Excitatory amino acids and central synaptic transmission

T I P S - September 1984 I

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Excitatory amino a c id s a n d c e n t r a l synaptic transmission J. C. Watkins

lklmtmtmt of PImTnacologv.",~leMedimlScheol. Unh~utr WMk,Brb~. R~81TD.UK. Different types of excitawry amino acid receptors probably exettw specific fiaw~onal roles within the manunaban CNS. t..Glutamate may be the trammitter at all of these receptors, with extracellul+w Mg z + regulating the sensitivity of one particular receptorionophore system. As Jeff Watkins explains, this probable role of L-glutamme remains unproven. But progress is being made in characterizing the receptors with which it is proposed to interact. Thiv has led to the devdopment of pharmacological ligunds which may, in the future, form the basis of a new class of centrally .acting drugs. More than a quarter of a century has passed since Hayashi discovered th~ convulsant effects of intraventdculatly-

injected L-glutamate and L-aspartate. and the neuronal depolarizing action of these amino acids was reported by Curtis

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colleabmes. Dm-ing this tinu:, m~tt has accngd to support a transmitter role of the~ amino acids in the mammalian CNS n~. Yet such a role must stillbe considered unwoven. Wlmt d ~ ~ n certain, however, is that at leas, some of the r~l~lOrs activated ~ exogenously administered excitatory amim, acids arc imieed transtmtter receptors. If not L-glutamate and/or t,-aspattatc, the transmitters activating these receptors p h ~ l y must be of very similar chemical structure. While the debate continues as to the identity of the natural transmitters, considerable pro~'ess is being made in the characterization of the receptors ~ith which the) interact. This not only advances our understanding of central nervous function, but dso paves the ~ y for the development of rt~" centrallyactive drags.

it is now well established that there arc sevega] dasscs of reggptors for excitato~ amino acicls2J (Table t). The most easily chatactet~d of these ate ~ receptors, which ate activated 'Non-NMA' excitatmy amino aod m:aptcxs by N-methyl-aspattate (particularly the D form), and blocked specifica~, by D(-)HORN 2-amino-5-phosphonovalerate (AI~) &,d related phosphonates. These receptors are highly sensitive to the ex~racelh~at Mg "÷ concentration. Other receptors ate relatively insensitive to the phosphonates and Mg 2,. Such +non-NMA" receptors include those that ate activated by the naturally occuffing anthelmintics, quisqualate and kainate. Certain evidence sugg~ts that these two agonists preferentially activate diffe~nt subtypes of n o n - ~ receptors. Hog,ever, this evidence is not as strong as that kgkm by ~ ( 3 'N underlying the clear characterization of NMA receptors as a specific type. Exogenous L-glutamate and L-aspartate probably activate all the above types of excitatory amino acid receptors and possibly other types as well. This interoxatatory prctation is based on the finding that an~no ~ rematom responses induced by L-glutamate (especially) are depressed by all the antagonists yet tested, but often to a lesser degree than are responses to the other agonists, however, most investigators consider it unwarranted to sub-divide Fig, I, Schematicrepresentationof e.gcitatoryaminoacidrecepwrsin thespinalcord. Cholinergicreceptors on Renshaw cells and the glycinergic synapses of these cells with motoneurones also shown. For excitatory amino acid receptors further at this stage of our knowledge. abl~viatiom, see legend to Table I.

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T I P S - September 1984

374 TABLE !. Excitatory amino acid receptors 'NMA'

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Excitatory interneurones to dorsal and ventral horn neurones in spinal cord.

(AMPA)

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ASP-AMP GLU-AMP ,IDGG PDA Kynurenic acid

Synaptic pamways:

Oui~ualate

Some fast conducting primary afferents to dorsal and ventral horn neurones in spinal cord. Sensory nerves ac;ivated by hair movementsto neuvones in cuneate nucleus and trigeminal nucleus.

Schaffer collateral fibres in Perforant path to hippocampal dentate granule cells, hippocampus. (CA3-CAI)S Cerebral cortex to cuneate neurones. Cerebral cortex to dopaminergic caudate neurones. Optic tract fibres to LGN ce~Is.

Cerebellar parallel fibres to Purkinje cells.

*'i.~tqJ term potentiation' appeam to i~ mediated by ~ receptors and normal synaptic eggitation by non-NMA ~ (Ref. 4). Abb~viltklm: NMDA, N-methyi-D-aspagtate; NMLA, N-methyi.L.

a~mtate; AMPA, a-amino-3.hydm~-S-meth~tamlepmpionate; APS, 2-mino-5-phowhmopentanmte; APT,2 - a m i n o - 7 ~ t e ; ASP-AMP.~t~¢~Whniometbyl phmpho~te; GLU-AMP, "pt>$1utamylaminomethyl phoqphonate; ~/DGG, V-~glutamylgb¢ine; PDA, c/s-2,3pilgddine dicagboxylate; GAMS, V-~glutamylaminomethyl sulphonate: GLU-TAU, "eD-81utamylfamine. See Refs 2 and 3.

S~q~pathwm~ ~ t , ~ The potency and selectivity of AP5 and the longer chain analogue, AP7, as NMA receptor antagonists are such that a function of NMA receptors in synaptic excitation in the mammalian and amphibian spinal cords can easily be demonstrated. Thus, polysynaptic activation of single cells in the cat spinal cord following stimulation of low threshold primary afferent fibres is suppressed by the same iontophoretic concentrations of AP5 as those required to antagonize responses of the cells induced by Nmethyi-D-aspartate (NMDA). Moreover, in the frog spinal cord/n vitro, a parallel relation has been demonstrated between the relative potencies of substances as antagonists of NMDA-induced depolarization and as depressants of the polysynaptic excitation of motorneurones following dorsal root stimul-

ation. These results suggest that NMA receptors mediate the effects of an amino acid transmitter released by excitatory interneurones in the spinal cord (Fig. 1). A function of NMA receptors in higher centres of the brain also seems likely. Sensitivity to NMA antagonists has been reported for visual and auditory pathways and for discrete hippocampal synaptic systems. A particularly intriguing possibility, stemming from the work of McLennan and colleagues 4, is that NMA receptors mediate 'long term potentiation', a hippocampal phenomenon thought to be related to memory processes. However, many synaptic pathways in the CNS appear to be resistant to specific NMA antagonists, or are blocked only at relatively high concentrations of the antagonists which reduce the receptor selectivity of their actions. Monosynaptic

connections of low threshold primary afferents with spinal cells (Fig. I), and most of the well-defined pathways that have been studied in higher centres, appear to fall within this category (Table I). Several substances have proved more effective than specific NMA antagonists in suppressing synaptic excitation in these pathways. Such substances include cb2,3-piperidine dicarboxylic acid (PDA) 2. ~-o-glutamylgiycine (~DGG) 2 and the tryptophan metabolite, kynurenic acids. Although preferentially blocking NMA receptors, all these substances also suppress kainate/quisqualate receptors at higher concentrations. The two sulphonic dipeptides .¥-t>.giutamylaminomethyl sulphonate (GAMS) and "t-Dgiutamyltaurine (GLU-TAU) show a degree of kainateJquisqualate selectivity and may be forerunners to more specific agents of this t y p e 3. Recellter distrilmtiea Different central functions of specific excitatory amino acid receptors are probably reflected in the regional distribution patterns obtained with different radioactive ligunds. The striatum has the highest density of [3H]kainate binding sites6, while the hippocampus is the richest area for [3H]t>-AP5 binding?, indicative of NMA receptors. A four- to six-fold variation in different brain regions was found for each of these two ligands, with cerebellum, midbrain, thalamns and medulla pons having the lowest densities in both cases. Less variation has been found in the density of binding sites for [3H]AMPA, a quisqualate-like agonists. With this ligand, the area showing the highest deusity of binding sites (cerebral cortex) is enriched only 1.5 times compared with the area of lowest density (cerebellum). A detailed study of the densities of different excitatory amino acid receptors in the hippocampus was recently undertaken by Cotman and his colleagues9. They used quantitative autoradiography to demonstrate the localization of [3H]L-giutamate binding sites in the presence and absence of receptor specific inhibitors. Discrete Iocalizations of NMA, kainate and quisqualate receptors were found, consistent with specific synaptic pathways. In particular, dense regions of NMDA-sensitive [3Hlt.-giutamate binding sites corresponded to the termination fields of the Schaffer collateral and the C4-derived systems, while the more localized kainate-sensitive sites conresponded to mossy fibre terminations.

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Continuing development of selective antagonists is likely to lead to increas. ingly clear pharmacological characterization of the different types of receptors involved in amino acid.mediated synaptic excitation in the vertebrate CNS. However, it is unlikely to he of immediate aid in the identification of the natural transmitters that activate these receptors physiologically. For example, L-aspartate has been favoured as the transmitter released by excitatory intemeumnes on to NMA receptors in the spinal cord. Thus, polysynaptic excitation in the spinal cord can be depressed by concentrations of antagonists that depress L-aspaNate-induced responses more than L-glutamate-induced rrsponses of the same cells. Such an effect has been observed with a range of NMA antagonists including AP5. However, L-glutamate has a l~fold higher affinity than t.-aspartate for [3H]~AP5 binding sites in rat brain (Table II) 7. Indeed, t-glutamate has the highest affinity among all endogenous excitants so far tested, not only for [aH]~AP5 binding sites, but also for ['H]kainate and [aH]AMPA binding sites as well. Thus, it may well be that this amino acid is the transmitter that activates all the various excitatory amino acid receptors physiologically. It is unlikely that quinolinate (an endogenous NMA-type agonist t°) is the natural transmitter at NMA receptors in view of the low affinity of this substance for [3H]~AP5 binding sites7. However, several endogenous sulphur-containing amino acids have relatively high affinity for both [3H]Lglutamate t~ and [~H]D-AP5 binding sites~, and these substances, together with L-aspartate, must continue to be regarded as transmitter candidates. Regulatory role of Mg 2 + Apart from the identity of the transTABLE ll, Inhibition of [3HIwAP5 binding to cortical membranes by excitatoryaminoacid trans-

mitter candidates7 Excitant

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Mg2+{?) Fig. 2. H)'potl~t~al moremmts of ions foilos~ng acm'ation of NMA receptor~ The wmno acM zramrnme, (? I.-glutamawL releasedfi~m pre~ynaptic terminals, tnterac~ #~h NMA receptor~ m the po~ts~naptu membrane, inducb;g Na +~, ul Ca~" mflug msoc~ed K~th K" and po~ibh M~" e#hu Released.llg: " may produce nega~ve feed.back e~et't by damping d o ~ acutur oy NMA receptor~

mitter activating different excitatory amino acid receptors physiologically. another question that is currently evoking great interest is the role of Mg2. in amino acid-induced responses, it has been proposed that these ions regulate the sensitivity of NMA receptors to its transmitter '2. This proposal has received considerable recent support in intracellular studies of membrane conductance changes associated ~ith the activation of different excitatory amino acid receptors. Thus, it appears that a region of negative slope conductance in the current-voltage relation of the L-glutamate response t3 reflects a component of the response which is mediated by NMA receptors, and which is, in effect, subject to a voltage-dependent Mg2÷ block ~4 Nowak et ai. t4 suggest that Mg2÷ in th;: extracellular fluid "gates' the ion channels opened by the amino acid-receptor interaction, reducing the probability of channel opening. The effects of Mg2÷ observed b) Nowak et ai. were evident with concentrations of these ions as low as 10 ttM. similar to the threshold depressant effects of these ions on NMA receptors observed earlier uS. Half maximal depression of the activity of NMA receptors in these latter experiments occurred at about 200 ttM Mg2+. This figure is in acc~.rd with the fact that, in vivo, only low (e.g. 5 hA) iontophoretic ejection currents of Mg 2+ are required to be

passed from micropipettes in order t,, produce definite depression of NMDAinduced responses. Such ejection currents would not be expected to increase free extracellular M ~ " concentration by more than about 100-2110 ttM within 21) ttm of the tip of the micropipette, Yet the usually accepted level of Mg'* in extracellular fluid is around 1 mM or more. At this level, the sensiti~,ity of NMA receptors would alread~ be depressed by about 90%. and the slight increase in the extracellular concentrations of Mg'* effected by the low iontophoretic ejection currents would not be expected to produce much additkmal effect. A reasonable explanation could be that the actual concentration of Mg" in the extracellular fluid bathing NMA receptors is very much lower than that generally believed on the basis of the measured CSF concentration of the~ ions. Recent experiments have suggested the possibility that the mechanism of NMDA action may include the actixation of a voltage-dependent Ca-'* conductance '~'. it is indeed gell established that the extracellular Ca-'* concentration is lowered, and the intracellular concentration of these ions concomitantly increased, by the action of excitatoD' amino acids 17"'s. As yet, neither extranor intracellular concentrations of Mg2" have been measured during the actions of excitatory amino acids, lntraceUular

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376 stores of Mg 2+ are quite high and the concentration of free (diffusible) Mg 2+ may be increased by displacement from bound form(s) of these ions by Ca 2+. It is therefore conceivable that, along with the opposite transmembrane fluxes of Na + and K + generally atr,epted to be induced by excitatory amino acids ts, a concomitant Ca 2+ influx induced by these substances is as,~3ciated with a Mg 2+ efflux (Fig. 2). In the case of excitation mediated by NMA receptors, such an exchange could produce a negative feed back effect, the increase in extraceilular Mg2+ reducing the amino acid-induced response by the gating mechanism described above. Pump mechanisms may then restore the original membrane concentration gradients of the divalent ions, Drug potential of substances that modify the activity of excitatory amino acid transmitter systems There is little doubt that excitatory amino acid transmitter systems play a major role in central synaptic function. It is to be anticipated therefore that substances which modify the activity of such systems will produce discrete behavioural effects of possible clinical value. Indeed, several substances currently in medical and/or veterinary use differentiaily affect excitatory amino acid systems. and such actions may well contribute to their clinical effects. Ketamine t'~ and other disassociative anaesthetics selectively antagonize NMA receptors, as does the major tranquillizer, chlorpromazine'2. These iipophilic substances have structures that are quite different from those of excitatory amino acids and may produce their antagonist effects by actions at the ionophore rather than receptor level. More specific actions may be expected of those substances which act competitively with the transmitter at particular excitatory amino acid receptors. Of immediate interest in this respect are the recent observations of Meldrum and colleagues, that specific NMA antagonists have anticonvulsant activity against seizures of diverse origin'°. The most potent compounds tested so far are 2-

amino-7-phosphonoheptanoate (AP7), the pentanoate analogue (APS), and two related dipeptides, J3-D-aspartylaminomethyl phosphonate (ASP-AMP) and 3,-~glutamylaminomethyl phosphonate (GLU-AMP). Two less selective excitatory amino acid antagonists, ~-D-glutamylglycine (-/DGG) and c/s-2,3-piperidine dicarboxylic acid (PDA) have similar though weaker effects, Again the potency of these two substances correlated well with their NMA receptor antagonist activity, All these compounds are highly polar and would be expected to penetrate the blood-brain-barrier only very poorly. Indeed, further studies by the ~ m e group have confirmed this is the case for tritiated AP7, the most potent anticonvulsant. More lipophilic substances would be expected to cross the bloodbrain-barrier more easily and are currently under development. Cend~ Many questions remain, ranging from the function of different excitatory amino acid receptors, through the still uncertain identity of the transmitters activating these receptors physiologically, to the complex mechanism of their actions at the ionic level, Rapid advances in our understanding of these phenomena are. however, confidently to be anticipated, in particular, studies with ionselective electrodes may clarify the role of Mg 2+ at NMA receptors, and the relation between NMA and non-NMA receptors. New drugs, acting specifically on excitatory amino acid transmitter systems, are likely soon to be developed, Acknowledgements ! am grateful to the Medical Research Council and the Wellcome Trust for support. Special thanks are also due to my research colleagues Dr John Davies, Dr Dick Evans, Dr Arwel Jones, Dr Ken Mewett, Dr Harry Olverman, Mr Dave Smith and Mr Dan Oakes. ! also thank Dr Graham Collingridge for helpful discussion. RmUn8 list I curtis, D. R. and Johnston,G. A. R, (1974)

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September i 984

Ergebn. Physiol. Biol. Chem. Exp. Phannakol. 69, 97-188 2 Watkins, J. C. and Evans, R. H. (1981) Annu, Rev. Phannaw/. Tox/col. 21. 165-204 3 Davies, J., Evans, R. H., Jones, A. W., Mcwctt,

K. N,, Smith, D. A. S. and Watkim, J. C. (1983) in Excitotoxim, Wenner Gwn Intern. Syrup, ~ , Vo139(Fuxe,K., Robem, P. and Schwarcz, R., ech, pp. S3-M, Macmillan, London 4 Collingridge,G. I,., Kehl.S. J. and McLennan. H. (1983)J. Physiol (London) 334, 33-46 5 Perkins,M. N. and Stone,T. W. (1982)Brain Res. 263, 162-166 6 Simon, J. R., Contrera, J. F. and Kuhar, M. J. (1976) J. Neurochem. 26, 141-147 70lverman, H. J., Jones, A. W. and Watkins, J. C. (1984)Nature (London) ~)7, 460-462 8 Hono~, T., Lau~dscn, J. and KmgsgaardLarsen, P, 0982) J. Neurochem 38, 173-178 9 Monaghan,T. D., Holets, V. R,, Toy, D, W. and Carman, C. W. (19&'~)Nature ¢London) ,~6, 176-178 Stone,T. W, and Perkins,M. N. (1981)Eur. J. Pharmacol. 72, 411-412 Meweu, K, N., Oakes, D. J., Olverman,tl. J., Smith, D. A. S. and Watkins,J. C. (198,';)in CNS Receptors: From Molecular Phammcology to Behm,iour (Mandel, P. and deFeudis,

F. V., eds),pp. 163--174,RavenPress,NewYork 12 Watkins,J. C, (1980)Trends NeuroSci, 3, 61-66 13 MacDonald,J. F, and Wojtowicz,J, M. (1982) Can. J, P~ysiol. Pharmacol. 60, 282-296

14 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A, 0984) Nature (London) 307. 462.,465

15 Ault,B., Evans,R. H., Francis,A. A., Oakes, D. J. and Watkins, J. C, (1980) J. Physiol. (London) ,]07.412-428 16 Dingledine, R. (19&])J. Physiol. (London) M3, 385-405 17 Marciani,M. G., Laurel, J. and Heinemann, U. (1982) Brain Res. 238, 272-277 18 B0hrle,C. P, and Sonnhof,U. (1983)Pflugers Arch. 396, 154--162 19 Lodge, D, and Anis, N. A. (1982) Neurosci. Lea. 29, 281-286 20 Jones, A. W., Croucher,M. J., Meldrum,B. S. and Watkins,J. C. 0984) Neurosci. Leu. 45, 157-161 Dr J. C Watkins was born in Perth, Western Amtralia, in 1929. He obtainedan M.Sc. in Organic

Chem~st~ al the Universi~, of Western Amtralia in 1952 and a Ph.D. in Organic Chemistry m Cambridge University. UK in 1954. Subsequently he spent se~n years at the Amtmlian National Uni~wr. sity collaborating with D. R. Cuni~ in the Physiology Deparnnent under J. C. Eccles. For the past ten year~he hay been a Senior Research Fellowat the Universio, of Bristol. UK, in the Depanmen~ of Physiology and Pharmacology,