European Journal o f Pharrnacology, 50 (1978) 123--129
123
© Elsevier/North-Holland Biomedical Press
SPECIFIC ANTAGONISM OF EXCITANT AMINO ACIDS IN THE ISOLATED SPINAL CORD OF THE NEONATAL RAT RICHARD H. EVANS and J E F F R E Y C. WATKINS *
Department of Pharmacology and Physiology, University of Bristol, Medical School, University Walk, Bristol, BS8 1TD, U.K. Received 30 November 1977, revised MS received 8 March, 1978, accepted 7 April 1978
R.H. EVANS and J.C. WATKINS, Specific antagonism of excitant amino acids in the isolated spinal cord of the neonatal rat, European J. Pharmacol. 50 (1978) 123--129. The specificity of the neurodepressant actions of D-0~-aminoadipate, ~,e-diaminopimelic acid, HA-966 (HAP) and Mg2+ has been investigated. On the isolated spinal cord of the neonatal rat, ventral root depolarizations produced by kainate, substance P, carbachol and noradrenaline were relatively unaffected by the same concentrations (0.25--1 mM) of the agents as those which reduced synaptic activity and ventral root depolarizations produced by N-methyl-D-aspartate (eSpecially), L-aspartate and L-glutamate. The same or higher concentrations of the agents did not affect excitatory transmission in the isolated rat superior cervical ganglion. It is proposed that the agents specifically block synaptic transmission mediated by an excitatory amino acid. Amino acid antagonists a,e-Diaminopimelic acid
Spinal cord
D-~-Aminoadipate
1. Introduction Magnesium ions (Evans et al., 1977a; Davies and Watkins, 1977), a,e-diaminopimelic acid (DAPA) and D-a-aminoadipate (DAA) (Evans et al., 1978; Biscoe et al., 1977a, b; 1978) have recently been reported to block synaptic excitation and the excitatory effects of certain acidic amino acids in the amphibian and mammalian spinal cords. Of particular importance, DAA has been shown to reduce non-cholinergic dorsal root-evoked excitation of Renshaw cells without affecting cholinergic excitation of these cells produced by ventral root stimulation or iontophoretically applied acetylcholine (Biscoe et al., 1977b). In the present investigation the specificity of Mg2+, DAPA and DAA has been further examined by means of the rat isolated spinal * To whom correspondence should be addressed at the above address.
HA-966
Mg2+
cord preparation (Otsuka and Konishi, 1974) which responds to both amino acid and nonamino acid transmitter candidates (Konishi and Otsuka, 1974; Evans, 1978a). The effects of the antagonists on ventral root responses induced by a range of amino acids and by carbachol, noradrenaline and substance P have been examined and, in addition, the actions of the substances on synaptic transmission in isolated spinal cords and isolated superior cervical ganglia of rats have been compared. The effects of 1-hydroxy-3-aminopyrrolidone2 (HA-966, HAP), a previously reported amino acid antagonist (Davies and Watkins, 1973) which appears to act similarly to Mg2÷, DAPA and DAA on the isolated spinal cord of the frog (Biscoe et al., 1977a; Evans et al., 1978) have also been investigated on these two rat preparations. The results indicate that these substances are specific antagonists of responses of neoatal rat motoneurones to certain excitant amino
124 acids and that such amino acids, or like substances, probably function as transmitters in both monosynaptic and polysynaptic pathways of the neonatal rat spinal cord.
2. Materials and methods
2.1. Tissues and recording techniques Preparation o f hemisected spinal cords from 3--8 d a y old Wistar rats, the superfusion apparatus and the m e t h o d of electrical recording were as described by Evans (1978b), with the exception t h a t the hemicords were placed directly onto a stainless steel grid (1 mm mesh size) for superfusion instead of a layer of absorbent paper. Motoneurone responses to dorsal root stimulation and to chemical agents were recorded from ventral roots. The corresponding dorsal root was stimulated through two stainless steel wires with supramaximal pulses (0.05 msec, 30 V). Potentials were recorded between an electrode placed in contact with the distal end of a ventral root and another in contact with the proximal end of the ventral root via the superfusion solution. Increase in positivity of the distal electrode, shown by upward deflexion on the records, reflects depolarization of motoneuronal cell bodies a n d / o r processes. Isolated superior cervical ganglia were removed from rats (150--200 g b o d y weight) and superfused as described for hemisected spinal cords. The post-ganglionic nerve of the ganglion was placed in contact with the recording electrodes as for the ventral root of the spinal cord.
2.2. Superfusion media The composition of the standard superfusion medium was as follows (mM): NaC1 118, KC1 3, NaHCO3 24, CaC12 2.5, glucose 12; gassed with 95% 02--5% CO2; pH 7.4. The flow rate was 0.8 ml/mm. This solution was maintained at 15°C for the spinal cord and
R.H. EVANS, J.C. WATKINS 20°C for the superior cervical ganglion unless stated otherwise. The low temperature of the medium was considered to improve the stability o f agonist responses over 1--2 h periods. In some experiments, t e t r o d o t o x i n was included in the superfusion medium to block synaptically relayed activity and thereby minimize indirect actions o f perfused substances on motoneurones. Such preparations were pretreated with 4 pM TTX for 2 min, and the blockade which developed within a few seconds was maintained by 0.1 pM TTX in the medium t h r o u g h o u t the course of the experiment. Under these conditions no spontaneous electrical activity was recorded from dorsal or ventral roots and electrical stimulation of dorsal roots failed to evoke any detectable response in ventral roots. All acidic agonists and antagonists were dissolved in one equivalent of NaOH solution to form concentrated solutions of the Na salts (pH 7) which were added to the medium to produce the required concentration. HAP and DAPA were dissolved directly in the medium. None of these substances affected the pH of the medium. Agonist were applied in 2 ml test doses and antagonists were applied either simultaneously with the agonists (dissolved in the same 2 ml volume of medium) or were superfused continuously in the medium. The former m e t h o d allowed a shorter interval between control and test doses of agonist. The specificity of the antagonists was similar with either method.
2.3. Substances HAP was a gift from Professor I.L. Bonta (Rotterdam); N-methyl-D-aspartic acid was prepared by the m e t h o d of Watkins (1962). D-a-Aminoadipic acid ([a] D--23.5; C, 0.7 in 6 N (HC1) was isolated from the racemic mixture as its D-lysine salt, and recovered by ionexchange chromatography. Other chemicals and drugs were obtained from commercial sources.
EXCITATORY AMINO ACID ANTAGONISTS
125
3. Results 3.1. Effects o f antagonists on synaptic activity and on amino acid-induced responses o f the rat spinal cord The effects of DAA (0.25 mM), DAPA (0.5 mM), HAP (0.5 mM) and Mg 2+ ( l m M ) on responses of the rat spinal cord were generally similar to those observed in the frog spinal cord in vitro and on cat and mouse spinal neurones in vivo (Evans et al., 1977a, 1978; Biscoe et al., 1977a, b; 1978). Thus, responses to NMDA were always the most sensitive and responses to kainate the least sensitive to each of the antagonists. L-Aspartate- and L-glutamate-induced responses were generally both reduced to a moderate extent through responses to L-glutamate consistently appeared to be the more resistant. No difference was observed between the antagonists in respect of their effects on responses produced b y these latter two amino acids, though detailed quantitative data were not sought. Repesentative responses of the four amino acids in the presence and absence of DAA (0.25 mM) are shown in fig. 1A. Fig. 1B shows the effects of DAA on the dose--response curves for Lglutamate and L-aspartate, the differential effect of the antagonist on these two amino acids being quite marked in this particular case. Fig. 1C shows the depressant effects of DAA on spontaneous ventral root potentials, on ventral root potentials evoked b y dorsal root stimulation (DR-VRPs), and on NMDAand L-glutamate-induced responses of an unblocked preparation. Both fast and slow components of the DR-VRP were depressed; the former c o m p o n e n t has been suggested to arise as a result of monosynaptic EPSPs (Otsuka and Konishi, 1974) while the latter component probably involves EPSPs generated b y polysynaptic pathways. When applied in the absence of an agonist, none of the agents produced marked effects on ventral root polarity in TTX-blocked cords, while in unblocked preparations (e.g. fig. 1C), the depression of synaptic activity was associated with a small hyperpolarization.
0
0
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CONCENTRATION (raM) OAA
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Fig. 1. A, Effect of 250 pM D-a-aminoadipate (DAA) on depolarization of motoneurones evoked by 2 mM L-aspartate (AS), 2 mM L-glutamate (GL), 20 pM Nmethyl-D-aspartate (MA) and 20/~M kainate (KA). DAA was applied during the solid bar above and the agonists were applied during the open bars below the record. B, Dose--response plot from preparation shown in A. Open circles before and filled circles during perfusion with DAA (500 pM). C, Effect of DAA (250 pM) on depolarization of motoneurones produced by GL ( 5 0 0 p M ) and MA (5 pM). Dorsal root stimulation l/rain. Fast response of motoneurones to dorsal root stimulation was recorded at the times indicated by • and these records are shown in sequence below. Calibration. Vertical, 1 mV in records A and C, upper trace; 2 mV in C, lower trace. Horizontal, 10 rain in A and C, upper trace; 27 msec in C, lower trace. In A and B the superfusion medium contained 0.1 pM tetrodotoxin.
3.2. Effects o f the antagonists on ventral root responses to other types o f transmitter agonists The depression of synaptic transmission b y these substances could be postulated to arise
126 A
R.H. EVANS, J.C. WATKINS DAA
DAA
DAA
DAA
B
A
2.0
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o
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o
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.25 .5 1 1,5 AS ~
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o L7
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eL
SP
AS
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AS
GL
SP
Fig. 2. A, B, Effect of D-0~-aminoadipate, 250 tiM in A and 500 pM in B, on depolarizations of motoneutones produced by 6 tiM N-methyl-D-aspartate, 0.5 mM L-glutamate, 1 pM substance P (SP), 2.5 tiM noradrenaline (NO), 7.5 tiM carbachol (CA) and 1 mM L-aspartate, symbols as in fig. 1. D-(~-Aminoadipate was added simultaneously with the agonists. C, Ventral root recordings of effects of D-s-aminoadipate 250 pM on synaptic activity and on depolarization of motoneurones produced by 400 tiM L-aspartate, 250 pM L-glutamate and 1 tiM substance P. Dorsal root stimulated 1]min. Calibration. Vertical, 1 mV in A and B, 0.6 mV in C. Horizontal, 10 rain. The superfusion medium contained 0.1 pM tetrodotoxin in A and B.
b y an antagonism of the action of an acidic amino acid transmitter. This hypothesis would be strengthened if it could be shown that these agents do not antagonize responses to other types of putative transmitters. To this end, the actions of the substances were tested against the depolarizing responses of motoneurones to substance P (Konishi and Otsuka, 1974), carbachol (Evans, 1978a) and noradrenaline. The depolarizing action of noradrenaline on this preparation has not been reported previously. Fig. 2 shows representative responses o f the various depolarizing agents and indicates that DAA (0.25--0.5 mM) had little or no effect on responses to
20 50 100.25.5 1
25 5 1
25 .5 1
Fig. 3. Four preparations A, B, C and D showing the effect of 1 mM ~,e-diaminopimelie acid (filled circles and squares) on depolarization of immature rat motoneurones evoked by kainate (circles in A), Nmethyl-D-aspartate (squares in A), L-aspartate (squares in B, C and D), L-noradrenaline (circles in B) carbachol (circles in C) or substance P (circles in D). Other symbols as for fig. 2. The antagonist was applied for 4 rain at each point and agonists were applied for 2 min from 1 rain after commencement of the antagonist application. L-Aspartate concentrations in mM; all other agonist concentrations #M. Temperature of medium, 25°C. Ordinate: depolarization (mV); abscissa: concentration.
substance P, carbachol or noradrenaline at concentrations which depressed the responses of the same preparation to excitatory amino acids. Similar results were obtained with DAPA (1 mM), HAP (0.5 mM) and Mg 2÷ (0.5--1 raM). Dose--response plots for kainate, NMDA, L-aspartate, noradrenaline, carbachol and substance P in the presence and absence of DAPA (1 mM) are shown in fig. 3. 3.3. Effects o f the antagonists on transmission in the rat superior cervical ganglion Depression of synaptic activity may arise through less specific mechanisms than b y
EXCITATORY AMINO ACID ANTAGONISTS
antagonism of the post-junctional effect of an excitatory transmitter, for example, by an effect on transmitter release or through a local anaesthetic action. Indeed, the depressant action of Mg2÷ might be expected to be associated with the former phenomenon, though the well known action of Mg:÷ in reducing transmitter release (Del Castillo and Engbaek, 1954) is usually considered to require relatively high concentrations of the ions. To control for such unspecific effects of either type, the actions of the substances were tested on transmission in the isolated superior cervical ganglion of the rat. At concentrations similar to or higher than those required for marked depression of synaptic responses in the rat spinal cord, DAA (0.5 mM), DAPA (1 mM), HAP (1 mM) and Mg2÷ (0.5 mM) had no effect on the nicotinic component of gangionic transmission, which was marHEX O,lmM
DAP 1ram
Mg O.SmM
HEX 0.5ram
Fig. 4. Effect of antagonists on the fast synaptic response o f the rat isolated superior cervical ganglion to preganglionic nerve stimulation, extracellular recording. The time in m i n u t e s before, after t h e
entry, and after washout of antagonist solutions is s h o w n b e l o w each record. HEX, hexamethonium bromide; DAP, 0t,e-diaminopimelic acid; Mg, MgC12. Calibration, 2 mV, 20 msec.
127
kedly depressed by hexamethonium (0.1-0.5 mM). Representative responses of the ganglion are illustrated in fig. 4.
4. Discussion The depression of certain excitatory amino acid-induced responses by DAPA, DAA, HAP and Mg2÷ in the rat spinal cord contrasts with the lack of effect of these substances, at the same or higher concentrations, on the depolarizing responses of motoneurones to carbachol, noradrenaline and substance P. The antagonism shown by these agents in this preparation is thus highly specific with respect to motoneuronal depolarization induced by different putative transmitter agonists. Nevertheless, it should be pointed out that the insensitivity of the muscarinic (Evans, 1978a) receptors on rat motoneurones to each of the four amino acid antagonists contrasts with the sensitivity of the predominantly nicotinic (Curtis and Ryall, 1966) receptors on cat Renshaw cells to three of these agents, Mg2÷ (Davies and Watkins, 1977), HAP (Curtis et al., 1973) and DAPA (Biscoe et al., 1978). This latter difference in sensitivity may reflect an effect of the temperature difference between the in vitro and in vivo systems or some peculiarity of Renshaw cell receptors, since none of the four agents affected the nicotinic component of transmission through the rat superior cervical ganglion. With regard to the mode of action of these substances, the possibility that their depressant actions are produced by an action similar to that of GABA and related amino acids can be ruled out since these inhibitory amino acids depolarize motoneurones in the immature rat spinal cord (Otsuka and Konishi, 1976); DAPA, DAA, HAP and Mg2÷ produced little detectable change in the baseline level of ventral root polarization in TTX-blocked preparations and caused slight hyperpolarization is unblocked preparations. Moreover, GABA-related amino acids exert a relatively
128 uniform, rather than a differential depressant action on frog ventral root depolarizations produced by a range o f excitatory amino acids (Evans et al., 1977b). McLennan and Hall (1978) have made a similar observation in that GABA produced a non-selective depression o f the excitatory reponses of thalamic neurones to iontophoretically applied amino acids and acetyl choline whereas DAA had a selective depressant action similar to that observed in the present experiments. A local anaesthetic t y p e of mechanism can also be excluded, since such an action would have attenuated transmission through the rat superior cervical ganglion. While membrane conductance measurements will be necessary for more definitive evidence as to the mode of action of these antagonists, it would seem likely that t h e y depress synaptic activity by antagonizing EPSPs produced by an excitatory amino acid transmitter (Evans et al., 1977a, 1978; Biscoe et al., 1977a, b; 1978). Preliminary measurements of dose ratios for antagonisms at frog motoneurones indicate that DAA and DAPA produce their antagonism at a c o m m o n site but Mg2÷ acts at a separate site (Evans and Watkins, 1978). It is possible that the organic agents compete with a transmitter for receptor sites, and that these transmitter receptors, or associated ionophores, also have an affinity for Mg2÷. In this case, Mg2÷ may regulate the action of these receptors in vivo, especially if the concentration of the divalent ions in extracellular fluid is lower than that in mammalian cerebrospinal fluid, which is about 1.5 mM (Davson, 1967). A low concentration of Mg2÷ in extracelluiar fluid would seem likely in view of the marked depressant effects of low iontophoretic currents of Mg 2÷ on cat spinal neurones (Davies and Watkins, 1977). A blocking effect of the organic agents and Mg :÷ on excitatory amino acid transmitter receptors would readily explain the lack of effect of these agents on transmission through the sympathetic ganglion, where excitatory amino acids are not considered to have any transmitter function. It would also explain
R.H. EVANS, J.C. WATKINS the hyperpolarizing shift in base-line associated with depression of spontaneous ventral root potentials in the spinal cord if motoneurones were tonically depolarized by excitatory amino acid transmitter(s). If the agents do indeed act in this way, the fact that t h e y depress the fast c o m p o n e n t of the DR-VRP would support the hypothesis that the major transmitter released from primary afferent terminals on to motoneurones is an acidic amino acid (see Curtis and Johnston, 1974) and would render unlikely an alternative proposal that substance P fulfils such a function (Konishi and Otsuka, 1974; Saito et al., 1975). On the same basis, NMDA (the most readily antagonized amino acid), m a y act as a p o t e n t agonist of the transmitter at the antagonist-sensitive sites. Arguments t h a t L-aspartate may be the transmitter at these sites (Biscoe et al., 1977b, 1978; Evans et al., 1978) are not necessarily weakened by the moderate sensitivity to the antagonists of L-glutamate-induced responses since such sensitivity may reflect an action of exogenous L-glutamate on L-aspartate transmitter receptors rather than an action of the antagonists on L-glutamate transmitter receptors (Watkins, 1977). Nevertheless, neither L-glutamate, nor any other NMDAlike substance could be ruled out as such a transmitter on the basis of the present results.
Acknowledgements We thank Dr. L. Maitre, Ciba--Geigy, Basel, for a gift of substance P. This work was supported by the Medical Research Council.
References
Biscoe, T.J., J. Davies, A. Dray, R.H. Evans, A.A. Francis, M.R. Martin and J.C. Watkins, 1977a, Depression of synaptic excitation and of amino acid induced excitatory responses of spinal neurones by D-(~-aminoadipate, ~,e-diaminopimelic acid and HA-966, European J. Pharmacol. 45, 315. Biscoe, T.J., R.H. Evans, A.A. Francis, M.R. Martin, J.C. Watkins, J. Davies and A. Dray, 1977b, D-0~-
EXCITATORY AMINO ACID ANTAGONISTS Aminoadipate as a selective antagonist of amino acid-induced and synaptic excitation of mammalian spinal neurones, Nature (London) 270, 743. Biscoe, T.J., J. Davies, A. Dray, R.H. Evans, M.R. Martin and J.C. Watkins, 1978, D-~-Aminoadipate, a,e-diaminopimelic acid and HA-966 as antagonists of amino acid-induced and synaptic excitation of mammalian spinal neurones in vivo, Brain Res. (in press). Curtis, D.R. and G.A.R. Johnston, 1974, Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol. 69, 97. Curtis, D.R., G.A.R. Johnston, C.J.A. Game and R.M. McCulloch, 1973, Antagonism of neuronal excitation by 1-hydroxy-3-aminopyrrolidone-2, Brain Res. 4 9 , 4 6 7 . Curtis, D.R. and R.W. Ryall, 1966, The acetylcholine receptors of Renshaw cells, Exptl. Brain Res. 2, 66. Davies, J. and J.C. Watkins, 1973, Microelectrophoretic studies on the depressant action of HA-966 on chemically and synaptically excited neurones in the cat cerebral cortex and cuneate nucleus, Brain Res. 59,311. Davies, J. and J.C. Watkins, 1977, Effect of magnesium ions on the responses of spinal neurones to excitatory amino acids and acetylcholine, Brain Res. 130, 364. Davson, H., 1967, Physiology of the Cerebro-spinai Fluid (J. & A. Churchill Ltd., London). Del Castillo, J. and L. Engbaek, 1954, The nature of neuromuscular block produced by magnesium, J. Physiol. (London) 124,370. Evans, R.H., 1978a, Cholinoceptive properties of motoneurones of the immature rat spinal cord maintained in vitro, Neuropharmacology 1 7 , 2 7 7 . Evans, R.H., 1978b, The effects of amino acids and antagonists on the isolated hemisected spinal cord of the immature rat, Brit. J. Pharmacol. 62, 171. Evans, R.H., A.A. Francis and J.C. Watkins, 1977a, Selective antagonism b y Mg2+ of amino acid-
129 induced depolarization of spinal neurones, Experientia 33, 489. Evans, R.H., A.A. Francis and J.C. Watkins, 1977b, Differential antagonism b y chlorpromazine and diazepam of frog motoneurone depolarization induced by glutamate-related amino acids, European J. Pharmacol. 4 4 , 3 2 5 . Evans, R.H., A.A. Francis and J.C. Watkins, 1978, Mg2+-like selective antagonism of excitatory amino acid-induced r e s p o n s e s b y ~,e-diaminopimelic acid, D-~-aminoadipate and HA-966 in isolated spinal cord of frog and immature rat, Brain Res. (in press). Evans, R.H. and J.C. Watkins, 1978, Dual sites for antagonism of excitatory amino acid actions on central neurones, J. Physiol. (London) 277, 57P. Konishi, S. and M. Otsuka, 1974, Excitatory action o f hypothalamic substance P on spinal motoneutones of newborn rats, Nature 2 5 2 , 7 3 4 . McLennan, H. and J.G. Hall, 1978, The action of D-~-aminoadipate on excitatory amino acid receptors of rat thalamic neurones, Brain Res. (in press). Otsuka, M. and S. Konishi, 1974, Electrophysiology o f mammalian spinal cord in vitro, Nature 252, 733. Otsuka, M. and S. Konishi, 1976, in: GABA in Nervous System Function, Eds. E. Roberts, T.N. Chase and D.B. Tower (Raven Press, New York) p. 197. Saito, K., S. Konishi and M. Otsuka, 1975, Antagonisro between Lioresal and substance P in rat spinal cord, Brain Res. 9 7 , 1 7 7 . Watkins, J.C., 1962, The synthesis of some acidic amino acids possessing neuro-pharmacological activity, J. Med. Pharm. Chem. 5, 1187. Watkins, J.C., 1977, Transmitter identification and pharmacological interactions at specific synapses and the use of transmitter specific antagonists, in: Iontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, eds. R.W. Ryall and J.S. Kelly (Elsevier/North Holland, Amsterdam).
123
© Elsevier/North-Holland Biomedical Press
SPECIFIC ANTAGONISM OF EXCITANT AMINO ACIDS IN THE ISOLATED SPINAL CORD OF THE NEONATAL RAT RICHARD H. EVANS and J E F F R E Y C. WATKINS *
Department of Pharmacology and Physiology, University of Bristol, Medical School, University Walk, Bristol, BS8 1TD, U.K. Received 30 November 1977, revised MS received 8 March, 1978, accepted 7 April 1978
R.H. EVANS and J.C. WATKINS, Specific antagonism of excitant amino acids in the isolated spinal cord of the neonatal rat, European J. Pharmacol. 50 (1978) 123--129. The specificity of the neurodepressant actions of D-0~-aminoadipate, ~,e-diaminopimelic acid, HA-966 (HAP) and Mg2+ has been investigated. On the isolated spinal cord of the neonatal rat, ventral root depolarizations produced by kainate, substance P, carbachol and noradrenaline were relatively unaffected by the same concentrations (0.25--1 mM) of the agents as those which reduced synaptic activity and ventral root depolarizations produced by N-methyl-D-aspartate (eSpecially), L-aspartate and L-glutamate. The same or higher concentrations of the agents did not affect excitatory transmission in the isolated rat superior cervical ganglion. It is proposed that the agents specifically block synaptic transmission mediated by an excitatory amino acid. Amino acid antagonists a,e-Diaminopimelic acid
Spinal cord
D-~-Aminoadipate
1. Introduction Magnesium ions (Evans et al., 1977a; Davies and Watkins, 1977), a,e-diaminopimelic acid (DAPA) and D-a-aminoadipate (DAA) (Evans et al., 1978; Biscoe et al., 1977a, b; 1978) have recently been reported to block synaptic excitation and the excitatory effects of certain acidic amino acids in the amphibian and mammalian spinal cords. Of particular importance, DAA has been shown to reduce non-cholinergic dorsal root-evoked excitation of Renshaw cells without affecting cholinergic excitation of these cells produced by ventral root stimulation or iontophoretically applied acetylcholine (Biscoe et al., 1977b). In the present investigation the specificity of Mg2+, DAPA and DAA has been further examined by means of the rat isolated spinal * To whom correspondence should be addressed at the above address.
HA-966
Mg2+
cord preparation (Otsuka and Konishi, 1974) which responds to both amino acid and nonamino acid transmitter candidates (Konishi and Otsuka, 1974; Evans, 1978a). The effects of the antagonists on ventral root responses induced by a range of amino acids and by carbachol, noradrenaline and substance P have been examined and, in addition, the actions of the substances on synaptic transmission in isolated spinal cords and isolated superior cervical ganglia of rats have been compared. The effects of 1-hydroxy-3-aminopyrrolidone2 (HA-966, HAP), a previously reported amino acid antagonist (Davies and Watkins, 1973) which appears to act similarly to Mg2÷, DAPA and DAA on the isolated spinal cord of the frog (Biscoe et al., 1977a; Evans et al., 1978) have also been investigated on these two rat preparations. The results indicate that these substances are specific antagonists of responses of neoatal rat motoneurones to certain excitant amino
124 acids and that such amino acids, or like substances, probably function as transmitters in both monosynaptic and polysynaptic pathways of the neonatal rat spinal cord.
2. Materials and methods
2.1. Tissues and recording techniques Preparation o f hemisected spinal cords from 3--8 d a y old Wistar rats, the superfusion apparatus and the m e t h o d of electrical recording were as described by Evans (1978b), with the exception t h a t the hemicords were placed directly onto a stainless steel grid (1 mm mesh size) for superfusion instead of a layer of absorbent paper. Motoneurone responses to dorsal root stimulation and to chemical agents were recorded from ventral roots. The corresponding dorsal root was stimulated through two stainless steel wires with supramaximal pulses (0.05 msec, 30 V). Potentials were recorded between an electrode placed in contact with the distal end of a ventral root and another in contact with the proximal end of the ventral root via the superfusion solution. Increase in positivity of the distal electrode, shown by upward deflexion on the records, reflects depolarization of motoneuronal cell bodies a n d / o r processes. Isolated superior cervical ganglia were removed from rats (150--200 g b o d y weight) and superfused as described for hemisected spinal cords. The post-ganglionic nerve of the ganglion was placed in contact with the recording electrodes as for the ventral root of the spinal cord.
2.2. Superfusion media The composition of the standard superfusion medium was as follows (mM): NaC1 118, KC1 3, NaHCO3 24, CaC12 2.5, glucose 12; gassed with 95% 02--5% CO2; pH 7.4. The flow rate was 0.8 ml/mm. This solution was maintained at 15°C for the spinal cord and
R.H. EVANS, J.C. WATKINS 20°C for the superior cervical ganglion unless stated otherwise. The low temperature of the medium was considered to improve the stability o f agonist responses over 1--2 h periods. In some experiments, t e t r o d o t o x i n was included in the superfusion medium to block synaptically relayed activity and thereby minimize indirect actions o f perfused substances on motoneurones. Such preparations were pretreated with 4 pM TTX for 2 min, and the blockade which developed within a few seconds was maintained by 0.1 pM TTX in the medium t h r o u g h o u t the course of the experiment. Under these conditions no spontaneous electrical activity was recorded from dorsal or ventral roots and electrical stimulation of dorsal roots failed to evoke any detectable response in ventral roots. All acidic agonists and antagonists were dissolved in one equivalent of NaOH solution to form concentrated solutions of the Na salts (pH 7) which were added to the medium to produce the required concentration. HAP and DAPA were dissolved directly in the medium. None of these substances affected the pH of the medium. Agonist were applied in 2 ml test doses and antagonists were applied either simultaneously with the agonists (dissolved in the same 2 ml volume of medium) or were superfused continuously in the medium. The former m e t h o d allowed a shorter interval between control and test doses of agonist. The specificity of the antagonists was similar with either method.
2.3. Substances HAP was a gift from Professor I.L. Bonta (Rotterdam); N-methyl-D-aspartic acid was prepared by the m e t h o d of Watkins (1962). D-a-Aminoadipic acid ([a] D--23.5; C, 0.7 in 6 N (HC1) was isolated from the racemic mixture as its D-lysine salt, and recovered by ionexchange chromatography. Other chemicals and drugs were obtained from commercial sources.
EXCITATORY AMINO ACID ANTAGONISTS
125
3. Results 3.1. Effects o f antagonists on synaptic activity and on amino acid-induced responses o f the rat spinal cord The effects of DAA (0.25 mM), DAPA (0.5 mM), HAP (0.5 mM) and Mg 2+ ( l m M ) on responses of the rat spinal cord were generally similar to those observed in the frog spinal cord in vitro and on cat and mouse spinal neurones in vivo (Evans et al., 1977a, 1978; Biscoe et al., 1977a, b; 1978). Thus, responses to NMDA were always the most sensitive and responses to kainate the least sensitive to each of the antagonists. L-Aspartate- and L-glutamate-induced responses were generally both reduced to a moderate extent through responses to L-glutamate consistently appeared to be the more resistant. No difference was observed between the antagonists in respect of their effects on responses produced b y these latter two amino acids, though detailed quantitative data were not sought. Repesentative responses of the four amino acids in the presence and absence of DAA (0.25 mM) are shown in fig. 1A. Fig. 1B shows the effects of DAA on the dose--response curves for Lglutamate and L-aspartate, the differential effect of the antagonist on these two amino acids being quite marked in this particular case. Fig. 1C shows the depressant effects of DAA on spontaneous ventral root potentials, on ventral root potentials evoked b y dorsal root stimulation (DR-VRPs), and on NMDAand L-glutamate-induced responses of an unblocked preparation. Both fast and slow components of the DR-VRP were depressed; the former c o m p o n e n t has been suggested to arise as a result of monosynaptic EPSPs (Otsuka and Konishi, 1974) while the latter component probably involves EPSPs generated b y polysynaptic pathways. When applied in the absence of an agonist, none of the agents produced marked effects on ventral root polarity in TTX-blocked cords, while in unblocked preparations (e.g. fig. 1C), the depression of synaptic activity was associated with a small hyperpolarization.
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CONCENTRATION (raM) OAA
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Fig. 1. A, Effect of 250 pM D-a-aminoadipate (DAA) on depolarization of motoneurones evoked by 2 mM L-aspartate (AS), 2 mM L-glutamate (GL), 20 pM Nmethyl-D-aspartate (MA) and 20/~M kainate (KA). DAA was applied during the solid bar above and the agonists were applied during the open bars below the record. B, Dose--response plot from preparation shown in A. Open circles before and filled circles during perfusion with DAA (500 pM). C, Effect of DAA (250 pM) on depolarization of motoneurones produced by GL ( 5 0 0 p M ) and MA (5 pM). Dorsal root stimulation l/rain. Fast response of motoneurones to dorsal root stimulation was recorded at the times indicated by • and these records are shown in sequence below. Calibration. Vertical, 1 mV in records A and C, upper trace; 2 mV in C, lower trace. Horizontal, 10 rain in A and C, upper trace; 27 msec in C, lower trace. In A and B the superfusion medium contained 0.1 pM tetrodotoxin.
3.2. Effects o f the antagonists on ventral root responses to other types o f transmitter agonists The depression of synaptic transmission b y these substances could be postulated to arise
126 A
R.H. EVANS, J.C. WATKINS DAA
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Fig. 2. A, B, Effect of D-0~-aminoadipate, 250 tiM in A and 500 pM in B, on depolarizations of motoneutones produced by 6 tiM N-methyl-D-aspartate, 0.5 mM L-glutamate, 1 pM substance P (SP), 2.5 tiM noradrenaline (NO), 7.5 tiM carbachol (CA) and 1 mM L-aspartate, symbols as in fig. 1. D-(~-Aminoadipate was added simultaneously with the agonists. C, Ventral root recordings of effects of D-s-aminoadipate 250 pM on synaptic activity and on depolarization of motoneurones produced by 400 tiM L-aspartate, 250 pM L-glutamate and 1 tiM substance P. Dorsal root stimulated 1]min. Calibration. Vertical, 1 mV in A and B, 0.6 mV in C. Horizontal, 10 rain. The superfusion medium contained 0.1 pM tetrodotoxin in A and B.
b y an antagonism of the action of an acidic amino acid transmitter. This hypothesis would be strengthened if it could be shown that these agents do not antagonize responses to other types of putative transmitters. To this end, the actions of the substances were tested against the depolarizing responses of motoneurones to substance P (Konishi and Otsuka, 1974), carbachol (Evans, 1978a) and noradrenaline. The depolarizing action of noradrenaline on this preparation has not been reported previously. Fig. 2 shows representative responses o f the various depolarizing agents and indicates that DAA (0.25--0.5 mM) had little or no effect on responses to
20 50 100.25.5 1
25 5 1
25 .5 1
Fig. 3. Four preparations A, B, C and D showing the effect of 1 mM ~,e-diaminopimelie acid (filled circles and squares) on depolarization of immature rat motoneurones evoked by kainate (circles in A), Nmethyl-D-aspartate (squares in A), L-aspartate (squares in B, C and D), L-noradrenaline (circles in B) carbachol (circles in C) or substance P (circles in D). Other symbols as for fig. 2. The antagonist was applied for 4 rain at each point and agonists were applied for 2 min from 1 rain after commencement of the antagonist application. L-Aspartate concentrations in mM; all other agonist concentrations #M. Temperature of medium, 25°C. Ordinate: depolarization (mV); abscissa: concentration.
substance P, carbachol or noradrenaline at concentrations which depressed the responses of the same preparation to excitatory amino acids. Similar results were obtained with DAPA (1 mM), HAP (0.5 mM) and Mg 2÷ (0.5--1 raM). Dose--response plots for kainate, NMDA, L-aspartate, noradrenaline, carbachol and substance P in the presence and absence of DAPA (1 mM) are shown in fig. 3. 3.3. Effects o f the antagonists on transmission in the rat superior cervical ganglion Depression of synaptic activity may arise through less specific mechanisms than b y
EXCITATORY AMINO ACID ANTAGONISTS
antagonism of the post-junctional effect of an excitatory transmitter, for example, by an effect on transmitter release or through a local anaesthetic action. Indeed, the depressant action of Mg2÷ might be expected to be associated with the former phenomenon, though the well known action of Mg:÷ in reducing transmitter release (Del Castillo and Engbaek, 1954) is usually considered to require relatively high concentrations of the ions. To control for such unspecific effects of either type, the actions of the substances were tested on transmission in the isolated superior cervical ganglion of the rat. At concentrations similar to or higher than those required for marked depression of synaptic responses in the rat spinal cord, DAA (0.5 mM), DAPA (1 mM), HAP (1 mM) and Mg2÷ (0.5 mM) had no effect on the nicotinic component of gangionic transmission, which was marHEX O,lmM
DAP 1ram
Mg O.SmM
HEX 0.5ram
Fig. 4. Effect of antagonists on the fast synaptic response o f the rat isolated superior cervical ganglion to preganglionic nerve stimulation, extracellular recording. The time in m i n u t e s before, after t h e
entry, and after washout of antagonist solutions is s h o w n b e l o w each record. HEX, hexamethonium bromide; DAP, 0t,e-diaminopimelic acid; Mg, MgC12. Calibration, 2 mV, 20 msec.
127
kedly depressed by hexamethonium (0.1-0.5 mM). Representative responses of the ganglion are illustrated in fig. 4.
4. Discussion The depression of certain excitatory amino acid-induced responses by DAPA, DAA, HAP and Mg2÷ in the rat spinal cord contrasts with the lack of effect of these substances, at the same or higher concentrations, on the depolarizing responses of motoneurones to carbachol, noradrenaline and substance P. The antagonism shown by these agents in this preparation is thus highly specific with respect to motoneuronal depolarization induced by different putative transmitter agonists. Nevertheless, it should be pointed out that the insensitivity of the muscarinic (Evans, 1978a) receptors on rat motoneurones to each of the four amino acid antagonists contrasts with the sensitivity of the predominantly nicotinic (Curtis and Ryall, 1966) receptors on cat Renshaw cells to three of these agents, Mg2÷ (Davies and Watkins, 1977), HAP (Curtis et al., 1973) and DAPA (Biscoe et al., 1978). This latter difference in sensitivity may reflect an effect of the temperature difference between the in vitro and in vivo systems or some peculiarity of Renshaw cell receptors, since none of the four agents affected the nicotinic component of transmission through the rat superior cervical ganglion. With regard to the mode of action of these substances, the possibility that their depressant actions are produced by an action similar to that of GABA and related amino acids can be ruled out since these inhibitory amino acids depolarize motoneurones in the immature rat spinal cord (Otsuka and Konishi, 1976); DAPA, DAA, HAP and Mg2÷ produced little detectable change in the baseline level of ventral root polarization in TTX-blocked preparations and caused slight hyperpolarization is unblocked preparations. Moreover, GABA-related amino acids exert a relatively
128 uniform, rather than a differential depressant action on frog ventral root depolarizations produced by a range o f excitatory amino acids (Evans et al., 1977b). McLennan and Hall (1978) have made a similar observation in that GABA produced a non-selective depression o f the excitatory reponses of thalamic neurones to iontophoretically applied amino acids and acetyl choline whereas DAA had a selective depressant action similar to that observed in the present experiments. A local anaesthetic t y p e of mechanism can also be excluded, since such an action would have attenuated transmission through the rat superior cervical ganglion. While membrane conductance measurements will be necessary for more definitive evidence as to the mode of action of these antagonists, it would seem likely that t h e y depress synaptic activity by antagonizing EPSPs produced by an excitatory amino acid transmitter (Evans et al., 1977a, 1978; Biscoe et al., 1977a, b; 1978). Preliminary measurements of dose ratios for antagonisms at frog motoneurones indicate that DAA and DAPA produce their antagonism at a c o m m o n site but Mg2÷ acts at a separate site (Evans and Watkins, 1978). It is possible that the organic agents compete with a transmitter for receptor sites, and that these transmitter receptors, or associated ionophores, also have an affinity for Mg2÷. In this case, Mg2÷ may regulate the action of these receptors in vivo, especially if the concentration of the divalent ions in extracellular fluid is lower than that in mammalian cerebrospinal fluid, which is about 1.5 mM (Davson, 1967). A low concentration of Mg2÷ in extracelluiar fluid would seem likely in view of the marked depressant effects of low iontophoretic currents of Mg 2÷ on cat spinal neurones (Davies and Watkins, 1977). A blocking effect of the organic agents and Mg :÷ on excitatory amino acid transmitter receptors would readily explain the lack of effect of these agents on transmission through the sympathetic ganglion, where excitatory amino acids are not considered to have any transmitter function. It would also explain
R.H. EVANS, J.C. WATKINS the hyperpolarizing shift in base-line associated with depression of spontaneous ventral root potentials in the spinal cord if motoneurones were tonically depolarized by excitatory amino acid transmitter(s). If the agents do indeed act in this way, the fact that t h e y depress the fast c o m p o n e n t of the DR-VRP would support the hypothesis that the major transmitter released from primary afferent terminals on to motoneurones is an acidic amino acid (see Curtis and Johnston, 1974) and would render unlikely an alternative proposal that substance P fulfils such a function (Konishi and Otsuka, 1974; Saito et al., 1975). On the same basis, NMDA (the most readily antagonized amino acid), m a y act as a p o t e n t agonist of the transmitter at the antagonist-sensitive sites. Arguments t h a t L-aspartate may be the transmitter at these sites (Biscoe et al., 1977b, 1978; Evans et al., 1978) are not necessarily weakened by the moderate sensitivity to the antagonists of L-glutamate-induced responses since such sensitivity may reflect an action of exogenous L-glutamate on L-aspartate transmitter receptors rather than an action of the antagonists on L-glutamate transmitter receptors (Watkins, 1977). Nevertheless, neither L-glutamate, nor any other NMDAlike substance could be ruled out as such a transmitter on the basis of the present results.
Acknowledgements We thank Dr. L. Maitre, Ciba--Geigy, Basel, for a gift of substance P. This work was supported by the Medical Research Council.
References
Biscoe, T.J., J. Davies, A. Dray, R.H. Evans, A.A. Francis, M.R. Martin and J.C. Watkins, 1977a, Depression of synaptic excitation and of amino acid induced excitatory responses of spinal neurones by D-(~-aminoadipate, ~,e-diaminopimelic acid and HA-966, European J. Pharmacol. 45, 315. Biscoe, T.J., R.H. Evans, A.A. Francis, M.R. Martin, J.C. Watkins, J. Davies and A. Dray, 1977b, D-0~-
EXCITATORY AMINO ACID ANTAGONISTS Aminoadipate as a selective antagonist of amino acid-induced and synaptic excitation of mammalian spinal neurones, Nature (London) 270, 743. Biscoe, T.J., J. Davies, A. Dray, R.H. Evans, M.R. Martin and J.C. Watkins, 1978, D-~-Aminoadipate, a,e-diaminopimelic acid and HA-966 as antagonists of amino acid-induced and synaptic excitation of mammalian spinal neurones in vivo, Brain Res. (in press). Curtis, D.R. and G.A.R. Johnston, 1974, Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol. 69, 97. Curtis, D.R., G.A.R. Johnston, C.J.A. Game and R.M. McCulloch, 1973, Antagonism of neuronal excitation by 1-hydroxy-3-aminopyrrolidone-2, Brain Res. 4 9 , 4 6 7 . Curtis, D.R. and R.W. Ryall, 1966, The acetylcholine receptors of Renshaw cells, Exptl. Brain Res. 2, 66. Davies, J. and J.C. Watkins, 1973, Microelectrophoretic studies on the depressant action of HA-966 on chemically and synaptically excited neurones in the cat cerebral cortex and cuneate nucleus, Brain Res. 59,311. Davies, J. and J.C. Watkins, 1977, Effect of magnesium ions on the responses of spinal neurones to excitatory amino acids and acetylcholine, Brain Res. 130, 364. Davson, H., 1967, Physiology of the Cerebro-spinai Fluid (J. & A. Churchill Ltd., London). Del Castillo, J. and L. Engbaek, 1954, The nature of neuromuscular block produced by magnesium, J. Physiol. (London) 124,370. Evans, R.H., 1978a, Cholinoceptive properties of motoneurones of the immature rat spinal cord maintained in vitro, Neuropharmacology 1 7 , 2 7 7 . Evans, R.H., 1978b, The effects of amino acids and antagonists on the isolated hemisected spinal cord of the immature rat, Brit. J. Pharmacol. 62, 171. Evans, R.H., A.A. Francis and J.C. Watkins, 1977a, Selective antagonism b y Mg2+ of amino acid-
129 induced depolarization of spinal neurones, Experientia 33, 489. Evans, R.H., A.A. Francis and J.C. Watkins, 1977b, Differential antagonism b y chlorpromazine and diazepam of frog motoneurone depolarization induced by glutamate-related amino acids, European J. Pharmacol. 4 4 , 3 2 5 . Evans, R.H., A.A. Francis and J.C. Watkins, 1978, Mg2+-like selective antagonism of excitatory amino acid-induced r e s p o n s e s b y ~,e-diaminopimelic acid, D-~-aminoadipate and HA-966 in isolated spinal cord of frog and immature rat, Brain Res. (in press). Evans, R.H. and J.C. Watkins, 1978, Dual sites for antagonism of excitatory amino acid actions on central neurones, J. Physiol. (London) 277, 57P. Konishi, S. and M. Otsuka, 1974, Excitatory action o f hypothalamic substance P on spinal motoneutones of newborn rats, Nature 2 5 2 , 7 3 4 . McLennan, H. and J.G. Hall, 1978, The action of D-~-aminoadipate on excitatory amino acid receptors of rat thalamic neurones, Brain Res. (in press). Otsuka, M. and S. Konishi, 1974, Electrophysiology o f mammalian spinal cord in vitro, Nature 252, 733. Otsuka, M. and S. Konishi, 1976, in: GABA in Nervous System Function, Eds. E. Roberts, T.N. Chase and D.B. Tower (Raven Press, New York) p. 197. Saito, K., S. Konishi and M. Otsuka, 1975, Antagonisro between Lioresal and substance P in rat spinal cord, Brain Res. 9 7 , 1 7 7 . Watkins, J.C., 1962, The synthesis of some acidic amino acids possessing neuro-pharmacological activity, J. Med. Pharm. Chem. 5, 1187. Watkins, J.C., 1977, Transmitter identification and pharmacological interactions at specific synapses and the use of transmitter specific antagonists, in: Iontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, eds. R.W. Ryall and J.S. Kelly (Elsevier/North Holland, Amsterdam).
