European Journal of Pharmacology, 215 (1992) 127-133
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52427
The antinociceptive actions of d e x m e d e t o m i d i n e on dorsal horn neuronal responses in the anaesthetized rat A.F. Sullivan, E . A . Kalso a, H.J. M c Q u a y ~ a n d A . H . D i c k e n s o n Department of Pharmacology, Unit,ersity College London, Gower Street, London WC1 6BT, U.K. and ~ Nuffield Department of Anaesthetics, Unit'ersity of Oxford, Oxford, U.K.
Received 17 October 1991, revised MS received 6 February 1992, accepted 18 February 1992
The actions of the selective a2-adrenoceptor agonist dexmedetomidine were examined on the nociceptive C and innocuous A/3 fibre-evoked responses of dorsal horn neurones to transcutaneous electrical stimulation in the intact anaesthetized rat. C fibre-evoked responses were dose dependently reduced by intrathecal dexmedetomidine - to a maximum 86 + 6% inhibition by 10 /xg of the agonist. The EDs. for inhibition of C fibre responses was estimated to be 2.5 /xg. A/3-evoked responses were inhibited to a lesser degree - a maximum 54 + 8% inhibition after 10 /xg dexmedetomidine. The antinociceptive effects of dexmedetomidine were reversed by the a2-adrenoceptor antagonist atipamezole and the opioid antagonist naloxone. The results are discussed with reference to adrenergic and opioid mechanisms in the spinal cord. Antinociception; Spinal cord; Dorsal horn neurons; az-Adrenoccptor agonists; Dexmedetomidine; Opioids
1. Introduction The terminals of bulbospinal noradrenergic fibres and associated postsynaptic a2-adrenoceptors in the dorsal horn of the spinal cord are implicated in the modulation of nociceptive transmission in this area (for reviews see Yaksh, 1985; Fitzgerald, 1986). Behavioural antinociception is produced by the direct spinal administration of noradrenaline (NA) and more selective a2-adrenoceptor agonists (Yaksh, 1985) which also inhibit the nociceptive responses of convergent neurones after iontophoretic or intrathecal administration in the dorsal horn (Belcher et al., 1978; Davies and Quinlan, 1985; Fleetwood-Walker et al., 1985; Headley et al., 1978; Satoh et al., 1979; Sullivan et al., 1987). Medetomidine, used as a sedative/analgesic agent in veterinary practice, is an extremely potent and selective a2-adrenoceptor agonist (Virtanen, 1989) which produced behavioural analgesia in certain tests of nociception (acetic acid writhing, formalin) (Virtanen, 1989; Pertovaara et al., 1990). Although the sedative effects of this agonist were thought to contribute to the positive results (Pertovaara et al., 1990) an electrophysiological study clearly showed inhibitions of nociceptive
Correspondence to: A.F. Sullivan, Department of Pharmacology, University College London, Gower Street, London WCI 6BT, U.K.
responses of spinothalamic dorsal horn neurones by medetomidine (Pertovaara et al., 1991). Recent behavioural studies report potent antinociception after spinal administration of dexmedetomidine, the active d-enantiomer of medetomidine at sub-sedative doses (Kalso et al., 1991; Fisher et al., 1991) and this agonist also inhibited the slow ventral root potential in neonatal rat cord in vitro (Kendig et al., 1991). In the present electrophysiological study the actions of intrathecal dexmedetomidine were examined on the nociceptive responses of dorsal horn neurones in the intact anaesthetised rat. Inhibition of dexmedetomidine's actions by atipamezole, a selective a2-adrenoceptor antagonist (Virtanen 1989) and the opioid antagonist naloxone were examined and the results discussed with reference to the spinal antinociceptive effects of the adrenoceptor agonist clonidine and opioid agonists examined in previous studies on the same model (Sullivan et al., 1987; Dickenson and Sullivan, 1986).
2. Materials and methods Adult male Sprague-Dawley rats (200-250 g) were initially anaesthetized with 3% halothane in a 66% N20, 33% 0 2 gaseous mix and then maintained on 1.5-2% halothane for cannulation of the trachea and
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subsequent surgery. During the experiment the halothane concentration was held at 0 . 7 - 1 % maintaining complete areflexia and full surgical anaesthesia. The rat was placed in a stereotaxic frame and a laminectomy performed exposing the L 1 - L 3 region of the spinal cord. The vertebrae caudal and rostral to the exposed cord were supported by clamps to hold the cord rigid and a glass-coated tungsten electrode was lowered into the dorsal horn. Single unit extracellular recordings were made of dorsal horn neurones responding to both innocuous (brush) and noxious (pinch, heat) stimuli applied to the ipsilateral receptive fields in the plantar region of the hind paw. The depth from the surface of the cord of the neuronal recording site was established using a SCAT microdrive (Digitimer). Neurones were characterised by means of natural stimuli and only those with no spontaneous firing but showing a sustained response to innocuous brush and prod and noxious pinch were included in the study. Needles were then inserted into the centre of the receptive field in order to apply an electrical stimulus. The threshold for activation of a A/3 fibre-evoked response by electrical stimulation was detirmined as the lowest current at which the neurone fired action potentials at a latency less than 20 ms. The
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higher threshold for the C fibre-evoked response of the cell (a latency greater than 90 ms) was then measured. For a constant reproducible test stimulus for the experiment 16 electrical pulses (0.5 Hz, 2 ms wide) were given at 3 times the threshold current for C fibre activation and a post-stimulus histogram (PSTH) was built and displayed by means of a 1401 interface (C.E.D., UK) and M R A T E software. From the PSTH the C fibre-evoked response could be separated by latency and threshold from the A/3 response and quantified. A/3 fibre-evoked responses were measured separately again at 3 times the threshold for A/3 fibre activation. After at least three control tests at 5-rain intervals which did not differ by more than 10%, drugs were applied in a volume of 50 /xl 0.9% saline via a micropipette directly onto the surface of the cord. Tests were carried out 5 and 10 min after each application and subsequently every 10 min. The results were calculated as a percentage change from control values in each neurone and the overall results expressed as the means + S.E.M. Dexmedetomidine and atipamezole were kindly donated by Farmos Group Ltd., Finland. Naloxone HCI was obtained from Sigma, U.K.
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3. Results
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A total of 23 cells were studied, the majority located deep in the dorsal horn, a mean depth of 731 _+ 41 > m from the surface of the cord. Two ceils situated superficially at a depth less than 250/.tm were influenced by dexmedetomidine to a similar degree as the deep cells and were included in the overall results. The C fibre-evoked neuronal responses were clearly depressed by dexmedetomidine in a dose dependent manner. Whereas 0.5 /_tg of the a2-agonist had no significant effect on C fibre responses, producing a small facilitation in three out of four cells, 2.5 /.tg clearly inhibited the response which was abolished by 10 /.tg in two out of four cells and was profoundly inhibited in the remaining two cells. An example of dexmedetomidine's inhibitory effects on the responses of a single neurone is shown in fig. 1. From the dose-response curve the EDs0 for inhibition of C fibre-evoked responses was estimated to be 2.5 /xg (11 nmol) dexmedetomidine (fig. 2). Wind-up, the increase in the and fibre-evoked response per stimulus during a train of stimuli exhibited by most deep cells at 3 times threshold (see Dickenson and Sullivan, 1986), was not affected by dexmedetomidine. The basal response to each stimulus was reduced but wind-up was still apparent as shown in fig. 3. Aft-evoked responses were inhibited in parallel with and C fibre responses after 1 and 2.5/xg dexmedetomidine. However higher doses were more selective for nociceptive responses, only inhibiting Aft responses to a maximum 54 _+ 8%, compared to a maximum 86 _+ 6% inhibition of C fibre responses (fig. 2).
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single neurone before (o) and after dexmedetomidine (e). Note the reduction in the response to the first few stimuli in the presence of 5 /.tg dexmedetomidine but the subsequent wind-up still occurs. After atipamezole the response is similar to the control ( • ).
3.2. Reversal of dexmedetomidine's effects by selective eQ-adrenoceptor and opioid antagonists Once the maximal effect of dexmedetomidine was obtained, intrathecal administration of 1 5 / , g atipamezole, a selective c%-adrenoceptor antagonist, could rapidly reverse an otherwise prolonged inhibition back to control levels (figs. 1 and 4A). In a further four neurones the opiate antagonist intrathecal naloxone (20/zg) was administered instead of atipamezole. The responses inhibited by dexmedetomidine were restored to control levels in three out of four neurones (fig. 4A). In the remaining neurone which was unaffected by naloxone the inhibition by dexmedetomidine was antagonised by subsequent administration of atipamezole. Atipamezole did not reverse inhibitions of C fibreevoked responses by the #-opioid agonist morphine (10 /.tg) which were subsequently antagonised by 5 /.tg naloxone (fig. 4B). When administered in the absence of dexmedetomidine neither antagonist had any effect on neuronal responses.
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Fig. 2. The dose-response curve for inhibition of C (o) and A/3 (o) fibre-evoked responses by dexmedetomidine. Using the Students t-test, * P < 0.005.
In some neurones the presence of dexmedetomidine led to a reduction in the amplitude of the action potential (AP) (fig. 5). This was not related to depth of the cell, drug concentration or size of inhibition of the C or Aft-evoked response. The precise numbers of neurones affected in this way was difficult to assess and the changes are reported as observations rather than a quantitative study. In the initial experiments AP amplitude was not measured, although changes were noted. In five subsequent cells where changes were quantified the AP amplitude was reduced to 40 _+ 12% of control following the agonist, recovering after administration of the antagonist in all but one cell. However in other
130
neurones the AP amplitude was clearly unaltered even when a high dose of dexmedetomidine had profoundly inhibited the C fibre-evoked response. Since the neurones were not identified by for instance antidromic techniques the change in AP height could be attributed to the recording of a different neurone during the experiment. Although this possibility cannot be excluded without further experiments recovery of the AP height after the antagonist would argue against this. Furthermore the profile of the C fibre-evoked PSTH for each neurone is usually sufficiently distinct to identify that neurone throughout the experiment.
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In the present electrophysiological study dexmedetomidine produced a potent spinal antinociceptive ac-
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Fig. 4. (A) Reversal of the inhibitory effects of 5 # g dexmedetomidine on C fibre-evoked responses (DX) by 15 # g selective a 2antagonist atipamezole (AT) (n - 6) or 20/xg naloxone (NX) (n = 4). (B) Lack of effect of atipamezole in reversing inhibitions by the /x-opioid agonist morphine (MO) (n = 4) which are antagonised by 5 /zg naloxone (n = 4).
tion as measured by the inhibition of noxious dorsal horn neuronal responses following intrathecal administration of the agonist. Although non-nociceptive responses were also inhibited, with higher doses of dexmedetomidine there was a clear distinction between C fibre-evoked responses which were almost abolished compared with a maximal 50% reduction of A/3-evoked responses. This contrasts with an earlier study in the same preparation on the spinal antinociceptive actions of clonidine which only reduced C fibre-evoked responses to a maximal 52% (Sullivan et al., 1987) The antinociceptive potency of dexmedetomidine was also much greater than clonidine which required 50 ~ g to inhibit C fibre responses to 50% of control compared to a dose of 2.5 p~g of dexmedetomidine to achieve the same effect. This may represent varying degrees of access of both compounds to the receptors in the dorsal horn but could also reflect the partial agonist nature of clonidine. The profile of dexmedetomidine against C and A/3 responses is similar to that seen after intrathecal administration of opioid agonists in the same electrophysiological model. ~-Agonists such as morphine and the
131
3-agonist D S T B U L E T (Tyr-D-Ser(otbu)-Gly-Leu-Thr) also reduce C fibre-evoked responses by over 80% compared to maximal A/3 inhibitions of 40-50% (Dickenson and Sullivan, 1986; Sullivan et al., 1989). In addition the potency of dexmedetomidine is close to that of morphine and D S T B U L E T in this model. Thus the similarity of antinociceptive profile and potency leads to the conclusion that activation of a2-adrenoceptors in the spinal cord produces comparable antinociception to activation of the 'classic antinociceptive' opioid receptor. Behavioural studies lend support to the above conclusion. The antinociceptive doses of dexmedetomidine in the electrophysiological study correspond well to those (3-10 p~g) which produce behavioural antinociception in rats (Fisher et al., 1991; Kalso et al., 1991). However in conscious rats the sedative effects of 6-10 p~g, indicating leakage of the drug to the systemic circulation, highlight possible problems in extending the use of dexmedetomidine to clinical spinal analgesia. I.v. administration of 40 /xg/kg dexmedetomidine is sufficient to produce profound sedation in rats (Fisher et al., 1991) and in humans sedation was reported after bolus i.v. doses of 25-100 /zg medetomidine (Kauppila et al., 1991; Scheinin et al., 1987). A possible coml~cating factor in interpretation of the present series ot experiments is the interaction of dexmedetomidine with the anaesthetic halothane. Both medetomidine and dexmedetomidine have hypnotic or anaesthetic effects in animals, the latter reducing the halothane minimal alveolar concentration after both systemic and intrathecal administration via an interaction with a2-adrenoceptors (Segal et al., 1988; Nagasaka and Yaksh, 1990). That the inhibitory actions of dexmedetomidine on neuronal responses seen in the present study are due to an increase in level of anaesthesia and therefore an indirect general depression of neuronal activity must be considered. However this explanation seems unlikely since similar intrathecal doses in the absence of anaesthestic agents produce behavioural antinociception (Kalso et al., 1991; Fisher et al., 1991). The effect of dexmedetomidine on AP height is difficult to explain although it does appear to be common to ce2-adrenoceptor agonists in general since intrathecal clonidine also produced this effect in a small proportion of neurones (unpublished observations). The AP amplitude could change for reasons other than a drug effect, for instance overdepolarisation of a particulary responsive neurone or drifting of the neurone away from the recording electrode. However overdepolarisation of neurones in our electropysiological model is seen as a progessive reduction in the action potential height during the train of 16 electrical stimuli not as a constant reduced AP height apparent from the first stimulus onwards as was seen after dexmedetomidine.
Furthermore overdepolarisation is unlikely to appear in a neurone depressed by an agonist when absent in the control response. Drifting of the neurone also seems an unlikely explanation in view of the restoration of AP height following administration of the antagonist which reversed dexmedetomdine's inhibitory effect. Interestingly the opposite, an increase in AP amplitude of dorsal horn neurones has been noted after inontophoretic NA. This was attributed to hyperpolarisation of the neurone by the agonist (Howe and Zieglg~insberger, 1987). However depression of neuronal responses by dexmedetomidine presumably involving hyperpolarisation was independent of any change in AP height since the former could be elicited in the absence of the latter, and AP amplitude was decreased rather than increased. Therefore it is difficult to apply the above explanation to the effects of dexmedetomidine on AP amplitude in the present study. The antagonism of the antinociceptive effects of dexmedetomidine by intrathecal atipamazole confirmed the involvement of an a2-adrenoceptor. Autoradiographical studies clearly show the presence of c~2-adrenoceptors in the superfical layers of the dorsal horn (Sullivan et al., 1987, Unnerstail et at., 1984). These receptors are postsynaptic to descending noradrenergic fibre terminals because spinalisation or 6-hydroxydopamine treatment did not decrease receptor numbers (Yaksh, 1985). However precisely where a 2adrenoceptors are located, on nociceptive afferent terminals or postsynaptic to these terminals on dorsal horn neurones is not clear. Whilst a presynaptic locus on primary afferents has been suggested (Calvillo and Guignone, 1986; Kuraishi et al., 1985) destruction of C fibres with neonatal capsaicin does not reduce [3H]clonidine binding sites in mice (Wikberg and Hajos, 1987). Furthermore immunohistochemical studies show no synaptic contact between descending noradrenergic fibre terminals and primary afferent terminals suggesting that a2-adrenoceptors are located on dorsal horn neurones postsynaptic to primary afferents (Hagihira et al., 1990). Direct catecholaminergic innervation of primate spinothalamie tract neurones has recently been demonstrated (Westlund et al., 1990). In vivo nociceptive neuronal responses of dorsal horn neurones including those characterised as spinothalamic are selectively depressed by iontophoretic or intrathecal administration of NA and more selective c~2-adrenoceptor agonists including medetomidine (Davies and Quinlan, 1985; FleetwoodWalker et al., 1985; Headley et al., 1978; Pertovaara et al., 1991; Sullivan et al., 1987). In addition dexmedetomidine inhibited the neuronal response evoked by exogenous substance P, implicated in spinal nociceptive transmission (Kendig et al., 1991). In vitro ion-
132 tophoretic NA hyperpolarised substantia gelatinosa n e u r o n e s via an i n c r e a s e in p o t a s s i u m c o n d u c t a n c e ( N o r t h a n d Y o s h i m u r a , 1984) a n d this m a y r e p r e s e n t a direct p o s t s y n a p t i c m e c h a n i s m of action for the d e p r e s sion of n e u r o n a l r e s p o n s e s by d e x m e d e t o m i d i n e in the d o r s a l horn. In a d d i t i o n to the activation o f c~2-adrenoceptors by d e x m e d e t o m i d i n e t h e a n t a g o n i s m of this a g o n i s t ' s actions by n a l o x o n e in most of the n e u r o n e s t e s t e d in the p r e s e n t study suggests t h e i n v o l v e m e n t o f o p i o i d r e c e p tors, which occur b o t h p r e s y n a p t i c a l l y on p r i m a r y afferents a n d p o s t s y n a p t i c a l l y on d o r s a l horn n e u r o n e s (Besse et al., 1990 a n d r e f e r e n c e s within). It is unlikely that d e x m e d e t o m i d i n e acts directly at o p i o i d r e c e p t o r s ( V i r t a n e n , 1988) b u t i n t e r a c t i o n s b e t w e e n o p i o i d a n d a d r e n e r g i c spinal m e c h a n i s m s involving i n d e p e n d e n t r e c e p t o r activation a n d resulting in synergism of the spinal a n t i n o i c e p t i v e effect a f t e r c o a d m i n i s t r a t i o n of e x o g e n o u s o p i o i d a n d a d r e n o c e p t o r agonists have b e e n widely r e p o r t e d (Sullivan et al., 1987; Wilcox et al., 1987; Yaksh, 1985). T h e i n v o l v e m e n t of e n d o g e n o u s o p i o i d m e c h a n i s m s in the action o f e x o g e n o u s a d r e n o c e p t o r agonists a d m i n i s t e r e d a l o n e has not b e e n universally seen; n a l o x o n e h a d no effect on the antinocic e p t i o n m e d i a t e d by o t h e r a ~ - a d r e n o c e p t o r agonists in most p r e v i o u s b e h a v i o u r a l a n d e l e c t r o p h y s i o l o g i c a l studies (Yaksh, 1985). H o w e v e r n a l o x o n e reversibility o f N A - m e d i a t e d a n t i n o c i c e p t i o n in the tail-flick test has b e e n r e p o r t e d by L o o m i s et al. (1987). F u r t h e r m o r e c l o n i d i n e - i n d u c e d inhibitions of n o c i c e p t i v e responses w e r e r e v e r s e d by n a l o x o n e in a p r o p o r t i o n of d o r s a l h o r n n e u r o n e s t e s t e d ( O m o t e et al., 1991) a n d the d e p r e s s i o n o f the v e n t r a l root p o t e n t i a l by d e x m e d e t o m i d i n e in the i s o l a t e d n e o n a t a l c o r d was p a r t i a l l y b l o c k e d by n a l o x o n e ( K e n d i g et al., 1991). It is h a r d to see how the inhibitory actions of end o g e n o u s o p i o i d s could be e n t i r e l y r e s p o n s i b l e for the p r o f o u n d n e u r o n a l d e p r e s s i o n s e v o k e d by h i g h e r doses o f d e x m e d e t o m i d i n e since p r o t e c t i o n o f e n d o g e n o u s o p i o i d s by the e n k e p h a l i n a s e i n h i b i t o r k e l a t o r p h a n only p r o d u c e d a 50% m a x i m a l inhibition of C f i b r e - e v o k e d r e s p o n s e s in the s a m e m o d e l ( D i c k e n s o n et al., 1987). H o w e v e r this d o e s not p r e c l u d e a synergistic interaction b e t w e e n e n d o g e n o u s o p i o i d a n d e x o g e n o u s a d r e n ergic i n h i b i t o r y m e c h a n i s m s m e d i a t i n g the p o t e n t effects of d e x m e d e t o m i d i n e . M i l l a r a n d W i l l i a m s (1989) o b s e r v e d excitation o f l a m i n a II and III n e u r o n e s by p e r i a q u e d u c t a l gray s t i m u l a t i o n a n d i o n t o p h o r e t i c nor a d r e n a l i n e which i n h i b i t e d d e e p e r W D R (wide dynamic range) neurones. Opioid interneurones located in the s u b s t a n t i a g e l a t i n o s a m a y be involved in b o t h this a n d the action of d e x m e d e t o m i d i n e in the p r e s e n t study. T h e effects o f d e x m e d e t o m i d i n e on w i n d - u p a r e also similar to those m e d i a t e d by opioids. W i n d - u p involves the activation of N M D A r e c e p t o r s , a n d can be inhib-
ited by selective a n t a g o n i s t s (Davies a n d L o d g e , 1987; D i c k e n s o n a n d Sullivan, 1987). O p i o i d s whilst r e d u c i n g the initial r e s p o n s e to a stimulus d o not p r e v e n t the w i n d - u p itself ( D i c k e n s o n a n d Sullivan, 1986) a n d this is now also d e m o n s t r a t e d for d e x m e d e t o m i d i n e suggesting a similarity in the site of action for o p i o i d a n d c~2-adrenoceptor agonists. In conclusion, d e x m e d e t o m i d i n e could c o m p l e t e l y inhibit C f i b r e - e v o k e d r e s p o n s e s in spinal dorsal horn n e u r o n e s s u p p o r t i n g b e h a v i o u r a l e v i d e n c e for a a n t i n o c i c e p t i v e role of this a d r e n o c e p t o r agonist in the spinal cord. A l t h o u g h the actions of d e x m e d e t o m i d i n e w e r e clearly r e v e r s e d by a t i p a m e z o l e c o n f i r m i n g t h e involvement of a 2 - a d r e n o c e p t o r s t h e r e was also evid e n c e for o p i o i d m e c h a n s i m s c o n t r i b u t i n g to d e x m e d e t o m i d i n e ' s effect.
Acknowledgements This work was supported by the Medical Research Council, UK and the European Academy of Anaesthesiology.
References Belcher, G., R.W. Ryall and R. Shaffner, 1978, The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurones in the cat, Brain Res. 151,307. Besse. D., M.C. Lombard, J.M. Zajac, B.P. Roques and J.M. Besson, 1990, Pre- and postsynaptic distribution of p~, 6 and K opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord, Brain Res. 521, 15. Calvillo, O. and M. Ghignone, 1986, Presynaptic effect of clonidine on unmyelinated afferent fibres in the spinal cord of the cat, Neurosci. Len. 64, 335. Davies, S.N. and D. Lodge, 1987, Evidence for involvement of N-methyl-D-aspartate receptors in wind-up of class 2 neurones in the dorsal horn of the rat, Brain Res. 424, 402. Davies, J. and J.E. Quinlon, 1985, Selective inhibition of responses of feline dorsal horn neurones to noxious cutaneous stimuli by tizanidine (DS 103-282) and noradrenaline: involvement of a 2adrenoceptors, Neuroscience 16, 673. Dickenson, A.H. and A.F. Sullivan, 1986, Electrophysiological studies on the effects of intrathecal morphine on nociceptive neurones in the rat dorsal horn, Pain 24, 211. Dickenson, A.H. and A.F. Sullivan, 1987, Evidence for a role of the NMDA receptor in the frequency dependent potentiation of deep dorsal horn nociceptive neurones following C fibre stimulation, Neuropharmacology 26, 1235. Dickenson, A.H., A.F. Sullivan, M.C. Fournie-Zaluski and B.P. Roques, 1987, Prevention of degradation of endogenous enkephalins produces inhibition of nociceptive neurones in rat spinal cord, Brain Res. 408, 185. Fisher, B., M.N. Zornow, T.L. Yaksh and B.M. Peterson, 1991, Antinociceptive properties of intrathecal dexmedetomidine in rats, Eur. J. Pharmacol. 192, 221. Fitzgerald, M., 1986, Monoamines and descending control of nociception, Trends Neurosci. 9, 51. Fleetwood-Walker, S.M., R. Mitchell, P.O. Hope, V. Maloney and A. Iggo, 1985, An o~2 receptor mediates the selective inhibition
133 by noradrenaline of nociceptive responses of identified dorsal horn neurones, Brain Res. 334, 243. Hagihira, S., E. Senba, S. Yoshida, M. Tohyama and I. Yoshiya, 1990, Fine structure of noradrenergic terminals and their synapses in the rat spinal dorsal horn: an immunohistochemical study, Brain Res. 526, 73. Headley, P.M., A.W. Duggan and ST. Griersmith, 1978, Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptire responses of cat dorsal horn neurones, Brain Res. 145, 185. Howe, J.R. and W. Zieglg~nsberger, 1987, Responses of rat dorsal horn neurons to natural stimulation and to iontophoretically applied norepinephrine, J. Comp. Neurol. 255, 1. Kalso, E.A., R. Poyhia and ~.H. Rosenberg, 1991, Spinal antinociception by dexmedetomidine, a highly selective o~2-adrenergic agonist, Pharmacol. Toxicol. 68, 140. Kauppila, T., P. Kemppainen, N. Tanila and A. Pertovaara, 1991, Effect of systemic medetomidine, an alpha-2-adrenoceptor agonist, on experimental pain in humans, Anesthesiology 74, 3. Kendig, J.J, M.K. Savola, S.J. Woodley and M. Maze, 1991, a 2Adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord, Eur. J. Pharmacol. 192, 293. Kuraishi, Y., N. Hirota, Y. Sato, S. Kaneko, M. Satoh and H. Takagi, 1985, Noradrenergic inhibition of the release of substance P from primary afferents in the rabbit spinal dorsal horn, Brain Res. 359, 177. Loomis, C.W., K. Jhamandas, B. Milne and F. Cervenko, 1987, Monoamine and opioid interactions in spinal analgesia and tolerance, Pharmacol. Biochem. Behav. 26, 445. Millar, J. and G.V. Williams, 1989, Effects of iontophoresis of noradrenaline and stimulation of the periaqueductal gray on single-unit activity in the rat superficial dorsal horn, J. Comp. Neurol. 287, 119. Nagasaka, H. and T.L. Yaksh, 1990, Pharmacology of intrathecal adrenergic agonists: cardiovascular and nociceptive reflexes in halothane-anesthetised rats, Anesthesiology 73, 1198. North, R.A. and M. Yoshimura, 1984, The actions of noradrenaline on neurones of the rat substantia gelatinosa in vitro, J. Physiol. 349, 43. Omote, K., L.M. Kitahata, J.G. Collins, K. Nakatani and I. Nakagawa, 1991, Interaction between opiate subtype and apha-2 adrenergic agonists in suppression of noxiously evoked activity of W D R neurons in the spinal dorsal horn, Anesthesiology 74, 737. Pertovaara, A., T. Kauppila and T. Tukeva, 1990, The effect of medetomidine, an a2-adrenoceptor agonist, in various pain tests, Eur. J. Pharmacol. 179, 323. Pertovaara, A., T. Kauppila, E. Jyvasjarvi and E. Kalso, 199l, Involvement of supraspinal and spinal segmental alpha-2-adren-
ergic mechananisms in the medetomidine-induced antinociception, Neuroscience 44, 705. Satoh, M., S.I. Kawajiri, Y. Ukai and M. Yamamoto, 1979, Selective and non-selective inhibition by enkephalins and noradrenaline of nociceptive responses of lamina V type neurons in the spinal dorsal horn of the rabbit, Brain Res. 177, 384. Scheinin, M., A. Kallio, M. Koulu, J. Viikari and H. Scheinin. 1987, Sedative and cardiovascular effects of medetomidine, a novel selective c~2-adrenoceptor agonist, in healthy volunteers, Br. J. Clin. Pharmacol. 24, 443. Segal, I.S., R.G. Vickery, J.K. Walton, V.A. Doze and M. Maze, 1988, Dexmedetomidine diminishes halothane anesthetic requirements in rats through a postsynaptic alpha2-adrenergic receptor. Anesthesiology 69, 818. Sullivan, A.F., M.R. Dashwood and A.H. Dickenson, 1987, o~2Adrenoceptor modulation of nociception in rat spinal cord: location, effects and interactions with morphine, Eur. J. Pharmacol. 138, 169. Sullivan, A.F., A.H. Dickenson and B.P. Roques, 1989, 6-Opioid mediated inhibtions of acute and prolonged noxious-evoked responses in rat dorsal horn neurones, Br. J. Pharmacol. 98, 1039. Unnerstall, J.R., T.A. Kopajtic and M.J. Kuhar, 1984, Distribution of c~2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev. 7, 69. Virtanen, R., 1988, Characterisation of the selectivity, specificity and potency of medetomidine as an a2-adrenoceptor agonist, Eur. J. Pharmacol. 150, 9. Virtanen, R., 1989, Pharmacological profiles of medetomidine and its antagonist, atipamezole, Acta Vet. Scand. 85, 29. Westlund, K.N., S.M. Carlton, D. Zhang and W.D. Willis, 1990, Direct catecholaminergic innervation of primate spinothalamic tract neurons, J. Comp. Neurol. 299, 178. Wikberg, J.E.S. and M. Hajos, 1987, Spinal cord a2-adrenoceptors may be located postsynaptically with respect to primary sensory neurons: destruction of primary C-afferents with neonatal capsaicin does not affect the number of [3H]clonidine binding sites in mice, Neurosci.. Lett. 76, 63. Wilcox, G.L., K.H. Carlsson, A. Jochim and I. Jurna, 1987, Mutual potentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord, Brain Res. 405, 84. Yaksh, T.L., 1985, Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing, Pharmacol. Biochem. Behav. 22 845.
127
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52427
The antinociceptive actions of d e x m e d e t o m i d i n e on dorsal horn neuronal responses in the anaesthetized rat A.F. Sullivan, E . A . Kalso a, H.J. M c Q u a y ~ a n d A . H . D i c k e n s o n Department of Pharmacology, Unit,ersity College London, Gower Street, London WC1 6BT, U.K. and ~ Nuffield Department of Anaesthetics, Unit'ersity of Oxford, Oxford, U.K.
Received 17 October 1991, revised MS received 6 February 1992, accepted 18 February 1992
The actions of the selective a2-adrenoceptor agonist dexmedetomidine were examined on the nociceptive C and innocuous A/3 fibre-evoked responses of dorsal horn neurones to transcutaneous electrical stimulation in the intact anaesthetized rat. C fibre-evoked responses were dose dependently reduced by intrathecal dexmedetomidine - to a maximum 86 + 6% inhibition by 10 /xg of the agonist. The EDs. for inhibition of C fibre responses was estimated to be 2.5 /xg. A/3-evoked responses were inhibited to a lesser degree - a maximum 54 + 8% inhibition after 10 /xg dexmedetomidine. The antinociceptive effects of dexmedetomidine were reversed by the a2-adrenoceptor antagonist atipamezole and the opioid antagonist naloxone. The results are discussed with reference to adrenergic and opioid mechanisms in the spinal cord. Antinociception; Spinal cord; Dorsal horn neurons; az-Adrenoccptor agonists; Dexmedetomidine; Opioids
1. Introduction The terminals of bulbospinal noradrenergic fibres and associated postsynaptic a2-adrenoceptors in the dorsal horn of the spinal cord are implicated in the modulation of nociceptive transmission in this area (for reviews see Yaksh, 1985; Fitzgerald, 1986). Behavioural antinociception is produced by the direct spinal administration of noradrenaline (NA) and more selective a2-adrenoceptor agonists (Yaksh, 1985) which also inhibit the nociceptive responses of convergent neurones after iontophoretic or intrathecal administration in the dorsal horn (Belcher et al., 1978; Davies and Quinlan, 1985; Fleetwood-Walker et al., 1985; Headley et al., 1978; Satoh et al., 1979; Sullivan et al., 1987). Medetomidine, used as a sedative/analgesic agent in veterinary practice, is an extremely potent and selective a2-adrenoceptor agonist (Virtanen, 1989) which produced behavioural analgesia in certain tests of nociception (acetic acid writhing, formalin) (Virtanen, 1989; Pertovaara et al., 1990). Although the sedative effects of this agonist were thought to contribute to the positive results (Pertovaara et al., 1990) an electrophysiological study clearly showed inhibitions of nociceptive
Correspondence to: A.F. Sullivan, Department of Pharmacology, University College London, Gower Street, London WCI 6BT, U.K.
responses of spinothalamic dorsal horn neurones by medetomidine (Pertovaara et al., 1991). Recent behavioural studies report potent antinociception after spinal administration of dexmedetomidine, the active d-enantiomer of medetomidine at sub-sedative doses (Kalso et al., 1991; Fisher et al., 1991) and this agonist also inhibited the slow ventral root potential in neonatal rat cord in vitro (Kendig et al., 1991). In the present electrophysiological study the actions of intrathecal dexmedetomidine were examined on the nociceptive responses of dorsal horn neurones in the intact anaesthetised rat. Inhibition of dexmedetomidine's actions by atipamezole, a selective a2-adrenoceptor antagonist (Virtanen 1989) and the opioid antagonist naloxone were examined and the results discussed with reference to the spinal antinociceptive effects of the adrenoceptor agonist clonidine and opioid agonists examined in previous studies on the same model (Sullivan et al., 1987; Dickenson and Sullivan, 1986).
2. Materials and methods Adult male Sprague-Dawley rats (200-250 g) were initially anaesthetized with 3% halothane in a 66% N20, 33% 0 2 gaseous mix and then maintained on 1.5-2% halothane for cannulation of the trachea and
128
subsequent surgery. During the experiment the halothane concentration was held at 0 . 7 - 1 % maintaining complete areflexia and full surgical anaesthesia. The rat was placed in a stereotaxic frame and a laminectomy performed exposing the L 1 - L 3 region of the spinal cord. The vertebrae caudal and rostral to the exposed cord were supported by clamps to hold the cord rigid and a glass-coated tungsten electrode was lowered into the dorsal horn. Single unit extracellular recordings were made of dorsal horn neurones responding to both innocuous (brush) and noxious (pinch, heat) stimuli applied to the ipsilateral receptive fields in the plantar region of the hind paw. The depth from the surface of the cord of the neuronal recording site was established using a SCAT microdrive (Digitimer). Neurones were characterised by means of natural stimuli and only those with no spontaneous firing but showing a sustained response to innocuous brush and prod and noxious pinch were included in the study. Needles were then inserted into the centre of the receptive field in order to apply an electrical stimulus. The threshold for activation of a A/3 fibre-evoked response by electrical stimulation was detirmined as the lowest current at which the neurone fired action potentials at a latency less than 20 ms. The
AP
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higher threshold for the C fibre-evoked response of the cell (a latency greater than 90 ms) was then measured. For a constant reproducible test stimulus for the experiment 16 electrical pulses (0.5 Hz, 2 ms wide) were given at 3 times the threshold current for C fibre activation and a post-stimulus histogram (PSTH) was built and displayed by means of a 1401 interface (C.E.D., UK) and M R A T E software. From the PSTH the C fibre-evoked response could be separated by latency and threshold from the A/3 response and quantified. A/3 fibre-evoked responses were measured separately again at 3 times the threshold for A/3 fibre activation. After at least three control tests at 5-rain intervals which did not differ by more than 10%, drugs were applied in a volume of 50 /xl 0.9% saline via a micropipette directly onto the surface of the cord. Tests were carried out 5 and 10 min after each application and subsequently every 10 min. The results were calculated as a percentage change from control values in each neurone and the overall results expressed as the means + S.E.M. Dexmedetomidine and atipamezole were kindly donated by Farmos Group Ltd., Finland. Naloxone HCI was obtained from Sigma, U.K.
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129
3. Results
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A total of 23 cells were studied, the majority located deep in the dorsal horn, a mean depth of 731 _+ 41 > m from the surface of the cord. Two ceils situated superficially at a depth less than 250/.tm were influenced by dexmedetomidine to a similar degree as the deep cells and were included in the overall results. The C fibre-evoked neuronal responses were clearly depressed by dexmedetomidine in a dose dependent manner. Whereas 0.5 /_tg of the a2-agonist had no significant effect on C fibre responses, producing a small facilitation in three out of four cells, 2.5 /.tg clearly inhibited the response which was abolished by 10 /.tg in two out of four cells and was profoundly inhibited in the remaining two cells. An example of dexmedetomidine's inhibitory effects on the responses of a single neurone is shown in fig. 1. From the dose-response curve the EDs0 for inhibition of C fibre-evoked responses was estimated to be 2.5 /xg (11 nmol) dexmedetomidine (fig. 2). Wind-up, the increase in the and fibre-evoked response per stimulus during a train of stimuli exhibited by most deep cells at 3 times threshold (see Dickenson and Sullivan, 1986), was not affected by dexmedetomidine. The basal response to each stimulus was reduced but wind-up was still apparent as shown in fig. 3. Aft-evoked responses were inhibited in parallel with and C fibre responses after 1 and 2.5/xg dexmedetomidine. However higher doses were more selective for nociceptive responses, only inhibiting Aft responses to a maximum 54 _+ 8%, compared to a maximum 86 _+ 6% inhibition of C fibre responses (fig. 2).
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3.2. Reversal of dexmedetomidine's effects by selective eQ-adrenoceptor and opioid antagonists Once the maximal effect of dexmedetomidine was obtained, intrathecal administration of 1 5 / , g atipamezole, a selective c%-adrenoceptor antagonist, could rapidly reverse an otherwise prolonged inhibition back to control levels (figs. 1 and 4A). In a further four neurones the opiate antagonist intrathecal naloxone (20/zg) was administered instead of atipamezole. The responses inhibited by dexmedetomidine were restored to control levels in three out of four neurones (fig. 4A). In the remaining neurone which was unaffected by naloxone the inhibition by dexmedetomidine was antagonised by subsequent administration of atipamezole. Atipamezole did not reverse inhibitions of C fibreevoked responses by the #-opioid agonist morphine (10 /.tg) which were subsequently antagonised by 5 /.tg naloxone (fig. 4B). When administered in the absence of dexmedetomidine neither antagonist had any effect on neuronal responses.
3.3. Change in action potential amplitude INHIBITION 1001-
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In some neurones the presence of dexmedetomidine led to a reduction in the amplitude of the action potential (AP) (fig. 5). This was not related to depth of the cell, drug concentration or size of inhibition of the C or Aft-evoked response. The precise numbers of neurones affected in this way was difficult to assess and the changes are reported as observations rather than a quantitative study. In the initial experiments AP amplitude was not measured, although changes were noted. In five subsequent cells where changes were quantified the AP amplitude was reduced to 40 _+ 12% of control following the agonist, recovering after administration of the antagonist in all but one cell. However in other
130
neurones the AP amplitude was clearly unaltered even when a high dose of dexmedetomidine had profoundly inhibited the C fibre-evoked response. Since the neurones were not identified by for instance antidromic techniques the change in AP height could be attributed to the recording of a different neurone during the experiment. Although this possibility cannot be excluded without further experiments recovery of the AP height after the antagonist would argue against this. Furthermore the profile of the C fibre-evoked PSTH for each neurone is usually sufficiently distinct to identify that neurone throughout the experiment.
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In the present electrophysiological study dexmedetomidine produced a potent spinal antinociceptive ac-
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tion as measured by the inhibition of noxious dorsal horn neuronal responses following intrathecal administration of the agonist. Although non-nociceptive responses were also inhibited, with higher doses of dexmedetomidine there was a clear distinction between C fibre-evoked responses which were almost abolished compared with a maximal 50% reduction of A/3-evoked responses. This contrasts with an earlier study in the same preparation on the spinal antinociceptive actions of clonidine which only reduced C fibre-evoked responses to a maximal 52% (Sullivan et al., 1987) The antinociceptive potency of dexmedetomidine was also much greater than clonidine which required 50 ~ g to inhibit C fibre responses to 50% of control compared to a dose of 2.5 p~g of dexmedetomidine to achieve the same effect. This may represent varying degrees of access of both compounds to the receptors in the dorsal horn but could also reflect the partial agonist nature of clonidine. The profile of dexmedetomidine against C and A/3 responses is similar to that seen after intrathecal administration of opioid agonists in the same electrophysiological model. ~-Agonists such as morphine and the
131
3-agonist D S T B U L E T (Tyr-D-Ser(otbu)-Gly-Leu-Thr) also reduce C fibre-evoked responses by over 80% compared to maximal A/3 inhibitions of 40-50% (Dickenson and Sullivan, 1986; Sullivan et al., 1989). In addition the potency of dexmedetomidine is close to that of morphine and D S T B U L E T in this model. Thus the similarity of antinociceptive profile and potency leads to the conclusion that activation of a2-adrenoceptors in the spinal cord produces comparable antinociception to activation of the 'classic antinociceptive' opioid receptor. Behavioural studies lend support to the above conclusion. The antinociceptive doses of dexmedetomidine in the electrophysiological study correspond well to those (3-10 p~g) which produce behavioural antinociception in rats (Fisher et al., 1991; Kalso et al., 1991). However in conscious rats the sedative effects of 6-10 p~g, indicating leakage of the drug to the systemic circulation, highlight possible problems in extending the use of dexmedetomidine to clinical spinal analgesia. I.v. administration of 40 /xg/kg dexmedetomidine is sufficient to produce profound sedation in rats (Fisher et al., 1991) and in humans sedation was reported after bolus i.v. doses of 25-100 /zg medetomidine (Kauppila et al., 1991; Scheinin et al., 1987). A possible coml~cating factor in interpretation of the present series ot experiments is the interaction of dexmedetomidine with the anaesthetic halothane. Both medetomidine and dexmedetomidine have hypnotic or anaesthetic effects in animals, the latter reducing the halothane minimal alveolar concentration after both systemic and intrathecal administration via an interaction with a2-adrenoceptors (Segal et al., 1988; Nagasaka and Yaksh, 1990). That the inhibitory actions of dexmedetomidine on neuronal responses seen in the present study are due to an increase in level of anaesthesia and therefore an indirect general depression of neuronal activity must be considered. However this explanation seems unlikely since similar intrathecal doses in the absence of anaesthestic agents produce behavioural antinociception (Kalso et al., 1991; Fisher et al., 1991). The effect of dexmedetomidine on AP height is difficult to explain although it does appear to be common to ce2-adrenoceptor agonists in general since intrathecal clonidine also produced this effect in a small proportion of neurones (unpublished observations). The AP amplitude could change for reasons other than a drug effect, for instance overdepolarisation of a particulary responsive neurone or drifting of the neurone away from the recording electrode. However overdepolarisation of neurones in our electropysiological model is seen as a progessive reduction in the action potential height during the train of 16 electrical stimuli not as a constant reduced AP height apparent from the first stimulus onwards as was seen after dexmedetomidine.
Furthermore overdepolarisation is unlikely to appear in a neurone depressed by an agonist when absent in the control response. Drifting of the neurone also seems an unlikely explanation in view of the restoration of AP height following administration of the antagonist which reversed dexmedetomdine's inhibitory effect. Interestingly the opposite, an increase in AP amplitude of dorsal horn neurones has been noted after inontophoretic NA. This was attributed to hyperpolarisation of the neurone by the agonist (Howe and Zieglg~insberger, 1987). However depression of neuronal responses by dexmedetomidine presumably involving hyperpolarisation was independent of any change in AP height since the former could be elicited in the absence of the latter, and AP amplitude was decreased rather than increased. Therefore it is difficult to apply the above explanation to the effects of dexmedetomidine on AP amplitude in the present study. The antagonism of the antinociceptive effects of dexmedetomidine by intrathecal atipamazole confirmed the involvement of an a2-adrenoceptor. Autoradiographical studies clearly show the presence of c~2-adrenoceptors in the superfical layers of the dorsal horn (Sullivan et al., 1987, Unnerstail et at., 1984). These receptors are postsynaptic to descending noradrenergic fibre terminals because spinalisation or 6-hydroxydopamine treatment did not decrease receptor numbers (Yaksh, 1985). However precisely where a 2adrenoceptors are located, on nociceptive afferent terminals or postsynaptic to these terminals on dorsal horn neurones is not clear. Whilst a presynaptic locus on primary afferents has been suggested (Calvillo and Guignone, 1986; Kuraishi et al., 1985) destruction of C fibres with neonatal capsaicin does not reduce [3H]clonidine binding sites in mice (Wikberg and Hajos, 1987). Furthermore immunohistochemical studies show no synaptic contact between descending noradrenergic fibre terminals and primary afferent terminals suggesting that a2-adrenoceptors are located on dorsal horn neurones postsynaptic to primary afferents (Hagihira et al., 1990). Direct catecholaminergic innervation of primate spinothalamie tract neurones has recently been demonstrated (Westlund et al., 1990). In vivo nociceptive neuronal responses of dorsal horn neurones including those characterised as spinothalamic are selectively depressed by iontophoretic or intrathecal administration of NA and more selective c~2-adrenoceptor agonists including medetomidine (Davies and Quinlan, 1985; FleetwoodWalker et al., 1985; Headley et al., 1978; Pertovaara et al., 1991; Sullivan et al., 1987). In addition dexmedetomidine inhibited the neuronal response evoked by exogenous substance P, implicated in spinal nociceptive transmission (Kendig et al., 1991). In vitro ion-
132 tophoretic NA hyperpolarised substantia gelatinosa n e u r o n e s via an i n c r e a s e in p o t a s s i u m c o n d u c t a n c e ( N o r t h a n d Y o s h i m u r a , 1984) a n d this m a y r e p r e s e n t a direct p o s t s y n a p t i c m e c h a n i s m of action for the d e p r e s sion of n e u r o n a l r e s p o n s e s by d e x m e d e t o m i d i n e in the d o r s a l horn. In a d d i t i o n to the activation o f c~2-adrenoceptors by d e x m e d e t o m i d i n e t h e a n t a g o n i s m of this a g o n i s t ' s actions by n a l o x o n e in most of the n e u r o n e s t e s t e d in the p r e s e n t study suggests t h e i n v o l v e m e n t o f o p i o i d r e c e p tors, which occur b o t h p r e s y n a p t i c a l l y on p r i m a r y afferents a n d p o s t s y n a p t i c a l l y on d o r s a l horn n e u r o n e s (Besse et al., 1990 a n d r e f e r e n c e s within). It is unlikely that d e x m e d e t o m i d i n e acts directly at o p i o i d r e c e p t o r s ( V i r t a n e n , 1988) b u t i n t e r a c t i o n s b e t w e e n o p i o i d a n d a d r e n e r g i c spinal m e c h a n i s m s involving i n d e p e n d e n t r e c e p t o r activation a n d resulting in synergism of the spinal a n t i n o i c e p t i v e effect a f t e r c o a d m i n i s t r a t i o n of e x o g e n o u s o p i o i d a n d a d r e n o c e p t o r agonists have b e e n widely r e p o r t e d (Sullivan et al., 1987; Wilcox et al., 1987; Yaksh, 1985). T h e i n v o l v e m e n t of e n d o g e n o u s o p i o i d m e c h a n i s m s in the action o f e x o g e n o u s a d r e n o c e p t o r agonists a d m i n i s t e r e d a l o n e has not b e e n universally seen; n a l o x o n e h a d no effect on the antinocic e p t i o n m e d i a t e d by o t h e r a ~ - a d r e n o c e p t o r agonists in most p r e v i o u s b e h a v i o u r a l a n d e l e c t r o p h y s i o l o g i c a l studies (Yaksh, 1985). H o w e v e r n a l o x o n e reversibility o f N A - m e d i a t e d a n t i n o c i c e p t i o n in the tail-flick test has b e e n r e p o r t e d by L o o m i s et al. (1987). F u r t h e r m o r e c l o n i d i n e - i n d u c e d inhibitions of n o c i c e p t i v e responses w e r e r e v e r s e d by n a l o x o n e in a p r o p o r t i o n of d o r s a l h o r n n e u r o n e s t e s t e d ( O m o t e et al., 1991) a n d the d e p r e s s i o n o f the v e n t r a l root p o t e n t i a l by d e x m e d e t o m i d i n e in the i s o l a t e d n e o n a t a l c o r d was p a r t i a l l y b l o c k e d by n a l o x o n e ( K e n d i g et al., 1991). It is h a r d to see how the inhibitory actions of end o g e n o u s o p i o i d s could be e n t i r e l y r e s p o n s i b l e for the p r o f o u n d n e u r o n a l d e p r e s s i o n s e v o k e d by h i g h e r doses o f d e x m e d e t o m i d i n e since p r o t e c t i o n o f e n d o g e n o u s o p i o i d s by the e n k e p h a l i n a s e i n h i b i t o r k e l a t o r p h a n only p r o d u c e d a 50% m a x i m a l inhibition of C f i b r e - e v o k e d r e s p o n s e s in the s a m e m o d e l ( D i c k e n s o n et al., 1987). H o w e v e r this d o e s not p r e c l u d e a synergistic interaction b e t w e e n e n d o g e n o u s o p i o i d a n d e x o g e n o u s a d r e n ergic i n h i b i t o r y m e c h a n i s m s m e d i a t i n g the p o t e n t effects of d e x m e d e t o m i d i n e . M i l l a r a n d W i l l i a m s (1989) o b s e r v e d excitation o f l a m i n a II and III n e u r o n e s by p e r i a q u e d u c t a l gray s t i m u l a t i o n a n d i o n t o p h o r e t i c nor a d r e n a l i n e which i n h i b i t e d d e e p e r W D R (wide dynamic range) neurones. Opioid interneurones located in the s u b s t a n t i a g e l a t i n o s a m a y be involved in b o t h this a n d the action of d e x m e d e t o m i d i n e in the p r e s e n t study. T h e effects o f d e x m e d e t o m i d i n e on w i n d - u p a r e also similar to those m e d i a t e d by opioids. W i n d - u p involves the activation of N M D A r e c e p t o r s , a n d can be inhib-
ited by selective a n t a g o n i s t s (Davies a n d L o d g e , 1987; D i c k e n s o n a n d Sullivan, 1987). O p i o i d s whilst r e d u c i n g the initial r e s p o n s e to a stimulus d o not p r e v e n t the w i n d - u p itself ( D i c k e n s o n a n d Sullivan, 1986) a n d this is now also d e m o n s t r a t e d for d e x m e d e t o m i d i n e suggesting a similarity in the site of action for o p i o i d a n d c~2-adrenoceptor agonists. In conclusion, d e x m e d e t o m i d i n e could c o m p l e t e l y inhibit C f i b r e - e v o k e d r e s p o n s e s in spinal dorsal horn n e u r o n e s s u p p o r t i n g b e h a v i o u r a l e v i d e n c e for a a n t i n o c i c e p t i v e role of this a d r e n o c e p t o r agonist in the spinal cord. A l t h o u g h the actions of d e x m e d e t o m i d i n e w e r e clearly r e v e r s e d by a t i p a m e z o l e c o n f i r m i n g t h e involvement of a 2 - a d r e n o c e p t o r s t h e r e was also evid e n c e for o p i o i d m e c h a n s i m s c o n t r i b u t i n g to d e x m e d e t o m i d i n e ' s effect.
Acknowledgements This work was supported by the Medical Research Council, UK and the European Academy of Anaesthesiology.
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