Brain Research, 506 (1990) 31-39

31

Elsevier BRES 15080

Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat Anthony H. Dickenson and Ann F. Sullivan Department of Pharmacology, University College London, London (U.K.) (Accepted 30 May 1989)

Key words: Excitatory amino acid; Nociception; Glutamate; Spinal cord

The effects of two excitatory amino acid receptor antagonists y-D-glutamylglycine (DGG) and 2-amino-5-phosphonovaleric acid (AP5), applied onto the spinal cord surface, were tested on the responses of dorsal horn nociceptive neurones in the anaesthetized rat. DGG is a non-selective antagonist at both the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, whereas AP5 acid is selective for the NMDA receptor. DGG dose-dependently reduced the A and C fibre-evoked responses of neurones in all laminae of the dorsal horn and also inhibited the post-discharges of intermediate and deep neurones resulting from repeated C fibre stimulation. There was little difference in the effects of the antagonist on the intermediate neuronal population compared to superficial or deep cells in the dorsal horn. AP5 has little effect on C fibre-evoked activity in superficial cells but produced slight inhibitions of the C fibre-evoked responses and clear reductions in the post-discharge of the deep neurones. This contrasts with the excitatory effects of the antagonist on both types of responses in the intermediate cells. A fibre-evoked responses were unaffected by AP5. Taking into account the results with the two antagonists it appears that both A and C fibre-evoked responses of dorsal horn nociceptive neurones are mediated by non-NMDA receptors whilst the C fibre-evoked wind-up of deep dorsal horn cells involves the NMDA receptor which also seems to mediate, in a complex manner, C fibre responses of intermediate, presumed substantia gelatinosa neurones. The results are discussed with regard to nociceptive mechanisms in the dorsal horn. INTRODUCTION

the o u t e r laminae of the dorsal horn 21-32 where the nociceptive afferents terminate 3"5'6"9'26"2~'37-39'46'47. Be-

A great deal of interest has been directed towards studies on the transmitter candidates in p r i m a r y sensory afferent fibres, particularly those conveying nociceptive information from the p e r i p h e r y to the spinal cord (see ref. 3). N e u r o p e p t i d e s have been a source of much research but lack of selective antagonists has caused difficulties in assigning particular roles to peptides in sensory transmission. T h e converse now exists regarding the excitatory amino acids ( E A A ) , glutamate and aspartate since the use of antagonists can differentiate between the N M D A (N-methyl-D-aspartate) and the n o n - N M D A (quisqualate/kainate) subtypes of receptors for these amino acids 19"29"48"49.

havioural studies indicate that application of E A A and analogues onto the spinal cord p r o d u c e s aversive reactions in animals 1, whereas b l o c k a d e of amino acid receptors in vivo produces r e d u c e d responses of dorsal horn n e u r o n e s in the rat to cutaneous afferents 45. The actions of a variety of intrathecal opioids have been studied on nociceptive neuronal responses in the anaesthetized rat 14. The present study uses this model to investigate the effects of y-D-glutamylglycine ( D G G ) a non-selective E A A r e c e p t o r antagonist and 2-amino5-phosphonovaleric acid (AP5), a selective N M D A antagonist 12'1~'19"48"49, on the responses of dorsal horn

E v i d e n c e for a role of E A A in sensory transmission in the spinal cord is p r o v i d e d by electrophysiological studies in which g l u t a m a t e depolarizes spinal neurones 2,1°'17'4°,53 and a p p e a r s to m e d i a t e excitatory postsynaptic potentials (e.p.s.p.s) b e t w e e n dorsal root ganglion and spinal n e u r o n e s in co-culture 24. T h e r e is a higher concentration of g l u t a m a t e in dorsal roots c o m p a r e d to ventral roots and also evidence for glutamate in dorsal root ganglion cells 8'16'17. A n a t o m i c a l studies have shown that glutamate binding sites and N M D A sites are found in high levels in

neurones to A and C fibre stimulation. The intrathecal route of administration allows a comparison with the effects of opioids and is also a potential route of administration of clinical use since, due to the diversity of functions m e d i a t e d by E A A in the CNS 4A9"29"33"48, systemic administration is likely to be of no therapeutic relevance. Nociceptive neurones with ascending axons are found in the superficial and d e e p e r laminae of the dorsal horn 3'5'6'2°'27'5152 with the latter group also likely to be intercalated in nociceptive reflex pathways n5. A third group of p r e s u m e d i n t e r n e u r o n e s is located in an

Correspondence: A.H. Dickenson, Department of Pharmacology, University College London, Gower Street, London WCIE 6BT, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

32 i n t e r m e d i a t e z o n e , the substantia g e l a t i n o s a '~. We have r e c o r d e d f r o m n e u r o n e s t h r o u g h o u t the dorsal h o r n and m o n i t o r e d the s t e a d y r e s p o n s e s of t h e s e n e u r o n e s

to

e v o k e d a f f e r e n t input and also t h e increasing activity of the d e e p e r cells elicited by c o n s t a n t r e p e t i t i v e C fibre s t i m u l a t i o n ( w i n d - u p ) 15"3~'44. A p r e l i m i n a r y a c c o u n t of a p o r t i o n of this w o r k has b e e n p u b l i s h e d ~s. MATERIALS AND METHODS Single unit extraceilular recordings were made from dorsal horn neurones in the lumbar enlargement of the spinal cord of the rat. The rats were intact and anaesthetized with halothane (2% for surgery, 1% for recording) in a gaseous mix of 66% nitrous oxide

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and 33% oxygen. Following exposure ot the I.I--1.3 section of t!le spinal cord, the cord was held rigid by clamps caudal and rostral to the exposed section and a glass-coated tungsten electrode lowered into the dorsal horn. The neurones were identified by means o[ natural (brush, touch, prod, pinch and heat) stimuli and by their responses to transcutaneous electrical stimulation of the hmdpaw receptive field. Receptive field size was variable but generally extended from part of a toe to several toes, All cells included in the study responded to both innocuous and noxious stimuli and to A and C fibre afferents. Following identification of a single neurone the thresholds to electrical stimulation (2 ms wide pulse, 0.5 Hz) for the A and C fibre responses were determincd, each type of response separated on the basis of threshold and latency of response. The A fibre responses were taken as between 0 and 20 ms, C fibre responses between 90 and 300 ms and the post-discharge of those cells exhibiting this prolonged firing as from 300 to 800 ms (Fig. 1). Sixteen stimuli at 3 times the A and then the C fibre threshold made up a trial and the control stimuli were continued at 5-min intervals until the neuronal response varied by less than 10%; typically 4-5 controls were made and responses quantified by reference to the last two control measures. Data was captured and analysed using a CED 1401 interface coupled to a Victor VPCII computer and poststimulus histograms compiled for the quantification of the responses. Following the controls DL-AP5 or DGG (Sigma) were applied onto the exposed surface of the spinal cord in a volume of 50 #1 and the neurones monitored at 5 min post-drug and then at 10-rain intervals for 1-2 h. The depths of the cells in the dorsal horn was taken from the mean microdrive readings both on descent and then after returning to the cord surface. The largest discrepancy encountered was 50 /~m difference between the two readings. Results were calculated as percentages of the mean control values with the maximum change following application of a drug being used for analysis. The Student's t-test was used for calculation of the statistical values.

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T h e effects o f A P 5 a n d D G G w e r e s t u d i e d o n the A ~ and C f i b r e - e v o k e d r e s p o n s e s o f 56 n e u r o n e s l o c a t e d t h r o u g h o u t t h e dorsal h o r n . T h e m o d u l a t i o n of e v o k e d

L,

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n e u r o n a l activity by A P 5 d i f f e r e d a c c o r d i n g to the d e p t h of the cell in t h e dorsal h o r n . T h e cells w e r e t h e r e f o r e d i v i d e d into 3 g r o u p s ,

t h o s e f o u n d v e r y superficially

( 0 - 2 5 0 / l m ) , t h o s e o c c u r r i n g in the d e e p l a m i n a e of the cord (550-1000/~m)

and the r e m a i n i n g g r o u p of cells

i n t e r m e d i a t e b e t w e e n the o t h e r two ( 2 5 0 - 5 5 0 ¢¢m). T h e

I,!

m e a n t h r e s h o l d s for e l e c t r i c a l l y - e v o k e d a c t i v a t i o n of Aft and C f i b r e - e v o k e d r e s p o n s e s in e a c h g r o u p of n e u r o n e s is s h o w n in Fig. 2A. T h e m e a n t h r e s h o l d s for b o t h Aft and C fibre a c t i v a t i o n d e c r e a s e d with i n c r e a s i n g d e p t h of the cells f r o m the surface of t h e spinal cord. T h e d e e p cells also d i s p l a y e d a g r e a t e r e v o k e d r e s p o n s e to C fibre s t i m u l a t i o n at 3 times the t h r e s h o l d for that p a r t i c u l a r cell c o m p a r e d to t h e m o r e superficial cells (Fig. 2B). Very L:. ......... i . . . . . . . . a . . . . . . . . i . . . . . . .

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Fig. 1. Single sweep histograms of a single deep dorsal horn neurone following electrical stimulation of the receptive field. A: the response for the first stimulus illustrating the short latency Aft fibre response followed by the long latency C fibre response. B: the response to the sixteenth stimulus illustrating the augmented C fibre response and later post-discharge of the cell resulting from wind-up of the neurone.

few superficial cells e x h i b i t e d a p o s t - d i s c h a r g e r e s p o n s e at 3 times t h e t h r e s h o l d s t i m u l u s for C fibre a c t i v a t i o n , w h e r e a s in the i n t e r m e d i a t e and d e e p cells this constit u t e d a c o n s i d e r a b l e p r o p o r t i o n of t h e n e u r o n a l r e s p o n s e f o l l o w i n g C fibre stimulation. A l t h o u g h the d e e p e r n e u r o n e s t e n d e d to exhibit a h i g h e r c o n t r o l C fibre and p o s t - d i s c h a r g e r e s p o n s e to t h e s t i m u l a t i o n c o m p a r e d to

33

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the more superficial cells the response to Aft fibre activation was similar in all 3 groups of neurones (Fig. 2B).

2-Amino-5-phosphonovaleric acid The effects of AP5 varied considerably depending on the location of the cell in the dorsal horn (Fig. 3B).

7- D-Glutam y lglycine D G G produced dose-dependent inhibitions of both Aft and C fibre-evoked activity and post-discharges in the majority of dorsal horn cells tested (n = 29) (Fig. 3A). In the superficial and deep cells 1000 pg D G G profoundly inhibited the C fibre-evoked response (96 +_ 6% inhibition, n = 5; 75 + 12%, n = 6 respectively) and abolished the post-discharge in the deep cells (100%, n = 6) (Fig. 4). Aft fibre-evoked activity was reduced to a similar extent in deep (75 + 11%) and superficial cells (76 + 7%). D G G had a slightly lesser effect on the intermediate neurones, as both Aft and C fibre-evoked activity were inhibited by 68% after 1000 pg of the antagonist (Fig. 4). In 2 out of 8 superficial and 1 out of 11 intermediate neurones C fibre-evoked activity was increased only by 500pg D G G to a mean 124 + 5% of control

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When the maximal inhibitory effect of D G G occurred the neurones were insensitive to activation by natural peripheral stimuli. Following block of the electricallyevoked C fibre response a cell no longer responded to pinch. Likewise a weak or absent response to touch or prod accompanied the profound Aft fibre inhibition. The onset of effect of D G G was rapid (5-10 min) and the inhibition of the Aft and C fibre responses were in parallel, but the maximum effect of D G G on C fibreevoked responses in intermediate and deep cells tended to occur later (30 ___ 6 min) than on the superficial cells (18 _ 8 min). The time at which maximum inhibition of the Aft fibre-evoked activity occurred did not vary with depth (32 _ 7 min). Once the effects of the antagonist had reached a maximal plateau the drug was washed off the surface of the cord with repeated administration of saline. The neurones recovered from the action of D G G to a variable extent but for all cells the control responses were almost or completely regained at 1 h following washing.

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u a l dorsal horn neurones in this study. T h e results for D G G (1000 yg) are shown in A and these with AP5 (500 ktg) in B. The percentage changes from control values are expressed against depth of the cell in the dorsal horn. S, superficial cells; I, intermediate; D, deep neurones. T h e closed circles represent the C fibre-evoked responses, triangles the Aft fibre-evoked responses, and the squares the post-discharge.

34 Generally cells recorded very superficially in the dorsal horn were not influenced by AP5 (n = 8). The lack of effect of AP5 on these neurones served as a control for the acid nature of the solution of AP5 (pH 3.5) since the superficial cells would be exposed to the highest concentration of the solution and no alteration in neuronal responses nor spike amplitude was yet observed. In deep cells both Aft and C fibre-evoked responses and postdischarges were inhibited by AP5 but to a far lesser extent than seen for D G G . The greatest effect of the antagonist was on the post-discharges which were inhibited by 51 _+ 6% after 500/~g AP5 (Fig. 5). The action of AP5 on neurones located in the

intermediate laminae of the dorsal horn were complex and clearly differed from the effects seen on the superficial and deep located neurones studied• A/5 fibreevoked activity was inhibited by AP5 in 4 out of 7 cells but elevated in the remaining 3 cells. Thus the mean effect of the antagonist on this response did not differ from the controls (Fig. 5). The lowest dose of AP5 (250 ¢tg) had no effect on C fibre responses in the intermediate cells but caused a marked increase in the post-discharge of these cells (n = 3). When 500 ~tg of the antagonist was tested on a further 4 cells a greater excitation of the post-discharge of these neurones occurred (243 _+7% of controls) accompanied by an increase in the C fibreevoked response to 153 _+ 14% of control. The mean

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Fig. 4. Overall results with DGG on the populations of superficial, intermediate and deep neurones. The results are given for both doses, the hatched column for 500 pg, the cross-hatched column for 1000 ~g. A: C fibre responses. B: Aft fibre responses. C: post-discharges (not exhibited by superficial cells). Note the dose-dependency of the effects and the similar magnitude of effects on the Aft and C fibres.

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35 wind-up was markedly or completely abolished for 8 of the 11 cells. Either the onset of the increase in response to repetitive stimuli was delayed and/or the slope of the wind-up response was reduced. The converse was true for wind-up observed in the intermediate cells (n = 7) in which the slope was not altered but the peak values achieved by wind-up were considerably greater than control values (Fig. 6). Since the post-discharges occurring in these cells are consequence of the increased excitability of the neurones as it winds up we would attribute the change in the post-discharge response to the alteration of wind-up by AP5. Because wind-up contributes to the overall C fibre-evoked responses in the cell and not just the post-discharge the change in wind-up is also likely to be the basis for the increase and decrease in C fibre-evoked responses after AP5 in, respectively, the intermediate and deep cells. If the number of C fibre spikes evoked by the first stimulus in the trial of 16 was taken as a measure of C fibre input onto the cell there was no difference between the controls and post-AP5 in deep neurones. Thus it appears that the reduction of C fibre-evoked and post-discharge responses observed is not a consequence of reduced afferent input to these cells. In the population of intermediate neurones tested with both doses of AP5, increases in post-discharges of individual cells could be accompanied by either a decrease or an increase in the Aft and C fibre-evoked responses. Therefore it is clear

maximal increase in post-discharge of the cells occurred about one hour after the application of the antagonist. Thus the effect of AP5 on the intermediate neurones in the dorsal horn was generally the reverse to that observed on the deep neurones, namely an excitation of C fibre-evoked activity and particularly the post-discharge of the intermediate neurones, as opposed to the slight inhibitions of the C fibre-evoked response in the deep neurones (Fig. 3B). Wind-up was analysed for all the cells in the study. The superficial cells do not generally exhibit any wind-up nor post-discharges at the standard 3x threshold stimulation used in these experiments. However 3 neurones did exhibit wind-up and post-discharges after increasing the stimulation to 6-9-times threshold for C fibre activation. In two of these cells the wind-up was sufficient for AP5 to be tested. The wind-up was abolished by 250 ktg of the antagonist without any change in Aft and C fibre-evoked responses. In the case of the intermediate and deep dorsal horn nociceptive neurones both wind-up and post-discharge reliably occur with 3x threshold stimulation (Fig. 6). All cells exhibiting wind-up to the electrical stimulation also had prolonged responses to natural noxious stimuli such as pinch and noxious heat which outlasted the period of stimulation. Both the reduction in post-discharge of the deep cells and enhancement in the intermediate cells mediated by AP5 were associated with changes in the wind-up of the cell. In the deep cells

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Fig 6 Four individual examples of the effect of AP5 on wind-up A, B: two deep neurones C, D: two intermediate neurones The spikes per stimulus are plotted against time for the sixteen stimuli trial (one stimulus per 2 s) The controls are the open circles and the post-AP5 results are represented by the closed circles

6 that changes in afferent input are not a prerequisite for alteration in the responses of the cell to that input. DISCUSSION Interpretation of the present results depends on the presumption that D G G is acting at both N M D A and non-NMDA receptors whereas AP5 is acting only at the N M D A receptor. We would assume that non-selective effects of AP5 can be ruled out as can effects on neuronal excitability independent of amino acid antagonism despite the high doses required for the following reasons. The Aft and C fibre-evoked responses of the superficial neurones exposed to the highest concentrations of AP5 were not influenced in any way by the antagonist which, however, clearly reduced the wind-up of the deep cells. By contrast, D G G markedly reduced all responses, including wind-up, of all neurones. It would also therefore appear that the antagonists penetrate at effective concentrations to the appropriate receptors for all cells since selective effects on the superficial cells are not observed with either compound. Thus the interpretation of the results is that non-NMDA antagonism reduces both A and C fibre-evoked activity of these dorsal horn nociceptive neurones whereas N M D A blockade by AP5 prevents the consequences of wind-up. Since we applied the drugs intrathecally it would have been impractical to have tested the two antagonists against a variety of agonists in order to gauge the degree of N M D A and non-NMDA selectivity in this same model. However this is not likely to prejudice the interpretation since nonspecific effects can be ruled out, as discussed above, and the dose-dependent effects argue for receptor specific actions. Furthermore, it is generally agreed that D G G is non-selective for the non-NMDA and N M D A receptors whereas AP5 is selective for the latter12l~'19"48"49; the present results bear this out. Applying these antagonists intrathecally on to the exposed surface of the cord has one advantage over iontophoresis in that the drug can diffuse to a wide area of the cord. The dorsal horn is an area where the dendritic trees of the neurones can be extensive s'6'9'53 and where receptors can be distant from the neuronal soma so that this topical application overcomes the problems of site of ejection of the drug. Binding studies using autoradiography reveal that both glutamate binding sites and the N M D A receptor are located most densely in the substantia gelatinosa of the dorsal horn 21"32. The evidence from several studies on spinal neurones and excitatory amino acid transmission indicates that nonN M D A receptors are involved in monosynaptic events since antagonists are able to reduce monosynaptic excitation of neurones and fast e.p.s.p.s evoked by D R G

stimulation in co-cultures of ganglion and dorsal horn celis~°" L2.13.1s,24. By contrast N M D A receptors have been implicated in polysynaptic events ~' ~2.~3,4s,4~J.53. Although without intracellular marking of the cells exact locations cannot be given, it would seem that the cells we have described as intermediate cells are likely to be substantia gelatinosa cells. This assumption is supported by the location of these cells between the superficial and deep laminae of the dorsal horn, and the differential effect of AP5 on these cells compared to the other two groups of neurones. Few studies have examined the modulation of nociceptive transmission in the dorsal horn by excitatory amino acids. However, topical D G G has been shown to depress Aft and C fibre-evoked activity in the rat dorsal horn45; whereas ketamine, the non-competitive N M D A antagonist, does not influence input onto the neurones but blocks wind-up of deep dorsal horn cells 11. Nociceptive reflexes and motoneurone activity are also reduced by ketamine 23 and similarly glutamate-induced and C fibre-evoked activity can be reduced by phencyclidine 52. Furthermore AP5 has been shown to reduce the long latency e.p.s.p, and the composite e.p.s.p, recorded from deep dorsal horn cells whilst sparing the fast e.p.s.p. produced by afferent stimulation 53. These results, taken with our present data would indicate that N M D A receptors are involved in polysynaptic pathways such as the consequences of the C fibre input onto deeper cord cells, whereas non-NMDA are concerned with monosynaptic responses. However this may not be entirely the case since in our experiments superficial cells could be persuaded to exhibit 'wind up' by increasing the strength of C fibre stimulation. Thus the N M D A component of the response of these cells may not be apparent under normal conditions, possibly due to the relatively low level of firing produced by the C fibre inputs in superficial cells compared to deep cells. This C fibre input may be too small to depolarize the cells sufficiently to overcome the Mg 2+ block of the N M D A channel, present at resting conditions 29'34. Increasing the C fibre input by raising the stimulation strength may enhance the depolarisation in some cells resulting in the activation of the N M D A receptor, akin to the situation in the hippocampus 22. The involvement of N M D A receptors in the consistent wind-up of deep cells is of great interest to nociceptive processes in the spinal cord. Wind-up is frequency dependent, only elicited by C fibre stimulation, and results in an increased excitability of the deep neurones since both A and C inputs increase ~-s3~4453. The sudden increase in response to a steady repeated input is characteristic of N M D A events in terms of the ligand and voltage gating of the receptor-channel complex. Wind-up is observed in human psychophysical studies 3°'35~36 and

37 has been suggested to be critical in allowing prolonged firing of central neurones to C fibre inputs even as these afferent inputs are starting to fail 43. Finally the flexion reflex in response to noxious stimuli also appears dependent on wind-up of these deep cells 44 and ketamine reduces these reflexes 23. It is interesting to note that morphine 14 and other kt and 6 opioids (unpublished observations) reduce the inputs onto these neurones and so delay rather than inhibit wind-up, in direct contrast to AP5 which spared the inputs but markedly reduced wind-up. There are some similarities between wind-up and long term potentiation in the hippocampus, in terms of frequency dependence and reduction by N M D A antagonism 4"14'22'31'33 although the spinal events are of considerably shorter duration, lasting minutes rather than days. The C fibre potential in the dorsal horn also exhibits wind-up but this continues for up to 100 stimuli 42 whereas the neuronal wind-up tends to plateau by about 16 stimuli. The neuronal wind-up may thus be countered by inhibitory mechanisms onto the deep neurones so curtailing their responses. The majority of neurones sensitive to excitatory amino acids are in the superficial areas of the spinal cord according to one study in a slice preparation 4~, although more recent work indicates that most .deep cells are responsive to excitatory amino acids as well 25. Since there is now accumulating evidence for glutamate as a transmitter in nociceptive C fibres (see Introduction) and the majority of C fibres terminate more superficially than the A fibres it is possible that some of the superficial cell bodies are depolarized by glutamate released from monosynaptic C fibre contacts and some deep cells may receive direct A fibre inputs. However it is also likely that certain deep cells receive C fibre inputs onto their dendrites which penetrate up into the C fibre terminal zone. The present results with D G G support this idea since the antagonist blocked C fibre afferents before A fibre inputs, which, as has been suggested, may result from the deeper terminal zones of the larger fibres such that longer periods of time are needed for diffusion of the antagonist to the Aft terminal zone 45. In behavioural experiments N M D A applied intrathecally in the mouse has been shown to produce biting and REFERENCES 1 Aanonsen, L.M. and Wilcox, G.L., Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and o agonists, J. Pharmacol, Exp. Ther., 243 (1987) 9-19. 2 Bernardi, G., Zieglgansberger, W., Herz, A. and Puil, A., Intracellular studies on action of L-glutamic acid on spinal neurones of the cat, Brain Research. 39 (1972) 523-525. 3 Besson. J.M. and Chaouch, A., Peripheral and spinal mecha-

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Acknowledgements.This work was supported by The Medical Research Council and The Wellcome Trust. We are grateful to V. Grant and D. Menday for their expert secretarial assistance.

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