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Effects of clonidine on morphine withdrawal signs in the rat
European Journal of Pharmacology 30 (1975) 93-99 O North-Holland Publishing Company
EFFECTS OF CLONIDINE ON MORPHINE WITHDRAWAL
SIGNS IN THE RAT
Liang-Fu TSENG, Horace H. LOH and Eddie T. WEI Department of Pharmacology, University of California, San Francisco, California 94143, and School o f Public Health, University o f California, Berkeley, California 94720, U.S.A.
Received 17 July 1974, revised MS received 1 October 1974, accepted 7 October 1974 L.-F. TSENG, H.H. LOH and E.T. WEt, Effects of elonidine on morphine withdrawal signs in the rat, European J. Pharmacol. 30 (1975) 93-99. The influence of clonidine on the naloxone-induced withdrawal signs, escape attempts and precipitated shakes, was studied in morphine-dependent rats. Clonidine injected i.p. or intraventricularly (i. vent.) inhibited precipitated shakes and potentiated escape attempts induced by naloxone in morphine-dependent rats. Under pentobarbital anesthesia, precipitated shakes and ice water-induced wet shakes were inhibited by clonidine and norepinephrine. Clonidine injected i.vent, reduced body temperatures in morphine-implanted rats and in placebo pellet-implanted rats. Naloxone reduced body temperatures only in morphine-dependent rats but not in placebo pellet-implanted rats. We suggest that clonidine modulates morphine withdrawal signs by potentiating the behavior associated with heat dissipation (escape attempts) and inhibiting the behavior associated with heat gain mechanisms (precipitated shakes). These effects may occur via stimulation of central noradrenergic mechanisms. Thermoregulatory behavior
Clonidine
Norepinephrine
1. Introduction The relationship of catecholamines to morphine analgesia, tolerance and physical dependence has been the subject of many studies (see reviews by Gunne, 1963; Way and Shen, 1971). Recently, several investigations have focused on the role of catecholamines in the manifestation of the withdrawal syndrome (Matilla et al., 1968; Schwartz and Eidelberg, 1970; Maruyama and Takemori, 1973). In these investigations, one commonly used technique was the administration of a-methyltyrosine to inhibit catecholamine synthesis and then to observe the withdrawal syndrome after depletion of brain catecholamines. Information on the Converse condition, that is, the effects of adrenergic stimulation on withdtawal behavior, has been lacking because peripherally administered catecholamines do not gain ready access to brain cells. Clonidine, a centrally acting antihypertensive agent (Schmitt and Schmitt, 1969; Bousquet and Guertzenstein, 1973) which is effective when administered by the peripheral route, is thought to be a
Morphine abstinence signs
central norepinephrine-like stimulant (Hoefke and Kobinger, 1966; Anddn et al., 1970). In this paper, we report the effects of central administration of clonidine and norepinephrine on the naloxone-precipitated abstinence syndrome in morphine-dependent rats.
2. Materials and methods 2.1. Materials
Male Sprague-Dawley rats (Horton Laboratories, Oakland, Calif.) weighing 2 2 0 - 2 7 0 g were used throughout these experiments. Clonidine (2-(2,6dichlorophenylamino)-2-imidazoline • HC1) was obtained from Boehringer Ingelheim, GMBH, Ingelheim, Germany; naloxone • HC1 from Endo Laboratories, Garden City, N.Y., and 1-norepinephrine hydrochloride from Sigma Chemical Co., St. Louis, Missouri. All doses refer to the salt forms of the drug.
94
L..F. Tseng et aL, Clonidine and morphine abstinence signs
2.2. Methods 2.2.1. Observation o f naloxone-precipitated withdrawal in morphine-implanted rats To produce dependence on morphine, a morphine pellet, containing 75 mg of morphine base (Gibson and Tingstad, 1970), was implanted s.c. in the lower abdominal wall. 3 days after the pellet implant, animals were weighed and placed in one gallon jars. The abstinence syndrome was precipitated by injecting naloxone • HC1, 4 mg/kg s.c. Clonidine - HC1 or saline was administered i.p. 10 min before the injection of naloxone. After injection of naloxone, the withdrawal signs of escape attempts and precipitated shakes were counted continuously for 10 min according to Wei et al. (1973a). 2.2.2. L vent. and intracerebral {i.c.) injections For the i.vent, injections, rat brains were stereotaxically implanted with 20 gauge stainless steel guide cannulae 2 days before morphine pellet implantation. 3 days after the morphine implant, 5/al of saline or a saline solution of clonidine - HC1 (5/ag and I0 /ag total dose) was injected unilaterally into the lateral ventricle of unanesthetized rats. Natoxone-precipirated withdrawal was then induced as described in section 2.2.1. For i.vent, and i.c. microinjections, we used the methods described by Hedge et al. (1966). Animals were anesthetized with sodium pentobarbital, 50 mg/kg i.p. 1-Norepinephrine • HC1, dissolved in saline was injected bilaterally in a volume of 0.5/al per side into the periaqueductal grey. Norepinephrine was injected in the periaqueductal grey because recent investigations indicate that this brain area may be an important site for the primary pharmacological actions of morphine (Yaksh and Pert, 1974; Mayer and Liebeskind, 1974). The stereotaxic coordinates for the periaqueductal grey, based on the atlas of KiSnig and Klippel (1963) were posterior, 0.5 mm; lateral, 0.5 mm; and vertical from the brain surface, 5.5 mm. 2.2.3. 'Precipitated shakes' and 'wet shakes'in pentobarbital anes'thetized rats A withdrawal sign, characterized by repetitive shaking movements of the body and termed 'wet dog shakes' by many investigators (Martin et al., 1963), can be elicited by naloxone in morphine-dependent
rats anesthetized with sodium pentobarbital (Wei, 1973). A similar shaking behavior can also be elicited in naive sodium pentobarbital-anesthetized rats, after immersion in ice water (Wei et al., 1973b). The effect of clonidine and norepinephrine on naloxone precipitated shakes in morphine-dependent rats hereafter termed 'precipitated shakes', and ice water-induced shakes, hereafter termed 'wet shakes', was studied. Morphine-implanted rats were injected with sodium pentobarbital, 40 mg/kg s.c., 30 min before the injection of clonidine. 10 min after the injections of clonidine or norepinephrine, animals were injected with naloxone - HC1, 4 mg/kg i.p. and the number of precipitated shakes counted for 10 rain. For observing wet shakes, naive rats were injected with sodium pentobarbital, 50 mg/kg s.c. 30 min before the injection of clonidine. 10 min after clonidine, rats were immersed in ice water and the number of wet shakes counted for 5 min (Wei et al., 1973b). 2.2.4. Measurement o f body temperature The rats were restrained in small animal holders 30 min before drug injection. Clonidine • HC1, in a dose of 15/ag, was injected i.vent. 10 min before the injection of naloxone - HC1 (4 mg/kg i.p.). The core temperature was measured with a thermistor probe inserted at least 6 cm into the rectum, taped to the base of the tail and connected to a telethermometer. The body temperatures were recorded at 5 - 1 0 min intervals for 2 hr after naloxone injection. 2.2.5. Statistical evaluation The Spearman Rank Correlation test was used to see if a correlation between the precipitated shakes and escape attempts was obtained. The MannWhitney U-test was used for the statistical evaluation of the significance of the observed differences.
3. Results
3.1. Withdrawal signs after naloxone injection in morphine-dependent rats Untreated morphine-dependent rats placed in jars did not manifest any shaking behavior or escape attempts. The injection of naloxone, 4 mg/kg s.c. elicited the abstinence signs of precipitated shakes and
L.-F. Tseng et al., Clonidine and morphine abstinence signs
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Fig. 1. Lack of correlation between the escape attempts and precipitated shake behavior after the injection of naloxone in morphine-dependent rats. Rats were rendered dependent on morphine by the implantation of a morphine pellet for 3 days. Animals were injected with naloxone • HCI, 4 mg/kg s.c., and observed for withdrawal signs for 10 min. Each dot represents the frequency of precipitated shakes and escape attempts of each animal. 12s = - 0.016,p > 0.05.
escape attempts. Other abstinence signs such as increased locomotor activity, blanching of the ears, teeth chattering, seminal emissions and diarrhea were also observed. The effect of clonidine on the number of precipitated shakes and escape attempts was measured in the present study. These withdrawal signs may be readily quantified and are highly characteristic of the morphine abstinence syndrome in the rat (Wei et al., 1973a). Moreover, the occurrence of the precipitated shakes and escape attempts in a given rat appears to be an independent event, as shown by the data in fig. I. 3.2. Effect o f clonidine on the naloxone-precipitated withdrawal signs
Fig. 2. Effect of clonidine injected i.p. on the frequency of naloxone-induced precipitated shakes and escape attempts. Clonidine or saline was administered (i.p.) 10 min before the injection o f naloxone. The withdrawal signs were observed for 10 min after the injection o f naloxone • HC1 (4 mg/kg s.c.). Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the frequency of the withdrawal signs. *p < 0.05; **p < 0.01; ***p < 0.002. 50 50
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Fig. 3. Effect of clonidine injected i.vent, on the frequency of naloxone-induced precipitated shakes and escape attempts. Clonidine or saline was administered i.vent. 10 min before the injection o f naloxone • HCI (4 mg/kg s.c.). The withdrawal signs were observed for 10 min after the injection of naloxone. Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the frequency of the withdrawal signs. *p < 0.02; **p < 0.01.
L.-F. Tseng et al., Clonidine and morphine abstinence signs
96
served following n a l o x o n e injection were p o t e n t i a t e d by clonidine p r e t r e a t m e n t . In order to d e t e r m i n e if the effect o f clonidine on w i t h d r a w a l was a central or peripheral p h e n o m e n o n , clonidine (5 and 15/~g) was injected i.vent, and withdrawal precipitated w i t h n a l o x o n e . Clonidine injected i.vent, decreased the f r e q u e n c y o f precipitated shakes and p o t e n t i a t e d the n u m b e r of escape a t t e m p t s (fig. 3). Thus, the effects o f clonidine on withdrawal behavior was qualitatively similar when clonidine was administered by either the i.p. or the i.venr, route.
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Fig. 5. Effect of norepinephrine on naloxone-induced precipitated shakes and ice water-induced wet shakes. Rats were anesthetized with sodium pentobarbital 20-30 min before the injection of norepinephrine. Norepinephrine (5 ~g) was injected into the periaqueductal grey (PAG) 10 min before the injection of naloxone • HCI (4 mg/kg i.p.) or bathing in ice water. The precipitated shakes were observed for 10 min and wet shakes were observed for 5 min. Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the number of shakes. *p < 0.002. SAL = saline; NE = norepinephrine.
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Fig. 4. Effect of clonidine on naloxone-induced precipitated shakes and ice water-induced wet shakes. Rats were anesthetized with sodium pentobarbital 20-30 min before the injection of clonidine. Clonidine was injected 10 min before the injection of naloxone • HC1 (4 mg/kg s.c.) or bathing in ice water. The precipitated shakes were observed for 10 min and wet shakes were observed for 5 min. Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the number of shakes, i.p. = intraperitoneal injection; i.vent. = intraventricular injection. *p < 0.01 ; **p < 0.002. SAL = saline; CLO = clonidine.
inhibited by clonidine, administered i.vent, or i.p. (fig. 4). Clonidine injected i.vent, or i.p. also antagonized the wet shakes p r o v o k e d by ice water in naive rats anesthetized with s o d i u m p e n t o b a r b i t a l (fig. 4). A 5 ~tg dose o f norepinephrine injected into the periaq u e d u c t a l grey region also antagonized the naloxoneinduced-precipitated shakes and the wet shakes provoked by ice water (fig. 5). To determine if clonidine had morphine-like properties, we pretreated rats with a 5 m g / k g s.c. dose o f n a l o x o n e before the administration o f clonidine (0.4 m g / k g i.p.). N a l o x o n e p r e t r e a t m e n t did not reverse the i n h i b i t o r y effects o f clonidine on the wet shake response (clonidine = 0, clonidine + n a l o x o n e = 0 shakes, n = 5).
L.-F. Tseng et al., Clonidine and morphine abstinence signs
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Fig. 6. Changes in body temperature following the injection of clonidine and naloxone. (a) morphine implanted rats; (b) placebo-implanted rats; S = saline; Nx = naloxone; C = clonidine; n = 5-6. ~c~ injected i.vent, with clonidine (15 ,g) or saline.~ injected i.p. with naloxone (4 mg/kg) or saline. 3.4. Effect o f clonidine and naloxone on body temperature
Clonidine • HCI (15/ag) injected i.vent, produced hypothermia in morphine-implanted rats and in placebo-implanted rats (fig. 6). Naloxone • HCI (4 mg/kg) reduced the body temperature in morphine-dependent rats but did not change the body temperature in placebo-implanted rats. The hypothermia produced by clonidine in morphine-implanted rats and in placebo-implanted rats was not affected by naloxone.
4. Discussion
We have recently suggested that certain morphine withdrawal signs resemble the behavior consequent to a central derangement of thermoregulatory mechanisms (Wei et al., 1974). In particular, shaking was considered to be a form of shivering or heat gain behavior, whereas escape attempts were interpreted as heat loss behavior (Hainsworth, 1967). Furthermore, we speculated that drugs which make animals 'feel hot' may potentiate escape and antagonize the shakes of morphine withdrawal.
97
Clonidine and, by inference, other central adrenergic stimulants, may be an example of a drug which modulates morphine withdrawal signs by increasing the likelihood of behavior associated with heat dissipation. The results of this study, at least, clearly indicate that shaking was inhibited by clonidine, whereas the frequency of escape attempts was potentiated. It is likely that the inhibitory effects of clonidine on shaking were mediated via a central noradrenergic mechanism, since the inhibition of shaking could also be obtained by intracerebral injection of norepinephfine. In the following sections, we summarize some of the evidence which is consistent with this interpretation of clonidine's effects on morphine withdrawal. The neuroanatomical pathways of precipitated shakes and wet shakes appear to originate from the medial portions of the brainstem (Wei, 1973; Wei et al., 1973c). This brain region is similar to the 'shivering center' described by Hemingway (1963). Shaking may be linked to shivering mechanisms because maneuvers which elevate body temperature, such as placing rats in a 3 4 - 3 7 °C environment or on a hot blanket or in warm water, inhibit the shaking response precipitated by naloxone or elicited by water. Domer and Feldberg (1960) showed that norepinephrine injections into the lateral ventricles of cats inhibited shivering produced by i.p. injection of pentobarbitone sodium or by i.m. injection of chlorpromazinc. Other workers have also found that i.vent. administration of norepinephrine suppresses shivering in the chick (Allen et al., 1970), rat (Stone and Mendlinger, 1974) and pigeon (Hissa and Rautenberg, 1974). Thus, it appears plausible that clonidine suppresses shivering by activating central noradrenergic mechanisms and that shaking behavior, which shares common neural pathways with shivering, is also concomitantly suppressed. Inhibition of brain monoamine oxidase by pargyline results in a potentiation of jumping behavior during naloxone-precipitated withdrawal in mice (Iwamoto et al., 1971). The potentiation of jumping has been attributed to the elevation of biogenic amines in brain produced by monoarnine oxidase inhibition. Chiosa et al. (1968) have also reported that imipramine, a drug which inhibits the reuptake of norepinephrine into nerve endings, potentiates morphine withdrawal activity in mice. Thus, our finding that clonidine potentiates the escape behavior of pre-
98
L.-F. Tseng et al., Clonidine and morphine abstinence signs
cipitated withdrawal is consistent with the notion that adrenergic stimulation results in an enhancement of one withdrawal sign: the urge to escape or jump. Wei et al. (1973b) showed that morphine inhibited the wet shakes provoked by ice water in naive rats. Naloxone reversed the inhibitory effects of morphine. Since the inhibitory effects of clonidine on shaking was not antagonized by naloxone, it appears clear that the adrenergic receptors involved in clonidine effects and opiate receptors fundamentally differ in their response to naloxone. The central actions o f catecholamines may be anatomically specific. Behavioral arousal has been observed in rats after i.vent, or intrahypothalamic injection of norepinephrine (Benkert, 1969; Segal and Mandell, 1970). However, other investigators found predominantly depressant effects such as sedation, analgesia, stupor and catalepsy alter i.c. or i.ci. injection of catecholamines (Marley and Stephenson, 1972). Bousquet and Guerzenstein (1973) showed that the hypotensive effect of i.vent, administered clonidine changed to a hypertensive effect when the cerebral aqueduct was cannulated. These results suggest that adrenergic stimulation o f forebrain elements may result in excitatory effects whereas depressant effects occur mainly in the brain stem. It would be of interest to determine if the effects of clonidine on the two withdrawal signs may be dissociated according to brain region, that is, potentiation o f escape arising from activation o f adrenergic receptors in the forebrain and suppression of wet shakes due to activation of adrenergic receptors in areas around the periaqueductal grey.
Acknowledgements This work was supported by Grants DA 00091 and DA 00564 from the National Institutes of Health. We are grateful for the generous supply of clonidine from Boehringer Ingelhelm and of naloxone • HC1 from Endo Laboratories. The authors also gratefully acknowledge the excellent technical assistance of Ms. Sheelah Sigel and Mr. J.B. Cunningham and the assistance of Barbara Halperin in preparation of the manuscript.
References Allen, D.J., K.N. Garg and E. Marley, 1970, Mode of action of c~-methyl-noradrenahne on temperature and oxygen consumption in young chickens, Brit. J. Pharmacol. 38, 667. And6n, N.E., H. Corrodi, K. Fuxe, B. H6kfelt, T. H6kfelt, T. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9, 513. Benkert, O., 1969, Measurement of hyperactivity in rats in a dose-response curve after intrahypothalamic norepinephrine injection, Life Sci. 8,943. Bousquet, P. and P.G. Guertzenstein, 1973, Localization of the central cardiovascular action of clonidine, Brit. J. Pharmacol. 49,573. Chiosa, L., S. Dumitrascu and A. Banaru, 1968, The inlluence of imipramine on the "abstinence syndrome" to morphine in mice, Intern. J. Neuropharmacol. 7,161. Domer, F.R. and W. Feldberg, 1960, Tremor in cats: the effect of administration of drugs into the cerebral ventricles, Brit. J. Pharmacol. 15,578. Gibson, R.D. and J.E. Tingstad, 1970, Formulation of a morphine implantation pellet suitable for tolerance-physical dependence studies in mice, J. Pharm. Sci. 59,426. Gunnc, L.M., 1963, Catecholamines and 5-hydroxytryptamine in morphine tolerance and withdrawal, Acta Physiol. Scand. 58, Suppl. 204, 1. Hainsworth, F.R., 1967, Saliva spreading activity and body temperature regulation in the rat, Amer. J. Physiol. 212, 1288. Hedge, G.A., M.B. Yates, R. Marcus and F.E. Yates, 1966, Site of action of vasopressin in causing corticotropin release, Endocrinol. 79,328. Hemingway, A., 1963, Shivering, Physiol. Rev. 43,397. Hissa, R. and W. Rautenberg, 1974, The influence of centrally applied noradrenaline on shivering and body temperature in the pigeon, J. Physiol. 238,421. Hoefke, W. and W. Kobinger, 1966, Pharmacologische Wirkungen des 2-(2,6-Dichlorphenylamino)-2-imidazolin-hydrochlorides, einer neuen, antihypertcnsiven Substanz, Arzneim. Forsch. 16, 1038. lwamoto, E.T., F.H. Shen, H.H. Loh and E.L. Way, 1971, The effects of pargyline on morphine tolerant-dependent mice, Federation Proc. 30,278. K6nig, J.F.R. and R.A. Klippel, 1963, The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem (The Williams and Wilkins Co., Baltimore). Marley, E. and J.D. Stephenson, 1972, Central actions of catecholamines, in: Catecholamines, eds. H. Blaschko and E. Muscholl (Springer-Verlag, Berlin) p. 463. Martin, W.R., A. Wikler, C.G. Eades and F.T. Pescor, 1963, Tolerance to and physical dependence on morphine in rats, Psychopharmacologia 4,247.
L.-F. Tseng et al., Clonicline and morphine abstinence signs Maruyama, Y. and A.E. Takemori, 1973, The role of dopamine and norepinephrine in the naloxone-induced abstinence of morphine-dependent mice, J. Pharmacol. ExptL Therap. 185,602. Matilla, M.J., E. Alhava and R. Mansner, 1968, Modification of disulfiram or methyltyrosine methylester (H 44/68) of abstinence symptoms in morphine-tolerant rats, Axzneim. Forsch. 19, 1163. Mayer, D.J. and J.C. Liebeskind, 1974, Pain reduction by local electrical stimulation of the brain: An anatomical and behavioral analysis, Brain Res. 68, 79. Schmitt, H. and H. Schmitt, 1969, Localization of the hypotensive effect of 2-(2,6-dichlorphenylamino-2-imidazoline hydrochloride (St. 155, Catepresan), European J. Pharmacol. 6, 8. Schwartz, A.S. and E. Eidelberg, 1970, Role of biogenic amines in morphine dependence, Life Sci. 9,613. Segal, D.S. and A.J. Mandell, 1970, Behavioral activation of rats during intraventricular infusion of norepinephrine, Proc. Nat. Acad. Sci. 66,289. Stone, E.A. and S. Mendlinger, 1974, Effect of intraventrieutar amines on motor activity in hypothermic rats, Res. Commun. Chem. Pathol. Pharmacol. 7,549.
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Way, E.L. and F.H. Shen, 1971, Catecholamines and 5-hydroxytryptamine in: Narcotic Drugs: Biochemical Pharmacology, ed. D.H. Clouet (Plenum Press, New York) p. 229. Wei, E.~ 1973, Brain lesions attenuating 'wet shake' behavior in morphine-abstinent rats, Life Sci. 12,385. Wei, E., H.H. Loh and E.L. Way, 1973a, Quantitative aspects of precipitated abstinence in morphine-dependent rats, J. Pharmacol. Exptl. Therap. 184, 398. Wei, E., H.H. Loh and E.L. Way, 1973b, Neuroanatomical correlates of wet shake behavior in the rat, Life Sci. 12, 489. Wei, E., H.H. Loh and E.L. Way, 1973c, Brain sites of precipitated abstinence in morphine-dependent rats, J. Pharmacol. Exptl. Therap. 185,108. Wei, E., L.F. Tseng, H.H. Loh and E.L. Way, 1974, Morphine abstinence signs: similarity to thermoregulatory behavior, Nature 347,398. Yaksh, T. and A. Pert, 1974, Microinjection mapping in the primate brain of the antinociceptive sites of action of morphine, Federation Proc. 33,488.
EFFECTS OF CLONIDINE ON MORPHINE WITHDRAWAL
SIGNS IN THE RAT
Liang-Fu TSENG, Horace H. LOH and Eddie T. WEI Department of Pharmacology, University of California, San Francisco, California 94143, and School o f Public Health, University o f California, Berkeley, California 94720, U.S.A.
Received 17 July 1974, revised MS received 1 October 1974, accepted 7 October 1974 L.-F. TSENG, H.H. LOH and E.T. WEt, Effects of elonidine on morphine withdrawal signs in the rat, European J. Pharmacol. 30 (1975) 93-99. The influence of clonidine on the naloxone-induced withdrawal signs, escape attempts and precipitated shakes, was studied in morphine-dependent rats. Clonidine injected i.p. or intraventricularly (i. vent.) inhibited precipitated shakes and potentiated escape attempts induced by naloxone in morphine-dependent rats. Under pentobarbital anesthesia, precipitated shakes and ice water-induced wet shakes were inhibited by clonidine and norepinephrine. Clonidine injected i.vent, reduced body temperatures in morphine-implanted rats and in placebo pellet-implanted rats. Naloxone reduced body temperatures only in morphine-dependent rats but not in placebo pellet-implanted rats. We suggest that clonidine modulates morphine withdrawal signs by potentiating the behavior associated with heat dissipation (escape attempts) and inhibiting the behavior associated with heat gain mechanisms (precipitated shakes). These effects may occur via stimulation of central noradrenergic mechanisms. Thermoregulatory behavior
Clonidine
Norepinephrine
1. Introduction The relationship of catecholamines to morphine analgesia, tolerance and physical dependence has been the subject of many studies (see reviews by Gunne, 1963; Way and Shen, 1971). Recently, several investigations have focused on the role of catecholamines in the manifestation of the withdrawal syndrome (Matilla et al., 1968; Schwartz and Eidelberg, 1970; Maruyama and Takemori, 1973). In these investigations, one commonly used technique was the administration of a-methyltyrosine to inhibit catecholamine synthesis and then to observe the withdrawal syndrome after depletion of brain catecholamines. Information on the Converse condition, that is, the effects of adrenergic stimulation on withdtawal behavior, has been lacking because peripherally administered catecholamines do not gain ready access to brain cells. Clonidine, a centrally acting antihypertensive agent (Schmitt and Schmitt, 1969; Bousquet and Guertzenstein, 1973) which is effective when administered by the peripheral route, is thought to be a
Morphine abstinence signs
central norepinephrine-like stimulant (Hoefke and Kobinger, 1966; Anddn et al., 1970). In this paper, we report the effects of central administration of clonidine and norepinephrine on the naloxone-precipitated abstinence syndrome in morphine-dependent rats.
2. Materials and methods 2.1. Materials
Male Sprague-Dawley rats (Horton Laboratories, Oakland, Calif.) weighing 2 2 0 - 2 7 0 g were used throughout these experiments. Clonidine (2-(2,6dichlorophenylamino)-2-imidazoline • HC1) was obtained from Boehringer Ingelheim, GMBH, Ingelheim, Germany; naloxone • HC1 from Endo Laboratories, Garden City, N.Y., and 1-norepinephrine hydrochloride from Sigma Chemical Co., St. Louis, Missouri. All doses refer to the salt forms of the drug.
94
L..F. Tseng et aL, Clonidine and morphine abstinence signs
2.2. Methods 2.2.1. Observation o f naloxone-precipitated withdrawal in morphine-implanted rats To produce dependence on morphine, a morphine pellet, containing 75 mg of morphine base (Gibson and Tingstad, 1970), was implanted s.c. in the lower abdominal wall. 3 days after the pellet implant, animals were weighed and placed in one gallon jars. The abstinence syndrome was precipitated by injecting naloxone • HC1, 4 mg/kg s.c. Clonidine - HC1 or saline was administered i.p. 10 min before the injection of naloxone. After injection of naloxone, the withdrawal signs of escape attempts and precipitated shakes were counted continuously for 10 min according to Wei et al. (1973a). 2.2.2. L vent. and intracerebral {i.c.) injections For the i.vent, injections, rat brains were stereotaxically implanted with 20 gauge stainless steel guide cannulae 2 days before morphine pellet implantation. 3 days after the morphine implant, 5/al of saline or a saline solution of clonidine - HC1 (5/ag and I0 /ag total dose) was injected unilaterally into the lateral ventricle of unanesthetized rats. Natoxone-precipirated withdrawal was then induced as described in section 2.2.1. For i.vent, and i.c. microinjections, we used the methods described by Hedge et al. (1966). Animals were anesthetized with sodium pentobarbital, 50 mg/kg i.p. 1-Norepinephrine • HC1, dissolved in saline was injected bilaterally in a volume of 0.5/al per side into the periaqueductal grey. Norepinephrine was injected in the periaqueductal grey because recent investigations indicate that this brain area may be an important site for the primary pharmacological actions of morphine (Yaksh and Pert, 1974; Mayer and Liebeskind, 1974). The stereotaxic coordinates for the periaqueductal grey, based on the atlas of KiSnig and Klippel (1963) were posterior, 0.5 mm; lateral, 0.5 mm; and vertical from the brain surface, 5.5 mm. 2.2.3. 'Precipitated shakes' and 'wet shakes'in pentobarbital anes'thetized rats A withdrawal sign, characterized by repetitive shaking movements of the body and termed 'wet dog shakes' by many investigators (Martin et al., 1963), can be elicited by naloxone in morphine-dependent
rats anesthetized with sodium pentobarbital (Wei, 1973). A similar shaking behavior can also be elicited in naive sodium pentobarbital-anesthetized rats, after immersion in ice water (Wei et al., 1973b). The effect of clonidine and norepinephrine on naloxone precipitated shakes in morphine-dependent rats hereafter termed 'precipitated shakes', and ice water-induced shakes, hereafter termed 'wet shakes', was studied. Morphine-implanted rats were injected with sodium pentobarbital, 40 mg/kg s.c., 30 min before the injection of clonidine. 10 min after the injections of clonidine or norepinephrine, animals were injected with naloxone - HC1, 4 mg/kg i.p. and the number of precipitated shakes counted for 10 rain. For observing wet shakes, naive rats were injected with sodium pentobarbital, 50 mg/kg s.c. 30 min before the injection of clonidine. 10 min after clonidine, rats were immersed in ice water and the number of wet shakes counted for 5 min (Wei et al., 1973b). 2.2.4. Measurement o f body temperature The rats were restrained in small animal holders 30 min before drug injection. Clonidine • HC1, in a dose of 15/ag, was injected i.vent. 10 min before the injection of naloxone - HC1 (4 mg/kg i.p.). The core temperature was measured with a thermistor probe inserted at least 6 cm into the rectum, taped to the base of the tail and connected to a telethermometer. The body temperatures were recorded at 5 - 1 0 min intervals for 2 hr after naloxone injection. 2.2.5. Statistical evaluation The Spearman Rank Correlation test was used to see if a correlation between the precipitated shakes and escape attempts was obtained. The MannWhitney U-test was used for the statistical evaluation of the significance of the observed differences.
3. Results
3.1. Withdrawal signs after naloxone injection in morphine-dependent rats Untreated morphine-dependent rats placed in jars did not manifest any shaking behavior or escape attempts. The injection of naloxone, 4 mg/kg s.c. elicited the abstinence signs of precipitated shakes and
L.-F. Tseng et al., Clonidine and morphine abstinence signs
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escape attempts. Other abstinence signs such as increased locomotor activity, blanching of the ears, teeth chattering, seminal emissions and diarrhea were also observed. The effect of clonidine on the number of precipitated shakes and escape attempts was measured in the present study. These withdrawal signs may be readily quantified and are highly characteristic of the morphine abstinence syndrome in the rat (Wei et al., 1973a). Moreover, the occurrence of the precipitated shakes and escape attempts in a given rat appears to be an independent event, as shown by the data in fig. I. 3.2. Effect o f clonidine on the naloxone-precipitated withdrawal signs
Fig. 2. Effect of clonidine injected i.p. on the frequency of naloxone-induced precipitated shakes and escape attempts. Clonidine or saline was administered (i.p.) 10 min before the injection o f naloxone. The withdrawal signs were observed for 10 min after the injection o f naloxone • HC1 (4 mg/kg s.c.). Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the frequency of the withdrawal signs. *p < 0.05; **p < 0.01; ***p < 0.002. 50 50
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L.-F. Tseng et al., Clonidine and morphine abstinence signs
96
served following n a l o x o n e injection were p o t e n t i a t e d by clonidine p r e t r e a t m e n t . In order to d e t e r m i n e if the effect o f clonidine on w i t h d r a w a l was a central or peripheral p h e n o m e n o n , clonidine (5 and 15/~g) was injected i.vent, and withdrawal precipitated w i t h n a l o x o n e . Clonidine injected i.vent, decreased the f r e q u e n c y o f precipitated shakes and p o t e n t i a t e d the n u m b e r of escape a t t e m p t s (fig. 3). Thus, the effects o f clonidine on withdrawal behavior was qualitatively similar when clonidine was administered by either the i.p. or the i.venr, route.
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Fig. 4. Effect of clonidine on naloxone-induced precipitated shakes and ice water-induced wet shakes. Rats were anesthetized with sodium pentobarbital 20-30 min before the injection of clonidine. Clonidine was injected 10 min before the injection of naloxone • HC1 (4 mg/kg s.c.) or bathing in ice water. The precipitated shakes were observed for 10 min and wet shakes were observed for 5 min. Each dot represents the frequency of withdrawal signs of each animal. Histogram represents the mean of the number of shakes, i.p. = intraperitoneal injection; i.vent. = intraventricular injection. *p < 0.01 ; **p < 0.002. SAL = saline; CLO = clonidine.
inhibited by clonidine, administered i.vent, or i.p. (fig. 4). Clonidine injected i.vent, or i.p. also antagonized the wet shakes p r o v o k e d by ice water in naive rats anesthetized with s o d i u m p e n t o b a r b i t a l (fig. 4). A 5 ~tg dose o f norepinephrine injected into the periaq u e d u c t a l grey region also antagonized the naloxoneinduced-precipitated shakes and the wet shakes provoked by ice water (fig. 5). To determine if clonidine had morphine-like properties, we pretreated rats with a 5 m g / k g s.c. dose o f n a l o x o n e before the administration o f clonidine (0.4 m g / k g i.p.). N a l o x o n e p r e t r e a t m e n t did not reverse the i n h i b i t o r y effects o f clonidine on the wet shake response (clonidine = 0, clonidine + n a l o x o n e = 0 shakes, n = 5).
L.-F. Tseng et al., Clonidine and morphine abstinence signs
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Fig. 6. Changes in body temperature following the injection of clonidine and naloxone. (a) morphine implanted rats; (b) placebo-implanted rats; S = saline; Nx = naloxone; C = clonidine; n = 5-6. ~c~ injected i.vent, with clonidine (15 ,g) or saline.~ injected i.p. with naloxone (4 mg/kg) or saline. 3.4. Effect o f clonidine and naloxone on body temperature
Clonidine • HCI (15/ag) injected i.vent, produced hypothermia in morphine-implanted rats and in placebo-implanted rats (fig. 6). Naloxone • HCI (4 mg/kg) reduced the body temperature in morphine-dependent rats but did not change the body temperature in placebo-implanted rats. The hypothermia produced by clonidine in morphine-implanted rats and in placebo-implanted rats was not affected by naloxone.
4. Discussion
We have recently suggested that certain morphine withdrawal signs resemble the behavior consequent to a central derangement of thermoregulatory mechanisms (Wei et al., 1974). In particular, shaking was considered to be a form of shivering or heat gain behavior, whereas escape attempts were interpreted as heat loss behavior (Hainsworth, 1967). Furthermore, we speculated that drugs which make animals 'feel hot' may potentiate escape and antagonize the shakes of morphine withdrawal.
97
Clonidine and, by inference, other central adrenergic stimulants, may be an example of a drug which modulates morphine withdrawal signs by increasing the likelihood of behavior associated with heat dissipation. The results of this study, at least, clearly indicate that shaking was inhibited by clonidine, whereas the frequency of escape attempts was potentiated. It is likely that the inhibitory effects of clonidine on shaking were mediated via a central noradrenergic mechanism, since the inhibition of shaking could also be obtained by intracerebral injection of norepinephfine. In the following sections, we summarize some of the evidence which is consistent with this interpretation of clonidine's effects on morphine withdrawal. The neuroanatomical pathways of precipitated shakes and wet shakes appear to originate from the medial portions of the brainstem (Wei, 1973; Wei et al., 1973c). This brain region is similar to the 'shivering center' described by Hemingway (1963). Shaking may be linked to shivering mechanisms because maneuvers which elevate body temperature, such as placing rats in a 3 4 - 3 7 °C environment or on a hot blanket or in warm water, inhibit the shaking response precipitated by naloxone or elicited by water. Domer and Feldberg (1960) showed that norepinephrine injections into the lateral ventricles of cats inhibited shivering produced by i.p. injection of pentobarbitone sodium or by i.m. injection of chlorpromazinc. Other workers have also found that i.vent. administration of norepinephrine suppresses shivering in the chick (Allen et al., 1970), rat (Stone and Mendlinger, 1974) and pigeon (Hissa and Rautenberg, 1974). Thus, it appears plausible that clonidine suppresses shivering by activating central noradrenergic mechanisms and that shaking behavior, which shares common neural pathways with shivering, is also concomitantly suppressed. Inhibition of brain monoamine oxidase by pargyline results in a potentiation of jumping behavior during naloxone-precipitated withdrawal in mice (Iwamoto et al., 1971). The potentiation of jumping has been attributed to the elevation of biogenic amines in brain produced by monoarnine oxidase inhibition. Chiosa et al. (1968) have also reported that imipramine, a drug which inhibits the reuptake of norepinephrine into nerve endings, potentiates morphine withdrawal activity in mice. Thus, our finding that clonidine potentiates the escape behavior of pre-
98
L.-F. Tseng et al., Clonidine and morphine abstinence signs
cipitated withdrawal is consistent with the notion that adrenergic stimulation results in an enhancement of one withdrawal sign: the urge to escape or jump. Wei et al. (1973b) showed that morphine inhibited the wet shakes provoked by ice water in naive rats. Naloxone reversed the inhibitory effects of morphine. Since the inhibitory effects of clonidine on shaking was not antagonized by naloxone, it appears clear that the adrenergic receptors involved in clonidine effects and opiate receptors fundamentally differ in their response to naloxone. The central actions o f catecholamines may be anatomically specific. Behavioral arousal has been observed in rats after i.vent, or intrahypothalamic injection of norepinephrine (Benkert, 1969; Segal and Mandell, 1970). However, other investigators found predominantly depressant effects such as sedation, analgesia, stupor and catalepsy alter i.c. or i.ci. injection of catecholamines (Marley and Stephenson, 1972). Bousquet and Guerzenstein (1973) showed that the hypotensive effect of i.vent, administered clonidine changed to a hypertensive effect when the cerebral aqueduct was cannulated. These results suggest that adrenergic stimulation o f forebrain elements may result in excitatory effects whereas depressant effects occur mainly in the brain stem. It would be of interest to determine if the effects of clonidine on the two withdrawal signs may be dissociated according to brain region, that is, potentiation o f escape arising from activation o f adrenergic receptors in the forebrain and suppression of wet shakes due to activation of adrenergic receptors in areas around the periaqueductal grey.
Acknowledgements This work was supported by Grants DA 00091 and DA 00564 from the National Institutes of Health. We are grateful for the generous supply of clonidine from Boehringer Ingelhelm and of naloxone • HC1 from Endo Laboratories. The authors also gratefully acknowledge the excellent technical assistance of Ms. Sheelah Sigel and Mr. J.B. Cunningham and the assistance of Barbara Halperin in preparation of the manuscript.
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L.-F. Tseng et al., Clonicline and morphine abstinence signs Maruyama, Y. and A.E. Takemori, 1973, The role of dopamine and norepinephrine in the naloxone-induced abstinence of morphine-dependent mice, J. Pharmacol. ExptL Therap. 185,602. Matilla, M.J., E. Alhava and R. Mansner, 1968, Modification of disulfiram or methyltyrosine methylester (H 44/68) of abstinence symptoms in morphine-tolerant rats, Axzneim. Forsch. 19, 1163. Mayer, D.J. and J.C. Liebeskind, 1974, Pain reduction by local electrical stimulation of the brain: An anatomical and behavioral analysis, Brain Res. 68, 79. Schmitt, H. and H. Schmitt, 1969, Localization of the hypotensive effect of 2-(2,6-dichlorphenylamino-2-imidazoline hydrochloride (St. 155, Catepresan), European J. Pharmacol. 6, 8. Schwartz, A.S. and E. Eidelberg, 1970, Role of biogenic amines in morphine dependence, Life Sci. 9,613. Segal, D.S. and A.J. Mandell, 1970, Behavioral activation of rats during intraventricular infusion of norepinephrine, Proc. Nat. Acad. Sci. 66,289. Stone, E.A. and S. Mendlinger, 1974, Effect of intraventrieutar amines on motor activity in hypothermic rats, Res. Commun. Chem. Pathol. Pharmacol. 7,549.
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Way, E.L. and F.H. Shen, 1971, Catecholamines and 5-hydroxytryptamine in: Narcotic Drugs: Biochemical Pharmacology, ed. D.H. Clouet (Plenum Press, New York) p. 229. Wei, E.~ 1973, Brain lesions attenuating 'wet shake' behavior in morphine-abstinent rats, Life Sci. 12,385. Wei, E., H.H. Loh and E.L. Way, 1973a, Quantitative aspects of precipitated abstinence in morphine-dependent rats, J. Pharmacol. Exptl. Therap. 184, 398. Wei, E., H.H. Loh and E.L. Way, 1973b, Neuroanatomical correlates of wet shake behavior in the rat, Life Sci. 12, 489. Wei, E., H.H. Loh and E.L. Way, 1973c, Brain sites of precipitated abstinence in morphine-dependent rats, J. Pharmacol. Exptl. Therap. 185,108. Wei, E., L.F. Tseng, H.H. Loh and E.L. Way, 1974, Morphine abstinence signs: similarity to thermoregulatory behavior, Nature 347,398. Yaksh, T. and A. Pert, 1974, Microinjection mapping in the primate brain of the antinociceptive sites of action of morphine, Federation Proc. 33,488.