 227 ± 83 nM). These depressant effects were reversed by idazoxan (1 AM). Baclofen (EC25 28.7 ± 10.0, n = 3) depressed the postganglionic response to a maximum level of 71.8 + 2.4% (n = 4) control at a concentration of 100 JAM. The latter depressant action was reversed by the GABAB receptor antagonist, CGP35348 (1 mM). Diazepam (1 JAM) had no significant effect on ganglionic transmission. 8 It is concluded that the activation of benzodiazepine or M2-noradrenaline receptors can modulate NMDA receptor-mediated excitatory synaptic pathways whereas synaptic excitation from primary afferent terminals, mediated by non-NMDA receptors, appears to lack the propensity for this type of modulation. The results show also that the isolated spinal preparation can be used to identify central myorelaxant actions that are mediated through functional benzodiazepine or X2-noradrenaline receptors. Keywords: Myorelaxant; spinal cord; NMDA receptors
Introduction Baclofen, diazepam and tizanidine are centrally acting myorelaxant drugs in current use. These three drugs have differing sites of action which may contribute to their therapeutic properties. These sites are respectively GABAB receptors, benzodiazepine receptors and M2-adrenoceptors. The purpose of the present investigation was to compare the action of these agents on the synaptic input to motoneurones of the in vitro spinal cord preparation. The immature rat spinal cord preparation is of special interest for such comparison because a long duration (> 20 s) ventral root reflex (DR-VRP) of (1 to 5 day old) rat spinal cord preparations has been associated with nociceptive pathways (Akagi et al., 1985). The GABAB receptor agonist, baclofen (Akagi & Yanagisawa, 1987), diazepam (Akagi & Yanagisawa, 1987) and M2-adrenoceptor agonists (Kendig et I
Author for correspondence.
al., 1991) have all been shown to depress this long duration reflex. Tizanidine has been shown to have M2-adrenoceptor agonist properties at dorsal horn neurones in vivo (Davies & Quinlan, 1985). Thus in the present investigation the action of the myorelaxant, tizanidine, has been compared with the prototypic M2-adrenoceptor agonist, clonidine. Baclofen differs from diazepam and tizanidine in being a potent depressant of monosynaptic transmission from 1 a primary afferent fibres (Edwards et al., 1989). Flumazenil, idazoxan and the GABAB antagonist, CGP35348 (P-[3-aminopropyl]-P-diethoxymethylphosphinic acid; Bittiger et al., 1990) have been used to produce rightward shifts in the concentration effect plots to respective agonists and thus obtain apparent Kd values at the receptors involved in the depressant effects of these myorelaxants. An important feature of the long duration reflex of the in vitro spinal preparation is that it is mediated via neurotransmitter(s) which act at NMDA receptors (Brugger et al.,
MYORELAXANTS AND SPINAL CORD
1990). Thus in the present investigation the effects of an NMDA receptor antagonist have been compared with those of the other drugs. Because (X2-adrenoceptors, benzodiazepine receptors and GABAB receptors are not confined to the central nervous system, the in vitro superior cervical ganglion preparation has been used in the present study in order to compare the synaptic depressant potency and selectivity of the drugs at this peripheral site with the data from the spinal cord. Some of these results have been reported in preliminary form (Siarey et al., 1991a,b).
Methods The methods of superfusion, stimulation and recording were as described by Evans et al. (1982). Hemisected spinal cord preparations from rats of 2 to 5 days post partum age were superfused with medium containing (mM) MgSO4 1.25, CaCl2 1.5, NaCl 118, KCI 3, NaHCO3 24 and dextrose 12. Supramaximal electrical stimulation (10 to 20 times threshold) of a dorsal root (L4 or 5), cycle time 90s, was used to evoke synaptic potentials in the corresponding ventral root (DRVRP). Superior cervical ganglion and rat vagus nerve preparations were superfused with the same medium as for spinal cord preparations excepting for the MgSO4 concentration which was 0.75 mm. The presynaptic nerves of ganglia and the vagus nerves were also stimulated supramaximally but at a rate of 0.033 Hz. All preparations were maintained at 250C. Depressant effects of drugs on the electrically evoked responses were measured as the reduction in peak amplitude or area after integration over the complete time-course. Depressant potencies were estimated as the concentration required to depress synaptic responses to 50% of control values (EC50). This procedure was simple for those agents that could completely abolish synaptic responses. However, when the maximal depressant effect was close to 50% of control, potency was recorded as the concentration required to produce a 25% depression (EC25). The latter procedure was chosen because non-specific depressant effects produced at high concentrations, as described for tizanidine below, made it difficult or impossible to determine the maximal depressant action and thus normalize the depressant resa
ponses as fractions of the maximum. Unless stated otherwise, mean values are presented ± s.e.mean.
Results Involvement of NMDA receptors Figure la illustrates the depressant effect of RS-2-amino-5phosphonopentanoate (AP5) on the long duration component of the DR-VRP. Under control conditions the mean integrated area measured from 2 to 32 s was 19.5 ± 1.2 mV.s and the mean time of decay to half area of these potentials was 9.67 ± 0.49 s (n = 31). As expected from previous work (Evans et al., 1982; Long et al., 1990) the early component of the reflex was resistant to this antagonist whereas the long duration component (assessed from the area between 2 and 32 s) was depressed completely by concentrations of 100 gM or higher (EC50 8.13±0.92cM, n=3).
Action of c2-adrenoceptor agonists. Investigation of the properties of tizanidine, in relation to its myorelaxant action, was a major interest of this study. Because tizanidine has been characterized as an M2-adrenoceptor agonist, it was of interest to compare the properties of clonidine as a prototypic a2-adrenoceptor agonist with those of tizanidine. Figure lb illustrates the depressant action of clonidine. The early component of the synaptic response was resistant to clonidine. The long duration DR-VRP was partially resistant to clonidine as shown by the maximal depression to 18.8 ± 3.8% of control (n = 4) produced by concentrations of 100 nm or higher (EC50 26.0 ± 2.2 nM, n = 4). It can be seeen from Figure lb that recovery of the synaptic response on removal of clonidine was very slow compared to recovery from the depressant action of AP5 (Figure la). Thus after 60 min washout of clonidine, the long duration DR-VRP had recovered to only 24.5 ± 3.4% (n = 4) of the control area. However, the depressant action of clonidine was reversed rapidly on introduction of idazoxan (1 ILM) such that full recovery to the control area of synaptic response occurred within 17 ± 6 min (n = 4) (Figure lb). The selectivity of idazoxan, in reversing clonidine-induced depression, was demonstrated following the depressant action of b
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Figure 1 (a) Shows RS-2-amino-5-phosphonopentanoate (AP5)-sensitivity of long duration ipsilateral DR-VRP. Note return to control size of potential on washout of AP5. AP5 1, 5, 10 and 100 pM was introduced cumulatively as indicated by the bar above the plot. (b) Separate preparation showing depressant effect of clonidine and reversal by idazoxan. Clonidine was applied
cumulatively at 10, 20, 100 and 300 nm. The filled bar above the plot indicates introduction of idazoxan I pm. Single records before, during and after the depressant effects are shown above. Each point plotted below is the mean ± s.e.mean (vertical bars) of three consecutive recordings. Vertical scale bars 0.5 mV in (a) and 1 mV in (b). Horizontal scale bars 10 s.
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diazepam and baclofen (see below). Figure 2 illustrates that the effect of clonidine was mimicked, in every respect examined, by tizanidine which depressed the long duration DR-VRP to 23.2 ± 1.4% of control levels (EC50 for depression of the long duration DRVRP 135 nM ± 33, n = 7). As illustrated in Figure 2 doseratios for antagonism of the depressant effect by tizanidine by idazoxan were obtained in five preparations (apparent Kd 9.57 nM ± 2.14). The M2-adrenoceptor antagonist, prazosin (0. 5 JM), illustrated in Figure 2, and the opiate antagonist, naloxone (1 JM), did not reverse the depressant effect of tizanidine.
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The maximal depressant effect of diazepam (decrease in control DR-VRP area to 47.6 ± 1.6% produced by 1 JAM, EC25, 114 nM ± 12, n = 4) was less than that of the a2-adrenoceptor agonists. On all five preparations tested this depressant effect was not reversed by idazoxan (1 gM) (Figure 3). However, as illustrated in Figure 3, the depressant effect of diazepam was quickly reversed following introduction of flumazenil (1 JM).
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As illustrated by Figure 1, the component of the DRP-VRP which was most sensitive to clonidine, tizanidine and diazepam is characteristically mediated by NMDA receptors (Brugger et al., 1990). Therefore the myorelaxant drugs were tested for NMDA receptor antagonist properties, particularly because of previously reported postjunctional depressant actions of tizanidine (Curtis et al., 1983; Villanueva et al., 1988) and diazepam (Evans et al., 1977). In tetrodotoxinblocked preparations, clonidine (1O JM), tizanidine (1 00 JM) and diazepam (10 gM) produced mean dose-ratios of 0.97 ± 0.03 (n = 8), 1.00 ± 0.003 (n = 3) and 1.01 ± 0.03 (n = 3) respectively for antagonism of depolarizations produced by 10 to 30,M NMDA. In this series of experiments AP5 (1O JM) or kynurenate (100IM) produced mean doseratios of 2.29 ± 0.08 (n = 6) and 2.10 ± 0.08 (n = 6) respectively. Consistent with the resistance of the initial component of the DR-VRP, clonidine (1O JM), tizanidine (100 JM) and diazepam (10 JM) did not alter RS-a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA)-induced depolarization of motoneurones in the presence of tetrodotoxin. The respective dose-ratios were 0.97 ± 0.03 (n = 7), 0.83 ± 0.10 (n = 3) and 1.02 ± 0.07 (n = 3). In this series of experiments kynurenate (100 JM) produced a mean dose-ratio of 1.93 + 0.02 (n = 6) against the depolarizations induced by AMPA.
Action of baclofen The potent depressant effect of baclofen on spinal transmission from primary afferent nerves has been reported on several occasions and the role of GABA receptors in mediating this depressant action has been controversial (see review by Evans, 1989). Availability of the GABAB receptor antagonist, CGP35348 for the present experiments has allowed some resolution of this controversy. In the present experiments baclofen completely abolished all the components of the DR-VRP at concentrations higher than 2 JM. The early and long duration phases of the reflex had differing sensitivities to baclofen as shown by respective EC50 values of 237 ± 68 nM (n = 7) and 57 ± 10 nM (n = 7). However, the sensitivity of these components to antagonism by CGP35348 was not significantly different. Thus the effects of baclofen on short and long duration components of the reflex were reversed in the presence of CGP35348, yielding respective apparent Kd values of 16.7 ± 6.4 JM (n = 3) and 14.3 ± 3.9 JM (n = 6). The latter values are not significantly different. Idazoxan (1 JAM) failed to reverse the depressant effects of baclofen. These effects are illustrated in Figure 4.
MYORELAXANTS AND SPINAL CORD
Effects on the monosynaptic population e.p.s.p. from primary afferents The initial component of the DR-VRP consists mainly of the population spike of motoneurones. Therefore it is possible that the resistance of this component to the M2-adrenoceptor agonists and diazepam was apparent rather than real because the underlying excitatory postsynaptic potential (e.p.s.p.) could have been depressed to a level insufficient to prevent firing of motoneurones. To test this possibility the short latency population spike of the DR-VRP was blocked with kynurenic acid. In the presence of 1 mM kynurenate the initial component of the reflex then consisted of a smooth population e.p.s.p. (Figure 5) of mean latency 5.99 ± 0.62 ms, time to peak 21.7 ± 1.1 ms, peak amplitude 0.84 ± 0.12 mV and slope of 0.084 ± 0.018 V s-' (n = 10). Clonidine (300 nM, n = 3), tizanidine (1 jM, n = 3) (Figure 5) and diazepam (1 tM, n = 4) (Figure 5) had no significant effect on the rising slope of these potentials. However, as illustrated by the lower traces in Figure 4, baclofen decreased the rising slope of the e.p.s.p. when applied at concentrations insufficient to block it completely. This depressant effect of baclofen was reversed also by CGP35348 (200 pM).
Effects on ganglionic transmission Clonidine has been reported previously to depress transmission in the superior cervical ganglion by an action at &2adrenoceptors (Brown & Caulfield, 1979; Medgett, 1983). Thus it was of interest to compare depressant potencies of the x2-adrenoceptor agonists at this peripheral site with their potencies for depression of the spinal preparation. The postganglionic response of ganglion preparations had a mean latency of 12.9 ± 0.5 ms (n = 19). Clonidine and
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Figure 5 Effect of tizanidine (a), diazepam (b) and baclofen (c) on the population e.p.s.p. of motoneurones. The recordings were made in the presence of kynurenate (I mM) in order to block the compound action potential of the motoneurones. Each trace is the average of four consecutive recordings. (a), (b) and (c) are from different preparations. The first column of traces was obtained before introduction of drug. The second column was obtained 40 min after introduction of tizanidine (1 pM) or diazepam (1 #M) or 15 min after introduction of baclofen (0.3 pM). The third column of traces was obtained 10 min following the respective application of (in descending order) idazoxan (1 pM), flumazenil (1 pM) or CGP35348 (0.2mM). Only baclofen depressed the e.p.s.p. and the depression was reversed by CGP35348. Vertical scale bar 0.4 mV in (a) and (c), 0.22 mV in (b). Horizontal scale bar 10 ms.
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Figure 4 (b) Depressant action of baclofen on the initial monosynaptic (U, left hand ordinate scale) and the long duration (0, right hand ordinate scale) components of the DR-VRP. The recordings of the long duration DR-VRP in (a) are consecutively before introduction of baclofen, in the presence of baclofen (I pM) and in the presence of CGP35348 (1 mM) (vertical scale bar I mV). Cumulative introduction of 0.01, 0.03, 0.05, 0.1, 0.3, 1, 3, 10 and 30 pM baclofen is indicated above the time-course plot in (b) and the upper bar on the right indicates introduction of CGP35348 (I mM). Other details as for Figure 1.
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Figure 6 Effect of diazepam, baclofen and tizanidine on synaptic response of superior cervical ganglion. (a) Recordings obtained consecutively before introduction of diazepam, in the presence of diazepam (101pM), following washout of diazepam, in the presence of baclofen (100 gM), following washout of baclofen, in the presence of tizanidine (1 pM) and in the presence of idazoxan (I pM) as indicated above the time-course in (b) (vertical scale bar 2.5 mV, horizontal scale bar 5 ms). (b) The bars above the time-course indicate from the left introduction of diazepam (1 and 10 pM), baclofen (1 and 100 pM) and tizanidine (0.03, 0.3 and 1 pM). Introduction of idazoxan (1 pM) is indicated by the upper bar on the right above tizanidine. It can be seen that, unlike the other two drugs, diaztpam had no significant depressant action. (c) Concentration-effect plot of the depressant action of tizanidine on four preparations showing the rightward shift produced in the presence of idazoxan (I pM). Other details as for Figure 1.
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tizanidine (Figure 6) depressed the population spike of this response to plateau levels of 60.0 ± 5.2% (n =4) and 60.7 ± 5.6% (n = 5) respectively. As with the spinal cord, these depressant actions recovered very slowly on washout of drug but were quickly reversed on introduction of idazoxan (1 gM). These effects are illustrated in Figure 6. The preparation of Figure 6b was the one of the series on which tizanidine had the greatest depressant effect. The EC25 values (nM) for clonidine and tizanidine were 15.3 ± 3.0 (n = 4) and 227 ± 83 (n = 5) respectively. In contrast to clonidine and tizanidine, diazepam (1O0 M, n 6) (Figure 6) and AP5 (100 gM, n = 4), at concentrations which clearly depressed the long duration DRP-VRP, failed to depress ganglionic transmission. The apparent Kd values obtained for idazoxan in reversing the spinal (9.57 ± 2.14 nM, n = 5) and ganglionic (12.8 ± 2.0 nM, n = 5) depressant effects of tizanidine were not significantly different (P = 0.23 Wilcoxon-Mann-Whitney). In contrast to a previous report (Evans, 1979) the amplitude of postganglionic potentials was depressed by baclofen (EC25 28.7 ± 10.0 JAM, n = 3) to a mean maximal level of 71.8 ± 2.4% of control (100%) at 100 JM (n = 4). This depressant effect of 10 JAM baclofen was reversed in the presence of CGP35348 (1 mM). In three out of four ganglion preparations it was observed that application of baclofen (10 M) produced a mean postganglionic hyperpolarization of 0.020 ± 0.007 mV. The hyperpolarizations developed over a period of 3 to 8 min and were swiftly reversed on application of CGP35348 (1 mM). Tizanidine, at higher concentrations than those above, had a non-specific depressant action. This non-specific action was shown following application of 1 mM tizanidine to four preparations of rat vagus nerve. The amplitudes of A and C elevations in the compound action potential of the nerves were depressed to 63.3 ± 5.0% and 53.8 ± 4.0% of control (100%) levels respectively. This depression was not reversed by 1 JAM idazoxan. Clonidine and diazepam were not tested on the =
vagus nerve.
Discussion The long duration DR-VRP is mediated by NMDA receptors as indicated by the complete blockade which occurs in the presence of an appropriate concentration of AP5. The depressant effect of the M2-adrenoceptor agonists on this reflex reflects intrinsic inhibitory mechanisms which operate through M2-
adrenoceptors (Taylor et al., 1991). Similarly the depressant effect of diazepam is likely to reflect the operation of GABAA receptors in inhibitory pathways. These drugs failed to depress the rising slope of the dorsal root evoked population e.p.s.p. of motoneurones which is mediated through non-NMDA receptors (Long et al., 1990). These three drugs failed to antagonize NMDA- or AMPAinduced depolarizations at concentrations higher than those required for the depressant action on the long duration DRVRP. Therefore the depressant action was unlikely to have been due to postjunctional antagonism at excitatory amino acid receptors. Diazepam has some excitatory amino acid antagonist activity but only at concentrations much higher than those used in the present study (Evans et al., 1977). Thus the drugs appear to modulate selectively NMDA receptormediated excitatory transmission. It is implied from this that transmitters released on to NMDA receptors can be modulated separately from those released on to non-NMDA receptors. Such selectivity may be a consequence of the temporal difference between non-NMDA- and NMDA-receptormediated events. The n2-agonists produced a larger maximal depressant action than did diazepam suggesting that these two classes of drug may influence different populations of excitatory synapses.
The speedy and selective reversal of depressant effects produced by the antagonists idoxazan and flumazenil were important in demonstrating the effects of the respective agonists. The synaptic depression produced by these agonists is slow to reverse in the absence of antagonist, indicating persistence of the agonists in the tissue during the application of agonist-free wash solution. The effects of the a2-adrenoceptor agonists and diazepam contrast with the action of baclofen which depressed all components of the DR-VRP as expected from its action at the primary afferent synapse (Edwards et al., 1989). The apparent Kd of 10.6 pM reported here for the GABAB antagonist, CGP35348, in reversing the action of baclofen is not significantly different from the value of 11.2 gM for depression of e.p.s.ps of striatal neurones (Seabrook et al., 1990). In the present experiments the different components of the DR-VRP had different sensitivities from baclofen as shown by the four fold higher EC50 for depression of the initial monosynaptic as compared to the long duration component. However, the dose-ratios for reversal of these depressant actions of baclofen, produced by the antagonist CGP35348 were not significantly different. Thus the antagonist is able to define only one population of GABAB receptors in this preparation. Although in the present investigation baclofen was 500 times less potent at depressing ganglionic than it was at depressing spinal transmission, this effect of baclofen on the superior cervical ganglion preparations was unexpected since a previous study showed no action of baclofen at concentrations up to mM (Evans, 1979). The failure of baclofen to depress the postjunctional response of ganglia to less than approximately 70% of control levels suggests that postjunctional hyperpolarization of ganglionic neurones may be involved rather than the depression of transmitter release. The adrenoceptor agonists were distinguished from diazepam by their depressant action on the superior cervical ganglion preparation. The apparent Kd value of 11 nM for idazoxan-induced reversal of the effect of tizanidine applied to the ganglionic preparations was not significantly different from the value obtained with the spinal preparations or from the value of 9 nM obtained by Williams et al. (1985) at locus coeruleus neurones. Thus the affinities for idazoxan of spinal, cephalic and peripheral a2-adrenoceptors are very similar if not the same. In the present investigation, tizanidine and clonidine had a greater maximal depressant effect than diazepam on the spinal cord and baclofen and tizanidine depressed ganglionic transmission whereas diazepam did not. Thus benzodiazepine ligands can be differentiated from adrenoceptor ligands due to their lack of effect on ganglionic transmission as well as their sensitivity to flumazenil. Muscle spasm is painful. Thus it is helpful if myorelaxant drugs are also analgesic. Baclofen (Sawynok & Reid, 1988), benzodiazepines (Goodchild & Serrao, 1987) and M2-adrenoceptor agonists (Bonnet et al., 1989) are all reported to have analgesic actions at the spinal level. It is claimed that the long duration DR-VRP is a manifestation of activity in nociceptive pathways in the spinal cord (Akagi et al., 1985). This is certainly consistent with the analgesic action of intrathecally administered AP5 (Cahusac et al., 1984). Thus depression of the long duration reflex by these myorelaxant drugs is consistent with the proposed link between this reflex and nociception. Therefore depression of the long duration component of the DR-VRP may be a predictor for in vivo centrally acting myorelaxant activity. The myorelaxant action of AP5 and other NMDA receptor antagonists (Turski et al., 1984) is consistent with this expectation. R.H.E. was supported by the Medical Research Council, The Taberner Trust and The Wellcome Trust and R.J.S. was supported by a DUPHAR research scholarship.
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EVANS, R.H., FRANCIS, A.A. & WATKINS, J.C. (1977). Differential antagonism by chlorpromazine and diazepam of frog motoneurone depolarization induced by glutamate-related amino acids. Eur. J. Pharmacol., 44, 325-330. GOODCHILD, C.S. & SERRAO, J.M. (1987). Intrathecal midazolam in the rat: evidence for spinally-mediated analgesia. Br. J. Anaesth., 59, 1563-1570. KENDIG, J.J., SAVOLA, M.K.T., WOODLEY, S.J. & MAZE, M. (1991).
a2-Adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord. Eur. J. Pharmacol., 192, 293-300. LONG, S.K., SMITH, D.A.S., SIAREY, R.J. & EVANS, R.H. (1990).
Effect of 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) on dorsal root-, NMDA, kainate- and quisqualate-mediated depolarization of rat motoneurones in vitro. Br. J. Pharmacol., 100, 850-854. MEDGETT, I.C. (1983). Modulation of transmission in rat sympathetic ganglion by activation of presynaptic a- and fradrenoceptors. Br. J. Pharmacol., 78, 17-27. SAWYNOK, J. & REID, A. (1988). Role of ascending and descending serotonergic pathways in the antinociceptive effect of baclofen. Naunyn Schmiedebergs Arch. Pharmacol., 337, 359-365. SEABROOK, G.R., HOWSON, W. & LACEY, M.G. (1990), Electro-
physiological characterization of potent agonists and antagonists at pre- and postsynaptic GABAB receptors on neurones in rat brain slices. Br. J. Pharmacol., 101, 949-957. SIAREY, R.J., LONG, S.K. & EVANS, R.H. (1991a). Effect of clonidine on synaptic activity of rat in vitro spinal cord preparations. J. Physiol., 435, 70P. SIAREY, R.J., LONG, S.K. & EVANS, R.H. (1991b). Potentiation of synaptic reflexes by D-serine in the rat spinal cord in vitro. Eur. J. Pharmacol., 195, 241-244. TAYLOR, J.S., NEAL, R.I., HARRIS, J., FORD, T.W. & CLARKE, R.W.
tizanidine (DS103-282) and noradrenaline: involvement of M2adrenoceptors. Neuroscience, 16, 673-682.
(1991). Prolonged inhibition of a spinal reflex after intense stimulation of distant peripheral nerves in the decerebrated rabbit. J. Physiol., 437, 71-84.
EDWARDS, F.R., HARRISON, P.J., JACK, J.J.B. & KULLMAN, D.M.
TURSKI, L., SCHWARCZ, M., TURSKI, W.A., KLOCKGETHER, T.,
(1989). Reduction by baclofen of monosynaptic EPSPs in lumbosacral motoneurones of the anaesthetised cat. J. Physiol., 416, 539-556. EVANS, R.H. (1979). Baclofen: lack of effect on neurotransmission in the mouse vas deferens. J. Pharm. Pharmacol., 31, 642-643. EVANS, R.H. (1989). The pharmacology of segmental transmission in the spinal cord. Progr. Neurobiol., 33, 255-279.
SONTAG, K.-H. & COLLINS, J.F. (1984). Muscle relaxant action of excitatory amino acid antagonists. Neurosci. Lett., 53, 321-326. VILLANUEVA, L., CHITOUR, D. & LE BARS, D. (1988). Effects of tizanidine (DS103-282) on dorsal horn convergent neurones in the rat. Pain, 35, 187-197. WILLIAMS, J.T., HENDERSON, G. & NORTH, R.A. (1985). Characterization of U2-adrenoceptors which increase potassium conductance in rat locus coeruleus neurones. Neuroscience, 14, 95-101.
EVANS, R.H., FRANCIS, A.A., JONES, A.W., SMITH, D.A.S. & WATKINS, J.C. (1982). The effects of a series of w-phosphonic acarboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations. Br. J.
Pharmacol., 75, 65-75.
(Received June 10, 1992 Revised June 29, 1992 Accepted July 1, 1992)
'."
Macmillan Press Ltd, 1992
The effect of centrally acting myorelaxants on NMDA receptor-mediated synaptic transmission in the immature rat spinal cord in vitro R.J. Siarey, S.K. Long & ',*R.H. Evans Department of CNS-Pharmacology, SOLVAY DUPHAR BV, P 0 Box 900, 1380 DA Weesp, The Netherlands and *The Department of Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 ITD 1 The effect of the myorelaxant drugs baclofen, diazepam and tizanidine have been compared on in vitro preparations of baby rat spinal cord and adult rat superior cervical ganglion. 2 Dorsal root-elicited long duration (time to half decay 9.71 ± 0.29 s.e.mean, n = 31) ipsilateral ventral root reflexes (DR-VRP), measured as integrated area, of immature rat spinal cord preparations were abolished by RS-2-amino-5-phosphonopentanoate (AP5) (EC50 8.13 ± 0.92 ILM, n = 3). The initial short latency component of DR-VRP was resistant to AP5. 3 Baclofen abolished both components of the DR-VRP. Respective EC50 values for the AP5-insensitive and AP5-sensitive components were 237 ± 68 nM (n ± 7) and 57 ± 10 nM (n = 7). These effects of baclofen were reversed by the GABAB antagonist, CGP35348. The apparent Kd values (16.7 ± 6.4 pLM, n = 3 and 14.3 ± 3.9 JtM, n = 6 respectively) for this reversal were not significantly different. 4 Tizanidine, clonidine and diazepam had no effect on the AP5-insensitive component of the DR-VRP. 5 The AP5-sensitive long duration component of the DR-VRP was depressed to respective maximal levels of 23.2 ± 1.4% (n = 7), 18.8 ± 3.8% (n = 4) and 47.6 ± 1.6% (n = 5) of control (100%) levels by tizanidine (EC50 135 ± 33 nM), clonidine (EC50 26.0 ± 2.2 nM) and diazepam (EC25 114 ± 12 nM, n = 4). The depressant effects of tizanidine and clonidine were reversed by idazoxan (1 1AM). Flumazenil (I tiM) failed to reverse the depressant effect of tizanidine. The depressant effect of diazepam was reversed by flumazenil (1 jLM) but not by idazoxan (1 JAM). Naloxone 1 jAM did not reverse the effects of either tizanidine or diazepam. 6 In the presence of tetrodotoxin (0.1 SAM) which abolished synaptic activity, clonidine, tizanidine or diazepam (10, 100 and 101JM respectively) produced no significant antagonism of NMDA-induced depolarizations recorded from ventral roots. 7 Control (100%) synaptic responses of rat superior cervical ganglion preparations were depressed respectively to near maximal levels of 60.0 ± 5.2% (n = 4) and 60.7 ± 5.6% (n = 5) by clonidine (0.5 JAM, EC25 15.3 ± 3.0 nM) and tizanidine (1 JAM, EC25 227 ± 83 nM). These depressant effects were reversed by idazoxan (1 AM). Baclofen (EC25 28.7 ± 10.0, n = 3) depressed the postganglionic response to a maximum level of 71.8 + 2.4% (n = 4) control at a concentration of 100 JAM. The latter depressant action was reversed by the GABAB receptor antagonist, CGP35348 (1 mM). Diazepam (1 JAM) had no significant effect on ganglionic transmission. 8 It is concluded that the activation of benzodiazepine or M2-noradrenaline receptors can modulate NMDA receptor-mediated excitatory synaptic pathways whereas synaptic excitation from primary afferent terminals, mediated by non-NMDA receptors, appears to lack the propensity for this type of modulation. The results show also that the isolated spinal preparation can be used to identify central myorelaxant actions that are mediated through functional benzodiazepine or X2-noradrenaline receptors. Keywords: Myorelaxant; spinal cord; NMDA receptors
Introduction Baclofen, diazepam and tizanidine are centrally acting myorelaxant drugs in current use. These three drugs have differing sites of action which may contribute to their therapeutic properties. These sites are respectively GABAB receptors, benzodiazepine receptors and M2-adrenoceptors. The purpose of the present investigation was to compare the action of these agents on the synaptic input to motoneurones of the in vitro spinal cord preparation. The immature rat spinal cord preparation is of special interest for such comparison because a long duration (> 20 s) ventral root reflex (DR-VRP) of (1 to 5 day old) rat spinal cord preparations has been associated with nociceptive pathways (Akagi et al., 1985). The GABAB receptor agonist, baclofen (Akagi & Yanagisawa, 1987), diazepam (Akagi & Yanagisawa, 1987) and M2-adrenoceptor agonists (Kendig et I
Author for correspondence.
al., 1991) have all been shown to depress this long duration reflex. Tizanidine has been shown to have M2-adrenoceptor agonist properties at dorsal horn neurones in vivo (Davies & Quinlan, 1985). Thus in the present investigation the action of the myorelaxant, tizanidine, has been compared with the prototypic M2-adrenoceptor agonist, clonidine. Baclofen differs from diazepam and tizanidine in being a potent depressant of monosynaptic transmission from 1 a primary afferent fibres (Edwards et al., 1989). Flumazenil, idazoxan and the GABAB antagonist, CGP35348 (P-[3-aminopropyl]-P-diethoxymethylphosphinic acid; Bittiger et al., 1990) have been used to produce rightward shifts in the concentration effect plots to respective agonists and thus obtain apparent Kd values at the receptors involved in the depressant effects of these myorelaxants. An important feature of the long duration reflex of the in vitro spinal preparation is that it is mediated via neurotransmitter(s) which act at NMDA receptors (Brugger et al.,
MYORELAXANTS AND SPINAL CORD
1990). Thus in the present investigation the effects of an NMDA receptor antagonist have been compared with those of the other drugs. Because (X2-adrenoceptors, benzodiazepine receptors and GABAB receptors are not confined to the central nervous system, the in vitro superior cervical ganglion preparation has been used in the present study in order to compare the synaptic depressant potency and selectivity of the drugs at this peripheral site with the data from the spinal cord. Some of these results have been reported in preliminary form (Siarey et al., 1991a,b).
Methods The methods of superfusion, stimulation and recording were as described by Evans et al. (1982). Hemisected spinal cord preparations from rats of 2 to 5 days post partum age were superfused with medium containing (mM) MgSO4 1.25, CaCl2 1.5, NaCl 118, KCI 3, NaHCO3 24 and dextrose 12. Supramaximal electrical stimulation (10 to 20 times threshold) of a dorsal root (L4 or 5), cycle time 90s, was used to evoke synaptic potentials in the corresponding ventral root (DRVRP). Superior cervical ganglion and rat vagus nerve preparations were superfused with the same medium as for spinal cord preparations excepting for the MgSO4 concentration which was 0.75 mm. The presynaptic nerves of ganglia and the vagus nerves were also stimulated supramaximally but at a rate of 0.033 Hz. All preparations were maintained at 250C. Depressant effects of drugs on the electrically evoked responses were measured as the reduction in peak amplitude or area after integration over the complete time-course. Depressant potencies were estimated as the concentration required to depress synaptic responses to 50% of control values (EC50). This procedure was simple for those agents that could completely abolish synaptic responses. However, when the maximal depressant effect was close to 50% of control, potency was recorded as the concentration required to produce a 25% depression (EC25). The latter procedure was chosen because non-specific depressant effects produced at high concentrations, as described for tizanidine below, made it difficult or impossible to determine the maximal depressant action and thus normalize the depressant resa
ponses as fractions of the maximum. Unless stated otherwise, mean values are presented ± s.e.mean.
Results Involvement of NMDA receptors Figure la illustrates the depressant effect of RS-2-amino-5phosphonopentanoate (AP5) on the long duration component of the DR-VRP. Under control conditions the mean integrated area measured from 2 to 32 s was 19.5 ± 1.2 mV.s and the mean time of decay to half area of these potentials was 9.67 ± 0.49 s (n = 31). As expected from previous work (Evans et al., 1982; Long et al., 1990) the early component of the reflex was resistant to this antagonist whereas the long duration component (assessed from the area between 2 and 32 s) was depressed completely by concentrations of 100 gM or higher (EC50 8.13±0.92cM, n=3).
Action of c2-adrenoceptor agonists. Investigation of the properties of tizanidine, in relation to its myorelaxant action, was a major interest of this study. Because tizanidine has been characterized as an M2-adrenoceptor agonist, it was of interest to compare the properties of clonidine as a prototypic a2-adrenoceptor agonist with those of tizanidine. Figure lb illustrates the depressant action of clonidine. The early component of the synaptic response was resistant to clonidine. The long duration DR-VRP was partially resistant to clonidine as shown by the maximal depression to 18.8 ± 3.8% of control (n = 4) produced by concentrations of 100 nm or higher (EC50 26.0 ± 2.2 nM, n = 4). It can be seeen from Figure lb that recovery of the synaptic response on removal of clonidine was very slow compared to recovery from the depressant action of AP5 (Figure la). Thus after 60 min washout of clonidine, the long duration DR-VRP had recovered to only 24.5 ± 3.4% (n = 4) of the control area. However, the depressant action of clonidine was reversed rapidly on introduction of idazoxan (1 ILM) such that full recovery to the control area of synaptic response occurred within 17 ± 6 min (n = 4) (Figure lb). The selectivity of idazoxan, in reversing clonidine-induced depression, was demonstrated following the depressant action of b
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Figure 1 (a) Shows RS-2-amino-5-phosphonopentanoate (AP5)-sensitivity of long duration ipsilateral DR-VRP. Note return to control size of potential on washout of AP5. AP5 1, 5, 10 and 100 pM was introduced cumulatively as indicated by the bar above the plot. (b) Separate preparation showing depressant effect of clonidine and reversal by idazoxan. Clonidine was applied
cumulatively at 10, 20, 100 and 300 nm. The filled bar above the plot indicates introduction of idazoxan I pm. Single records before, during and after the depressant effects are shown above. Each point plotted below is the mean ± s.e.mean (vertical bars) of three consecutive recordings. Vertical scale bars 0.5 mV in (a) and 1 mV in (b). Horizontal scale bars 10 s.
R.J. SIAREY et al.
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diazepam and baclofen (see below). Figure 2 illustrates that the effect of clonidine was mimicked, in every respect examined, by tizanidine which depressed the long duration DR-VRP to 23.2 ± 1.4% of control levels (EC50 for depression of the long duration DRVRP 135 nM ± 33, n = 7). As illustrated in Figure 2 doseratios for antagonism of the depressant effect by tizanidine by idazoxan were obtained in five preparations (apparent Kd 9.57 nM ± 2.14). The M2-adrenoceptor antagonist, prazosin (0. 5 JM), illustrated in Figure 2, and the opiate antagonist, naloxone (1 JM), did not reverse the depressant effect of tizanidine.
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The maximal depressant effect of diazepam (decrease in control DR-VRP area to 47.6 ± 1.6% produced by 1 JAM, EC25, 114 nM ± 12, n = 4) was less than that of the a2-adrenoceptor agonists. On all five preparations tested this depressant effect was not reversed by idazoxan (1 gM) (Figure 3). However, as illustrated in Figure 3, the depressant effect of diazepam was quickly reversed following introduction of flumazenil (1 JM).
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As illustrated by Figure 1, the component of the DRP-VRP which was most sensitive to clonidine, tizanidine and diazepam is characteristically mediated by NMDA receptors (Brugger et al., 1990). Therefore the myorelaxant drugs were tested for NMDA receptor antagonist properties, particularly because of previously reported postjunctional depressant actions of tizanidine (Curtis et al., 1983; Villanueva et al., 1988) and diazepam (Evans et al., 1977). In tetrodotoxinblocked preparations, clonidine (1O JM), tizanidine (1 00 JM) and diazepam (10 gM) produced mean dose-ratios of 0.97 ± 0.03 (n = 8), 1.00 ± 0.003 (n = 3) and 1.01 ± 0.03 (n = 3) respectively for antagonism of depolarizations produced by 10 to 30,M NMDA. In this series of experiments AP5 (1O JM) or kynurenate (100IM) produced mean doseratios of 2.29 ± 0.08 (n = 6) and 2.10 ± 0.08 (n = 6) respectively. Consistent with the resistance of the initial component of the DR-VRP, clonidine (1O JM), tizanidine (100 JM) and diazepam (10 JM) did not alter RS-a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA)-induced depolarization of motoneurones in the presence of tetrodotoxin. The respective dose-ratios were 0.97 ± 0.03 (n = 7), 0.83 ± 0.10 (n = 3) and 1.02 ± 0.07 (n = 3). In this series of experiments kynurenate (100 JM) produced a mean dose-ratio of 1.93 + 0.02 (n = 6) against the depolarizations induced by AMPA.
Action of baclofen The potent depressant effect of baclofen on spinal transmission from primary afferent nerves has been reported on several occasions and the role of GABA receptors in mediating this depressant action has been controversial (see review by Evans, 1989). Availability of the GABAB receptor antagonist, CGP35348 for the present experiments has allowed some resolution of this controversy. In the present experiments baclofen completely abolished all the components of the DR-VRP at concentrations higher than 2 JM. The early and long duration phases of the reflex had differing sensitivities to baclofen as shown by respective EC50 values of 237 ± 68 nM (n = 7) and 57 ± 10 nM (n = 7). However, the sensitivity of these components to antagonism by CGP35348 was not significantly different. Thus the effects of baclofen on short and long duration components of the reflex were reversed in the presence of CGP35348, yielding respective apparent Kd values of 16.7 ± 6.4 JM (n = 3) and 14.3 ± 3.9 JM (n = 6). The latter values are not significantly different. Idazoxan (1 JAM) failed to reverse the depressant effects of baclofen. These effects are illustrated in Figure 4.
MYORELAXANTS AND SPINAL CORD
Effects on the monosynaptic population e.p.s.p. from primary afferents The initial component of the DR-VRP consists mainly of the population spike of motoneurones. Therefore it is possible that the resistance of this component to the M2-adrenoceptor agonists and diazepam was apparent rather than real because the underlying excitatory postsynaptic potential (e.p.s.p.) could have been depressed to a level insufficient to prevent firing of motoneurones. To test this possibility the short latency population spike of the DR-VRP was blocked with kynurenic acid. In the presence of 1 mM kynurenate the initial component of the reflex then consisted of a smooth population e.p.s.p. (Figure 5) of mean latency 5.99 ± 0.62 ms, time to peak 21.7 ± 1.1 ms, peak amplitude 0.84 ± 0.12 mV and slope of 0.084 ± 0.018 V s-' (n = 10). Clonidine (300 nM, n = 3), tizanidine (1 jM, n = 3) (Figure 5) and diazepam (1 tM, n = 4) (Figure 5) had no significant effect on the rising slope of these potentials. However, as illustrated by the lower traces in Figure 4, baclofen decreased the rising slope of the e.p.s.p. when applied at concentrations insufficient to block it completely. This depressant effect of baclofen was reversed also by CGP35348 (200 pM).
Effects on ganglionic transmission Clonidine has been reported previously to depress transmission in the superior cervical ganglion by an action at &2adrenoceptors (Brown & Caulfield, 1979; Medgett, 1983). Thus it was of interest to compare depressant potencies of the x2-adrenoceptor agonists at this peripheral site with their potencies for depression of the spinal preparation. The postganglionic response of ganglion preparations had a mean latency of 12.9 ± 0.5 ms (n = 19). Clonidine and
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Figure 5 Effect of tizanidine (a), diazepam (b) and baclofen (c) on the population e.p.s.p. of motoneurones. The recordings were made in the presence of kynurenate (I mM) in order to block the compound action potential of the motoneurones. Each trace is the average of four consecutive recordings. (a), (b) and (c) are from different preparations. The first column of traces was obtained before introduction of drug. The second column was obtained 40 min after introduction of tizanidine (1 pM) or diazepam (1 #M) or 15 min after introduction of baclofen (0.3 pM). The third column of traces was obtained 10 min following the respective application of (in descending order) idazoxan (1 pM), flumazenil (1 pM) or CGP35348 (0.2mM). Only baclofen depressed the e.p.s.p. and the depression was reversed by CGP35348. Vertical scale bar 0.4 mV in (a) and (c), 0.22 mV in (b). Horizontal scale bar 10 ms.
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Figure 4 (b) Depressant action of baclofen on the initial monosynaptic (U, left hand ordinate scale) and the long duration (0, right hand ordinate scale) components of the DR-VRP. The recordings of the long duration DR-VRP in (a) are consecutively before introduction of baclofen, in the presence of baclofen (I pM) and in the presence of CGP35348 (1 mM) (vertical scale bar I mV). Cumulative introduction of 0.01, 0.03, 0.05, 0.1, 0.3, 1, 3, 10 and 30 pM baclofen is indicated above the time-course plot in (b) and the upper bar on the right indicates introduction of CGP35348 (I mM). Other details as for Figure 1.
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Figure 6 Effect of diazepam, baclofen and tizanidine on synaptic response of superior cervical ganglion. (a) Recordings obtained consecutively before introduction of diazepam, in the presence of diazepam (101pM), following washout of diazepam, in the presence of baclofen (100 gM), following washout of baclofen, in the presence of tizanidine (1 pM) and in the presence of idazoxan (I pM) as indicated above the time-course in (b) (vertical scale bar 2.5 mV, horizontal scale bar 5 ms). (b) The bars above the time-course indicate from the left introduction of diazepam (1 and 10 pM), baclofen (1 and 100 pM) and tizanidine (0.03, 0.3 and 1 pM). Introduction of idazoxan (1 pM) is indicated by the upper bar on the right above tizanidine. It can be seen that, unlike the other two drugs, diaztpam had no significant depressant action. (c) Concentration-effect plot of the depressant action of tizanidine on four preparations showing the rightward shift produced in the presence of idazoxan (I pM). Other details as for Figure 1.
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tizanidine (Figure 6) depressed the population spike of this response to plateau levels of 60.0 ± 5.2% (n =4) and 60.7 ± 5.6% (n = 5) respectively. As with the spinal cord, these depressant actions recovered very slowly on washout of drug but were quickly reversed on introduction of idazoxan (1 gM). These effects are illustrated in Figure 6. The preparation of Figure 6b was the one of the series on which tizanidine had the greatest depressant effect. The EC25 values (nM) for clonidine and tizanidine were 15.3 ± 3.0 (n = 4) and 227 ± 83 (n = 5) respectively. In contrast to clonidine and tizanidine, diazepam (1O0 M, n 6) (Figure 6) and AP5 (100 gM, n = 4), at concentrations which clearly depressed the long duration DRP-VRP, failed to depress ganglionic transmission. The apparent Kd values obtained for idazoxan in reversing the spinal (9.57 ± 2.14 nM, n = 5) and ganglionic (12.8 ± 2.0 nM, n = 5) depressant effects of tizanidine were not significantly different (P = 0.23 Wilcoxon-Mann-Whitney). In contrast to a previous report (Evans, 1979) the amplitude of postganglionic potentials was depressed by baclofen (EC25 28.7 ± 10.0 JAM, n = 3) to a mean maximal level of 71.8 ± 2.4% of control (100%) at 100 JM (n = 4). This depressant effect of 10 JAM baclofen was reversed in the presence of CGP35348 (1 mM). In three out of four ganglion preparations it was observed that application of baclofen (10 M) produced a mean postganglionic hyperpolarization of 0.020 ± 0.007 mV. The hyperpolarizations developed over a period of 3 to 8 min and were swiftly reversed on application of CGP35348 (1 mM). Tizanidine, at higher concentrations than those above, had a non-specific depressant action. This non-specific action was shown following application of 1 mM tizanidine to four preparations of rat vagus nerve. The amplitudes of A and C elevations in the compound action potential of the nerves were depressed to 63.3 ± 5.0% and 53.8 ± 4.0% of control (100%) levels respectively. This depression was not reversed by 1 JAM idazoxan. Clonidine and diazepam were not tested on the =
vagus nerve.
Discussion The long duration DR-VRP is mediated by NMDA receptors as indicated by the complete blockade which occurs in the presence of an appropriate concentration of AP5. The depressant effect of the M2-adrenoceptor agonists on this reflex reflects intrinsic inhibitory mechanisms which operate through M2-
adrenoceptors (Taylor et al., 1991). Similarly the depressant effect of diazepam is likely to reflect the operation of GABAA receptors in inhibitory pathways. These drugs failed to depress the rising slope of the dorsal root evoked population e.p.s.p. of motoneurones which is mediated through non-NMDA receptors (Long et al., 1990). These three drugs failed to antagonize NMDA- or AMPAinduced depolarizations at concentrations higher than those required for the depressant action on the long duration DRVRP. Therefore the depressant action was unlikely to have been due to postjunctional antagonism at excitatory amino acid receptors. Diazepam has some excitatory amino acid antagonist activity but only at concentrations much higher than those used in the present study (Evans et al., 1977). Thus the drugs appear to modulate selectively NMDA receptormediated excitatory transmission. It is implied from this that transmitters released on to NMDA receptors can be modulated separately from those released on to non-NMDA receptors. Such selectivity may be a consequence of the temporal difference between non-NMDA- and NMDA-receptormediated events. The n2-agonists produced a larger maximal depressant action than did diazepam suggesting that these two classes of drug may influence different populations of excitatory synapses.
The speedy and selective reversal of depressant effects produced by the antagonists idoxazan and flumazenil were important in demonstrating the effects of the respective agonists. The synaptic depression produced by these agonists is slow to reverse in the absence of antagonist, indicating persistence of the agonists in the tissue during the application of agonist-free wash solution. The effects of the a2-adrenoceptor agonists and diazepam contrast with the action of baclofen which depressed all components of the DR-VRP as expected from its action at the primary afferent synapse (Edwards et al., 1989). The apparent Kd of 10.6 pM reported here for the GABAB antagonist, CGP35348, in reversing the action of baclofen is not significantly different from the value of 11.2 gM for depression of e.p.s.ps of striatal neurones (Seabrook et al., 1990). In the present experiments the different components of the DR-VRP had different sensitivities from baclofen as shown by the four fold higher EC50 for depression of the initial monosynaptic as compared to the long duration component. However, the dose-ratios for reversal of these depressant actions of baclofen, produced by the antagonist CGP35348 were not significantly different. Thus the antagonist is able to define only one population of GABAB receptors in this preparation. Although in the present investigation baclofen was 500 times less potent at depressing ganglionic than it was at depressing spinal transmission, this effect of baclofen on the superior cervical ganglion preparations was unexpected since a previous study showed no action of baclofen at concentrations up to mM (Evans, 1979). The failure of baclofen to depress the postjunctional response of ganglia to less than approximately 70% of control levels suggests that postjunctional hyperpolarization of ganglionic neurones may be involved rather than the depression of transmitter release. The adrenoceptor agonists were distinguished from diazepam by their depressant action on the superior cervical ganglion preparation. The apparent Kd value of 11 nM for idazoxan-induced reversal of the effect of tizanidine applied to the ganglionic preparations was not significantly different from the value obtained with the spinal preparations or from the value of 9 nM obtained by Williams et al. (1985) at locus coeruleus neurones. Thus the affinities for idazoxan of spinal, cephalic and peripheral a2-adrenoceptors are very similar if not the same. In the present investigation, tizanidine and clonidine had a greater maximal depressant effect than diazepam on the spinal cord and baclofen and tizanidine depressed ganglionic transmission whereas diazepam did not. Thus benzodiazepine ligands can be differentiated from adrenoceptor ligands due to their lack of effect on ganglionic transmission as well as their sensitivity to flumazenil. Muscle spasm is painful. Thus it is helpful if myorelaxant drugs are also analgesic. Baclofen (Sawynok & Reid, 1988), benzodiazepines (Goodchild & Serrao, 1987) and M2-adrenoceptor agonists (Bonnet et al., 1989) are all reported to have analgesic actions at the spinal level. It is claimed that the long duration DR-VRP is a manifestation of activity in nociceptive pathways in the spinal cord (Akagi et al., 1985). This is certainly consistent with the analgesic action of intrathecally administered AP5 (Cahusac et al., 1984). Thus depression of the long duration reflex by these myorelaxant drugs is consistent with the proposed link between this reflex and nociception. Therefore depression of the long duration component of the DR-VRP may be a predictor for in vivo centrally acting myorelaxant activity. The myorelaxant action of AP5 and other NMDA receptor antagonists (Turski et al., 1984) is consistent with this expectation. R.H.E. was supported by the Medical Research Council, The Taberner Trust and The Wellcome Trust and R.J.S. was supported by a DUPHAR research scholarship.
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(Received June 10, 1992 Revised June 29, 1992 Accepted July 1, 1992)
