Bra#z Re,ear, h, 513 (1990) ~- 14 Elsevier BRES 15321
Participation of different brain regions in the anti-narcotic withdrawal action of clonidine in the dependent rat Jerry J. Buccafusco Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300 (U.S.A.) and The Veterans Administration Medical Center, Augusta, GA 30910 (U.S.A.) (Accepted 22 August 1989) Key words: Morphine withdrawal; Clonidine; Brain; Spinal cord; Blood pressure; Behavior; Naloxone
The naloxone-precipitated withdrawal response in morphine dependent rats was quantitated by measuring the post-withdrawal increase in mean arterial pressure (MAP), heart rate and several characteristic behavioral changes. Pretreatment with intrathecal injection of clonidine produced a significant reduction in most of the autonomic symptoms of withdrawal, including the increase in MAP, salivation, and chromodacryorrhea, but did not effect the behavioral signs, body shakes, escape attempts or teeth chattering. Central i.c.v, injection of clonidine also reduced the autonomic symptoms of withdrawal. Two differences between the two routes of clonidine administration were with respect to (1) the degree of hind limb kicking where i.t., but not i.c.v, administration was effective at reducing this symptom; and (2) i.c.v., but not i.t. clonidine was effective in significantly reducing the incidence of diarrhea. In contrast, intracisternal (medullary level) injection of clonidine was without effect on the post-withdrawal increase in MAP, and inhibitory effects on the other symptoms included only salivation and diarrhea. Thus the effect of clonidine following intrathecal injection was not due to redistribution of drug to higher centers. Direct intrathecal injection of naloxone also precipitated withdrawal in morphine dependent rats. In this case, intrathecal pretreatment with clonidine did not inhibit the post-withdrawal increase in MAP. These results are consistent with a spinal component for the autonomic and behavioral symptoms of morphine withdrawal in the rat. Clonidine's antiwithdrawal response is mediated at least in part through a spinal action. However, this spinal antiwithdrawal action of clonidine is selective for withdrawal responses which originate from supraspinal centers.
INTRODUCTION The use of post-withdrawal arterial blood pressure increase as an index of the sympathetic c o m p o n e n t of the narcotic abstinence s y n d r o m e dates back to the original studies of H i m m e s l b a c h in the 1930's 17'~8. Perhaps due to t h e complexities of maintaining patent arterial catheters in small animals, the models d e v e l o p e d since that time d e p e n d e d primarily on subjective s t e r e o t y p e d withdrawal s y m p t o m s observed in rodents. Studies over the last several years in this l a b o r a t o r y have established that as in clinical subjects, the withdrawal associated increase in m e a n arterial pressure ( M A P ) can provide a reliable and sensitive index of both the intensity of abstinence as well as the degree of physical d e p e n d e n c e p r o d u c e d as a result of chronic m o r p h i n e administration in rats 5"8'9"22-25. F u r t h e r m o r e , this autonomic symptom of withdrawal has been e m p l o y e d to predict the antiwithdrawal potential of clonidine and the related drug guanfacine 8. Since these experiments can be carried out in unanesthetized, freely moving animals, the classical behavioral signs of morphine withdrawal can also be recorded simultaneously.
The pressor response associated with morphine withdrawal is quite m a r k e d following systemic injection of the narcotic antagonist naloxone, in d e p e n d e n t rats (there is no effect on blood pressure on obvious changes in behavior following similar injection of naloxone in n o n - d e p e n d e n t , naive animals). Behavioral and autonomic signs of withdrawal can be elicited following the intra-arterial (i.a.) injection 5'23 of naioxone as well as following the injection of the antagonist into localized areas of the CNS 22. O u r m o r e recent studies have d e m o n s t r a t e d that m a r k e d behavioral and autonomic withdrawal symptoms can be o b t a i n e d following intrathecal (i.t.) injection at the level of the spinal sympathetic (thoracic) outflow 9~22~25. The i m p o r t a n c e of the spinal cord in mediating autonomic symptoms of withdrawal was underscored by the observation that two consecutive withdrawal responses of e q u a l m a g n i t u d e could be elicited when the first injection of naloxone was m a d e into the lateral cerebral ventricle and the second into the intrathecal space. Conversely, once the spinal cord was withdrawn (through i.t. injection of naloxone), a second withdrawal response could not be p r o d u c e d by injection
Correspondence." J.J. Buccafusco, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
o f n a l o x o n e via a n y o t h e r r o u t e 22. A l s o , t h e s p i n a l c o r d in i s o l a t i o n
was
capable
of m e d i a t i n g
a withdrawal
response, i.e., a marked pressor response was observed in s p i n a l t r a n s e c t e d (C1), m o r p h i n e d e p e n d e n t r a t s ( b u t n o t in s p i n a l t r a n s e c t e d n o n - d e p e n d e n t Enhanced
a n i m a l s ) 24.
s y m p a t h e t i c a c t i v i t y (as m e a s u r e d b y p o s t -
withdrawal blood pressure increase) can be elicited from cardiovascular
centers
at d i f f e r e n t l e v e l s o f t h e n e u -
r a x i s 22, t h a t is f o l l o w i n g i n t r a c e r e b r o v e n t r i c u l a r , f o u r t h v e n t r i c l e a n d i n t r a t h e c a l i n j e c t i o n o f n a l o x o n e in d e p e n dent
rats.
This finding suggests a redundancy
in t h e
m e c h a n i s m o f d e p e n d e n c e / w i t h d r a w a l m a y exist. I n fact, withdrawal enhanced activity of sympathetic autonomic neurons
originating within the spinal cord
has been
i d e n t i f i e d as a p o s s i b l e site f o r t h e a n t i w i t h d r a w a l effect o f c l o n i d i n e 12. S i n c e c l o n i d i n e m a y h a v e a c t i o n s at b o t h b r a i n a n d s p i n a l sites, t h e p r e s e n t s t u d y was d e s i g n e d t o determine
w h i c h o f t h e s e sites m a y p a r t i c i p a t e in t h e
anti-withdrawal action of clonidine.
MATERIALS AND METHODS This study was performed using conscious, freely, moving rats in their home cage environment. Typical behavioral assessments of morphine withdrawal were made simultaneously with the sympathetic measures. Male, Wistar rats (250-300 g) were obtained from Harlan Sprague-Dawley, Indianapolis, IN, and housed in an environmentally controlled room having a 12 h light-12 h dark cycle. Standard rat chow (Wayne Rodent Blox) and tap water was supplied on an unlimited basis.
Indwelling intrathecal (i.t.) and intracisternal (i.c.) catheters Rats were anesthetized with 65 mg/kg, i.p., of sodium methohexital and placed in a stereotaxic frame. Catheterization of the spinal subarachnoid space was performed as described previously 29 by inserting a sterile, normal saline-filled polyethylene (PE 10) catheter 5.5 cm caudal to a midline nick in the atlanto-occipital membrane and terminating in the TT-T 8 level of the spinal cord. The rostral end of the catheter was plugged with a 30 gauge stainless-steel wire and anchored to the skull with acrylic cement. Animals were allowed 5 days to recover prior to any additional procedures. Only normally moving, healthy animals were employed in subsequent experiments. Intrathecal (i.t.) injections (5/A) were delivered via a 50/A syringe over 15 s using a constant speed syringe pump. The injection was followed with a 10/A saline flush to clear the catheter of drug. Upon completion of the experiments, catheter placement was confirmed by dye injection and dorsal laminectomy. For intracisternal injection, a catheter was permanently placed in the cisterna magna according a procedure modified from Kiser 19. Rats were placed in a stereotaxic frame with the surface of the skull in a horizontal position. A midline incision was made to expose the posterior part of the skull and a burr hole placed at the sagittal midline approximately 0.5 mm rostral to the suture between the intraparietal and supraoccipital bones. A loose knot was placed in a length of PE 10 tubing. The distal end of the tubing was lowered freehand 6 mm below the surface of the skull. The proximal end of the tubing was stabilized to a stainless steel screw (previously mounted in the skull) using dental acrylic. The tubing was plugged and tunneled subcutaneously to emerge at the nape of the neck. At the completion of the experiment dye administration revealed distribution to the dorsal and ventral surface of the medulla and the posterior portion of the cerebellum, with little distribution to the cervical spinal cord.
lntracerebroventricular (i. c. v,) cannula guide Rats were anesthetized with methohexital and placed in a stereotaxic instrument in a fiat skull orientation26. A 10 mm, 26-gauge stainless-steel cannula guide was placed 1.5 mm below the cortical surface so that its tip was directly above the surface of the left lateral cerebral ventricle (0.8 mm caudal and 1.4 mm lateral to bregma). The guide was anchored in place via a stainless steel screw and covered with acrylic cement and plugged with 32 gauge wire. Rats were allowed to recover for at least 5 days prior to the next procedure. For i.c.v, injections in freely moving rats, a 32-gauge stainless-steel injection cannula was connected to a 50/A syringe using polyethylene tubing. The cannula was lowered through the guide (4.5 mm below cortex) to rest in the ventricle. Drug solutions or vehicle (sterile saline) in a volume of 5 ~1 were delivered via a 50/d syringe over 15 s using a constant speed syringe pump. Upon completion of the experiments, cannula placement was confirmed by dye injection.
Preparation for arterial blood pressure recordings and intraarterial (i.a.) infusions Rats were anesthetized with methohexital and a midline abdominal incision made to expose the left iliac artery. A polyethylene (PE 50) catheter, filled with heparinized (20 U/ml) saline was inserted so that the tip of the catheter terminated in the base of the abdominal aorta, below the origin of the renal arteries. The opposite end of the catheter was plugged with a 22 gauge wire and directed subcutaneously to emerge at the back of the neck. Following surgery the animals were returned to their home, clear plastic cages (45 x 25 × 20 cm) and the catheter passed through a spring support and connected to a water tight swivel cannula mounted 30 cm above the cage floor. This method allowed the chronically catheterized rat unrestricted movement to all areas of the cage while a constant infusion (8 ml/day) of heparinized saline (20 U/ml) maintained the patency of the catheter. Animals were allowed to recover for 48 h prior to beginning the morphine infusion schedule.
Morphine infusion schedule and precipitated withdrawal Rats were made physically dependent upon morphine by the infusion of morphine sulfate through the arterial catheter at a rate of 8 ml/day to deliver a total dose of 35 mg/kg/day. The concentration of morphine was adjusted each morning for the next 4 days to deliver 50, 70 and 100 and 100 mg/kg/day, respectively. Morphine was dissolved in sterile heparinized (20 U/ml) saline. This infusion schedule was previously demonstrated by us to produce animals physically dependent upon morphine 5'23. Depending upon the experiment, morphine withdrawal was precipitated by the administration of naloxone hydrochloride through one of three routes of administration: (1) intraarterial (i.a.) bolus injection of 5 mg/kg, or, (2) intrathecal injection of 60/~g/5/A to elicit only spinal cord withdrawal. Withdrawal was always elicited between 10.00 and 14.00 h.
Cardiovascular and behavioral measures of withdrawal Blood pressure and heart rate were recorded through the arterial catheter connected to a Statham transducer coupled to a Coulbourn Instruments polygraph recorder. Heart rate was monitored by a tachometer triggered from the arterial pressure pulses. The following behavioral withdrawal signs were counted for 1 h following naloxone administration: escape attempts (vigorously attempting to leap from cage), withdrawal body shakes (or 'wet dog' shakes, brief episodes of rapid repetitive shaking of entire body from side to side). Counted signs were individually summed and expressed as the mean frequency of each behavior. The presence or absence of the following behavioral checked signs were also recorded: chromodacryorrhea (the secretion of red-brown, porphyrin containing tears, produced by the harderian glands in response to stress), diarrhea, (loosely or unformed stools), hind limb kicking, and teeth chattering (audible, involuntary clicking of teeth). The occurrence of the checked behavioral signs are expressed as the proportion of animals exhibiting the sign.
10 Statistic,~ Comparison between the m e a n s of several populations was performed using a one- or two-way A N O V A or an A N O V A for repeated measures, and the differences considered significant at the P < 0.05 level. Student's t-test (two tailed) was employed as a post hoc test and for the comparison of two groups of data. Multiple x
Participation of different brain regions in the anti-narcotic withdrawal action of clonidine in the dependent rat Jerry J. Buccafusco Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300 (U.S.A.) and The Veterans Administration Medical Center, Augusta, GA 30910 (U.S.A.) (Accepted 22 August 1989) Key words: Morphine withdrawal; Clonidine; Brain; Spinal cord; Blood pressure; Behavior; Naloxone
The naloxone-precipitated withdrawal response in morphine dependent rats was quantitated by measuring the post-withdrawal increase in mean arterial pressure (MAP), heart rate and several characteristic behavioral changes. Pretreatment with intrathecal injection of clonidine produced a significant reduction in most of the autonomic symptoms of withdrawal, including the increase in MAP, salivation, and chromodacryorrhea, but did not effect the behavioral signs, body shakes, escape attempts or teeth chattering. Central i.c.v, injection of clonidine also reduced the autonomic symptoms of withdrawal. Two differences between the two routes of clonidine administration were with respect to (1) the degree of hind limb kicking where i.t., but not i.c.v, administration was effective at reducing this symptom; and (2) i.c.v., but not i.t. clonidine was effective in significantly reducing the incidence of diarrhea. In contrast, intracisternal (medullary level) injection of clonidine was without effect on the post-withdrawal increase in MAP, and inhibitory effects on the other symptoms included only salivation and diarrhea. Thus the effect of clonidine following intrathecal injection was not due to redistribution of drug to higher centers. Direct intrathecal injection of naloxone also precipitated withdrawal in morphine dependent rats. In this case, intrathecal pretreatment with clonidine did not inhibit the post-withdrawal increase in MAP. These results are consistent with a spinal component for the autonomic and behavioral symptoms of morphine withdrawal in the rat. Clonidine's antiwithdrawal response is mediated at least in part through a spinal action. However, this spinal antiwithdrawal action of clonidine is selective for withdrawal responses which originate from supraspinal centers.
INTRODUCTION The use of post-withdrawal arterial blood pressure increase as an index of the sympathetic c o m p o n e n t of the narcotic abstinence s y n d r o m e dates back to the original studies of H i m m e s l b a c h in the 1930's 17'~8. Perhaps due to t h e complexities of maintaining patent arterial catheters in small animals, the models d e v e l o p e d since that time d e p e n d e d primarily on subjective s t e r e o t y p e d withdrawal s y m p t o m s observed in rodents. Studies over the last several years in this l a b o r a t o r y have established that as in clinical subjects, the withdrawal associated increase in m e a n arterial pressure ( M A P ) can provide a reliable and sensitive index of both the intensity of abstinence as well as the degree of physical d e p e n d e n c e p r o d u c e d as a result of chronic m o r p h i n e administration in rats 5"8'9"22-25. F u r t h e r m o r e , this autonomic symptom of withdrawal has been e m p l o y e d to predict the antiwithdrawal potential of clonidine and the related drug guanfacine 8. Since these experiments can be carried out in unanesthetized, freely moving animals, the classical behavioral signs of morphine withdrawal can also be recorded simultaneously.
The pressor response associated with morphine withdrawal is quite m a r k e d following systemic injection of the narcotic antagonist naloxone, in d e p e n d e n t rats (there is no effect on blood pressure on obvious changes in behavior following similar injection of naloxone in n o n - d e p e n d e n t , naive animals). Behavioral and autonomic signs of withdrawal can be elicited following the intra-arterial (i.a.) injection 5'23 of naioxone as well as following the injection of the antagonist into localized areas of the CNS 22. O u r m o r e recent studies have d e m o n s t r a t e d that m a r k e d behavioral and autonomic withdrawal symptoms can be o b t a i n e d following intrathecal (i.t.) injection at the level of the spinal sympathetic (thoracic) outflow 9~22~25. The i m p o r t a n c e of the spinal cord in mediating autonomic symptoms of withdrawal was underscored by the observation that two consecutive withdrawal responses of e q u a l m a g n i t u d e could be elicited when the first injection of naloxone was m a d e into the lateral cerebral ventricle and the second into the intrathecal space. Conversely, once the spinal cord was withdrawn (through i.t. injection of naloxone), a second withdrawal response could not be p r o d u c e d by injection
Correspondence." J.J. Buccafusco, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
o f n a l o x o n e via a n y o t h e r r o u t e 22. A l s o , t h e s p i n a l c o r d in i s o l a t i o n
was
capable
of m e d i a t i n g
a withdrawal
response, i.e., a marked pressor response was observed in s p i n a l t r a n s e c t e d (C1), m o r p h i n e d e p e n d e n t r a t s ( b u t n o t in s p i n a l t r a n s e c t e d n o n - d e p e n d e n t Enhanced
a n i m a l s ) 24.
s y m p a t h e t i c a c t i v i t y (as m e a s u r e d b y p o s t -
withdrawal blood pressure increase) can be elicited from cardiovascular
centers
at d i f f e r e n t l e v e l s o f t h e n e u -
r a x i s 22, t h a t is f o l l o w i n g i n t r a c e r e b r o v e n t r i c u l a r , f o u r t h v e n t r i c l e a n d i n t r a t h e c a l i n j e c t i o n o f n a l o x o n e in d e p e n dent
rats.
This finding suggests a redundancy
in t h e
m e c h a n i s m o f d e p e n d e n c e / w i t h d r a w a l m a y exist. I n fact, withdrawal enhanced activity of sympathetic autonomic neurons
originating within the spinal cord
has been
i d e n t i f i e d as a p o s s i b l e site f o r t h e a n t i w i t h d r a w a l effect o f c l o n i d i n e 12. S i n c e c l o n i d i n e m a y h a v e a c t i o n s at b o t h b r a i n a n d s p i n a l sites, t h e p r e s e n t s t u d y was d e s i g n e d t o determine
w h i c h o f t h e s e sites m a y p a r t i c i p a t e in t h e
anti-withdrawal action of clonidine.
MATERIALS AND METHODS This study was performed using conscious, freely, moving rats in their home cage environment. Typical behavioral assessments of morphine withdrawal were made simultaneously with the sympathetic measures. Male, Wistar rats (250-300 g) were obtained from Harlan Sprague-Dawley, Indianapolis, IN, and housed in an environmentally controlled room having a 12 h light-12 h dark cycle. Standard rat chow (Wayne Rodent Blox) and tap water was supplied on an unlimited basis.
Indwelling intrathecal (i.t.) and intracisternal (i.c.) catheters Rats were anesthetized with 65 mg/kg, i.p., of sodium methohexital and placed in a stereotaxic frame. Catheterization of the spinal subarachnoid space was performed as described previously 29 by inserting a sterile, normal saline-filled polyethylene (PE 10) catheter 5.5 cm caudal to a midline nick in the atlanto-occipital membrane and terminating in the TT-T 8 level of the spinal cord. The rostral end of the catheter was plugged with a 30 gauge stainless-steel wire and anchored to the skull with acrylic cement. Animals were allowed 5 days to recover prior to any additional procedures. Only normally moving, healthy animals were employed in subsequent experiments. Intrathecal (i.t.) injections (5/A) were delivered via a 50/A syringe over 15 s using a constant speed syringe pump. The injection was followed with a 10/A saline flush to clear the catheter of drug. Upon completion of the experiments, catheter placement was confirmed by dye injection and dorsal laminectomy. For intracisternal injection, a catheter was permanently placed in the cisterna magna according a procedure modified from Kiser 19. Rats were placed in a stereotaxic frame with the surface of the skull in a horizontal position. A midline incision was made to expose the posterior part of the skull and a burr hole placed at the sagittal midline approximately 0.5 mm rostral to the suture between the intraparietal and supraoccipital bones. A loose knot was placed in a length of PE 10 tubing. The distal end of the tubing was lowered freehand 6 mm below the surface of the skull. The proximal end of the tubing was stabilized to a stainless steel screw (previously mounted in the skull) using dental acrylic. The tubing was plugged and tunneled subcutaneously to emerge at the nape of the neck. At the completion of the experiment dye administration revealed distribution to the dorsal and ventral surface of the medulla and the posterior portion of the cerebellum, with little distribution to the cervical spinal cord.
lntracerebroventricular (i. c. v,) cannula guide Rats were anesthetized with methohexital and placed in a stereotaxic instrument in a fiat skull orientation26. A 10 mm, 26-gauge stainless-steel cannula guide was placed 1.5 mm below the cortical surface so that its tip was directly above the surface of the left lateral cerebral ventricle (0.8 mm caudal and 1.4 mm lateral to bregma). The guide was anchored in place via a stainless steel screw and covered with acrylic cement and plugged with 32 gauge wire. Rats were allowed to recover for at least 5 days prior to the next procedure. For i.c.v, injections in freely moving rats, a 32-gauge stainless-steel injection cannula was connected to a 50/A syringe using polyethylene tubing. The cannula was lowered through the guide (4.5 mm below cortex) to rest in the ventricle. Drug solutions or vehicle (sterile saline) in a volume of 5 ~1 were delivered via a 50/d syringe over 15 s using a constant speed syringe pump. Upon completion of the experiments, cannula placement was confirmed by dye injection.
Preparation for arterial blood pressure recordings and intraarterial (i.a.) infusions Rats were anesthetized with methohexital and a midline abdominal incision made to expose the left iliac artery. A polyethylene (PE 50) catheter, filled with heparinized (20 U/ml) saline was inserted so that the tip of the catheter terminated in the base of the abdominal aorta, below the origin of the renal arteries. The opposite end of the catheter was plugged with a 22 gauge wire and directed subcutaneously to emerge at the back of the neck. Following surgery the animals were returned to their home, clear plastic cages (45 x 25 × 20 cm) and the catheter passed through a spring support and connected to a water tight swivel cannula mounted 30 cm above the cage floor. This method allowed the chronically catheterized rat unrestricted movement to all areas of the cage while a constant infusion (8 ml/day) of heparinized saline (20 U/ml) maintained the patency of the catheter. Animals were allowed to recover for 48 h prior to beginning the morphine infusion schedule.
Morphine infusion schedule and precipitated withdrawal Rats were made physically dependent upon morphine by the infusion of morphine sulfate through the arterial catheter at a rate of 8 ml/day to deliver a total dose of 35 mg/kg/day. The concentration of morphine was adjusted each morning for the next 4 days to deliver 50, 70 and 100 and 100 mg/kg/day, respectively. Morphine was dissolved in sterile heparinized (20 U/ml) saline. This infusion schedule was previously demonstrated by us to produce animals physically dependent upon morphine 5'23. Depending upon the experiment, morphine withdrawal was precipitated by the administration of naloxone hydrochloride through one of three routes of administration: (1) intraarterial (i.a.) bolus injection of 5 mg/kg, or, (2) intrathecal injection of 60/~g/5/A to elicit only spinal cord withdrawal. Withdrawal was always elicited between 10.00 and 14.00 h.
Cardiovascular and behavioral measures of withdrawal Blood pressure and heart rate were recorded through the arterial catheter connected to a Statham transducer coupled to a Coulbourn Instruments polygraph recorder. Heart rate was monitored by a tachometer triggered from the arterial pressure pulses. The following behavioral withdrawal signs were counted for 1 h following naloxone administration: escape attempts (vigorously attempting to leap from cage), withdrawal body shakes (or 'wet dog' shakes, brief episodes of rapid repetitive shaking of entire body from side to side). Counted signs were individually summed and expressed as the mean frequency of each behavior. The presence or absence of the following behavioral checked signs were also recorded: chromodacryorrhea (the secretion of red-brown, porphyrin containing tears, produced by the harderian glands in response to stress), diarrhea, (loosely or unformed stools), hind limb kicking, and teeth chattering (audible, involuntary clicking of teeth). The occurrence of the checked behavioral signs are expressed as the proportion of animals exhibiting the sign.
10 Statistic,~ Comparison between the m e a n s of several populations was performed using a one- or two-way A N O V A or an A N O V A for repeated measures, and the differences considered significant at the P < 0.05 level. Student's t-test (two tailed) was employed as a post hoc test and for the comparison of two groups of data. Multiple x
