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The action of K-agonists on the nociceptive responses of neurones in the medullary dorsal horn of the anaesthetized rat
Life Sciences, Vol. 33, Sup. I, 1983, pp. 541-544 Printed in the U.S.A.
Pergamon Press
THE ACTION OF K-AGONISTS ON THE NOCICEPTIVE RESPONSES OF NEURONES IN T H E M E D U L L A R Y DORSAL HORN OF THE ANAESTHETIZED RAT 3 . C a l t h r o p * and R.G.HiII+ * D e p t of Pharmacology, University of Bristol Medical School, Bristol BS8 1TD, U.K. +Parke-Davis Research Unit, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, U.K. (Received in final form June 26, 1983) Summary Responses of medullary dorsal horn neurones to both mechanical and thermal noxious stimuli were recorded in urethane anaesthetized rats. Opiates w i t h reported a c t i v i t y at K receptors (tifluadom, BL 5572M, and U50488) were found to reduce responses to both noxious stimuli, and in this respect, were indistinguishable from the I~ agonist fentanyl. These observations are in contrast to the behaviourat antinociceptive effects of K agonists as these substances are active in tests using mechanical noxious stimuli but in those using thermal stimuli have l i t t l e effect. It is therefore possible that the m o d a l i t y decoding seen in behavioural experiments occurs at a supraspinal level. Evidence is now emerging that K agonists possess a distinct pharmacological profile having properties, such as the a b i l i t y to promote diuresis (1), that are not shared by other opiates. Behavioural tests in rodents have shown t h a t K agonists are e f f e c t i v e against mechanical or chemical noxious stimuli but are not active against thermal noxious stimuli until doses producing impairment of gross motor function are given (2,3,4), This observation is paradoxical, as peripheral nociceptive afferents are thought to be polymodal (596) and points to a decoding process w i t h i n the central nervous system. The most obvious site for this is within the dorsal horn of the spinal cord and accordingly we have examined the action of systemically administered K agonists on the nociceptive responses of neurones in the medullary dorsal horn of the anaesthetized rat. Methods A d u l t male rats anaesthetized w i t h urethane (1.25g/kg) were used in all experiments. A tracheal cannula was inserted to allow a r t i f i c i a l respiration and one e x t e r n a l jugular vein was cannulated for the administration of drugs. A f t e r f i x a t i o n of the head in a stereotaxic frame, the dorsal caudal medulla was exposed by removal of the a t l a n t o o c c i p i t a l membrane and dura mater. E x t r a c e l l u l a r recordings of neuronal a c t i v i t y were made with single barrelled glass micropipettes f i l l e d w i t h 2.5% w/v pontamine sky blue (PSB) in acetate b u f f e r and deposition of PSB was used to mark recording positions w i t h i n the medulla.
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
542
K-agonists and Dorsal Horn Neurones
Vol. 33, Sup. I, 1983
Solutions of fentanyl citrate (Janssen), tifluadom (Sandoz), BL5572M (Bristol Myers), US0488H (Upjohn) and nalbuphine (Endo) were made in normal saline and given as intravenous bolus injections. Noxious stimuli were applied to the face ipsilateral to the site of recording. Mechanical stimuli consisted of a 5 second pinch with a pair of plastic forceps that locked at a reproducible pressure found to be noxious when tested on the experimenter. Thermal stimuli were provided by a fine jet of water directed at the face so as to produce a temperature ramp rising from ambient to a maximum of 52°C. Temperature was continuously monitored by a miniature thermocouple. Results Recordings were obtained f r o m 16 neurones in 13 rats. All neurones studied to both types of noxious stimulus, 15 being wide dynamic range neurones, the 1 being exclusively n o c i c e p t i v e (7). Post e x p e r i m e n t histology revealed neurones studied were located in laminae V and V[ but 2 were found to m a r g i n a l layer.
responded remaining t h a t most be in the
Fentanyl (3 to 30 Ilgm/kg) was found to a t t e n u a t e both mechanical and t h e r m a l n o c i c e p t i v e response of all 5 neurones tested. T i f l u a d o m , BL5772M and US0488H also a t t e n u a t e d both n o c i c e p t i v e responses. Na]buphine was i n e f f e c t i v e over the l i m i t e d dose range (0.3 - 0.45 m g / k g ) tested.
Mec.homcol Stlmut at ion • Fento nyl
o Tifluodom • BL 557"2bl o Nolbuphine U50488H o
t
50
i"Y
.c_
g ~o @J n."
6
11.
,
Dose.
i
,
i
,
i
0.1mg/kg
Fig.1 Percentage reduction in response to mechanical noxious s t i m u l i p l o t t e d against dose of drug in m g / k g . V e r t i c a l bars represent standard e r r o r (n=5 for fentanyl, 4 for BL5572M, 5 for US0488H) and those points w i t h o u t bars represent single trials.
Vol. 33, Sup. I, 1983
K-agonists and Dorsal Horn Neurones
543
[n order to exclude the possibility that K agonists were appearing to be none selective between the two noxious stimuli used in these experiments solely because excessively high doses were being used, a range of doses were used across a number of drug trials, [n order to compare data from d i f f e r e n t experiments the action of drug t r e a t m e n t on response size (measure'd from analogue rate meter traces as previously described (7)) was expressed as a percentage reduction, Results from all 22 drug trials made were pooled,
Drug dose against reduction of the response to mechanical stimulation is shown in Figure i and drug dose against the reduction of the response to thermal stimulation is shown in Figure 2. [t is apparent that a clear dose response realtionship exists against both sensory modalities for all drugs tested, with the exception of nalbuphine, but there is no instance in which a low dose of a K agonist is more e f f e c t i v e against mechanical than against thermal noxious stimulation. In addition to their effects on nociceptive responses, in some experiments BL5572M and US0488H caused an increase in background neuronal firing rate. Thermo!. Stimutotion
• Fentonyl o Tifluodom • BL5572M o Nolbuphine U50Z.8BH
50el mr t-
g
~.
"
i
i
i
i
Dose.
i
i
!
i
i
0-1rng/kg
Fig.2. A similar graph to Fig. 1 for percentage reduction in response to thermal noxious stimuli. Note that the individual doses of each drug produce a similar reduction in both mechanical and thermal nociceptive responses.
Discussion These experiments failed to show the expected separation in a b i l i t y to attenuate responses to thermal and mechanical noxious stimuli, that behavioural experiments w i t h K agonists would predict (2,3,4). One reason for this result could be the presence of anaesthesia, although in animals under exactly the same e x p e r i m e n t a l conditions i t is possible to show that e x c i t a t o r y amino acid antagonists w i l l selectively attenuate mechanical, but not thermal, nociceptive responses (7). A d d i t i o n a l l y , it is possible to demonstrate that morphine w i l l reduce responses to mechanical noxious but not to mechanical non-noxious stimuli (8).
544
K-agonists
and Dorsal Horn Neurones
Vol. 33, Sup. I, 1983
It is possible that the m o d a l i t y decoding seen in behavioural experiments is due to the a c t i v i t y of a group of dorsal born neurones that were not recorded from in the present study, i t is not likely to be a t t r i b u t a b l e to an action on marginal neurones as the 2 neurones of this type recorded in the present study behaved in a similar fashion to neurones found in deeper laminae. [t may be due to i n f o r m a t i o n processing w i t h i n the substantia gelatinosa, however. It is conceivable that K agonists have a supra-spinal action that can distinguish mechanical and thermal noxious stimuli (e.g. see 4) but this explanation must be examined in the light of our knowledge that the rat has c o m p a r a t i v e l y few non-spinal K receptors (9) and the independent experimental evidence for a spinal locus of action for K analgesia (i0,II). References 1,
2. 3,
4. 5. 6. 7. 8.
9,
i0. ii.
J.D.LEANDER, 3. Pharm. Exp. Ther 224 89-94 (1983) A.G.HAYES, R.G.HILL, M.SKiNGLE and M.B.TYERS, Br. J. Pharmacol. 78 47P (1983). M.B.TYERS, Br.3. Pharmacol. 69 503-512 (1980). R . M . B R Y A N T , J.E.OLLEY and M.B.TYERS, Br. J. Pharmacol. 78 659-663. B . L Y N N and S.F-. C A R P E N T E R , Brain Res. 238 29-45 (1982). R.F. BE|TEL and R. D U B N E R , J. Neurophysiol 59 1160-1175 (1976). T.E.SALT and P,.G.HILL, Brain Res 263 167-171 (1985). R.MORRIS, R.G.H[LL, P . M . B . C A H U S A C and T.E.SALT, in Anatomical Physiological and Pharmaco|ogical Aspects of Trigeminal Pain eds. B. MATTHEWS and R.G.HILL, Excerpta Medica, Amsterdam pp 195-206 (1982). M.G.C.GII_LAN and H.W. KOSTERLITZ, Br. J. Pharmac. 77 461-469 (1982). J-S.HAN and C-W. XIE, L i f e Sciences 31 1781-1784 (19827. M.PIERCEY, R.A. LAHT[, L.A.SCHROEDER, F.J. EINSPAHR and C. BARSUHN, L i f e Sciences 31 1197-1200 (1982).
Pergamon Press
THE ACTION OF K-AGONISTS ON THE NOCICEPTIVE RESPONSES OF NEURONES IN T H E M E D U L L A R Y DORSAL HORN OF THE ANAESTHETIZED RAT 3 . C a l t h r o p * and R.G.HiII+ * D e p t of Pharmacology, University of Bristol Medical School, Bristol BS8 1TD, U.K. +Parke-Davis Research Unit, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, U.K. (Received in final form June 26, 1983) Summary Responses of medullary dorsal horn neurones to both mechanical and thermal noxious stimuli were recorded in urethane anaesthetized rats. Opiates w i t h reported a c t i v i t y at K receptors (tifluadom, BL 5572M, and U50488) were found to reduce responses to both noxious stimuli, and in this respect, were indistinguishable from the I~ agonist fentanyl. These observations are in contrast to the behaviourat antinociceptive effects of K agonists as these substances are active in tests using mechanical noxious stimuli but in those using thermal stimuli have l i t t l e effect. It is therefore possible that the m o d a l i t y decoding seen in behavioural experiments occurs at a supraspinal level. Evidence is now emerging that K agonists possess a distinct pharmacological profile having properties, such as the a b i l i t y to promote diuresis (1), that are not shared by other opiates. Behavioural tests in rodents have shown t h a t K agonists are e f f e c t i v e against mechanical or chemical noxious stimuli but are not active against thermal noxious stimuli until doses producing impairment of gross motor function are given (2,3,4), This observation is paradoxical, as peripheral nociceptive afferents are thought to be polymodal (596) and points to a decoding process w i t h i n the central nervous system. The most obvious site for this is within the dorsal horn of the spinal cord and accordingly we have examined the action of systemically administered K agonists on the nociceptive responses of neurones in the medullary dorsal horn of the anaesthetized rat. Methods A d u l t male rats anaesthetized w i t h urethane (1.25g/kg) were used in all experiments. A tracheal cannula was inserted to allow a r t i f i c i a l respiration and one e x t e r n a l jugular vein was cannulated for the administration of drugs. A f t e r f i x a t i o n of the head in a stereotaxic frame, the dorsal caudal medulla was exposed by removal of the a t l a n t o o c c i p i t a l membrane and dura mater. E x t r a c e l l u l a r recordings of neuronal a c t i v i t y were made with single barrelled glass micropipettes f i l l e d w i t h 2.5% w/v pontamine sky blue (PSB) in acetate b u f f e r and deposition of PSB was used to mark recording positions w i t h i n the medulla.
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
542
K-agonists and Dorsal Horn Neurones
Vol. 33, Sup. I, 1983
Solutions of fentanyl citrate (Janssen), tifluadom (Sandoz), BL5572M (Bristol Myers), US0488H (Upjohn) and nalbuphine (Endo) were made in normal saline and given as intravenous bolus injections. Noxious stimuli were applied to the face ipsilateral to the site of recording. Mechanical stimuli consisted of a 5 second pinch with a pair of plastic forceps that locked at a reproducible pressure found to be noxious when tested on the experimenter. Thermal stimuli were provided by a fine jet of water directed at the face so as to produce a temperature ramp rising from ambient to a maximum of 52°C. Temperature was continuously monitored by a miniature thermocouple. Results Recordings were obtained f r o m 16 neurones in 13 rats. All neurones studied to both types of noxious stimulus, 15 being wide dynamic range neurones, the 1 being exclusively n o c i c e p t i v e (7). Post e x p e r i m e n t histology revealed neurones studied were located in laminae V and V[ but 2 were found to m a r g i n a l layer.
responded remaining t h a t most be in the
Fentanyl (3 to 30 Ilgm/kg) was found to a t t e n u a t e both mechanical and t h e r m a l n o c i c e p t i v e response of all 5 neurones tested. T i f l u a d o m , BL5772M and US0488H also a t t e n u a t e d both n o c i c e p t i v e responses. Na]buphine was i n e f f e c t i v e over the l i m i t e d dose range (0.3 - 0.45 m g / k g ) tested.
Mec.homcol Stlmut at ion • Fento nyl
o Tifluodom • BL 557"2bl o Nolbuphine U50488H o
t
50
i"Y
.c_
g ~o @J n."
6
11.
,
Dose.
i
,
i
,
i
0.1mg/kg
Fig.1 Percentage reduction in response to mechanical noxious s t i m u l i p l o t t e d against dose of drug in m g / k g . V e r t i c a l bars represent standard e r r o r (n=5 for fentanyl, 4 for BL5572M, 5 for US0488H) and those points w i t h o u t bars represent single trials.
Vol. 33, Sup. I, 1983
K-agonists and Dorsal Horn Neurones
543
[n order to exclude the possibility that K agonists were appearing to be none selective between the two noxious stimuli used in these experiments solely because excessively high doses were being used, a range of doses were used across a number of drug trials, [n order to compare data from d i f f e r e n t experiments the action of drug t r e a t m e n t on response size (measure'd from analogue rate meter traces as previously described (7)) was expressed as a percentage reduction, Results from all 22 drug trials made were pooled,
Drug dose against reduction of the response to mechanical stimulation is shown in Figure i and drug dose against the reduction of the response to thermal stimulation is shown in Figure 2. [t is apparent that a clear dose response realtionship exists against both sensory modalities for all drugs tested, with the exception of nalbuphine, but there is no instance in which a low dose of a K agonist is more e f f e c t i v e against mechanical than against thermal noxious stimulation. In addition to their effects on nociceptive responses, in some experiments BL5572M and US0488H caused an increase in background neuronal firing rate. Thermo!. Stimutotion
• Fentonyl o Tifluodom • BL5572M o Nolbuphine U50Z.8BH
50el mr t-
g
~.
"
i
i
i
i
Dose.
i
i
!
i
i
0-1rng/kg
Fig.2. A similar graph to Fig. 1 for percentage reduction in response to thermal noxious stimuli. Note that the individual doses of each drug produce a similar reduction in both mechanical and thermal nociceptive responses.
Discussion These experiments failed to show the expected separation in a b i l i t y to attenuate responses to thermal and mechanical noxious stimuli, that behavioural experiments w i t h K agonists would predict (2,3,4). One reason for this result could be the presence of anaesthesia, although in animals under exactly the same e x p e r i m e n t a l conditions i t is possible to show that e x c i t a t o r y amino acid antagonists w i l l selectively attenuate mechanical, but not thermal, nociceptive responses (7). A d d i t i o n a l l y , it is possible to demonstrate that morphine w i l l reduce responses to mechanical noxious but not to mechanical non-noxious stimuli (8).
544
K-agonists
and Dorsal Horn Neurones
Vol. 33, Sup. I, 1983
It is possible that the m o d a l i t y decoding seen in behavioural experiments is due to the a c t i v i t y of a group of dorsal born neurones that were not recorded from in the present study, i t is not likely to be a t t r i b u t a b l e to an action on marginal neurones as the 2 neurones of this type recorded in the present study behaved in a similar fashion to neurones found in deeper laminae. [t may be due to i n f o r m a t i o n processing w i t h i n the substantia gelatinosa, however. It is conceivable that K agonists have a supra-spinal action that can distinguish mechanical and thermal noxious stimuli (e.g. see 4) but this explanation must be examined in the light of our knowledge that the rat has c o m p a r a t i v e l y few non-spinal K receptors (9) and the independent experimental evidence for a spinal locus of action for K analgesia (i0,II). References 1,
2. 3,
4. 5. 6. 7. 8.
9,
i0. ii.
J.D.LEANDER, 3. Pharm. Exp. Ther 224 89-94 (1983) A.G.HAYES, R.G.HILL, M.SKiNGLE and M.B.TYERS, Br. J. Pharmacol. 78 47P (1983). M.B.TYERS, Br.3. Pharmacol. 69 503-512 (1980). R . M . B R Y A N T , J.E.OLLEY and M.B.TYERS, Br. J. Pharmacol. 78 659-663. B . L Y N N and S.F-. C A R P E N T E R , Brain Res. 238 29-45 (1982). R.F. BE|TEL and R. D U B N E R , J. Neurophysiol 59 1160-1175 (1976). T.E.SALT and P,.G.HILL, Brain Res 263 167-171 (1985). R.MORRIS, R.G.H[LL, P . M . B . C A H U S A C and T.E.SALT, in Anatomical Physiological and Pharmaco|ogical Aspects of Trigeminal Pain eds. B. MATTHEWS and R.G.HILL, Excerpta Medica, Amsterdam pp 195-206 (1982). M.G.C.GII_LAN and H.W. KOSTERLITZ, Br. J. Pharmac. 77 461-469 (1982). J-S.HAN and C-W. XIE, L i f e Sciences 31 1781-1784 (19827. M.PIERCEY, R.A. LAHT[, L.A.SCHROEDER, F.J. EINSPAHR and C. BARSUHN, L i f e Sciences 31 1197-1200 (1982).