- Email: [email protected]
Tolerance to the antinociceptive effect of morphine in the spinal rat
Nurlropharnlu‘olog, Prmted in Great
Vol. 20. pp. 653 to 657. 198, Britain. All rlghlc reserved
Copyright
002X-3908 XI !070653-05502.00/O 0 1981 Pergamon Pres Ltd
TOLERANCE TO THE ANTINOCICEPTIVE EFFECT MORPHINE IN THE SPINAL RAT
OF
O.-G. BERGE and K. HOLE Institute of Physiology, University of Bergen, Bergen, Norway (Accepted 3 February 1981) Summary-Tolerance and withdrawal were studied in spinal and intact rats receiving morphine hydrochloride (l&20 mg/kg, i.p.) every 12 hr for 10 days. All rats developed tolerance to morphine-induced antinociception measured by the tail-flick method. Tolerance was not reversed by D,L-Shydroxytryptophan (200 mg/kg, i.p.). Naloxone (l-2 mg/kg) induced signs of withdrawal both in spinal and intact rats. Thus, functional changes in the aminergic systems and in brain and brainstem structures appear not to be necessary for tolerance to morphine-induced antinociception and for dependence on morphine. The experiment does not support the hypothesis that conditioning to environmental stimuli and changes in cognitive functions play a major role in tolerance development in the rat.
The development of tolerance to and dependence on morphine are accompanied by changes in the metabolism of dopamine, noradrenaline and serotonin, and in the function of monoaminergic (Way, Ho and Loh, 1974; Bllsig, 1978; Van Loon, De Souza and Kim, 1978; Rastogi, Merali and Singhal, 1980) and GABAergic (Contreras, Tamayo and Quijada, 1979) neurones. It has been shown that structures in the vicinity of the fourth ventricle are of importance for dependence and withdrawal (Herz, 1978), and Lagowska, Calvin0 and Ben-Ari (1978) reported strong withdrawal signs when naloxone was injected into the amygdala and caudate putamen in morphinedependent rats. These observations suggest that certain brain and brainstem structures are particularly important for certain behavioural expressions of dependence and withdrawal. Spinal mechanisms are also involved (Wikler and Frank, 1948; Martin and Eades, 1964), and tolerance and dependence may be related to changes in the sensitivity of opiate receptors (Takemori, Oka and Nishiyama, 1973; Kitano and Takemori, 1979) and in cyclic AMP formation (Way et a/., 1974; Ho, Wong and Wen, 1979). Previous studies have therefore shown that the development of tolerance and dependence is accompanied by functional changes at different levels of the central nervous system and in several transmitter systems. It is less clear which of these changes are necessary for the various behavioural aspects of tolerance and dependence. In the present study, the antinociceptive effect of chronic morphine administration has been investigated in spinal and in intact rats. Morphine depresses the nociceptive tail-flick response to radiant heat in spinal rats, although to a lesser extent than in intact animals (Irwin, Houde, Bennett, Hendershot and Key words: morphine, tolerance. spinal rats, S-hydroxytryptophan. 653
Seevers, 1951). If tolerance to the antinociceptive effect of morphine is due to changes in the sensitivity of opiate receptors or postsynaptic mechanisms, the development of tolerance to morphine might be expected in the tail-flick response of spinal as well as intact rats. If functional changes in monoaminergic systems are required, tolerance should develop in the intact animals only, since descending monoaminergic pathways to the spinal cord are completely destroyed by spinal transection (Carlsson, Magnusson and Rosengren, 1963). The effect of 5-hydroxytryptophan (S-HTP) administration on morphine antinociception in tolerant rats was also investigated. Previous reports have indicated that tolerance to the antinociceptive effect of /Y-endorphin (Hosobuchi, Meglio, Adams and Li, 1977) and electrical stimulation of the nucleus raphe magnus (Oliveras, Hosobuchi, Guilbaud and Besson, 1978) in cats may be reversed by treatment with 5-HTP. De-. creased (Contreras, Tamayo, Quijada and Silva, 1973) as well as accelerated (Way et al., 1974) development of tolerance to morphine has been found in connection with S-HTP administration in mice but these studies did not take into account the peripheral effects of 5-hydroxytryptamine (S-HT) accumulation which might have interacted with the actions of morphine (Sarnivaara, 1969). Excessive peripheral formation of 5-HT after 5-HTP administration was therefore prevented by pretreating with an inhibitor of peripheral decarboxylation (Carter, Dykstra and Leander, 1978).
METHODS
Sixty male Wistar rats (250g) were individually housed with free access to food and water. Room temperature was kept at 23 + 1°C. Testing took place in the middle of the light phase of a 12/12 hr light/dark cycle. Pentobarbital (40 mg/kg) and chloral hydrate (130 mg/kg) were given intraperitoneally (i.p.) to
O.-G.
654
BERGE and K. HOLE
induce surgical anaesthesia. Vertebra Thlo was penetrated with a dental burr and 1.5 mm of the spinal cord removed by excavation. At least 14 days were allowed for recovery. During this period, the rats were handled and trained daily in the tail-flick test situation. Testing of nociceptive response The spinal reflex to noxious stimulation was measured by the tail-flick method using an IITC Export Inc. Model 33 Analgesia Meter. Radiant heat was focused on a spot l-2 cm from the tip of the tail with beam intensity adjusted to give a reaction time of 6.5-7.5 set in control rats. A cut-off time of 14 set was employed in order to limit tissue damage. During each test, tail-flick latency was determined at 25min intervals. A pre-injection basal level was established for each rat as the mean latency of trials 2 and 3. During the tolerance experiment, morphine was injected immediately after the third trial and the percentage inhibition of the tail-flick response was calculated according to the formula T - BL %I = ___ x 100 14 - BL where T = tail-flick latency 50min after and BL = basal level as defined above.
injection,
Induction of tolerance Tolerance was induced in 5 transected and 8 intact rats by intraperitoneal injections of morphine hydrochloride (10 mg/kg) every 12 hr for 10 days. The same numbers of lesioned and intact rats received saline injections. Tail-flick latency was tested on days 1, 3, 5 and 7 after the first injection of the day. On day 9 the effect of 5-HTP on morphine analgesia in tolerant D,L-5-Hydroxytryptophan investigated. rats was (200 mg/kg, i.p.) was injected 25 min after the first morphine injection of the day. To inhibit peripheral decarboxylation of 5-HTP, the animals were pretreated with carbidopa (Merck Sharp & Dohme, 75 mg/kg, i.p. 25 min prior to 5-HTP). This procedure prevents the formation of excess 5-HT outside the central nervous system (Carter et al., 1978). The percentage inhibition was calculated as above, the tailflick latency 50 min after the 5-HTP injection being used to evaluate the drug response. Separate groups of 8 spinal and 12 intact rats were given carbidopa and 5-HTP only. These two groups were pretrained, but had not previously been injected. Withdrawal was precipitated on day 10. Naloxone (1 mg/kg subcutaneously in the neck) was injected 90min after morphine in 4 spinal and 4 intact tolerant rats, and in the same numbers of non-tolerant (saline-treated) animals. Each rat was then placed in a plexiglass cylinder (30 x 30cm) and observed for 5 min by means of a television camera and monitor. The occurrence of escape responses (defined as jumping to the top of the cylinder) in intact rats, and the
occurrence and duration of reflex activity (vigorous movements of the hindlimbs and the tail) in spinal animals were scored blindly from video recordings. Defaecation during the 5 min observation period was registered. In addition, 7 spinal rats received morphine hydrochloride intraperitoneally every 12 hr according to the following schedule: day l-2: lOmg/kg, day 335: 15 mg/kg, day 610: 20 mg/kg. Another group of 7 spinal rats received saline injections. On day 10, both groups were given 2 mg/kg naloxone subcutaneously 90 min after 20 mg/kg morphine hydrochloride. The animals were observed and scored as above. RESULTS
As previously observed (Berge and Hole, 1979). sectioning of the spinal cord caused a facilitation of the tail-flick response by approx. 25”, compared to the intact animals. Tolerance Initially, the injection of 10 mg/kg morphine hydrochloride strongly depressed the tail-flick response in intact as well as spinal animals, although the effect was somewhat less pronounced in the latter group (Fig. 1). All animals developed tolerance to morphine (P < 0.015 for both groups, Friedmans analysis of variance for nonparametric data). In both groups, the
IOOr
l\
= e
40
z .-s
C
.$
20
:
z n=5 n- 8 “- 5
0
-IOL
IS,
3rd
5th
7th
Day of injection
Fig. 1. Development of tolerance to morphine. Mean f SEM (application of cut off time does not permit calculation of SEM for the morphine treated groups). Animals were injected every 12 hr: M = intact rats, AA = spinal rats, lOmg/kg morphine hydrochloride per injection; O---O = intact rats, A--A = spinal rats, saline injected. Testing took place 50min after injection. Percent inhibition was calculated on basis of pre-injection
scores for each rat as described in the text.
Effect of morphine Table
1. The effect of 5-HTP on tolerance tail-flick response, Saline
Intact rats Spinal rats
1.0 + 4.6 (8) -2.0 + 1.3 (5)
to morphine, percentage mean f SEM
inhibition
of
5-HTP and morphine (Tolerant rats)
S-HTP rats)
(Non-tolerant
655
in the spinal rat
22.3 + 3.9*** (12) 19.3 + 6.2*** (8)
32.8 &- 13.3* (8) 26.2 _+ 15.6** (5)
* Significantly greater than saline group (P < 0.03), not significantly different from 5-HTP group (P > 0.45). ** Significantly greater than saline group (P < 0.01). not significantly different from 5-HTP group (P > 0.99). *** Significantly greater than saline group (P < 0.01, Mann-Whitney U-test). Number of rats in parentheses
effect of morphine
was
greatly
attenuated
by
treat-
to 4 and 31% inhibition for the spinal and intact groups respectively. On day 9 the corresponding figures where 0 and 4%, the response being measured immediately before the injection of 5-HTP. The tolerance-inducing treatment had no significant effect on the basal level tail-flick response, as the pre-injection scores of morphine-treated and salinetreated rats did not differ statistically during the period of tolerance development (intact rats: P > 0.05, spinal rats: P > 0.20, analysis of variance for repeated measures). ment-day
5 and
SHydroxytryptophan
by day
7 reduced
treatment
The effect of 5-HTP treatment on day 9 is shown in Table 1. The combination of morphine and 5-HTP significantly increased the tail-flick latency in tolerant intact (P < 0.03) and tolerant transected rats (P < 0.01, Mann-Whitney U-test). The increase was however not significantly different from the effect of 5-HTP alone in non-tolerant rats (spinal rats: P > 0.99. intact rats: P > 0.45, Mann-Whitney U-test), and was very much weaker than the effect of 10 mg/kg morphine in non-tolerant rats (Fig. 1, day 1). Withdrawal
Injection of naloxone precipitated acute withdrawal symptoms in the tolerant rats. In the first withdrawal experiment, intact as well as spinal tolerant rats showed increased defaecation compared to the nontolerant rats. All tolerant rats showed signs of withdrawal: jumping behaviour in the intact rats and
vigorous hindlimb and tail reflex activity in the spinal animals. None of the non-tolerant rats responded to the morphine/naloxone treatment with withdrawal behaviour according to the criteria described above. This difference between the tolerant and non-tolerant rats was statistically significant (P < 0.025, Fisher exact probability test). The results of the second withdrawal experiment, where tolerance was established in spinal rats through a somewhat stronger exposure to morphine, are given in Table 2. There was again no effect on the recorded behaviour of the morphine/naloxone treatment in non-tolerant rats. All the tolerant rats however exhibited very strong reflex activity commencing within 2min of the naloxone injection and continuing throughout the 5 min observation period (P < 0.002, Mann-Whitney U-test, compared to non-tolerant rats). Defaecation (number of boli) was also significantly greater in the tolerant group (P < 0.005, Mann-Whitney U-test). DISCUSSION
This study demonstrates that tolerance to the antinociceptive action of morphine may develop in the transected spinal cord. Although humoural influences on the spinal cord from the brain or hypophysis cannot be excluded, it seems likely that the tolerance and dependence observed in the spinal rats were due to functional changes in the spinal cord itself. Tolerance to morphine antinociception has previously been demonstrated after intrathecal administration of the drug (Yaksh, Kohl and Rudy, 1977),
Table 2. Naloxone-precipitated Defecationt Tolerant rats (n = 7) Non-tolerant rats (n = 7)
withdrawal
in spinal rats
Hindlimb
4.9 * 1.3’ 0.0 + 0.0
* Significantly greater than non-tolerant U-test). t Number of boli, mean + SEM. $ Seconds of reflex activity within the + SEM.
and tail reflexes: 96.0 + 12.0* 2.0 _t 1.2
group,
P < 0.005 (Mann-Whitney
observation
period
(5 min),
mean
656
O.-G.
BERGE
strongly suggesting that tolerance develops in the spinal cord. The technique employed did not, however, exclude the possibility of morphine acting on synaptic transmission in descending pathways. It has been proposed that morphine may induce tolerance by stimulating presynaptic opiate receptors on noradrenergic neurones (Llorens, Martres, Baudry and Schwartz, 1978). The present results show that functional changes in the monoaminergic systems and in other brain and brainstem structures proiecting to the ._ spinal cord are not necessary for the development of tolerance to morphine antinociception measured by the tail-flick test. It has also been proposed that morphine tolerance involves a conditioning process where environmental cues represent the conditioning stimulus and the drug effect serves as the unconditioned stimulus (Siegel, 1975). This hypothesis has been supported by other experiments in which the tail-flick test was used to assess analgesia and tolerance (Advocat, 1980). It seems unlikely however, that a cognitive model may account for these results where tolerance developed similarly in spinal and intact rats. Tolerance and dependence have been demonstrated in several tissues containing opiate receptors (for references. see Collier, 1980). For tolerance to the analgesic effect of morphine, spinal mechanisms may be of considerable importance, since morphine has a potent analgesic effect at the sninal level fWiller and Bussell, 1980; Yaksh and Rudy, 1977). For the complete symptomatology of dependence and withdrawal, however. several regions of the central nervous system may be involved. S-Hydroxytryptophan in combination with morphine significantly prolonged the tail-flick reaction time in tolerant spinal and intact rats, but the effect was not significantly greater than the effect of 5-HTP alone in non-tolerant rats. Thus 5-HTP administration did not abolish tolerance to morphine in the rat as it has been reported to do for tolerance to /?-endorphin (Hosobuchi, et al., 1977) and stimulation of nucleus raDhe maenus (Oliveras er ul.. 1978) in the cat. Evidently, tolerance to morphine does not produce cross tolerance to the antinociceptive effect of 5-HTP, indicating that the antinociceptive effect of increased 5-HT stimulation is not mediated via spinal opiate receptors, in agreement with the observation that naloxone does not block the analgesic effect of the 5-HT agonist 5-methoxy-N,N-dimethyltryptamine (Berge and Hole, 1980). 1
Y
,
,
Acknowledgements-This investigation was supported in part by the Norwegian Research Council for Science and the Humanities and by Norsk Medisinaldepots Fond. We wish to thank MS Anita Thomassen and MS Ida Haugan for their excellent technical assistance.
REFERENCES C. (1980). Evidence for conditioned tolerance tail-flick reflex. Behac. Neural Biol. 29: 385-389.
Advokat,
the
of
and K.
HOLE
Berge. O.-G. and Hole, K. (1979). Influence of descending 5-hydroxytryptamine (5-HT) pathways on a reflex re: sponse to radiant heat. Neurosci. Lefr. Suppl. 3: S257. Berge, O.-G. and Hole, K. (1980). Morphine analgesia measured by the tail-flick (TF) test is not reduced bvi 5-hydroxyt&tamine (5-HT) dlockers. Neurosci. Lrtr. Suppl. S:~SiG6. Bllsig. J. (1978). On the role of brain catecholamines in acute and chronic opiate action. In: Dr&opments in Opiate Research (Herz, A., Ed.). pp. 279-356. Marcel Dekker. New York. Carlsson. A.. Magnusson. T. and Roseneren. E. 119631. 5-Hydroxytryptamine in the spinal cordYnormally anh after transection. Experientia 19: 359. Carter. R. B., Dykstra. L. A. and Leander, J. D. (1978). Role of peripheral mechanisms in the behavioral effects of 5-hydroxytryptophan. Pharmuc. Biochem. Behac. 9: 249-253. Collier. H. 0. J. (1980). Cellular site of opiate dependence, Nature
283: 625-629.
Contreras. E., Tamayo. L. and Quijada. L. (1979). Effects of the irreversible inhibition of GABA transaminase upon some morphine effects. Neuropharmucoloyp 18: 309-313. Contreras. E., Tamayo, L.. Quijada, L. and Silva. E. (1973). Decrease of tolerance development to morphine by 5-hydroxytryptophan and some related drugs. Eur. J. Pharmac.
22: 339-343.
Herz. A. (1978). Sites of opiate action in the central nervous system. In: Development in Opiate Research (Herz, A.. Ed.). pp. 153-191. Marcel Dekker, New York. Ho, W. K. K., Wong, H. K. and Wen, H. L. (1979). The influence of electro-acupuncture on naloxone-induced morphine withdrawal-III. The effect of cyclic-AMP. Neuronharmacoloav
18: 865-869.
Hosobudhi, Y., Me&o, M., Adams. J. E. and Li. C. H. (1977). fi-Endorphin: development of tolerance and its reversal by 5-hydroxytryptophan in cats. Proc. natn. Acad. Sci. U.S.A. 74: 40174019.
Irwin, S., Houde. R. W.. Bennett. D. R.. Hendershot, L. C. and Seevers. M. H. (1951). The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J. Pharmac. exp. Ther. 101: 132-143. Kitano. T. and Takemori, A. E. (1979). Further studies on the enhanced affinity of opioid receptors for naloxone in morphine-dependent mice. J. Pharmac. exp. Ther. 209: 45ti61. . Lagowska, J., Calvino. B. and Ben-Ari. Y. (1978). lntraamygdaloid applications of naloxone elicits severe withdrawal signs in morphine dependent rats. Neurosci. Left. 8: 241-245. Llorens. C.. Martres. M. P.. Baudry. M. and Schwartz, J. C. (1978). Hypersensitivity to noradrenaline in cortex after chronic morphine: relevance to tolerance and dependence. Nature 274: 603-605. Martin. W. R. and Eades. C. G. (1964). A comparison between acute and chronic physical dependence in the chronic spinal dog. J. Phurmac. exp. Ther. 146: 385-394. Oliveras, J. L., Hosobuchi. Y.. Guilbaud. G. and Besson. J. M. (1978). Analgetic electrical stimulation of the feline nucleus raphe magnus: development of tolerance and its reversal by 5-HTP. Brain Res. 146: 404-409. Rastogi. R. B.. Merali. Z. and Singhal. R. L. (1980). Studies on catecholamine and 5-hydroxytryptamine metabolism in discrete brain areas of rats during morphine dependence and withdrawal. Cm. Pharmac. II: 201-205. Sarnivaara, L. (1969). Effect of 5-hydroxytryptamine on morphine analgesia in rabbits. Ann. Med. rup. Fenn. 47: 113-123. Siegel. S. (1975). Evidence from rats that morphine tolerance is a learned response. 1. camp. physiol. Pswhol. 89: 498-506.
Takemori. A. E.. Oka. T. and Nishiyama. N. (1973). Alter-
Etfect of morphine ation of analgesic receptor antagonist interaction induced by morphine. J. Pharmac. exp. Ther. 186: 261-265. Van Loon, G. R., De Souza, E. B. and Kim, C. (1978). Alteration in brain dopamine and serotonin metabolism during the development of tolerance to human b-endornhin in rats. Can. J. Phvsiol. Pharmac. 56: 1067-1071. Way, E. L., Ho, I. K. and Loh, H. H. (1974). Brain 5-hydroxytryptamine and cyclic AMP in morphine tolerance and dependence. Ado. Biochem. Psychopharmac. 10: 219-231. Wikler, A. and Frank, K. (1948). Hindlimb reflexes of chronic spinal dogs during cycles of addiction to mor-
in the spinal
rat
657
phine and methadon. J. Pharmac. exp. Ther. 94: 382-400. Wilier, J. C. and Bussell, B. (1980). Evidence for a direct spinal mechanism in morphine-induced inhibition of nociceptive reflexes in humans. Brain Res. 187: 212-21s. Yaksh. T. L. and Rudv, T. A. (1977). Studies on the direct spinal action of narcotics in‘the production of analgesia in the rat. J. Phartnac. exp. Ther. 202: 411428. Yaksh, T. L., Kohl, R. L. and Rudy, T. A. (1977). Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space. Eur. J. Pharmac. 42: 215-284.
Vol. 20. pp. 653 to 657. 198, Britain. All rlghlc reserved
Copyright
002X-3908 XI !070653-05502.00/O 0 1981 Pergamon Pres Ltd
TOLERANCE TO THE ANTINOCICEPTIVE EFFECT MORPHINE IN THE SPINAL RAT
OF
O.-G. BERGE and K. HOLE Institute of Physiology, University of Bergen, Bergen, Norway (Accepted 3 February 1981) Summary-Tolerance and withdrawal were studied in spinal and intact rats receiving morphine hydrochloride (l&20 mg/kg, i.p.) every 12 hr for 10 days. All rats developed tolerance to morphine-induced antinociception measured by the tail-flick method. Tolerance was not reversed by D,L-Shydroxytryptophan (200 mg/kg, i.p.). Naloxone (l-2 mg/kg) induced signs of withdrawal both in spinal and intact rats. Thus, functional changes in the aminergic systems and in brain and brainstem structures appear not to be necessary for tolerance to morphine-induced antinociception and for dependence on morphine. The experiment does not support the hypothesis that conditioning to environmental stimuli and changes in cognitive functions play a major role in tolerance development in the rat.
The development of tolerance to and dependence on morphine are accompanied by changes in the metabolism of dopamine, noradrenaline and serotonin, and in the function of monoaminergic (Way, Ho and Loh, 1974; Bllsig, 1978; Van Loon, De Souza and Kim, 1978; Rastogi, Merali and Singhal, 1980) and GABAergic (Contreras, Tamayo and Quijada, 1979) neurones. It has been shown that structures in the vicinity of the fourth ventricle are of importance for dependence and withdrawal (Herz, 1978), and Lagowska, Calvin0 and Ben-Ari (1978) reported strong withdrawal signs when naloxone was injected into the amygdala and caudate putamen in morphinedependent rats. These observations suggest that certain brain and brainstem structures are particularly important for certain behavioural expressions of dependence and withdrawal. Spinal mechanisms are also involved (Wikler and Frank, 1948; Martin and Eades, 1964), and tolerance and dependence may be related to changes in the sensitivity of opiate receptors (Takemori, Oka and Nishiyama, 1973; Kitano and Takemori, 1979) and in cyclic AMP formation (Way et a/., 1974; Ho, Wong and Wen, 1979). Previous studies have therefore shown that the development of tolerance and dependence is accompanied by functional changes at different levels of the central nervous system and in several transmitter systems. It is less clear which of these changes are necessary for the various behavioural aspects of tolerance and dependence. In the present study, the antinociceptive effect of chronic morphine administration has been investigated in spinal and in intact rats. Morphine depresses the nociceptive tail-flick response to radiant heat in spinal rats, although to a lesser extent than in intact animals (Irwin, Houde, Bennett, Hendershot and Key words: morphine, tolerance. spinal rats, S-hydroxytryptophan. 653
Seevers, 1951). If tolerance to the antinociceptive effect of morphine is due to changes in the sensitivity of opiate receptors or postsynaptic mechanisms, the development of tolerance to morphine might be expected in the tail-flick response of spinal as well as intact rats. If functional changes in monoaminergic systems are required, tolerance should develop in the intact animals only, since descending monoaminergic pathways to the spinal cord are completely destroyed by spinal transection (Carlsson, Magnusson and Rosengren, 1963). The effect of 5-hydroxytryptophan (S-HTP) administration on morphine antinociception in tolerant rats was also investigated. Previous reports have indicated that tolerance to the antinociceptive effect of /Y-endorphin (Hosobuchi, Meglio, Adams and Li, 1977) and electrical stimulation of the nucleus raphe magnus (Oliveras, Hosobuchi, Guilbaud and Besson, 1978) in cats may be reversed by treatment with 5-HTP. De-. creased (Contreras, Tamayo, Quijada and Silva, 1973) as well as accelerated (Way et al., 1974) development of tolerance to morphine has been found in connection with S-HTP administration in mice but these studies did not take into account the peripheral effects of 5-hydroxytryptamine (S-HT) accumulation which might have interacted with the actions of morphine (Sarnivaara, 1969). Excessive peripheral formation of 5-HT after 5-HTP administration was therefore prevented by pretreating with an inhibitor of peripheral decarboxylation (Carter, Dykstra and Leander, 1978).
METHODS
Sixty male Wistar rats (250g) were individually housed with free access to food and water. Room temperature was kept at 23 + 1°C. Testing took place in the middle of the light phase of a 12/12 hr light/dark cycle. Pentobarbital (40 mg/kg) and chloral hydrate (130 mg/kg) were given intraperitoneally (i.p.) to
O.-G.
654
BERGE and K. HOLE
induce surgical anaesthesia. Vertebra Thlo was penetrated with a dental burr and 1.5 mm of the spinal cord removed by excavation. At least 14 days were allowed for recovery. During this period, the rats were handled and trained daily in the tail-flick test situation. Testing of nociceptive response The spinal reflex to noxious stimulation was measured by the tail-flick method using an IITC Export Inc. Model 33 Analgesia Meter. Radiant heat was focused on a spot l-2 cm from the tip of the tail with beam intensity adjusted to give a reaction time of 6.5-7.5 set in control rats. A cut-off time of 14 set was employed in order to limit tissue damage. During each test, tail-flick latency was determined at 25min intervals. A pre-injection basal level was established for each rat as the mean latency of trials 2 and 3. During the tolerance experiment, morphine was injected immediately after the third trial and the percentage inhibition of the tail-flick response was calculated according to the formula T - BL %I = ___ x 100 14 - BL where T = tail-flick latency 50min after and BL = basal level as defined above.
injection,
Induction of tolerance Tolerance was induced in 5 transected and 8 intact rats by intraperitoneal injections of morphine hydrochloride (10 mg/kg) every 12 hr for 10 days. The same numbers of lesioned and intact rats received saline injections. Tail-flick latency was tested on days 1, 3, 5 and 7 after the first injection of the day. On day 9 the effect of 5-HTP on morphine analgesia in tolerant D,L-5-Hydroxytryptophan investigated. rats was (200 mg/kg, i.p.) was injected 25 min after the first morphine injection of the day. To inhibit peripheral decarboxylation of 5-HTP, the animals were pretreated with carbidopa (Merck Sharp & Dohme, 75 mg/kg, i.p. 25 min prior to 5-HTP). This procedure prevents the formation of excess 5-HT outside the central nervous system (Carter et al., 1978). The percentage inhibition was calculated as above, the tailflick latency 50 min after the 5-HTP injection being used to evaluate the drug response. Separate groups of 8 spinal and 12 intact rats were given carbidopa and 5-HTP only. These two groups were pretrained, but had not previously been injected. Withdrawal was precipitated on day 10. Naloxone (1 mg/kg subcutaneously in the neck) was injected 90min after morphine in 4 spinal and 4 intact tolerant rats, and in the same numbers of non-tolerant (saline-treated) animals. Each rat was then placed in a plexiglass cylinder (30 x 30cm) and observed for 5 min by means of a television camera and monitor. The occurrence of escape responses (defined as jumping to the top of the cylinder) in intact rats, and the
occurrence and duration of reflex activity (vigorous movements of the hindlimbs and the tail) in spinal animals were scored blindly from video recordings. Defaecation during the 5 min observation period was registered. In addition, 7 spinal rats received morphine hydrochloride intraperitoneally every 12 hr according to the following schedule: day l-2: lOmg/kg, day 335: 15 mg/kg, day 610: 20 mg/kg. Another group of 7 spinal rats received saline injections. On day 10, both groups were given 2 mg/kg naloxone subcutaneously 90 min after 20 mg/kg morphine hydrochloride. The animals were observed and scored as above. RESULTS
As previously observed (Berge and Hole, 1979). sectioning of the spinal cord caused a facilitation of the tail-flick response by approx. 25”, compared to the intact animals. Tolerance Initially, the injection of 10 mg/kg morphine hydrochloride strongly depressed the tail-flick response in intact as well as spinal animals, although the effect was somewhat less pronounced in the latter group (Fig. 1). All animals developed tolerance to morphine (P < 0.015 for both groups, Friedmans analysis of variance for nonparametric data). In both groups, the
IOOr
l\
= e
40
z .-s
C
.$
20
:
z n=5 n- 8 “- 5
0
-IOL
IS,
3rd
5th
7th
Day of injection
Fig. 1. Development of tolerance to morphine. Mean f SEM (application of cut off time does not permit calculation of SEM for the morphine treated groups). Animals were injected every 12 hr: M = intact rats, AA = spinal rats, lOmg/kg morphine hydrochloride per injection; O---O = intact rats, A--A = spinal rats, saline injected. Testing took place 50min after injection. Percent inhibition was calculated on basis of pre-injection
scores for each rat as described in the text.
Effect of morphine Table
1. The effect of 5-HTP on tolerance tail-flick response, Saline
Intact rats Spinal rats
1.0 + 4.6 (8) -2.0 + 1.3 (5)
to morphine, percentage mean f SEM
inhibition
of
5-HTP and morphine (Tolerant rats)
S-HTP rats)
(Non-tolerant
655
in the spinal rat
22.3 + 3.9*** (12) 19.3 + 6.2*** (8)
32.8 &- 13.3* (8) 26.2 _+ 15.6** (5)
* Significantly greater than saline group (P < 0.03), not significantly different from 5-HTP group (P > 0.45). ** Significantly greater than saline group (P < 0.01). not significantly different from 5-HTP group (P > 0.99). *** Significantly greater than saline group (P < 0.01, Mann-Whitney U-test). Number of rats in parentheses
effect of morphine
was
greatly
attenuated
by
treat-
to 4 and 31% inhibition for the spinal and intact groups respectively. On day 9 the corresponding figures where 0 and 4%, the response being measured immediately before the injection of 5-HTP. The tolerance-inducing treatment had no significant effect on the basal level tail-flick response, as the pre-injection scores of morphine-treated and salinetreated rats did not differ statistically during the period of tolerance development (intact rats: P > 0.05, spinal rats: P > 0.20, analysis of variance for repeated measures). ment-day
5 and
SHydroxytryptophan
by day
7 reduced
treatment
The effect of 5-HTP treatment on day 9 is shown in Table 1. The combination of morphine and 5-HTP significantly increased the tail-flick latency in tolerant intact (P < 0.03) and tolerant transected rats (P < 0.01, Mann-Whitney U-test). The increase was however not significantly different from the effect of 5-HTP alone in non-tolerant rats (spinal rats: P > 0.99. intact rats: P > 0.45, Mann-Whitney U-test), and was very much weaker than the effect of 10 mg/kg morphine in non-tolerant rats (Fig. 1, day 1). Withdrawal
Injection of naloxone precipitated acute withdrawal symptoms in the tolerant rats. In the first withdrawal experiment, intact as well as spinal tolerant rats showed increased defaecation compared to the nontolerant rats. All tolerant rats showed signs of withdrawal: jumping behaviour in the intact rats and
vigorous hindlimb and tail reflex activity in the spinal animals. None of the non-tolerant rats responded to the morphine/naloxone treatment with withdrawal behaviour according to the criteria described above. This difference between the tolerant and non-tolerant rats was statistically significant (P < 0.025, Fisher exact probability test). The results of the second withdrawal experiment, where tolerance was established in spinal rats through a somewhat stronger exposure to morphine, are given in Table 2. There was again no effect on the recorded behaviour of the morphine/naloxone treatment in non-tolerant rats. All the tolerant rats however exhibited very strong reflex activity commencing within 2min of the naloxone injection and continuing throughout the 5 min observation period (P < 0.002, Mann-Whitney U-test, compared to non-tolerant rats). Defaecation (number of boli) was also significantly greater in the tolerant group (P < 0.005, Mann-Whitney U-test). DISCUSSION
This study demonstrates that tolerance to the antinociceptive action of morphine may develop in the transected spinal cord. Although humoural influences on the spinal cord from the brain or hypophysis cannot be excluded, it seems likely that the tolerance and dependence observed in the spinal rats were due to functional changes in the spinal cord itself. Tolerance to morphine antinociception has previously been demonstrated after intrathecal administration of the drug (Yaksh, Kohl and Rudy, 1977),
Table 2. Naloxone-precipitated Defecationt Tolerant rats (n = 7) Non-tolerant rats (n = 7)
withdrawal
in spinal rats
Hindlimb
4.9 * 1.3’ 0.0 + 0.0
* Significantly greater than non-tolerant U-test). t Number of boli, mean + SEM. $ Seconds of reflex activity within the + SEM.
and tail reflexes: 96.0 + 12.0* 2.0 _t 1.2
group,
P < 0.005 (Mann-Whitney
observation
period
(5 min),
mean
656
O.-G.
BERGE
strongly suggesting that tolerance develops in the spinal cord. The technique employed did not, however, exclude the possibility of morphine acting on synaptic transmission in descending pathways. It has been proposed that morphine may induce tolerance by stimulating presynaptic opiate receptors on noradrenergic neurones (Llorens, Martres, Baudry and Schwartz, 1978). The present results show that functional changes in the monoaminergic systems and in other brain and brainstem structures proiecting to the ._ spinal cord are not necessary for the development of tolerance to morphine antinociception measured by the tail-flick test. It has also been proposed that morphine tolerance involves a conditioning process where environmental cues represent the conditioning stimulus and the drug effect serves as the unconditioned stimulus (Siegel, 1975). This hypothesis has been supported by other experiments in which the tail-flick test was used to assess analgesia and tolerance (Advocat, 1980). It seems unlikely however, that a cognitive model may account for these results where tolerance developed similarly in spinal and intact rats. Tolerance and dependence have been demonstrated in several tissues containing opiate receptors (for references. see Collier, 1980). For tolerance to the analgesic effect of morphine, spinal mechanisms may be of considerable importance, since morphine has a potent analgesic effect at the sninal level fWiller and Bussell, 1980; Yaksh and Rudy, 1977). For the complete symptomatology of dependence and withdrawal, however. several regions of the central nervous system may be involved. S-Hydroxytryptophan in combination with morphine significantly prolonged the tail-flick reaction time in tolerant spinal and intact rats, but the effect was not significantly greater than the effect of 5-HTP alone in non-tolerant rats. Thus 5-HTP administration did not abolish tolerance to morphine in the rat as it has been reported to do for tolerance to /?-endorphin (Hosobuchi, et al., 1977) and stimulation of nucleus raDhe maenus (Oliveras er ul.. 1978) in the cat. Evidently, tolerance to morphine does not produce cross tolerance to the antinociceptive effect of 5-HTP, indicating that the antinociceptive effect of increased 5-HT stimulation is not mediated via spinal opiate receptors, in agreement with the observation that naloxone does not block the analgesic effect of the 5-HT agonist 5-methoxy-N,N-dimethyltryptamine (Berge and Hole, 1980). 1
Y
,
,
Acknowledgements-This investigation was supported in part by the Norwegian Research Council for Science and the Humanities and by Norsk Medisinaldepots Fond. We wish to thank MS Anita Thomassen and MS Ida Haugan for their excellent technical assistance.
REFERENCES C. (1980). Evidence for conditioned tolerance tail-flick reflex. Behac. Neural Biol. 29: 385-389.
Advokat,
the
of
and K.
HOLE
Berge. O.-G. and Hole, K. (1979). Influence of descending 5-hydroxytryptamine (5-HT) pathways on a reflex re: sponse to radiant heat. Neurosci. Lefr. Suppl. 3: S257. Berge, O.-G. and Hole, K. (1980). Morphine analgesia measured by the tail-flick (TF) test is not reduced bvi 5-hydroxyt&tamine (5-HT) dlockers. Neurosci. Lrtr. Suppl. S:~SiG6. Bllsig. J. (1978). On the role of brain catecholamines in acute and chronic opiate action. In: Dr&opments in Opiate Research (Herz, A., Ed.). pp. 279-356. Marcel Dekker. New York. Carlsson. A.. Magnusson. T. and Roseneren. E. 119631. 5-Hydroxytryptamine in the spinal cordYnormally anh after transection. Experientia 19: 359. Carter. R. B., Dykstra. L. A. and Leander, J. D. (1978). Role of peripheral mechanisms in the behavioral effects of 5-hydroxytryptophan. Pharmuc. Biochem. Behac. 9: 249-253. Collier. H. 0. J. (1980). Cellular site of opiate dependence, Nature
283: 625-629.
Contreras. E., Tamayo. L. and Quijada. L. (1979). Effects of the irreversible inhibition of GABA transaminase upon some morphine effects. Neuropharmucoloyp 18: 309-313. Contreras. E., Tamayo, L.. Quijada, L. and Silva. E. (1973). Decrease of tolerance development to morphine by 5-hydroxytryptophan and some related drugs. Eur. J. Pharmac.
22: 339-343.
Herz. A. (1978). Sites of opiate action in the central nervous system. In: Development in Opiate Research (Herz, A.. Ed.). pp. 153-191. Marcel Dekker, New York. Ho, W. K. K., Wong, H. K. and Wen, H. L. (1979). The influence of electro-acupuncture on naloxone-induced morphine withdrawal-III. The effect of cyclic-AMP. Neuronharmacoloav
18: 865-869.
Hosobudhi, Y., Me&o, M., Adams. J. E. and Li. C. H. (1977). fi-Endorphin: development of tolerance and its reversal by 5-hydroxytryptophan in cats. Proc. natn. Acad. Sci. U.S.A. 74: 40174019.
Irwin, S., Houde. R. W.. Bennett. D. R.. Hendershot, L. C. and Seevers. M. H. (1951). The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J. Pharmac. exp. Ther. 101: 132-143. Kitano. T. and Takemori, A. E. (1979). Further studies on the enhanced affinity of opioid receptors for naloxone in morphine-dependent mice. J. Pharmac. exp. Ther. 209: 45ti61. . Lagowska, J., Calvino. B. and Ben-Ari. Y. (1978). lntraamygdaloid applications of naloxone elicits severe withdrawal signs in morphine dependent rats. Neurosci. Left. 8: 241-245. Llorens. C.. Martres. M. P.. Baudry. M. and Schwartz, J. C. (1978). Hypersensitivity to noradrenaline in cortex after chronic morphine: relevance to tolerance and dependence. Nature 274: 603-605. Martin. W. R. and Eades. C. G. (1964). A comparison between acute and chronic physical dependence in the chronic spinal dog. J. Phurmac. exp. Ther. 146: 385-394. Oliveras, J. L., Hosobuchi. Y.. Guilbaud. G. and Besson. J. M. (1978). Analgetic electrical stimulation of the feline nucleus raphe magnus: development of tolerance and its reversal by 5-HTP. Brain Res. 146: 404-409. Rastogi. R. B.. Merali. Z. and Singhal. R. L. (1980). Studies on catecholamine and 5-hydroxytryptamine metabolism in discrete brain areas of rats during morphine dependence and withdrawal. Cm. Pharmac. II: 201-205. Sarnivaara, L. (1969). Effect of 5-hydroxytryptamine on morphine analgesia in rabbits. Ann. Med. rup. Fenn. 47: 113-123. Siegel. S. (1975). Evidence from rats that morphine tolerance is a learned response. 1. camp. physiol. Pswhol. 89: 498-506.
Takemori. A. E.. Oka. T. and Nishiyama. N. (1973). Alter-
Etfect of morphine ation of analgesic receptor antagonist interaction induced by morphine. J. Pharmac. exp. Ther. 186: 261-265. Van Loon, G. R., De Souza, E. B. and Kim, C. (1978). Alteration in brain dopamine and serotonin metabolism during the development of tolerance to human b-endornhin in rats. Can. J. Phvsiol. Pharmac. 56: 1067-1071. Way, E. L., Ho, I. K. and Loh, H. H. (1974). Brain 5-hydroxytryptamine and cyclic AMP in morphine tolerance and dependence. Ado. Biochem. Psychopharmac. 10: 219-231. Wikler, A. and Frank, K. (1948). Hindlimb reflexes of chronic spinal dogs during cycles of addiction to mor-
in the spinal
rat
657
phine and methadon. J. Pharmac. exp. Ther. 94: 382-400. Wilier, J. C. and Bussell, B. (1980). Evidence for a direct spinal mechanism in morphine-induced inhibition of nociceptive reflexes in humans. Brain Res. 187: 212-21s. Yaksh. T. L. and Rudv, T. A. (1977). Studies on the direct spinal action of narcotics in‘the production of analgesia in the rat. J. Phartnac. exp. Ther. 202: 411428. Yaksh, T. L., Kohl, R. L. and Rudy, T. A. (1977). Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space. Eur. J. Pharmac. 42: 215-284.