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Periaqueductal grey matter stimulation produces naloxone sensitive inhibition of reticular neurones in rat caudal medulla
Life Sciences, Vol. 31, pp. 2323-2325 Printed in the U.S.A.
Pergamon Press
PERIAQUEDUCTAL GREY MATTER STIMULATION PRODUCES NALOXONE SENSITIVE I N H I B I T I O N OF RETICULAR NEURONES IN RAT CAUDAL MEDULLA R.G.
Rill,
R. M o r r i s
a n d M.V.
Sofroniew
Department of Pharmacology, University of Bristol University Walk, Bristol BS8 1TD, U .K .
Medical
School,
(Received in final form June 14, 1982) Summary Neurones in the caudal medulla reticular formation (CRF) a r e excited by p e r i p h e r a l noxious stimuli a n d t h u s may h a v e a r o l e in nociceptive information processing. Stimulation of sites within the dorsal periaqueductal grey matter (PAG) p r o d u c e d a prolonged inhibition o f CRF n e u r o n e s . On 11 o f 16 n e u r o n e s tested mieroiontophoretic application of naloxone was found to attenuate these inhibitions suggesting that the neurotransmitter i n v o l v e d may h a v e b e e n a n e n d o g e n o u s o p i o i d . Histoehemical localization of ~endorphin-like immunoreactivity (BELI) showed fibres containing BELI i n t h e v i c i n i t y o f PAG stimulation sites and scattered individual fibres with short collateral branches in the vicinity of recording sites i n CRF. It has been demonstrated by a number of workers (1,2,3,4) that reticular formation neurones are excited by n o x i o u s s t i m u l i and therefore may h a v e a noeiceptive role, perhaps relaying information between the dorsal horn of the spinal cord and thalamus (2). Electrical stimulation w i t h i n t h e PAG i s k n o w n to produce analgesia (5) and reticular neurones are inhibited following PAG stimulation (3). As l i t t l e is known of the pharmacology of these inhibitions we h a v e u s e d m i c r o i o n t o p h o r e t i c techniques to examine the possibility that a descending inhibitory system utilizing endogenous opioids may b e i n v o l v e d . Method Adult male rats were anaesthetized with urethane (1.25G/Kg) and prepared for microelectrode recording from the caudal medulla reticular formation (CRF), in the vicinity of nucleus reticularis ventralis, as previously described (3,4). Extracellular action potentials were recorded from single CRF n e u r o n e s using the centre barrel of a multibarrel micropipette that was also used for microiontophoretic application of Bendorphin, met-enkephalin and naloxone (6,7). Recording positions within the medulla were verified by location of dye spots, deposited from the micropipette.
(45-55°C)
Noxious stimuli were applied to the hind limbs and tail by heating or by pinching with haemostats. PAG s t i m u l a t i n g electrodes were implanted chronically some t i m e p r e v i o u s l y to the electrophysiological experiments, under pentobarbital anaesthesia. Behavioural effects o f PAG s t i m u l a t i o n were assessed (see 7 for details) and all animals used in this study showed behavioural analgesia. At the end of the electrophysiological experiment a DC c u r r e n t lesion w a s m a d e t h r o u g h t h e PAG e l e c t r o d e so that stimulus sites could be mapped.
0024-3205/82/202323-03503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2324
Naloxone
and Reticular
Neurones
Vol.
31, No.s 20 & 21, 1982
Immunocytochemical techniques used (8) were conventional. An a n t i s e r u m was raised using synthetic B e n d o r p h i n (SERVA) c o n j u g a t e d and injected into rabbits, a n d ~ELI w a s v i s u a l i z e d using the immunoperoxidase technique. Photographs of stained sections w e r e t a k e n a n d m a p s o f BELI d i s t r i b u t i o n constructed.
Results I n 14 a n a e s t h e t i z e d animals previously shown to display analgesia after PAG stimulation, the responses o f 56 CRF n e u r o n e s w e r e e x a m i n e d . These neurones responded to peripheral stimuli in a characteristic manner (1,3,4) showing no response to tapping or brushing but being excited by noxious stimuli applied to the hindlimbs and/or the tail. Thirty f i v e (62%) o f t h e s e n e u r o n e s were inhibited by stimulation o f t h e PAG u s i n g c u r r e n t s ( 2 5 - 3 0 0 ~A, 0 . 5 ms pulses, 50Hz t r a i n s , 5-30s) previously shown to produce analgesia. Thirteen neurones (23%) w e r e e x c i t e d following PAG s t i m u l a t i o n and the remaining 8 (14~) were unaffected.
Microiontophoretic application of Bendorphin (10-60 nA) depressed iO CRF neurones, excited i, and 2 of the 13 tested were unaffected. Met-enkephalin (20-lOOnA) depressed 16 neurones out of 17 tested, the remaining 1 being unaffected. Naloxone neurones
(2-40nA) and that
reversed produced
the depression produced by 8endorphin by m e t - e n k e p h a l i n on 3 n e u r o n e s .
on 2
The effect o f n a l o x o n e on i n h i b i t i o n o f CRF n e u r o n e s p r o d u c e d by FAG stimulation was tested o n 16 n e u r o n e s a n d o n 12 o f t h e s e r e d u c t i o n in the inhibition was seen. T h e m o s t common o b s e r v a t i o n was a reduction in the time course of the inhibition rather than complete abolition. This parallels the observations made on t h e a c t i o n o f s y s t e m i c n a l o x o n e i n r e d u c i n g the inhibition o f CRF n e u r o n e s p r o d u c e d by p e r i p h e r a l stimulation (4). The l o c a t i o n o f ~ELI c o n t a i n i n g fibres was plotted onto standard transverse sections of the rat brain, at appropriate AP c o - o r d i n a t e s m a t c h i n g CRF recording sites and stimulation sites w i t h i n PAG. BELI c o n t a i n i n g fibres were found to have a patchy distribution w i t h i n t h e CRF b u t n o t t o s p r e a d laterally into the caudal trigeminal nucleus to any great extent. Only scattered individual fibres w e r e f o u n d , a n d no d e n s e b e d s o f t e r m i n a l s , although these individual fibres seemed to give off a large number of short collateral or terminal branches. A b u n d a n t 8ELI c o n t a i n i n g fibres were found w i t h i n PAG, a s p r e v i o u s l y described (9), and there was considerable overlap with the position of those stimulus sites needing the lowest current to produce analgesia ( 2 5 - 1 0 0 ~A) a n d 8 E L I .
Discussion I n t h i s s t u d y we h a v e o b t a i n e d evidence that neurones within t h e CRF o f anaesthetized rats are inhibited by stimuli applied t o FAG, a n d t h a t t h e s e inhibitions are reduced by microlontophoretic naloxone. Application of n a l o x o n e i n t h i s way r e s t r i c t s the action of the drug to the area close to the tip of the micropipette. It is thus likely that any endogenous oploid substance involved is acting locally rather t h a n r e m o t e l y w i t h i n PAG o r a t a spinal relay. It is important t o r e m e m b e r , h o w e v e r , t h a t t h e r e i s some e v i d e n c e t h a t n a l o x o n e may a l s o a n t a g o n i s e the actions o f GABA ( 1 0 ) a n d u n t i l this possibility has been eliminated these results should be interpreted with caution. Assuming that an endogenous optotd released within t h e CRF i s o p e r a t i n g these inhibitions, then the presence o f BELI f i b r e s in the region of both stimulating
Vol. 31, No.s 20 & 21, 1982
Naloxone and Reticular Neurones
2325
and recording sites is of interest. Injection of horse radish peroxidase w i t h i n t h e CRF r e s u l t s in retrograde transport of this marker to cell bodies in the arcuate nucleus (11). It is therefore possible that the naloxone sensitive inhibitory effects o f PAG s t i m u l a t i o n o n CRF n e u r o n e s a r e d u e t o activation o f ~ELI c o n t a i n i n g fibres passing t h r o u g h PAG, f r o m t h e a r c u a t e nucleus, towards the caudal medulla. It is possible that other opioids with a distribution t h a t we, a t p r e s e n t , have an incomplete k n o w l e d g e o f may a l s o be involved. It is unlikely that endorphin operated inhibition within t h e CRF w o u l d provide an adequate neuronal substrate for the analgesia p r o d u c e d b y PAG stimulation. However, when such a process is combined with the inhibition of more rostral reticular n e u r o n e s a n d o f PAG a n d p e r i v e n t r i c u l a r thalamic neurones then a powerful net analgesic effect ma y r e s u l t . A failure to demonstrate an opioid operated descending inhibition acting on dorsal horn neurones (7,12) is perhaps more understandable if such a system operates preferentially at secondary relays within the reticular formation and midbrain. Acknowledgements Supported by grants from the Wellcome Trust Endo for the gift of naloxone.
and Royal
Society.
We t h a n k
References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12.
R.M. BENJAMIN, B r a i n R e s . 24 5 2 5 - 5 2 9 ( 1 9 7 0 ) D. BOWSHER, P a i n 2 3 6 1 - 3 7 8 (1976) R . G . HILL a n d R. MORRIS, J . P h y s i o l . (Lond.) 301 38-39P (1980) M.L. MAYER a n d R . G . H I L L , N e u r o p h a r m a c o l . 17 5 3 3 - 5 3 9 ( 1 9 7 8 ) D . J . MAYER a n d D.D. P R I C E , P a i n 2 3 7 9 - 4 0 4 (1976) R . G . HILL a n d R. MORRIS, B r . J . P h a r m a c . 7 0 147P ( 1 9 8 0 ) R. MORRIS, R.G. H I L L , P . M . B . CAHUSAC a n d T . E . SALT, Anatomical Physiological and Pharmacological aspects of Trigeminal Pain, E d s . B. M a t t h e w s a n d R . G . H i l l , Elsevier, Amsterdam (1982) in press. M.V. SOFRONIEW, Am. J . A n a t . 154 2 8 3 - 2 8 9 ( 1 9 7 9 ) F. BLOOM, E. BATTENBERG, J . ROSSIER, N. LING a n d R. GUILLEMIN, Proc. Nat. Acad. Sci. (USA) 75 1 5 9 1 - 1 5 9 5 ( 1 9 7 8 ) R.G. HILL, Neurosci. Lett. 21 2 1 7 - 2 2 2 ( 1 9 8 1 ) M.V. SOFRONIEW a n d U. SCHRELL, Neurosci. Lett. 19 2 5 7 - 2 6 3 ( 1 9 8 0 ) E. CARSTENS, D. KLU~[PP a n d M. ZIMMERMAN, N e u r o s c i . Lett. 11 3 2 3 - 3 2 7 (1979)
Pergamon Press
PERIAQUEDUCTAL GREY MATTER STIMULATION PRODUCES NALOXONE SENSITIVE I N H I B I T I O N OF RETICULAR NEURONES IN RAT CAUDAL MEDULLA R.G.
Rill,
R. M o r r i s
a n d M.V.
Sofroniew
Department of Pharmacology, University of Bristol University Walk, Bristol BS8 1TD, U .K .
Medical
School,
(Received in final form June 14, 1982) Summary Neurones in the caudal medulla reticular formation (CRF) a r e excited by p e r i p h e r a l noxious stimuli a n d t h u s may h a v e a r o l e in nociceptive information processing. Stimulation of sites within the dorsal periaqueductal grey matter (PAG) p r o d u c e d a prolonged inhibition o f CRF n e u r o n e s . On 11 o f 16 n e u r o n e s tested mieroiontophoretic application of naloxone was found to attenuate these inhibitions suggesting that the neurotransmitter i n v o l v e d may h a v e b e e n a n e n d o g e n o u s o p i o i d . Histoehemical localization of ~endorphin-like immunoreactivity (BELI) showed fibres containing BELI i n t h e v i c i n i t y o f PAG stimulation sites and scattered individual fibres with short collateral branches in the vicinity of recording sites i n CRF. It has been demonstrated by a number of workers (1,2,3,4) that reticular formation neurones are excited by n o x i o u s s t i m u l i and therefore may h a v e a noeiceptive role, perhaps relaying information between the dorsal horn of the spinal cord and thalamus (2). Electrical stimulation w i t h i n t h e PAG i s k n o w n to produce analgesia (5) and reticular neurones are inhibited following PAG stimulation (3). As l i t t l e is known of the pharmacology of these inhibitions we h a v e u s e d m i c r o i o n t o p h o r e t i c techniques to examine the possibility that a descending inhibitory system utilizing endogenous opioids may b e i n v o l v e d . Method Adult male rats were anaesthetized with urethane (1.25G/Kg) and prepared for microelectrode recording from the caudal medulla reticular formation (CRF), in the vicinity of nucleus reticularis ventralis, as previously described (3,4). Extracellular action potentials were recorded from single CRF n e u r o n e s using the centre barrel of a multibarrel micropipette that was also used for microiontophoretic application of Bendorphin, met-enkephalin and naloxone (6,7). Recording positions within the medulla were verified by location of dye spots, deposited from the micropipette.
(45-55°C)
Noxious stimuli were applied to the hind limbs and tail by heating or by pinching with haemostats. PAG s t i m u l a t i n g electrodes were implanted chronically some t i m e p r e v i o u s l y to the electrophysiological experiments, under pentobarbital anaesthesia. Behavioural effects o f PAG s t i m u l a t i o n were assessed (see 7 for details) and all animals used in this study showed behavioural analgesia. At the end of the electrophysiological experiment a DC c u r r e n t lesion w a s m a d e t h r o u g h t h e PAG e l e c t r o d e so that stimulus sites could be mapped.
0024-3205/82/202323-03503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2324
Naloxone
and Reticular
Neurones
Vol.
31, No.s 20 & 21, 1982
Immunocytochemical techniques used (8) were conventional. An a n t i s e r u m was raised using synthetic B e n d o r p h i n (SERVA) c o n j u g a t e d and injected into rabbits, a n d ~ELI w a s v i s u a l i z e d using the immunoperoxidase technique. Photographs of stained sections w e r e t a k e n a n d m a p s o f BELI d i s t r i b u t i o n constructed.
Results I n 14 a n a e s t h e t i z e d animals previously shown to display analgesia after PAG stimulation, the responses o f 56 CRF n e u r o n e s w e r e e x a m i n e d . These neurones responded to peripheral stimuli in a characteristic manner (1,3,4) showing no response to tapping or brushing but being excited by noxious stimuli applied to the hindlimbs and/or the tail. Thirty f i v e (62%) o f t h e s e n e u r o n e s were inhibited by stimulation o f t h e PAG u s i n g c u r r e n t s ( 2 5 - 3 0 0 ~A, 0 . 5 ms pulses, 50Hz t r a i n s , 5-30s) previously shown to produce analgesia. Thirteen neurones (23%) w e r e e x c i t e d following PAG s t i m u l a t i o n and the remaining 8 (14~) were unaffected.
Microiontophoretic application of Bendorphin (10-60 nA) depressed iO CRF neurones, excited i, and 2 of the 13 tested were unaffected. Met-enkephalin (20-lOOnA) depressed 16 neurones out of 17 tested, the remaining 1 being unaffected. Naloxone neurones
(2-40nA) and that
reversed produced
the depression produced by 8endorphin by m e t - e n k e p h a l i n on 3 n e u r o n e s .
on 2
The effect o f n a l o x o n e on i n h i b i t i o n o f CRF n e u r o n e s p r o d u c e d by FAG stimulation was tested o n 16 n e u r o n e s a n d o n 12 o f t h e s e r e d u c t i o n in the inhibition was seen. T h e m o s t common o b s e r v a t i o n was a reduction in the time course of the inhibition rather than complete abolition. This parallels the observations made on t h e a c t i o n o f s y s t e m i c n a l o x o n e i n r e d u c i n g the inhibition o f CRF n e u r o n e s p r o d u c e d by p e r i p h e r a l stimulation (4). The l o c a t i o n o f ~ELI c o n t a i n i n g fibres was plotted onto standard transverse sections of the rat brain, at appropriate AP c o - o r d i n a t e s m a t c h i n g CRF recording sites and stimulation sites w i t h i n PAG. BELI c o n t a i n i n g fibres were found to have a patchy distribution w i t h i n t h e CRF b u t n o t t o s p r e a d laterally into the caudal trigeminal nucleus to any great extent. Only scattered individual fibres w e r e f o u n d , a n d no d e n s e b e d s o f t e r m i n a l s , although these individual fibres seemed to give off a large number of short collateral or terminal branches. A b u n d a n t 8ELI c o n t a i n i n g fibres were found w i t h i n PAG, a s p r e v i o u s l y described (9), and there was considerable overlap with the position of those stimulus sites needing the lowest current to produce analgesia ( 2 5 - 1 0 0 ~A) a n d 8 E L I .
Discussion I n t h i s s t u d y we h a v e o b t a i n e d evidence that neurones within t h e CRF o f anaesthetized rats are inhibited by stimuli applied t o FAG, a n d t h a t t h e s e inhibitions are reduced by microlontophoretic naloxone. Application of n a l o x o n e i n t h i s way r e s t r i c t s the action of the drug to the area close to the tip of the micropipette. It is thus likely that any endogenous oploid substance involved is acting locally rather t h a n r e m o t e l y w i t h i n PAG o r a t a spinal relay. It is important t o r e m e m b e r , h o w e v e r , t h a t t h e r e i s some e v i d e n c e t h a t n a l o x o n e may a l s o a n t a g o n i s e the actions o f GABA ( 1 0 ) a n d u n t i l this possibility has been eliminated these results should be interpreted with caution. Assuming that an endogenous optotd released within t h e CRF i s o p e r a t i n g these inhibitions, then the presence o f BELI f i b r e s in the region of both stimulating
Vol. 31, No.s 20 & 21, 1982
Naloxone and Reticular Neurones
2325
and recording sites is of interest. Injection of horse radish peroxidase w i t h i n t h e CRF r e s u l t s in retrograde transport of this marker to cell bodies in the arcuate nucleus (11). It is therefore possible that the naloxone sensitive inhibitory effects o f PAG s t i m u l a t i o n o n CRF n e u r o n e s a r e d u e t o activation o f ~ELI c o n t a i n i n g fibres passing t h r o u g h PAG, f r o m t h e a r c u a t e nucleus, towards the caudal medulla. It is possible that other opioids with a distribution t h a t we, a t p r e s e n t , have an incomplete k n o w l e d g e o f may a l s o be involved. It is unlikely that endorphin operated inhibition within t h e CRF w o u l d provide an adequate neuronal substrate for the analgesia p r o d u c e d b y PAG stimulation. However, when such a process is combined with the inhibition of more rostral reticular n e u r o n e s a n d o f PAG a n d p e r i v e n t r i c u l a r thalamic neurones then a powerful net analgesic effect ma y r e s u l t . A failure to demonstrate an opioid operated descending inhibition acting on dorsal horn neurones (7,12) is perhaps more understandable if such a system operates preferentially at secondary relays within the reticular formation and midbrain. Acknowledgements Supported by grants from the Wellcome Trust Endo for the gift of naloxone.
and Royal
Society.
We t h a n k
References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12.
R.M. BENJAMIN, B r a i n R e s . 24 5 2 5 - 5 2 9 ( 1 9 7 0 ) D. BOWSHER, P a i n 2 3 6 1 - 3 7 8 (1976) R . G . HILL a n d R. MORRIS, J . P h y s i o l . (Lond.) 301 38-39P (1980) M.L. MAYER a n d R . G . H I L L , N e u r o p h a r m a c o l . 17 5 3 3 - 5 3 9 ( 1 9 7 8 ) D . J . MAYER a n d D.D. P R I C E , P a i n 2 3 7 9 - 4 0 4 (1976) R . G . HILL a n d R. MORRIS, B r . J . P h a r m a c . 7 0 147P ( 1 9 8 0 ) R. MORRIS, R.G. H I L L , P . M . B . CAHUSAC a n d T . E . SALT, Anatomical Physiological and Pharmacological aspects of Trigeminal Pain, E d s . B. M a t t h e w s a n d R . G . H i l l , Elsevier, Amsterdam (1982) in press. M.V. SOFRONIEW, Am. J . A n a t . 154 2 8 3 - 2 8 9 ( 1 9 7 9 ) F. BLOOM, E. BATTENBERG, J . ROSSIER, N. LING a n d R. GUILLEMIN, Proc. Nat. Acad. Sci. (USA) 75 1 5 9 1 - 1 5 9 5 ( 1 9 7 8 ) R.G. HILL, Neurosci. Lett. 21 2 1 7 - 2 2 2 ( 1 9 8 1 ) M.V. SOFRONIEW a n d U. SCHRELL, Neurosci. Lett. 19 2 5 7 - 2 6 3 ( 1 9 8 0 ) E. CARSTENS, D. KLU~[PP a n d M. ZIMMERMAN, N e u r o s c i . Lett. 11 3 2 3 - 3 2 7 (1979)