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Dorsal root lesions block the expression of morphine withdrawal elicited from the rat spinal cord
Neuroscience Letters, 59 (1985) 319-324 Elsevier Scientific Publishers Ireland Ltd.
319
NSL 03500 DORSAL ROOT LESIONS BLOCK THE EXPRESSION OF MORPHINE
WITHDRAWAL
ELICITED FROM THE RAT SPINAL CORD
JERRY J. BUCCAFUSCO I-3,*and DENNIS C. MARSHALL l'** Departments oflPharmacology and Toxicology, and2Psychiatry, Medical College of Georgia, Augusta, GA 30912; and 3Medical Research Service, Veterans Administration Medical Center, Augusta, GA 30904 (U.S.A.)
(Received December 17th, 1984; Revised version received April 3rd, 1985; Accepted May 22nd, 1985)
Key words: morphine withdrawal - spinal cord - naioxone - blood pressure - intrathecal - dorsal root - rat
Evidence has been accumulating to indicate that the spinal cord plays an important role in the expression of several narcotic abstinence signs in the intact animal. One characteristic and reproducible withdrawal sign which can be elicited in both intact and spinal-transected dependent rats is the naloxoneinduced increase in arterial blood pressure. Employing this model, this autonomic component of narcotic withdrawal was quantitated in dependent spinal (C l)-transeeted rats with intact dorsal roots and in those with surgical lesions of the dorsal roots from T3 to L4. The withdrawal-associated hypertension observed in animals with intact dorsal roots was abolished in the rats having the lesioned roots. The central spinal location of the opiate receptors mediating the naloxone response was confirmed by experiments demonstrating the failure of a selective peripherally acting narcotic antagonist to elicit a comparable withdrawal response. It was concluded that continuous afferent input and spinal opiate receptors are requirements for the expression of spinal narcotic withdrawal. Several studies in this a n d o t h e r l a b o r a t o r i e s [4, 10, 11, 13, 19, 20] h a v e i n d i c a t e d t h a t the spinal c o r d p l a y s a n i m p o r t a n t role in the e x p r e s s i o n o f several signs o f m o r p h i n e w i t h d r a w a l . I n d e p e n d e n t rats b e a r i n g p r e v i o u s l y i m p l a n t e d i n t r a t h e c a l c a t h eters, we h a v e f o u n d t h a t injection o f n a l o x o n e via these c a t h e t e r s elicits a c o m p l e t e w i t h d r a w a l s y n d r o m e c h a r a c t e r i z e d b y b e h a v i o r a l ( w i t h d r a w a l , ' w e t - d o g ' shakes, escape a t t e m p t s , teeth c h a t t e r i n g , etc.) as well as a u t o n o m i c (increase in a r t e r i a l b l o o d p r e s s u r e a n d h e a r t rate, a n d h y p o t h e r m i a ) signs. T h e m a g n i t u d e o f the increase in m e a n a r t e r i a l p r e s s u r e ( M A P ) following n a l o x o n e injection in the d e p e n d e n t r a t p r o v i d e s a p a r t i c u l a r l y useful index o f b o t h the intensity o f w i t h d r a w a l a n d the degree o f p h y s i c a l d e p e n d e n c e . This r e s p o n s e is n o t o b s e r v e d in n o n - d e p e n d e n t animals, is o n l y w e a k l y p r e s e n t after a c u t e a d m i n i s t r a t i o n o f m o r p h i n e , is reversed b y high d o s e s o f m o r p h i n e a n d is directly related to the w i t h d r a w a l intensity, i.e. the
*Author for all correspondence at: Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, U.S.A. **Present address: Department of Pharmacology, Berlex Laboratories, Inc., 110 East Hanover Avenue, Cedar Knolls, NJ 07927, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
320 dose of naloxone [2, 12]. We also have observed this withdrawal-associated hypertension following intrathecal injection of naloxone in spinal-transected (CI) unanesthetized, dependent rats [10, 19]. Other signs of withdrawal have been observed in spinal dependent animals [4, 13, 20]; however, the change in postwithdrawal MAP provides a sensitive, objective, ongoing measure of the autonomic component of withdrawal. Since the transected spinal cord is free from tonic supraspinal influences we sought to determine whether local spinal circuits contributed to the expression of withdrawal-associated hypertension. In view of the fact that the dorsal horn is rich in endogenous enkephalin [5, 7, 8, 18] and in opiate receptors [1, 15], we examined the possibility that afferent dorsal root fibers might play a role in this naloxone-induced abstinence sign. Male Wistar rats (Harland Industries, Indianapolis, IN) weighing 300-380 g at the time of the experiment were anesthetized and the left iliac artery catheterized. The arterial line was exteriorized at the back of the neck and connected to a water-tight swivel mounted above the home cage. Rats were maintained on a constant light-dark cycle (12 h by 12 h) and had unlimited access to food and water. On the day following surgery, rats were administered a saline solution of morphine sulfate by continuous infusion via the arterial line. A schedule of increasing concentrations of morphine was employed to produce maximal physical dependence over 5 days [2, 12]. Dependent rats were then anesthetized with halothane and artifically ventilated via a tracheal cannula using a mixture of halothane vapors and 100~ 02 at a stroke volume of 1.8 ml and a rate of 90 inspirations/min. Arterial pressure was recorded continuously via the arterial catheter, while body temperature was recorded and maintained at a colonic temperature of 37.0 + 0.1 °C. Rats then were placed in a stereotaxic frame and a complete spinal cord transection made at the C1 level. Following a partial laminectomy the dorsal roots emerging from T3 to L4 were identified under a stereomicroscope gently lifted on a glass (200/~m) hook and severed. In some experiments, a dorsal laminectomy was not performed, but prior to transection a saline-filled polyethylene (PE 10) catheter was inserted at a distance of 5.5 cm into the intrathecal space to place its tip between segments 7 and 8 of the thoracic cord. After spinal section, the halothane was discontinued and the rat allowed to recover for 30 min prior to any subsequent treatment. At the completion of each experiment, the exact placement of the intrathecal cannula was verified by visual inspection following dye injection, and the spinal cord was examined in situ, for the completeness and extent of all lesions. In rats in which the dorsal roots were lesioned, the spinal cord was histologically fixed in formalin, and thin sections were prepared and stained with thionine. Intraarterial (i.a.) injection of 0.5 mg/kg of naloxone-HCl to morphine-dependent, spinal-transected rats produced an immediate increase in MAP which peaked within 5 min after injection (Fig. 1). At this time MAP increased to 76 + 3 mmHg over preinjection levels. This was followed by a gradual decline over the next 60 min to 24 mmHg above baseline. In addition to the MAP increase, heart rate (HR) also increased by 137__+8 beats/min following naloxone (Table I). Dorsal root lesions (T3 to L4) did not affect the resting levels of MAP and HR in dependent, spinal-tran-
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Minutes After Naloxone Fig. 1. Change in MAP as a function of time following precipitated withdrawal at time 0 in morphine dependent, spinal (Cl)-transeeted rats. CON NAL, intraarterial (i.a.) injection of 0.5 mg/kg naloxoneHC1; ITM, morphine 1200 #g by intrathecal injection was administered 4 min prior to i.a. injection of 0.5 mg/kg naloxone-HCl; NMB, i.a. injection of 0.56 mg/kg naloxone methobromide; DRL, dorsal roots (T3 to L4) were lesioned 30 min prior to i.a. injection of 0.5 mg/kg of naloxone. The control curve (CON NAL) was significantly different from the other 3 curves for between-treatments component by ANOVA for repeated measures (P < 0.01).
TABLE I WITHDRAWAL IN CONTROL AND DORSAL ROOT LESIONED RATS Values for resting MAP and HR and values for the magnitude of the increase in MAP and HR following intraarterial (i.a.) or intrathecal (i.t.) injection of naloxone-HCl or naloxone methobromide in morphinedependent, spinal (C 1)-transected rats. In some animals the dorsal roots from T3 to L4 were also lesioned. *Significantly different with respect to naloxone-HCl, 0.5 mg/kg, i.a., mean by Student's t-test. *P<0.05; **P<0.01 Treatment
Naloxone-HCl, 0.5 mg/kg, i.a. Dorsal root lesion then naloxone-HCl, 0.5 mg/kg, i.a. Morphine 1200/~g, i.t., 4 rain later naloxone-HC1, 0.5 mg/kg, i.a. Naloxone methobromide, 0.56 mg/kg i.a. Naloxone methobromide, 6.7 pg, i.t.
MAP (mmHg)
HR (beats/min)
n
Pre-naloxone Maximal resting level increase
Pre-naloxone Maximal resting level increase
674- 7
76___3
252___18
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322 sected rats, but the withdrawal-associated hypertension following naloxone-HC1 was completely abolished (Fig. 1). In contrast, the tachycardic response in dorsal rootlesioned animals was not significantly affected (Table I). Two approaches were taken to determine whether the site of action of naloxone-HCl was peripheral or central. In the first, 1200 #g of morphine was infused into the intrathecal space 4 min prior to naloxone-HC1 in dependent, spinal rats with intact dorsal roots. As indicated in Fig. 1 and Table I, intrathecal morphine pretreatment significantly reduced the naloxone-HCl-induced pressor response in both magnitude and duration, i.a. injection of 1200 pg of morphine, however, failed to produce any significant effect on the response. In the second approach we sought to determine whether a quarternary derivative of naloxone, naloxone methobromide, could elicit a characteristic withdrawal response following i.a. administration. This was examined initially in several dependent intact rats and compared with naloxone-HC1. Fig. 2 illustrates the data from these experiments, which reveal a characteristic postwithdrawal hypertensive response to 0.5 mg/kg of naloxone-HC1. The withdrawal period was also associated with several behavioral signs of withdrawal, including withdrawal body shakes, escape attempts, teeth chattering, defecation, diarrhea and chromodacryorrhea, which were quite pronounced in all 6 animals. In contrast, 0.56 mg/kg (an equimolar dose) of naloxone methobromide evoked only a weak transient pressor response, and only an occasional withdrawal sign was observed in one or two of the animals. Therefore, in intact
•
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Mimmm Fig. 2. Change in MAP as a function of time following precipitated withdrawal at time 0 in intact, morphine-dependent rats. CON NAL, i.a. injection of 0.5 mg/kg naloxone-HC1;NMB, i.a. injection of 0.56 mg/kg naloxone methobromide. The two curves were significantly different by ANOVA for repeated measures.
323 dependent rats we observed, as others previously [17], that the quartcrnary naloxone derivative was considerably less effective at precipitating withdrawal following peripheral administration. As in the intact animals, naloxone methobromide elicited only a weak increase in MAP and H R in the dependent, spinal transccted rats (Fig. 1, Table I). In contrast, central (intrathecal) injection of 6.7 gg of naloxone methobromide produced a withdrawal-associated hypertensive and tachycardic response which was similar to that following i.a. injection of naloxone-HCl. The present results confirm our earlier studies with this model [2, 10-12, 19] whereby these spinally mediated, withdrawal-associated increases in MAP and H R can be inhibited or reversed with classical autonomic blocking drugs or by pithing the spinal cord [11]. Also, selective surgical lesion of the dorsal roots at the level of the vasoconstrictor fiber (T3 to T13) outflow completely prevents the naloxone-HCl-induced increase in MAP. Therefore, blockade of either the efferent or afferent components of the sympathetic spinal reflex arc prevents the expression of the withdrawal-associated increase in MAP. Our histological examinations revealed no gross injury to the spinal cord or dorsal horn. Injection of the ganglionic stimulant dimethylphenylpiperazinum (DMPP) (200 #g/kg, i.v.) evoked a brief characteristic increase in MAP and HR, indicating that efferent postganglionic transmission also was not affected by the surgical lesions of the dorsal roots. Furthermore, the associated increase in H R was not affected by dorsal root lesion (Table I). The cardiac sympathetic efferents arise from the superior cervical ganglion and as such were quite rostral to the thoracic level lesions. Lesion of dorsal roots emerging from lower segments (TI3 to L5) also were without affect on the naloxone-HCl-induced responses. There are at least two implications for our finding of the effect of dorsal root lesions on MAP: (1) that the opiate receptors in question are located in the peripheral nervous system and/or (2) that in the absence of tonic descending input from supraspinal centers, continuous afferent input from peripheral receptors to the spinal cord is necessary for the expression of withdrawal signs. The present findings are not consistent with the former hypothesis since pretreatment with intrathecal morphine reduced the naloxone-HCl-induced pressor response, and intrathecal (central), but not peripheral, injection of the quaternary naloxone derivative was capable of eliciting withdrawal. Anatomically and functionally [3, 14] there exist neuronal connections between the dorsal root and autonomic preganglionic neurons. Such connections were easily demonstrated in these spinal preparations such that foot pinch or tail pinch reliably produced a brief increase in MAP and HR. The neuronal circuits within the spinal cord involved in the integration of afferent information from peripheral receptors appear to require ongoing stimulation for the expression of withdrawal signs. These results provide direct evidence supporting the concept previously put forth on the basis of anatomical [1, 15, 16] and electrophysiological [6, 9] studies that the spinal opiate system can interact with cells of the preganglionic sympathetic neurons in the expression of augmented sympathetic activity during withdrawal. We thank Drs. William Dewey and Herbert Merz for providing the naioxone
324 m e t h o b r o m i d e a n d E n d o L a b s . for s u p p l y i n g n a l o x o n e - H C 1 , a n d Ms. J e n n i e D o b y f o r t y p i n g this m a n u s c r i p t . T h i s s t u d y w a s s u p p o r t e d b y t h e M e d i c a l R e s e a r c h Service o f the V e t e r a n s A d m i n i s t r a t i o n . 1 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Res., 124 (1977) 53-67. 2 Buccafusco, J.J., Cardiovascular changes during morphine withdrawal in the rat: effects of clonidine, Pharmacol. Biochem. Behav., 18 (1983) 209-215. 3 DeGroat, W.C., Nadelhaft, I., Milne, R.J., Booth, A.M., Morgan, C. and Thor, K., Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine, J. Autonom. Nerv. Syst., 3 (1981) 135-160. 4 Delander, G.E. and Takemori, A.E., Spinal antagonism of tolerance and dependence induced by systemically administered morphine, Eur. J. Pharmacol., 94 (1983) 35-42. 5 Elde, R., H6kfelt, T., Johansson, O. and Terenius, L., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 6 Franz, D.N., Hare, B.D. and McCloskey, K.L., Spinal sympathetic neurons: possible sites of opiate withdrawal suppression by clonidine, Science, 215 (1982) 1643-1645. 7 Gibson, S.J., Polak, J., Bloom, S.R. and Wall, P.O., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal, J. Comp. Neurol., 201 (1981) 65-79. 8 Glazer, E.J. and Basbaum, A.I., Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn, Neuroscience, 10 (1983) 357-376. 9 Madsen, P.W., Hare, B.D. and Franz, D.N., Contrasting effects of opiate agonists and agonist-antagonists on adenylate cyclase and excitability in sympathetic preganglionic neurons, Soc. Neurosci. Abstr., 10 (1984) 1108. 10 Marshall, D.C. and Buccafusco, J.J., Role of cholinergic neurons in the spinal component of morphine withdrawal, Pharmacologist, 26 (1984) 197. 11 Marshall, D.C. and Buccafusco, J.J., Mechanism of morphine withdrawal in spinal-transected rats, Soc. Neurosci. Abstr., 10 (1984) 930. 12 Marshall, D.C. and Buccafusco, J.J., Cardiovascular and behavioral changes associated with narcotic withdrawal during the development of physical dependence, Proc. Soc. Exp. Biol. Med., 176 (t984) 216. 13 Martin, W.R. and Eades, C.G., A comparison between acute and chronic physical dependence in the chronic spinal dog, J. Pharmacol. Exp. Tiler., 146 (1964) 385-394. 14 Mawe, G.M., Bresnahan, J.C. and Beattie, M.S., Primary afferent projections from dorsal and ventral roots to autonomic preganglionic neurons in the cat sacral spinal cord: light and electron microscopic observations, Brain Res., 290 (1984) 152-157. 15 Pert, C.B., Kuhar, M.J. and Snyder, S.H., Autoradiographic localization of the opiate receptor in rat brain, Life Sci., 16 (1975) 1849-1853. 16 Romagnano, M.A. and Hamill, R.W., Spinal sympathetic pathway: an enkephalin ladder, Science, 225 (1984) 737-739. 17 Russell, J., Bass, P., Goldberg, L.I., Schuster, C.R. and Merz, H , Antagonism of gut, but not central effects of morphine with quarternary narcotic antagonists, Eur. J. Pharmacol., 78 (1982) 255-261, 18 Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.J. and Cuatrecacas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. Comp. Neurol., 182 (1978) 17-3% 19 Turner, R.M., Marshall, D.C. and Buccafusco, J.J., Supraspinal and spinal components of naloxone induced morphine withdrawal, Pharmacologist, 26 (1984) 197. 20 Wikler, A. and Frank, K., Hindlimb reflexes of chronic spinal dogs during cycles of addiction to mor, phine and methadone, J. Pharmaeol. Exp. Ther., 94 (1984) 382--400.
319
NSL 03500 DORSAL ROOT LESIONS BLOCK THE EXPRESSION OF MORPHINE
WITHDRAWAL
ELICITED FROM THE RAT SPINAL CORD
JERRY J. BUCCAFUSCO I-3,*and DENNIS C. MARSHALL l'** Departments oflPharmacology and Toxicology, and2Psychiatry, Medical College of Georgia, Augusta, GA 30912; and 3Medical Research Service, Veterans Administration Medical Center, Augusta, GA 30904 (U.S.A.)
(Received December 17th, 1984; Revised version received April 3rd, 1985; Accepted May 22nd, 1985)
Key words: morphine withdrawal - spinal cord - naioxone - blood pressure - intrathecal - dorsal root - rat
Evidence has been accumulating to indicate that the spinal cord plays an important role in the expression of several narcotic abstinence signs in the intact animal. One characteristic and reproducible withdrawal sign which can be elicited in both intact and spinal-transected dependent rats is the naloxoneinduced increase in arterial blood pressure. Employing this model, this autonomic component of narcotic withdrawal was quantitated in dependent spinal (C l)-transeeted rats with intact dorsal roots and in those with surgical lesions of the dorsal roots from T3 to L4. The withdrawal-associated hypertension observed in animals with intact dorsal roots was abolished in the rats having the lesioned roots. The central spinal location of the opiate receptors mediating the naloxone response was confirmed by experiments demonstrating the failure of a selective peripherally acting narcotic antagonist to elicit a comparable withdrawal response. It was concluded that continuous afferent input and spinal opiate receptors are requirements for the expression of spinal narcotic withdrawal. Several studies in this a n d o t h e r l a b o r a t o r i e s [4, 10, 11, 13, 19, 20] h a v e i n d i c a t e d t h a t the spinal c o r d p l a y s a n i m p o r t a n t role in the e x p r e s s i o n o f several signs o f m o r p h i n e w i t h d r a w a l . I n d e p e n d e n t rats b e a r i n g p r e v i o u s l y i m p l a n t e d i n t r a t h e c a l c a t h eters, we h a v e f o u n d t h a t injection o f n a l o x o n e via these c a t h e t e r s elicits a c o m p l e t e w i t h d r a w a l s y n d r o m e c h a r a c t e r i z e d b y b e h a v i o r a l ( w i t h d r a w a l , ' w e t - d o g ' shakes, escape a t t e m p t s , teeth c h a t t e r i n g , etc.) as well as a u t o n o m i c (increase in a r t e r i a l b l o o d p r e s s u r e a n d h e a r t rate, a n d h y p o t h e r m i a ) signs. T h e m a g n i t u d e o f the increase in m e a n a r t e r i a l p r e s s u r e ( M A P ) following n a l o x o n e injection in the d e p e n d e n t r a t p r o v i d e s a p a r t i c u l a r l y useful index o f b o t h the intensity o f w i t h d r a w a l a n d the degree o f p h y s i c a l d e p e n d e n c e . This r e s p o n s e is n o t o b s e r v e d in n o n - d e p e n d e n t animals, is o n l y w e a k l y p r e s e n t after a c u t e a d m i n i s t r a t i o n o f m o r p h i n e , is reversed b y high d o s e s o f m o r p h i n e a n d is directly related to the w i t h d r a w a l intensity, i.e. the
*Author for all correspondence at: Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, U.S.A. **Present address: Department of Pharmacology, Berlex Laboratories, Inc., 110 East Hanover Avenue, Cedar Knolls, NJ 07927, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
320 dose of naloxone [2, 12]. We also have observed this withdrawal-associated hypertension following intrathecal injection of naloxone in spinal-transected (CI) unanesthetized, dependent rats [10, 19]. Other signs of withdrawal have been observed in spinal dependent animals [4, 13, 20]; however, the change in postwithdrawal MAP provides a sensitive, objective, ongoing measure of the autonomic component of withdrawal. Since the transected spinal cord is free from tonic supraspinal influences we sought to determine whether local spinal circuits contributed to the expression of withdrawal-associated hypertension. In view of the fact that the dorsal horn is rich in endogenous enkephalin [5, 7, 8, 18] and in opiate receptors [1, 15], we examined the possibility that afferent dorsal root fibers might play a role in this naloxone-induced abstinence sign. Male Wistar rats (Harland Industries, Indianapolis, IN) weighing 300-380 g at the time of the experiment were anesthetized and the left iliac artery catheterized. The arterial line was exteriorized at the back of the neck and connected to a water-tight swivel mounted above the home cage. Rats were maintained on a constant light-dark cycle (12 h by 12 h) and had unlimited access to food and water. On the day following surgery, rats were administered a saline solution of morphine sulfate by continuous infusion via the arterial line. A schedule of increasing concentrations of morphine was employed to produce maximal physical dependence over 5 days [2, 12]. Dependent rats were then anesthetized with halothane and artifically ventilated via a tracheal cannula using a mixture of halothane vapors and 100~ 02 at a stroke volume of 1.8 ml and a rate of 90 inspirations/min. Arterial pressure was recorded continuously via the arterial catheter, while body temperature was recorded and maintained at a colonic temperature of 37.0 + 0.1 °C. Rats then were placed in a stereotaxic frame and a complete spinal cord transection made at the C1 level. Following a partial laminectomy the dorsal roots emerging from T3 to L4 were identified under a stereomicroscope gently lifted on a glass (200/~m) hook and severed. In some experiments, a dorsal laminectomy was not performed, but prior to transection a saline-filled polyethylene (PE 10) catheter was inserted at a distance of 5.5 cm into the intrathecal space to place its tip between segments 7 and 8 of the thoracic cord. After spinal section, the halothane was discontinued and the rat allowed to recover for 30 min prior to any subsequent treatment. At the completion of each experiment, the exact placement of the intrathecal cannula was verified by visual inspection following dye injection, and the spinal cord was examined in situ, for the completeness and extent of all lesions. In rats in which the dorsal roots were lesioned, the spinal cord was histologically fixed in formalin, and thin sections were prepared and stained with thionine. Intraarterial (i.a.) injection of 0.5 mg/kg of naloxone-HCl to morphine-dependent, spinal-transected rats produced an immediate increase in MAP which peaked within 5 min after injection (Fig. 1). At this time MAP increased to 76 + 3 mmHg over preinjection levels. This was followed by a gradual decline over the next 60 min to 24 mmHg above baseline. In addition to the MAP increase, heart rate (HR) also increased by 137__+8 beats/min following naloxone (Table I). Dorsal root lesions (T3 to L4) did not affect the resting levels of MAP and HR in dependent, spinal-tran-
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Minutes After Naloxone Fig. 1. Change in MAP as a function of time following precipitated withdrawal at time 0 in morphine dependent, spinal (Cl)-transeeted rats. CON NAL, intraarterial (i.a.) injection of 0.5 mg/kg naloxoneHC1; ITM, morphine 1200 #g by intrathecal injection was administered 4 min prior to i.a. injection of 0.5 mg/kg naloxone-HCl; NMB, i.a. injection of 0.56 mg/kg naloxone methobromide; DRL, dorsal roots (T3 to L4) were lesioned 30 min prior to i.a. injection of 0.5 mg/kg of naloxone. The control curve (CON NAL) was significantly different from the other 3 curves for between-treatments component by ANOVA for repeated measures (P < 0.01).
TABLE I WITHDRAWAL IN CONTROL AND DORSAL ROOT LESIONED RATS Values for resting MAP and HR and values for the magnitude of the increase in MAP and HR following intraarterial (i.a.) or intrathecal (i.t.) injection of naloxone-HCl or naloxone methobromide in morphinedependent, spinal (C 1)-transected rats. In some animals the dorsal roots from T3 to L4 were also lesioned. *Significantly different with respect to naloxone-HCl, 0.5 mg/kg, i.a., mean by Student's t-test. *P<0.05; **P<0.01 Treatment
Naloxone-HCl, 0.5 mg/kg, i.a. Dorsal root lesion then naloxone-HCl, 0.5 mg/kg, i.a. Morphine 1200/~g, i.t., 4 rain later naloxone-HC1, 0.5 mg/kg, i.a. Naloxone methobromide, 0.56 mg/kg i.a. Naloxone methobromide, 6.7 pg, i.t.
MAP (mmHg)
HR (beats/min)
n
Pre-naloxone Maximal resting level increase
Pre-naloxone Maximal resting level increase
674- 7
76___3
252___18
1374-8
6
74-+ 13
11+6"*
2454-30
113--+22
4
64-+ 7
46-+8**
258-+ 16
156-+26
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91-+4"
233-+9
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322 sected rats, but the withdrawal-associated hypertension following naloxone-HC1 was completely abolished (Fig. 1). In contrast, the tachycardic response in dorsal rootlesioned animals was not significantly affected (Table I). Two approaches were taken to determine whether the site of action of naloxone-HCl was peripheral or central. In the first, 1200 #g of morphine was infused into the intrathecal space 4 min prior to naloxone-HC1 in dependent, spinal rats with intact dorsal roots. As indicated in Fig. 1 and Table I, intrathecal morphine pretreatment significantly reduced the naloxone-HCl-induced pressor response in both magnitude and duration, i.a. injection of 1200 pg of morphine, however, failed to produce any significant effect on the response. In the second approach we sought to determine whether a quarternary derivative of naloxone, naloxone methobromide, could elicit a characteristic withdrawal response following i.a. administration. This was examined initially in several dependent intact rats and compared with naloxone-HC1. Fig. 2 illustrates the data from these experiments, which reveal a characteristic postwithdrawal hypertensive response to 0.5 mg/kg of naloxone-HC1. The withdrawal period was also associated with several behavioral signs of withdrawal, including withdrawal body shakes, escape attempts, teeth chattering, defecation, diarrhea and chromodacryorrhea, which were quite pronounced in all 6 animals. In contrast, 0.56 mg/kg (an equimolar dose) of naloxone methobromide evoked only a weak transient pressor response, and only an occasional withdrawal sign was observed in one or two of the animals. Therefore, in intact
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.
0"I
Mimmm Fig. 2. Change in MAP as a function of time following precipitated withdrawal at time 0 in intact, morphine-dependent rats. CON NAL, i.a. injection of 0.5 mg/kg naloxone-HC1;NMB, i.a. injection of 0.56 mg/kg naloxone methobromide. The two curves were significantly different by ANOVA for repeated measures.
323 dependent rats we observed, as others previously [17], that the quartcrnary naloxone derivative was considerably less effective at precipitating withdrawal following peripheral administration. As in the intact animals, naloxone methobromide elicited only a weak increase in MAP and H R in the dependent, spinal transccted rats (Fig. 1, Table I). In contrast, central (intrathecal) injection of 6.7 gg of naloxone methobromide produced a withdrawal-associated hypertensive and tachycardic response which was similar to that following i.a. injection of naloxone-HCl. The present results confirm our earlier studies with this model [2, 10-12, 19] whereby these spinally mediated, withdrawal-associated increases in MAP and H R can be inhibited or reversed with classical autonomic blocking drugs or by pithing the spinal cord [11]. Also, selective surgical lesion of the dorsal roots at the level of the vasoconstrictor fiber (T3 to T13) outflow completely prevents the naloxone-HCl-induced increase in MAP. Therefore, blockade of either the efferent or afferent components of the sympathetic spinal reflex arc prevents the expression of the withdrawal-associated increase in MAP. Our histological examinations revealed no gross injury to the spinal cord or dorsal horn. Injection of the ganglionic stimulant dimethylphenylpiperazinum (DMPP) (200 #g/kg, i.v.) evoked a brief characteristic increase in MAP and HR, indicating that efferent postganglionic transmission also was not affected by the surgical lesions of the dorsal roots. Furthermore, the associated increase in H R was not affected by dorsal root lesion (Table I). The cardiac sympathetic efferents arise from the superior cervical ganglion and as such were quite rostral to the thoracic level lesions. Lesion of dorsal roots emerging from lower segments (TI3 to L5) also were without affect on the naloxone-HCl-induced responses. There are at least two implications for our finding of the effect of dorsal root lesions on MAP: (1) that the opiate receptors in question are located in the peripheral nervous system and/or (2) that in the absence of tonic descending input from supraspinal centers, continuous afferent input from peripheral receptors to the spinal cord is necessary for the expression of withdrawal signs. The present findings are not consistent with the former hypothesis since pretreatment with intrathecal morphine reduced the naloxone-HCl-induced pressor response, and intrathecal (central), but not peripheral, injection of the quaternary naloxone derivative was capable of eliciting withdrawal. Anatomically and functionally [3, 14] there exist neuronal connections between the dorsal root and autonomic preganglionic neurons. Such connections were easily demonstrated in these spinal preparations such that foot pinch or tail pinch reliably produced a brief increase in MAP and HR. The neuronal circuits within the spinal cord involved in the integration of afferent information from peripheral receptors appear to require ongoing stimulation for the expression of withdrawal signs. These results provide direct evidence supporting the concept previously put forth on the basis of anatomical [1, 15, 16] and electrophysiological [6, 9] studies that the spinal opiate system can interact with cells of the preganglionic sympathetic neurons in the expression of augmented sympathetic activity during withdrawal. We thank Drs. William Dewey and Herbert Merz for providing the naioxone
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