Spinal substance p and N-methyl-d -aspartate receptors are coactivated in the induction of central sensitization of the nociceptive flexor reflex

0X%-45?1,9?$5.00+ 0.W PergamonPressLtd f 19921BRO

SPINAL

SUBSTANCE P AND ~-METHYL-D-ASPARTATE RECEPTORS ARE CUACTIVATED TN THE IN~~CTIUN OF CENTRAL SENSITIZATION OF THE NOCICEPTIVE FLEXOR REFLEX andZ. WIES~~FELD-HAI.~I~*~

X.-3. Xv,* C.-J. DALSGAARD? *Karolinska

Institute,

Department of Clinical Physiology. Section of Clinical Huddinge University Hospital, Huddinge, Sweden tAstra Pain Control AB, Sodertilje, Sweden

Neurophysiology,

Abstract-We have studied the effects and interactions of the neurokinin-1 receptor antagonist CP-96,345 and the N-methyl-n-aspartate receptor/channel blocker MK-801. both applied intravenously. on the flexor reflex and on the facilitation of the flexor reflex by conditioning stimulation of cutaneous C-afferents in decerebrate. spinafized, unanesthetized rats. The flexor reflex was evoked by subcutaneous electrical stimuli applied to the surai nerve innervation area I hnin at an intensity that activated C-fibers and uas recorded as e~e~tromyogram from the ipsilateral hamstring muscles. The magnitude of the baseline flexor reRex was usually highly stable in the course of the experiments without experimental manipulations. The same stimulus was used as a conditioning train (0.9 Hz, 20 shocks) and caused a brief facilitation of the IIexor rettex, which was maximal 0.5 and I min after stimulation (255.1 & 23.6% over baseline). During the course of the conditioning stimulus train. the refiex magnitude was gradually increased (wind-up). MK-801 (0.1 and OJmgjkg) consistently depressed the polysynaptic flexor reflex. At a dose of 0.5 mg/kg, but not 0. I mgjkg, MK-801 reduced the wind-up and blocked the facilitation of the flexor reflex induced by the conditioning stimulus by 90%. The facilitatory effect of 7 pmol intrathecal substance P was also partially reduced by MK-801. CP 96,345 (1 and 3 mg;kg) did not depress the flexor reflex. but dose-dependently antagonized reflex ~~~litation by the conditioning stimulus train, similarly to its antagonism of intrathecally apphed 7pmol substance P-induced facilitation. CP-96,345 had no effect upon the excitatory effect of intrathecally applied iV-methyl-u-aspartatc (6.8 nmol). The wind-up to the conditioning stimulation was also reduced by 3 mgikg CP-96,345. When subthreshold doses of MK-801 (0.1 mgikg) and CP-96,345 (0.3 mg/kg) were ~onlb~n~, both wind-up and ~ondjtioning stjmu~tioninduced flexor reflex facilitation were strongly blocked. The present results suggest that both substance P and N-methyl-I-I-aspartate receptors are involved in the deveiopment of wind-up during repetitive stimulatiotl of C-aIYerents, as well as in the prolonged increase of spinal cord excitability seen following stimulation. Thus, excitatory amino acids and substance P. which coexist in primary afferent terminals and may be co-released upon repetitive C-afferent stimulation. interact synergistically to induce central sensitization.

Stimulation of primary sensory afferents produces excitatory postsynaptic potentials (EPSP) lasting tens of milliseconds in dorsal and ventral harn neurons in the spinal cord.‘3.‘“.i7.33.5s In addition, prolonged synaptic potentiais. which can last tens of seconds. may he observed in dorsal horn neurons when the stimulus is strong enough to activate small diameter, unmyehnated (C) afferent fibers.“.‘9.‘5~55 Such prolonged excitatory synaptic potentials may underly the ability of C-afferents to produce activit~dependcnt central sensitization, demonstrated as increased excitability of neurons in the spinal cord and of spinal reRexes.z”,36 When C-fibers are repetitively stimulated

$To whom correspondence should be addressed. Ahhrez icx~ions:CP-Y&345, (f - cis - 2 - (diphenyimethyl) -N[(2-methoxyphenyl)-methyl]-l-azabicyclo~2~2.2]octan-3amine; CS, ~o~ldit~on~ng stimulus; EPSP, excitatory postsynaptic

potentials:

Lt., intrarhecal; MK-801. ( + j-5~,d]-cyclohepten-5,~~~-methyl-~-aspart~~te; SP, sub-

r~~~~hyl-l~,lI-dihydro-5H-di~n~o imine maleate; stance P.

NMDA.

641

at high frequencies, the phenomenon of “wind-up”, a progressive increase in the number of action potentials evoked per stimulus, occurs in dorsal horn neurons.” Such a phenomenon can also occur in flexor motoneurons 13i47.4Kresulting in a progressive increase of ffexor reflex magnitude during repetitive C-fiber stimulation. in addition to causing wind-up, brief repetitive stimulation of C-a&rents also induces prolonged increase of spinal cord excitabiiity.~~ The dur~~tion of such excitability changes depends on the type of peripheral nerves stimulated. A conditioning stimulus (CS) train (20 s at 1 Hz) administered to a cutaneous nerve facilitates the flexor reflex for a few minutes, while the same CS applied to a muscle nerve facilitates the reflex for about 1 h.” Prolonged hypersensitivity of the flexor reflex has been reported in animals after tissue injury,J4 Substantial evidence has accumulated which suggests a role for neuropeptides. particularly tachykinins, in the mediation of central sensitization

after repetitive C-fiber stimulation. Thus, it has been found that iontophoretically applied substance P (SP) typically causes a slowly developing, prolonged depolarization of dorsal horn neurons.‘4.‘h SP has also been shown to enhance the response of dorsal horn neurons to C-fiber input’*,56 and a SP antagonist prevented the development of wind-up in these neurons after repetitive C-fiber stimulation.‘x It has been reported that the slow depolarization potentials in dorsal horn neurons induced by repetitive electrical stimulation of dorsal roots is mimicked by SP and inhibited by a putative SP antagonist.35 Intrathecally (i.t.) applied SP also mimicked the facilitatory effect of the sural nerve CS on the flexor reflex in decerebrate, spinalized, unanesthetized rats”.“’ and a tachykinin antagonist. spantide IL4’ abolished reflex facilitation induced by both cutaneous and muscle nerve CS.39.40 In addition to tachykinins, the importance of the activation of the N-methyl-D-aspartate (NMDA) receptor by glutamate in the mediation of central sensitization is suggested by recent studies.4,Y.‘2.‘6.33.4* The wind-up phenomenon in dorsal and ventral horn neurons during repetitive C-fiber stimulation was shown to be inhibited by NMDA antagonists.4,y.‘3.4X Furthermore, the prolonged facilitation of the flexor reflex following C-afferent CS was reduced by NMDA receptor blockade.48 It has been established morphologically that SP and glutamate co-exist in dorsal root ganglion cells and in the dorsal horn.‘,” Co-existence of glutamate and SP in the terminals of primary sensory afferents has also been demonstrated.7.24 Co-expression of neurokinin- 1 receptors, for which SP is the endogenous ligand, and excitatory amino acid receptors has been reported in single dorsal horn neurons.30.43 There is considerable evidence suggesting an interaction between SP and glutamate in C-afferent mediated input. Activation of dorsal horn neurons by excitatory amino acids has been shown to be enhanced by SP.“.56 SP also potentiated glutamate-induced current in isolated dorsal horn neurons3” Moreover, spinally coadministered SP and glutamate receptor agonists interact synergistically to induce behavioural responses.” Based on these findings, it has been hypothesized that co-activation of SP and NMDA receptors may be important in inducing wind-up and central sensitization and, therefore, may contribute to a number of painful pathophysiological conditions.42.46 This hypothesis has not, however, been examined in studies with specific receptor antagonists. The present study was undertaken to study the possible interaction between SP and NMDA receptor activation in inducing the phenomenon of central sensitization in rat spinal cord by examining alterations of excitability of the rat hindlimb flexor reflex. The effects of applied CP-96,345, a novel non peptide neurokinin 1 receptor antagonist,‘2.32 and the noncompetitive NMDA receptor/channel blocker MK801 on flexor reflex excitability before, during and

after repetitive stimulation of cutaneous was examined. Some of the results communicated in abstract form.‘?

C’-afferents have been

EXPERIMENTAL PROCEDURES

Animal preparations Experiments were performed on 200-25Og Sprague Dawley rats of both sexes (ALAB, Stockholm). The animals were briefly anesthetized with methohexital (Brietal, Lilly. 70 mg/kg, i.p.) and a tracheal cannula was inserted. One jugular vein was cannulated for drug administration. The rats were decerebrated by aspiration of the forebrain and midbrain and were thereafter artificially ventilated. The spinal cord was exposed by a laminectomy at thoracic level and sectioned at Th8-9. In some experiments, an it. catheter (PE 10) was implanted caudally to the transection with its tip on the lumbar enlargement for injection of SP and NMDA. The flexor reflex was evoked by single (l/min) or trains of electric shocks applied to the sural nerve innervation area of the left foot via a pair of needle electrodes inserted subcutaneously approximately 0.5 cm apart. The stimuli (I ms, 5 mA) activated C-fibers.36 The reflex was recorded as electromyographic activity via stainless steel needle electrodes inserted in the ipsilateral biceps femoris/semitendinosus muscles. The number of action potentials elicited during the reflex evoked at l/min (baseline reflex) was integrated over 2 s. A CS train of 20 shocks at 0.9 Hz was used to produce wind-up and facilitation of the baseline reflex. During this CS, the electromyograph was integrated for l-s intervals to monitor reflex magnitude in order to evaluate wind-up. Rectal temperature was monitored and maintained within normal limits. The heart-rate was also monitored. Drugs CP-96,345 raccmate was synthesized at Astra Pain Control AB, dissolved in 1 M acetic acid and then diluted in saline to the desired concentration. MK-801 (Merck, Sharp and Dohme) was dissolved in saline. CP-96,345 and MK-801 were injected i.v. in a volume of 0.2ml. SP (Cambridge Research Biochemicals) was dissolved in 0.01 M acetic acid in saline and NMDA (Tocris Neuramin) was dissolved in saline. SP and NMDA were injected i.t. in a volume of IO ~1 followed by 10~1 of saline to flush the catheter. Data collectionand analysis A stable baseline reflex magnitude was established for at least 20 min before each drug injection or the CS. The effect of the drugs and the CS on the flexor reflex was expressed as percentage change in reflex magnitude compared to baseline. The facilitatory effects of the sural CS or it. SP or NMDA alone were taken as 100% and the antagonistic effect of CP-96,345 and MK-801 were expressed as a percentage of the control facilitation. All data are expressed as mean & S.E.M. and were analysed with ANOVA, followed by Dunnett’s test. RESULTS

Effects of CP-96,345 on the baseline jexor reflex and its facilitation by intrathecal substance P and N-methyl-D-aspartate CP-96,345 injected i.v. at 0.3, 1 and 3 mg/kg failed to alter baseline reflex magnitude (Fig. 1). The highest dose of CP-96,345 caused slight bradycardia (20-30% decrease in heart-rate) commencing immediately after injection and lasting IO-20 min. SP, 7 pmol (10 ng) i.t., facilitated the flexor reflex with a peak increase of

Substance

P and glutamate

in central Table

2. The wind-up of reflex magnitude during shock conditioning stimulus of C-alf‘erents

Stimulus

number

l-5 6-10 II I5 16-20

400

T

647

sensitization

“/o Increase

the 20

in reflex magnitude

116.3 255.1 353.7 3X6.7

* 13.0* *2h.It * 33.5: + 34.0

The data were collected from 20 experiments. The extent 01 wind-up is expressed as average increase in retlcx magntude over baseline responses for live consecutive stimuli. ANOVA showed a significant overall increase in rcllew magnitude over baseline (F,,,, = 20.636. P i 0.0001 ). Compared to baseline response. *P < 0.005; compared to Increase of reflex magnitude in previous li\c atlmuli. tF < 0.05, :P < 0.005.

cs

over baseline (n = 3). CP-96.345 (3 mgkg. i.v.) had no erect on the excitatory effect of Lt. NMDA in three experiments.

Time (min)

Fig. I. Illustration of the antagonistic effect of CP-96,345 (CP) on the i.t. SP (A) or the subcutaneous 0.9 Hz. 30.shock CS (B)-induced facilitation of the tlexor reflex in two experiments. The dose of SP injected was 7 pmol. The baseline reflex is defined as 100%. Note that CP-96,345 did not influence baseline reflex magnitude in either experiment. but slpnilicantly reduced reflex facilitation by SP or the CS. In B a transient depression was observed after the CS in the presence of CP-96,345. Similar findings were observed in about 80% of the experiments.

144. I + 20.6% over baseline for 6.1 & 0.7 min in eight experiments. The vehicle used to dissolve SP had no measurable effect on reflex magnitude and the facilitator) effect of Lt. SP was reproducible within each experiment. CP-96.345 administered 20-30 min prior to i.t. SP dose-dependently antagonized SP-induced reflex facilitation (Fig. IA. Table I). NMDA (i.t.. 6.8 nmol) excited the spinal cord, expressed as spontaneous motor discharges and facilitation of the flexor reflex with a peak increase of 174.0 k 68.0%

Table I. Antagonistic eH‘ect of CP-96.345 on 7 pmol i.t. substance P- or the subcutaneous conditioning stimulus-induced facilitation of the flexor reflex Dose ol (‘P-Oh.345 0.3 mg*kg I mfkg 3 n&kg

% Control SP 98.9 k 16.7 (4) 32.03 f 3.4 (4)* 2.2 + 2.3 (4)*

facilitation cs 86.8 k 5.0 (6) 34.9 i 97.7 (7)* 4.9 + 2. I (7)*

The number of experiments is indicated in brackets and variability is expressed as S.E.M. ANOVA revealed that I and 3 mg:kg CP-96,345 significantly antagonized both SP- and sural CS-induced facilitation of the flexor reflex (F, ,J = 75X.307 and F,,,, = 82.585, P < 0.0001). The mdividual comparisons were performed with Dunnett’s test. *P i 0.005 compared to control response.

The CS train (0.9 Hz. 20 shocks) applied to the sural nerve innervation area at an intensity that activated C-fibers induced a period of r&x facilitation after the CS had ceased. The facilitation \vas tnaximal between 30 s and I min after the CS and the reflex returned to baseline after 2-3 min (Fig. I B). The peak facilitation of the reflex after the CS to the sural nerve innervation area was 255.1 k 23.6°<1 with a duration of 2.5 i 0.2 min in 25 experiments. The eflect of C-fiber CS was reproducible within each cxperimcnt. Pretreatment with I or 3 mg:kg CP96.345 20-30 min prior to the CS dose-dependently reduced the CS-induced l’dcilitation, similarly to its antagonism of 7 pmol i.t. SP (Fig. IB. Tahlc I). Intercstingll, in the majority of the cxperimcnts. the flexor reflex was briefly depressed after C‘S in the prescncc of I and 3 mg:kg CP-96.345. as illu\tratcd in Fig. IB.

In addition to causing prolonged rcfcu facilitation. the CS in the sural ncrvc mncr\,ation ;II‘L’;Ialso produced a progressive increase in flcsor refjcx magnitude during the 20-pulse stimulus train (wind-up). The wind-up is presented in Table 2 as abcragc incrcasc in reflex magnitude to stimuli I 5. 6 IO. I I -15 and 16-20. compared to haselinc rcfcx magnitude in 20 experiments. Pretreatment with 0.3 mg’kp C‘PY6.345 20 30 min prior to the delivery of the nerve CS had no effect on wind-up (Fig. 2). However. I mg’kg CP-96.345 significantly depressed the incrcasc of reflex magnitude during stimuli I6 20 and 3 mg;kg of the drug depressed wind-up during stimuli I I I5 and 16~20 (Fig. 2).

644

;

80

s i? s! E E

60

40

8 s

20

1-5

6-10

11-15

16-20

Stimulus number Fig. 2. Effect of 0.3 n = 5) i.v. CP-96,345 the stimulus number. between the different of the reflex wind-up

mg/kg bars, = I mg/kg (hatched bars, n = 6) and 3 mg/kg (filled bars, on the wind-up of the flexor reflex during the CS. The figures under the bars indicate Two-way ANOVA indicated that there was a significant difference in the antagonism doses of CP-96,345 (F,,,,, = 12.658, P = O.OOOl), as well as between various phases Compared to control responses, *P < 0.05. ***P < 0.005. (FI,ox = 2.9, P -c0.05).

Effect of N-methyl-D-aspartate antagonist MK-801 on baselinej?exor rejex and its facilitation by intrathecul substance P

Both 0.1 and 0.5 mg/kg Lv. MK-801 depressed the polysynaptic flexor reflex (Fig. 3). The reflex depression became evident 5-10 min after i.v. injection and lasted for about an hour for the lower dose of MK-801. The duration of depression produced by the higher dose of MK-801 was longer than that caused by 0.1 mg/kg MK-801, but was not determined. The average reduction in flexor reflex magnitude 10 min after 0.1 mg/kg MK-801 was 34.5 k 5.6% (n = 8) and after 0.5mg/kg MK-801 was 49.4 + 9.6% (n = 6).

MK-801 at 0.5 mg/kg, but not at 0.1 mg/kg, also partially blocked the facilitatory effect of 7 pmol i,t. SP on flexor reflex excitability (34.5 + 5% of control peak facilitation, n = 3). Effect of’ MK-801 on wind-up and the prolonged facilitation caused by the conditioning stimuli

MK-801 at 0.1 mg/kg did not reduce the CSinduced wind-up, neither did it block reflex facilitation (Fig. 4). At 0.5mg/kgt MK-801 produced partial reduction of the wind-up (Fig. 4A). In contrast to the effect of CP-96,345, wind-up during the entire 20-p&e CS train was reduced by MK-801. This dose

0.1mglkg

0.5 mglkg c

0’

I

I

r

I

I

I

0

30

60

90

120

160

1

180

Time (min) Fig. 3. Illustration of the depressive effect of i.v. MK-801 on the flexor reflex. The doses and time of administration of MK-801 are indicated by the arrows. The baseline reflex magnitude is defined as 100%.

*** I

Substance P and glutamate in central sensitization

645

I_

r-

***

xxx

~_

I-5

6-10

if-15

i

16-20

(6)

(8)

Post-CS response

Stimulus number

Fig. 4. Effects of 0.1 mg/kg (open bars) and 0.5 mg/kg (filled bars) iv. MK-801 on reflex wind-up during the CS (A) and peak reflex facilitation after the CS (B). (A) Two-way ANOVA indicated a significant antagonism by 0.5 m&kg, but not 0.1 mgi’kg, MK-801 on wind-up (F2,hj= 109.163, P < 0.0001). which was uniform during the entire CS train (F,,, = 1.253, not significant). (B) ANOVA indicated a significant antagonism by 0.5 mgikg, but not 0.1 mg/kg, MK-801 of the Rexor reflex facilitation induced by the CS (F,,?,,= 169.221, P < 0.0001). Compared to control response. ***P < 0.005.

of MK-801 also virtually abolished

reflex facilitation

by the CS (Fig. 48). E@c?s ~?~~~administr~ti[)n qf‘CP-96.345 and MK-801 on wind-up and facilitation of the ,fZe.xor r~fks b_v the conditioning stimulation

The wind-up and reflex facilitation after the CS were significantly reduced by co-administration of 0.3 mg/kg CP-96.345 and 0.1 mg/kg MK-801, a combination of doses which alone produced no significant effect, when tested 20-30 min after drug administra-

tion (Fig. 5A, B). Further increasing the dose of CP-96,345 to 3mgikg and MK-SO1 to 0.5mgikg reduced both wind-up (Fig. SA) and peak reflex facilitation (Fig. 58) by over 80%.

DJSCUSSION

We have shown that CP-96,345 applied it. behaves as a selective SP antagonist. as it blocked the facilitatory effect of i.t. SP. but not i.t. neurokinin B

80

*

*

‘I I***

1

60

**

*** 1 ic ICL I

xxx

XI

-

~ l-5

**

It**

ii

6-10

11-15

Stimulus number

16-20

(6) (4) Post-CS response

Fig. 5. Effects of co-administration of 0.1 mg/kg MK-801 plus 0.3 mg/kg CP-96.345 (open bars) and 0.5 mg/kg MK-801 plus 3 mg/kg CP-96,345 (filled bars) on wind-up during the CS (A) and reflex facilitation after the CS (B). (A) T wo-way ANOVA indicated a significant antagonism by MK-801 plus CP-96,345 on the reflex wind-up with both dose combinations (jF2,4x = 24.982, P < 0.000 1), but not between different groups of stimuli (Fi,.,*= 0 122, P > 0.5). (B) ANOVA indicated a significant antagonism of the peak facilitation of the flexor reflex induced by the CS by both drug combinations (Fz,,l = 60.142, P = 0.0001). *P < 0.05, **P < 0.01 and ***P -c0.005. compared to control response.

A.” The present results demonstrate that i.v, CP96,345 antagonizes the facilitation of the flexor reflex induced by i.t. SP. but not i.t. NMDA, thus indicating that this drug is active in the CNS after systemic injection. Our results are therefore in agreement with previous studies which established the neurokinin 1 binding and antagonistic property of this compound jn rjl,o and jn L~~ro,?.X.2’.~l.3? Although the affinity of CP-96,345 to the neurokinin 1 receptor is lower in rats and mice than in some other species (such as man, cow, rabbit, guinea-pig and hamster),‘,3’ a number of laboratories have reported a significant antagonism of SP-mediated effects in rodents by this compound,“,‘0.“,54 in agreement with our previous”,” and present results. In a previous study, we found that the potency of CP-96,345 in blocking SP-induced facilitation of the flexor reflex was comparable to spantide II upon it. injection,“’ reflecting the relatively low potency of CP-96,345 in rats. In some other species the potency of CP-96,345 in displacing neurokinin 1 receptors is 100-1000 times stronger than the peptide SP antagonist.*,” CP-96.345 applied systemically did not depress the polysynaptic flexor reflex, but dose-dependently antagonized the facilitation of the reflex induced by CS of cutaneous C-afferents. At high doses. the windup of reflex magnitude during repetitive C-fiber stimulation was reduced by CP-96,345. These results suggest that the wind-up and central sensitization phenomena after repetitive C-afferent input are mediated via neurokinin 1 receptor activation, but that this receptor is not involved in the mediation of the baseline flexor reflex. Our findings are in agreement with recent reports by De Koninck and Henry in which they found that systemic CP-96,345 did not influence the response of cat dorsal horn neurons to single electrical stimuli of C-fibers, but abolished slow. prolonged excitatory postsynaptic potentials following a train of intense electrical stimuli.” In contrast to CP-96,345, systemic MK-801 depressed the polysynaptic flexor reflex. It is unlikely that this depressive effect was due to a direct depression of MK-801 on motoneuron activity, as previous studies have shown that the monosynaptic EPSPs evoked by primary afferents on motoneurons, as well as the monosynaptic reflex, are not depressed by NMDA antagonists.5,‘8,34 The effects of NMDA antagonists on dorsal horn neurons are controversial,g,‘O.‘y thus the exact site of the depressive effect of MK-801 on the flexor reflex is unclear. MK-801 at 0.5 mg/kg, but not 0.1 mg/kg, partially blocked wind-up and almost completely antagonized the prolonged facilitation of the flexor reflex after the CS. This result agrees with the report of Woolf and Thompson,48 indicating that activation of the NMDA receptor is essential for the induction of wind-up and central sensitization. Interestingly, the higher dose of MK-801 also reduced the facilitatory effect of i.t. SP, suggesting that i.t. SP-induced reflex facilitation is partially due to an amplification of NMDA receptor

activation-mediated excitation. The depressant c~fcct on wind-up and reflex facilitation by a Hugh dose ot MK-801 was apparently independent of its dcpressivs effect on the baseline reflex. as the lower dose of MK801 failed to block the wind-up and reflex facilitation. even though it depressed the reflex. Simil,~rly. 1.1. morphine and x2 adrenoceptor agonists effectively reduce the baseline reflex magnitude, but much higher doses are required for these drugs to have any blocking effect on wind-up and central sensttization (Wiesenfeld-Hallin et al.. unpublished observations). Low doses of CP-96,345 and MK-801, which by themselves had no effect, synergistically blocked windup and reflex facilitation. This result strongly suggests that there is an important interaction between the SP and the NMDA receptors in the development ot wind-up and subsequent central sensitization. The interaction between SP and NMDA may occur as an enhancement of the responsiveness of NMDA reccptot by SP, which had a long-lasting depolarizing effect upon dorsal horn neurons”,‘” and may be involved in removing the voltage-dependent block of the NMDA receptor-ion channel complex normally imposed by Mg’ ’ .‘? An interesting finding was that 3 mg/kg CP-96,345 only reduced the wind-up in response to stimuli 1 l- 20, but not to stimuli l--10. In contrast, MK-801 reduced wind-up during the entire 20-shock train. One possible explanation is that glutamate was released by every shock in the CS train, but SP only during the latter part of the CS. However, another possibility is that although SP was also released in response to the early part of the CS, its action was delayed and therefore the effects of the antagonist could only be detected in the later part of the CS. A brief depression of flexor refex excitability after C-fiber CS was observed in the majority of the experiments in the presence of CP-96,345. This result is similar to an earlier finding by Woolf and Wall who reported that after capsaicin treatment of the sciatic nerves, C-fiber CS no-longer induced a facilitation of the reflex. but rather a transient depression.” It is possible that this inhibition is mediated by largediameter A-fiber input which was concomitantly activated during the C-fiber CS4’ However, it is also possible that the C-fiber CS by itself had an inhibitory effect on spinal reflex excitability,45 directly by releasing the inhibitory peptide galanin’“,” and/or indirectly by activating other endogenous analgesic systems. CONCLUSION

The present study provides direct evidence for a synergistic interaction between NMDA and SP receptors in the mediation of the wind-up phenomenon during repetitive C-fiber stimulation and therefore in the induction of central sensitization. As the importance of such activity-dependent increase in spinal cord excitability is believed to play an important role in a

Substance P and glutamate in central sensitization number

of painful

states,46 antagonists

acting on the

NMDA or SP receptors may be useful for treating certain painful conditions, such as hy~ralgesia. However, due to the widespread presence and multiple functions of glutamate that such drugs will effects. Therefore, the doses of NMDA and

and SP in the CNS, it is likely

cause many undesirable

side-

synergistic effect of very low SP antagonists in depressing

647

spinal cord excitability may be particularly interesting from the clinical point of view. Acknowledgements-This study was supported by Astra Pain Control AB, the Swedish MRC (project no. 079139). the Bank of Sweden Tercentenary Foundation, the Wenner-Gren Center Foundation, the Marcus and Amalia Wallenberg Foundation, Tatjana and Jacob Kamras Foun~tion and research funds of the Karoiinska Institute.

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