Electrophysiological studies on the spinal roles of endogenous opioids in carrageenan inflammation

&in,

sJ(lYY4)

Elsevier

Science

185-191 B.V.

PAIN 024.10

studies on the spinal roles of endogenous opioids in carrageenan inflammation

Electrophysiological

Louise C. Stanfa Departmenr (Received

* and Anthony

of Phurmacology. Uniwrsity Collqy Ixdor~ 27 May 1993. revision

received

H. Dickenson f~or~dor~W’CIE hBT lllK1

25 July lYY3. accepted

2Y July 1903)

Summary This electrophysiological study uses the mixed peptidase inhibitor kelatorphan and the selective K-antagonist nor-binaltorphimine (nor-BNI) to investigate whether there is altered modulation of spinal nociceptive transmission by endogenous opioids 3 h after injection of carrageenan into the ipsilateral paw. lntrathecal kelatorphan (5-250 pg> inhibited the C-fibre evoked response of dorsal horn neurones in both normal and carrageenan animals, with no difference in this inhibitory effect found between the 2 groups of animals. In both groups of animals, this inhibition reached a plateau at 50%. Thus there was no change in the effects exerted by the spinal enkephalins at this point in the inflammatory state. Nor-BNI (10 and 100 pg) produced a bidirectional change in the C-fibre evoked response of dorsal horn neurones in both normal and carrageenan animals, facilitating the evoked response of some neuroncs whilst inhibiting others. The magnitude of the change in the neuronal response induced by nor-BNI in carrageenan animals was significantly greater than that seen in normal animals, suggesting a greater release of spinal dynorphin in the inflammatory state. Dorsal horn neurones showed a bidirectional change in response as carrageenan-induced inflammation dcvcloped, although the direction of this change did not correlate with the subsequent direction of effect of nor-BNI. There was, however, a significant correlation between the magnitude of the change in the C-fibre evoked response after the injection of carrageenan and the magnitude of change produced in the same cells by nor-BNI. This suggests that there is a strong link between the dynorphin system in the spinal cord and the system responsible for producing the changes in neuronal response seen post-carrageenan, possibly the N-methyl-I)-aspartatc (NMDA) receptor system. Key words:

Endogenous

opiate;

Inflammation;

Kelatorphan;

Introduction Endogenous opiates may play a role in the altered transmission of nociceptive information seen in inflammatory states. Many studies have investigated the changes that occur in the levels of mRNA for the endogenous opiate peptides and in the levels of the opiate peptides themselves in the spinal cord following peripheral inflammation (see refs. in Dubner and Ruda

* C‘orres~~orulin~ uuthor: Louise Stanfa, Department of Pharmacology, University College London. Gower Street, London WCIE hBT, UK. Tel.: (34-71) 387-7050: FAX: (44-71) 3072Y8.

Nor-Binaltorphimine;

Spinal

cord

lYY2). However, less is known about how alterations in the physiological release of these opioid peptides in inflammatory states affects the response of spinal ncurones involved in the transmission of nociceptive information. Behavioural studies have looked at the effects of opiate antagonists on the hyperalgesia that is associated with the development of peripheral inflammation (Kayser and Guilbaud 1981, lYY0, 1991; Millan ct al. 1987. 1988; Millan and Colpaert 1991). However, these studies have relied on systemic administration of the drugs and hence their site of action is not always clear, possibly involving actions at peripheral and supraspinal as well as spinal sites. Thus this study has focussed on 1 specific site of opioid modulation, the spinal cord. The aim of this

electrophysiological study is to examine whether there is increased modulation of spinal nociceptive transmission by endogenous opioids 3 h after the injection of carrageenan into the paw. This time point was chosen as it roughly corresponds to the time of peak inflammation and hyperalgesia seen in behavioural studies using this inflammatory agent (Hargreaves et al. 1988; Hylden et al. 1991b). In addition, we have previously shown in an electrophysiological study in carragecnan animals that the response of dorsal horn neuroncs to electrical stimulation at C-fibre intensity is significantly altered at this time point but in a complex bidirectional fashion (Stanfa et al. 1992; Stanfa and Dickenson 1993). Furthermore, the potency of exogenous p-opiates applied by the spinal route is enhanced 3 h after injection of carrageenan into the paw as shown by both behavioural (Hylden et al. 1991b) and electrophysiological (Stanfa et al. 1992) studies. Thus this study was undertaken in an attempt to gauge whether any increase in the physiological release of opiate peptides within the spinal cord occurs at this stage in the inflammatory process and hence whether endogenous opiate peptides play any role in this early central response to inflammation. Alterations in spinal enkephalinergic activity were gauged by using the mixed peptidase inhibitor kelatorphan (Fournie-Zaluski et al. 1984) applied directly to the spinal cord in animals with carrageenan inflammation and comparing these actions to those seen in normal animals. In addition we used the selective Knor-binaltorphimine (nor-BNI) opiate antagonist (Portoghesc et al. 1987) to examine the activity of dynorphin-containing neurones in the spinal cord.

Methods The technique

is essentially that described previously for compar-

ing the response

of convergent

neurones

with carrageenan

inflammation

to those in normal

effect of drugs thereon Dawley

rats (200-250

mixture

of 66%

halothane The

rat

N,O

and 33%

for the cannulation the

metal clamps for stability. sufficient A

Ll-L3

and then

to maintain

frame

region

maintained and

of the

to the exposed

complete tungsten

cord.

cord were

was lowered

single-unit

extracellular

pm) which responded

of neuronal

recordings

was

used

to

least

hind paw. The recording

10 times the background

stimuli/trial),

Electrical

stimulation

obtain

from deep dorsal horn neurones

to both non-noxious

be from a single unit if the action potentials, activity, (0.5

stimuli such as

applied via 2 needles inserted

fields

was judged

to

which were typicatly at

were Hz,

;tnd then i times the threshold each particular reproducible

neurone.

current

101

(

-ttbre it~t~\;~t~~,~l

for A,!&tihte

This type ot stimulatmn

test stimulus for the experiment

actrvallon

I<.$

provides a constant

and .noids the pnlhlert~

of altered baseline responses which could result ftcrm hyperatgera natural

stimuli

performed

as a consequence

rvcty

IO min

and wc

change

occurs in the thresholds

period

of the

experiment

ol the inflanimatton. have prevtously for C-fibre

(Stanfa

et al.

haats of threshold histograms.

( c IO’Z In

variation the

and latency. in the C-fibre

animals

A-carrageonan

with

3 h prior to drug administration.

inflammation.

this period,

The results are only taken ot the action

,Z(,

evoked response (11 with

potenttals

the 3 readings

from neurrmes whetc

revealed

no change

shape or height during the recording (0.34-17

100 ~1 of

used as controls iclr the ~uhscquenr

drug effect.

Kelatorphan

control tri,il\

region of the hind paw

The electrically

during

monitoring potential

,lC(-tihrc

cvokcd response).

peripheral

prior to the drug administration

n<, the

trom post-\ttmuius

following 3 \tahlc

were injected into the plantar

these cells was monitored

that

during

and

I(:

MCII’

from c‘rch othet on rhc

wcrc quantified

Drugs were administered

shown

activation

lY92.1. C’

evoked responses. which could be separated

Trial\

mM;

5-250

pg)

\i\ual

rn action

period.

and nor-BNI

(0.23-2.1

mM; IO- 100 kg) were applied directly to the surface of the cord in a volume

of 50 /II

subsequent trathecal

(equivalent

evoked

response

tcl an intrathecal

injection)

of the cell followed

and the

for 60 min.

In-

naloxone (0.05 mM: I pg) was used to reverse the effects ot

kelatorphan. The results are expressed as a percentage age inhibition

or as a percentage

change

of control,

as percrnt-

from contro1tS.E..

results were analysed using linear regression. the Mann-Whitney 01 Student’s

The te\t

I test.

Results Changes in evoked neuronal rageenan inflammation

responses following car-

The C-fibre evoked response of 17 single neurones was followed during the 3 h period following injection of carrageenan into the ipsilateral hind paw. The change in the C-fibre evoked response of the neurones 7 h after the onset of inflammation was bidirectional, with 8 neurones showing an enhanced response (mean: 131.9 _t 12.6% of control) whilst the other 9 neurones showed a decreased response to the electrical stimulation (mean: 85.0 + 3.5% of control). This pattern of changes in the neuronal responses is identical to that which we have reported previously in this model following carrageenan inflammation and where we have shown that the changes in the neuronal responses are not seen after peripheral injection of the saline vehicle (Stanfa et al. 1992: Stanfa and Dickenson 1993).

to 1.5% which was

light pressure and noxious stimuli such as pinch from receptive

amplitude.

The

held with

areflexia. microelectrode

(500-1000

on the toes of the ipsilateral

in a

on 2%

a laminectomy

spinal

Prior to the commencement

the level of halothane

glass-coated

male Sprague-

with 3% halothane

of the trachea and subsequent surgery.

rostra1 and caudal

recordings

0,

in a stereotaxic

to expose

animals and the

et al. 1992). Briefly,

g) were anaesthetized

was placed

performed vertebrae

(Stanfa

in the spinal cord of rats

was then gtvrn at 3 ttmrs the threshold current

of a single constant

2 msec wide

pulse,

into the receptive

lh

field.

Ejfect of intrathecal kelatorphan in normal animals

Three doses of kelatorphan (5, 50 and 250 pg) applied to the spinal cord were tested on the C- and A@-fibre evoked responses of 12 neurones. The lowest dose tested produced a significant inhibition of the C-fibre evoked response of 22.2 + 5.5% (P = 0.009, n = 5), which increased to 42.4 + 10.1%: (n = 5) with the next dose of 50 pg. However, when the dose of

IX7

cantly different to those seen in normal animals, although they tended to be slightly smaller. As in the normal animals, the inhibitory effects of kelatorphan appeared to reach a plateau with 250 pg of kelatorphan failing to produce a significantly greater inhibition than that seen with SO pg. The Ap-fibrc rcsponses were not affected by kelatorphan. with a dose of 50 pg producing an inhibition of 4.2 + 6.2%~. Thcsc results demonstrate that 3 h after the onset of carrageenan inflammation (the time of peak hyperalge$a in behavioural studies (Hargreavcs et al. lY8X: Hylden et al. 199lb)), kelatorphan is neither more potent (as judged by the effect of individual doses) nor capable of producing a greater response (as judged by the maximum effect) than in normal animals.

01 10

1

1000

100

pg Kelatorphan Fig.

I.

Dose-response

response of dorul kelatorphan rageenan

curve for the Inhibition

horn neurona

in normal

of the C-fibre

by the mixed peptidasr

itnimalb (~3

and 3 h after

Into the paw (0). Note that the inhibitory

phan reacha

a plateau

tions produced

in both groups of animals.

not exceeding W’i

injection

evoked inhibitor of car-

effect of kelatorwith the inhihi-

(n = J-X for each point).

kelatorphan was increased to 250 Fg, no further increase in the inhibitory effect of kelatorphan was seen with the inhibition of the C-fibre evoked response remaining at 42.6 i 9.2% (n = 5) (Fig. 1). These findings agree with an earlier study in this model where the effects of kelatorphan were found to plateau at a dose of 25-50 pg, with the inhibitions produced not exceeding 5W (Dickcnson et al. 1987). The AD-fibre rcsponses were almost insensitive to the effects of kclatorphan with a dose of 250 /lg producing a maximum inhibition of only 7.0 + S.fG. Effect of intrathecal kelatorphan in animals with carrageenan inflrrmmation The cffccts of the same 3 doses of kelatorphan (5, 50 and 250 pg) were tested on the C- and AP-fibre evoked responses of 10 neurones 3 h after injection of carragecnan into the receptive field. The lowest dose tested (5 pg) produced a significant inhibition of the C-fibre evoked response (15.1 + 4.5%, P = 0.015, n = 7) which is similar to that seen in normal animals. As the dose of kelatorphan was increased to 50 and then 350 pg, the inhibitions of the C-fibre evoked response increased to 32.1 + 5.4% (n = 8) and 39 i 12.5% (n = 4) (Fig. 1). respectively. The inhibitions produced by kelatorphan in the carrageenan animals are not signifi-

Effd of inrruthecal nor-BNI in r~ormal a~zimals The cffccts of 2 doses of intrathccal nor-BNI (IO and 100 /*g) were tested on the <‘- and AD-fibrc evoked responses of I I neurones. The response of the neurones to nor-BNI was not uniform, although the properties and locations of recording sites of the neurones were indistinguishable. The C-fibre evoked responses of 4 of 11 neurones wcrc facilitated by both doses of nor-BNI. whilst 3 of I I ncurones showed purely inhibitory responses following both doses of nor-BNI. The remaining 4 neuroncs showed a mixed response to nor-BNI, being inhibited by the low dose of IO pg and then facilitated by 100 pg of nor-BNI (Fig. 2. upper panel). Ten micrograms of nor-BNI produced a mean change (in either direction) from control of 15.7 f 2.7% (n = 11) with 100 pg of nor-BNI producing no further increase in effect (mean change from control: 15.6 & 3.3r/r, n = 1I ). The magnitude of the change in the C-fibre evoked response produced by both doses of nor-BNI was significantly grcatcr than that produced by the application of saline alone (mean change from control: 7.0 5 1.5? ). The changes produced in the Ab-fibre response by nor-BNI wcrc also bidirectional but of a smaller magnitude than those produced in the C-fibre response with IO pug producing a change from control of 6.2 & 2.6% (n = Y) which incrcascd to 10.7 f 2.3% with IO0 pg (n = 0). Efjlticr of intrathecal nor-BNI iti rrrlimuls with currugeenun inflummution The effects of the same 2 doses of nor-BNI (10 and 100 /Ig) were tested on the C- and AP-fibrc evoked responses of 14 ncurones 3 h after injection of carrageenan into the paw. As in normal animals. the response of the neuroncs to nor-BNI was not uniform. The C-fibre evoked responses of 3 of 14 neuroncs showed an enhanced response following both doses of nor-BNI; 6 of 14 neuroncs had a decreased response following both doses of nor-BNI. whilst the remaining 5 neurones had a decreased response following IO pg

NORMAL

change in the C-fibre evoked response ot 20.9 2 4. I ‘, tn = 14), whilst 100 pg produced a mcan change 01 26.1 & 4.5% from control (n r 14). which was significantly greater than that produced by 100 pg of nor-BNI in normal animals (P = 0.05). The change in the A/$ fibre evoked response produced in these animals by nor-BNI was again bidirectional. Unlike the C-fibre evoked response, the magnitude of the change in the A&fibre evoked response produced by nor-BNI was not enhanced in the inflammatory state (mean change from control 7.8 _t 2.2% and 7.9 + 1.8~~~with 10 and 100 pg of nor-BNI, respectively (n = 1 i )I.

160 1 140120loo60-

L__ CONTROL

10

Correlations of nor- BNI 100

pg nor-BNI

CARRAGEENAN 1601 140

-1

120100606040 1 20!

1

CONTROL

1

10

between

neuronal

responses uncl the efferct.v

The injection of carrageenan into the paw induces bidirectional changes in the evoked response of the ipsilateral convergent dorsal horn neurones (Stanfa et al. 1992; Stanfa and Dickenson 1993; this study). However, there was no correlation between the direction of change in the response of the cell in the 3 h following the injection of carrageenan, i.e., whether the neurone showed a facilitated or inhibited response, and the direction of the subsequent response to nor-BNI. Thus nor-BNI failed to return the altered neuronal responses induced by carrageenan to control levels. However, there was a significant correlation between the magnitude of the change in the neuronal response of a cell following the injection of carrageenan into the paw and the magnitude of the change in the neuronal response subsequently produced by nor-BNI (r = 0.722, P = 0.018 with 100 pg of nor-BNI). Fig. 3 shows that neurones exhibiting the largest change in response post-carrageenan also showed the largest change in response after 100 ,ug of nor-BNI.

I

100

Discussion

pg nor-BNI Fig. 2. Responses of individual neurones to the intrathecal administration of 10 and 100 kg of nor-BNI in normal animals (upper panel, n = 11) and 3 h after the intra-plantar injection of carrageenan (lower panel, n = 14). All doses of nor-BNI produced a significantly greater change in response than saline alone. One hundred micrograms of nor-BNI produced a significantly greater change in the evoked response of neurones in carrageenan animals than in normal animals (P = 0.05).

of nor-BNI, which was then facilitated above the control levels when the dose was increased to 100 pg (Fig. 2, lower panel). The magnitude of the change in the C-fibre evoked response produced by nor-BNI in the animals with carrageenan inflammation was greater than that seen in normal animals. In the animals with carrageenan inflammation, 10 pug of nor-BNI produced a mean

Although there have been many studies investigating changes in the levels of the endogenous opiate peptides, the enkephalins and dynorphins, in the spinal cord following peripheral inflammation (see Dubner and Ruda 1992), attempts to assign a physiological role to this up-regulation during inflammation have led to conflicting ideas. It has been suggested by some groups that dynorphin plays an analgesic role in the inflammatory state (Millan et al. 1987, 1988; Kayser et al. 1991; Millan and Colpaert 1991) although there are also suggestions that dynorphin may play a pro-nociceptive role (Hylden et al. 1991a; Millan and Colpaert 1991). In this study we have employed the mixed peptidase inhibitor kelatorphan to protect the enkephalins (which act spinally at the a-receptor under these conditions (Dickenson et al. 1986)) from breakdown, and the selective K-opiate receptor antagonist nor-BNI

0 0

a

0

Magnitude

# 10

20

30

40

of change post-carrageenan

(%I

Fig. 3. Correlation between the magnitude of the change in the neuronal response produced by carrageenan inflammation and the magnitude of the change subsequently produced in the same neurones by 100 ~g nor-BNI. A significant correlation exists between these 2 factors (r = 0.722, P = 0.018). suggesting a link exists between the causal mechanisms (see text for full details).

(Portoghese et al. 1987). We have tried to address the question of whether the spinal up-regulation in the levels of endogenous opiate peptides plays an integral part in the early dynamic changes in nociceptive transmission in the spinal cord or whether this represents a later adaptive response to the persistent pain state. The results presented for the actions of the peptidase inhibitor kelatorphan in carrageenan animals compared to normal animals show that 3 h into the development of carrageenan inflammation, there is no significant alteration in the enkephalinergic control of the transmission of nociception in the spinal cord. There was no increase in the maximum effect of kelatorphan and in addition, the ED,,, for the inhibitory effect of kelatorphan was unchanged in the carrageenan when compared to normal animals, suggesting that there has not been any enhancement of the potency of the enkephalins following inflammation. This lack of change in the potency of kelatorphan agrees with the results seen with the &opioid agonist DSTBULET in this carrageenan model which showed only a small enhancement in potency in carrageenan animals compared with the 30-fold enhancement in spinal potency seen with the p-opiates (Stanfa et al. 1992). Kelatorphan has been examined in a behavioural study in arthritic animals and in this study an enhanced

antinociceptive action was found (Kayser et al. 1989). However, this study used systemic administration of the drug which is thought to penetrate the blood-brain barrier only poorly. Thus these results cannot be compared to studies using spinal administration since they are likely to result at least partly from actions of the drug at sites in the periphery in the inflammatory state. The lack of change seen in the actions of spinal kelatorphan at this time point in the inflammatory process is not surprising since immunohistochemical studies find only a small increase in the staining for the mRNA for enkephalin even after several days of inflammation (Iadarola et al. 1988b; Draisci and ladarola 1989). Indeed it has been suggested that the high level of background staining for enkephalin may indicate that the pool of enkephalin is sufficient to cope with the extra demands of inflammation (Draisci and ladarola 1989). We then went on to examine the role of spinal dynorphin acting at K-opiate receptors in the modulation of nociceptive transmission following carrageenan inflammation. Although several behavioural studies have attempted to examine changes in the role of dynorphin in the inflammatory state by using high doses of naloxone which should be sufficient to block the K-opiate receptor (Kayser and Guilbaud 1991: MilIan and Colpaert 1991). naloxonc is a non-specific opiate antagonist and would thcrcforc also be simultaneously blocking p- and &receptors at the doses used. In addition, these studies used systemic administration of the drug and therefore actions may be due to interactions with opiate receptors at supraspinal and peripheral sites (which become functional in inflammatory states, Stein et al. 1988) as well as spinal sites. Dynorphin, via an action at K-receptors has been implicated in the modulation of behavioural hypcralgcsia associated with inflammation (Millan et al. 1987, 198X; Millan and Colpaert 1991). the expansion of the receptive fields of lamina I projection ncurones that has been reported to occur following inflammation (Hylden et al. 1989, 199la) and also in the spread of pathology to the contralateral side of the cord following unilateral inflammation (Millan and Colpaert I99 1). We have shown that the cvokcd response of deep convergent dorsal horn neuroncs is altered following the inflammation, with approximately half the cells showing enhanced responses as the intlammation develops and the other half show dccrcascd responses (Stanfa et al. 1992; Stanfa and Dickcnson 1993; this study). This bidirectional response of the ncuroncs to the developing inflammation is similar to that seen when dynorphin is applied to the spinal cord in normal animals when both facilitations and inhibitions of the neurones are seen (Knox and Dickenson 1987). Hylden et al. (199la) have also reported dual effects of dynorphin and other K-agonists on both the rcsponscs and

peripheral receptive field sizes of dorsal horn neurones. Immunocytochemical studies have demonstrated an increase in the levels of preprodynorphin mRNA in the cord as early as 4 h after the onset of inflammation (Draisci and Iadarola 1989), although the levels ot dynorphin itself do not start to rise until later (Iadarola et al. 1988a). Thus in view of this reported increase in the levels of spinal dynorphin following intlammation. albeit belatedly, and the similarity between the neuronal response to the spinal application of dynorphin and that seen post-carrageenan we hypothesized that an increase in spinal dynorphin in response to the inflammation might be driving the observed neuronal changes. The results with nor-BNI, however, do not appear to support this theory, at least in terms of an action of dynorphin at K-receptors. Nor-BNI produced a bidirectional change in the evoked response of the neurones, presumably by antagonizing the actions of endogenous dynorphin at K-receptors, the magnitude of which was significantly greater in the carrageenan animals than in normal animals. However, there was no correlation between the direction of change in the neuronal response post-carrageenan and the direction of the subsequent change in neuronal response produced by norBNI. Despite the similarities between the response of spinal neurones to exogenous dynorphin (Knox and Dickenson 1987; Hylden et al. 1991a) and the change in response arising from the development of inflammation, the absence of this critical correlation argues against an increased activation of K-receptors by dynorphin being responsible for the alterations in neuronal response seen post-carrageenan. There was, however, a significant correlation between the magnitude of the change in the C-fibre evoked neuronal response induced by carrageenan (but not the direction) and the magnitude of the change in response of the same cells produced by nor-BNI. Thus the neurones that showed the largest inhibition and those with the largest facilitation of their response with the development of carrageenan-induced inflammation then showed the largest change in their response after nor-BNI, whether or not this was an enhanced or decreased response. This correlation suggests that although dynorphin acting at K-ECeptOrS is unlikely to be driving the neuronal changes induced by carrageenan, there is likely to be a strong link between the spinal system responsible for driving both these changes and those in the spinal dynorphin system. The N-methyl-D-aspartate (NMDA) receptor system within the spinal cord is a likely candidate. We have previously shown a role of the NMDA receptor in these neuronal changes post-carrageenan based on evidence that the degree of wind-up of the

ncurones pre-carrageenan (an NMDA-mediated event i determines the direction and magnitude of the suhse. quent neuronal change (Stanfa ct al. 19911. It is therefore interesting to note that some 01 the effects oi dynorphin on C-fibre reflexes seem to involve nonopioid, possibly NMDA-mediated cvcnts (Caudlc and Isaac 1988). In view of this. the present study on the role of dynorphin in inflammation is restricted to actions at K-receptors as it relies on opiate receptor antagonists as tools. However. these may not reveal all the possible actions of dynorphin and this must bc borne in mind when interpreting these results. Thus it appears that dynorphin plays an increased although complex role in the control of spinal nociceptive transmission following inflammation. The incrcasc in the levels of dynorphin in the spinal cord, at least in terms of the modulation of spinal nociceptive transmission via K-opiate receptors, is most likely to represent a later adaptive response to the development of the peripheral inflammation rather than a driving force in the early central changes. In support of this is the finding that neonatal capsaicin treatment attenuates the increase in dynorphin gene expression following inflammation yet has little effect on the development of behavioural hyperalgesia (Hylden et al. 1992). Increases in dynorphin may. however. be responsible for the later central changes that take place following inflammation such as the transfer of the symptoms of intlammation to the contralateral side as has been shown by Millan and Colpaert (1991).

Acknowledgements This study was supported Council. L.C.S. holds an dentship.

by the Medical Research MRC postgraduate stu-

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Fournie-Zsluski.

M.C‘..

Waksman. of

Chaillet,

G.. Costcntin.

kelatorphan,

enkephalin

new

a

degrading

P.. Bouboutou.

J. and Roques, highly

potent

enzymes.

Eur.

R..

Coulard.

B.P.. Analgesic inhibitor

of

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kappa-opioid

nociceptivc

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I

in

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projection

Expamion

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in rats

the contribution

Trauh,

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of

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of

dorsal

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J.L.K..

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dorsal

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Guilhaud.

enkephalin-degrading and arthritic

limb

;IS a

313~34X.

and

time

ot nociceptive

in a rat model

Res.. ih7

( IYY I )

01

1’17-

<‘-lihre

Brain

evohcd rc-

Re\..

415

(IYX7)

Opioid

systems in the resptrnsr to

Neurosciencr.

Hiillt.

suggests role of K- hut not

in the modulation

V.

A..

42

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