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Dependence and withdrawal following intracerebroventricular and systemic morphine administration: functional anatomy and behavior
6
Brain Research, 518 (1990) 6-10 Elsevier
BRES 15509
Dependence and withdrawal following intracerebroventricular and systemic morphine administration" functional anatomy and behavior Robert E. Adams and G. Frederick Wooten Department of Neurology, University of Virginia School of Medicine, Charlottesville, VA 22908 (U.S.A.) (Accepted 7 November 1989) Key words: Morphine; Intracerebroventricular; 2-Deoxy-o-glucose; Autoradiography; Naloxone; Minipump; Regional cerebral glucose utilization
Regional cerebral glucose utilization (RCGU) and behavior during precipitated morphine withdrawal were studied in rats made dependent by either intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of morphine. [14C]2-deoxy-D-glucoseautoradiography revealed that RCGU increased in an anatomically related group of limbic and brainstem structures in rats that were in morphine withdrawal precipitated by naloxone administration compared to morphine-dependent controls that were not in precipitated withdrawal. Correlation of RCGU for 24 brain structures comparing i.c.v, vs s.c. morphine-treated rats was highly significant for groups in withdrawal and for controls (r values, 0.958 and 0.971, respectively). Withdrawal behaviors including autonomic signs of withdrawal, withdrawal jumping, and incidence of diarrhea were not different between the two groups in withdrawal (i.c.v. and s.c.), Weight loss during withdrawal increased (P < 0.05) in rats made dependent by s.c. morphine administration compared to rats that received morphine by the i.c.v, route. Taken together, these results indicate that RCGU changes during morphine withdrawal result solely from effects of chronic morphine in the central nervous system, not in peripheral sites. The increased weight loss of s.c.-treated, morphine-dependent rats in withdrawal suggests an independent peripheral effect perhaps mediated by visceral opiate receptors. INTRODUCTION Increases in
MATERIALS AND METHODS regional cerebral
glucose
utilization
( R C G U ) occur in an anatomically related group of limbic and brainstem structures during morphine withdrawal in the rat 5-7A4. The magnitude of these regionally selective increases in R C G U is directly related to both the total morphine dose administered to produce dependence and the naloxone dose used to precipitate withdrawal 5'6. The same structures are metabolically active whether withdrawal is precipitated by naloxone or occurs through abstinence 7. Behavioral and physiological (in vivo and in vitro) studies have suggested that at least some manifestations of opiate withdrawal may result in part from the effects of opiates at peripheral sites 2'4'~°-1z. For example, diarrhea during opiate withdrawal may result from not only central effects but also systemic effects of opiates on the gut 2"9"11,12. Previously, R C G U during withdrawal was only studied after systemic administration of morphine. To address the relative roles of central vs peripheral effects of chronic morphine administration on R C G U during withdrawal, we have also studied R C G U and behavior during naloxone-precipitated morphine withdrawal in rats made d e p e n d e n t upon morphine administered by the intracerebroventricular (i.c.v.) route.
Male Sprague-Dawley rats (250-300 g, Dominion Labs, Dublin, VA) were given food and water ad libitum and kept in a 12 h light-dark cycle throughout the study. Morphine dependence was produced in some groups by i.c.v, infusion of morphine sulfate (MS) (Mallinckrodt, St. Louis, MO), and in other groups by subcutaneous (s.c.) implantation of sustained-release, morphine-base pellets (NIDA, Rockville, MD). An effort was made to produce similar degrees of dependence (i.e. comparable degrees of behavioral withdrawal) in all dependent groups whether morphine was administered by the i.c.v, or s.c. route. 1.c. v. infusions Rats were anesthetized with a 1 ml/kg intraperitoneal injection of 1:1 ketamine/xylozine cocktail (100 mg/ml to 20 mg/ml) and placed in a Kopf stereotactic frame (incisor bar, -3.3 to -3.5 ram). The skull was exposed by making a mid-sagittal scalp incision, and a burr hole was made over the right lateral ventricle (bregma -0.9 mm, right -1.5 mm, ventrally 3.5 mm from brain surface) 13. The infusion apparatus consisted of an Alzet osmotic minipump (Model 2002) connected to a 26 gauge stainless steel blunt tube via PE 60 tubing. The entire apparatus, primed and filled with MS (80 mg/ml distilled water), was implanted subcutaneously between the scapulae with the tip of the blunt tube in the right lateral ventricle held in place by skull screws and dental acrylic. Morphine sulfate was then infused at the rate of 40/~g.0.5/d 1.h-1 for 3 days by the apparatus. A concentration of 40 ktg/0.5/~1 has proven to be well within the limits of solubility at in vivo temperatures. In addition, no precipitation of MS was found in any rats upon sectioning of infusion apparatus, and histologic examination of tissue. Infusions of higher concentrations of morphine sulfate (over 50 ~g.#l-l.h-~) resulted in definite precipitation and ventriculitis.
Correspondence: G.F. Wooten, Department of Neurology, Box 394, University of Virginia Medical Center, Charlottesville, VA 22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Morphine pellet implantation Under light halothane anesthesia (2%), a 1 cm midline incision dorsal to the mid thoracic spine was produced in the rat's trunk. An s.c. pocket was made, into which sustained-release, morphine-base pellets containing 75 mg of morphine per pellet were implanted (1 pellet/45 g body weight). Three days later, using the same technique, pellets were implanted into a second s.c. pocket (l pellet/70 g) and left in place 2 additional days for a total of 5 days of morphine pellet exposure.
measurements of each lateral structure or 4 total measurements of each midline structure were taken and expressed as an average for each brain.
Statistics Comparisons of OD values and weight loss among the 5 groups were made using Duncan's Multiple Range TesP. Analysis of jumping behavior, ANS, and presence of diarrhea was performed using Fisher's Exact Test 1.
Experimental groups Groups of rats in withdrawal consisted of rats made dependent by i.c.v, or s.c. morphine administration and given an intravenous (i.v.) injection of naloxone HCI (Dupont, Wilmington, DE) on the day of experimentation. Controls consisted of morphine-dependent rats (i.c.v. or s.c.) given i.v. normal saline on experimental day and rats that received a 3-day, chronic, i.c.v, infusion of sterile distilled water (dHzO) (using the same technique described for the infusion of MS above) and i.v. naloxone on the experimental day.
[~ I ~ [z23
= °e
~
A
SC PELLETS - NALOX SC PELLETS - NS ICV MS % NALOX ICY MS - NS
6
o L
Experimentation On the day of experimentation (day 3 for i.c.v.-administered groups, day 5 for s.c.-administered groups) under 2% halothane anesthesia, the right jugular vein was cannulated with PE 50 tubing and the other end was tunneled subcutaneously and exposed through a stab wound in the back. Rats were then placed in separate cages to recover for 2-4 h prior to experimentation. After recovery from general anesthesia, rats were placed in a cylindrical glass container (30 cm diameter, 37 cm height) and given a 1 mg/kg injection of either naloxone HCI 5 mg/ml in normal saline or normal saline alone, and, 1 min later, all groups were injected with 10 ItCi/lO0 g of [x4C]2-deoxy-D-glucose (American Radiolabelled Chemicals, St. Louis, MO). The following behavioral signs of withdrawal were then observed and recorded: jumps/20 min, weight loss/50 min, and the presence of autonomic signs (ANS) for 50 min. Autonomic signs recorded included tachypnea, teeth chatter, vocalization to touch, irritability, ptosis, writhing, forepaw tremor, diarrhea, urination, lacrimation, salivation, rhinorrhea, erection and ejaculation. Each ANS was assigned l/z point for a total potential ANS score of 7. After the 50 min observation period, rats were killed by decapitation, and the brains were removed, frozen and embedded. Twenty/tm coronal sections were taken and exposed to Kodak SB-5 X-ray film for 5 days to produce an autoradiogram. Optical densities of various structures were measured using a Leitz Ergolux variable aperture microdensitometer and expressed as a ratio of the optical density (OD) of that structure to the OD of corpus callosum (representing pure white matter). No fewer than 4 bilateral
g o P,
[EEE] S C P E L L E T S - N A L O X I SO PELLETS - NS ICY M S - N A L O X
"z
EZZ] ICY MS - NS I ~ ICY dH20 - NALOX
15-
10-
LSEPT
CNA
PVN
LHYPO
LHB
IPN
B
10~
w,
4
0 DBB
10
WSEPT
)
VMN H
DMN
1i11
PT
CENTR VMN TH
6
20
I
i
~ STR
I
S
W w
0i
LPOA
4
20.
E
0 MPOA
or--
o
2
r-
i
c
Fig. 1. Weight loss, mean + S.D., and statistical group assignment (A,B,C) according to the Duncan Multiple Range test. A, B, and C are defined by Duncan Multiple Range analysis to be groups of numbers (in this case, g of weight loss/rat) that are statistically unrelated to groups of different letter, and related to groups of same letter (P ~ 0.05).
GP
ACC
MMAM LIWAM AVAM
Zl
RAPHE SCSG
Fig. 2. Mean O D ratios (OD CNS structure/OD corpus callosum) and S.D. for 24 CMS structures measured. * Indicate significantly increased values (P ~< 0.05) for rats in withdrawal (i.c.v and/or s.c.) compared to their respective dependent controls not in withdrawal.
A: CNS structures in which OD ratios are significantly increased for rats of both groups in withdrawal compared to non-withdrawing controls. B: CNS structures in which OD ratios are significantly increased for rats of only one group in withdrawal (i.c.v. or s.c.) compared to their controls. C: CNS structures in which OD ratios are not significantly different between groups in withdrawal and those not in withdrawal. See Abbreviation List at end of article for abbreviations.
rats. In another study, 4 rats (300 g), implanted with 3 pellets (225 mg of morphine) and given naloxone 3 days later, manifested less pronounced withdrawal (data not shown) than the s.c. pellet or i.c.v. MS-treated rats used in this study; yet the amount of weight loss was still higher than in rats made dependent by i.c.v. MS infusion (11.0 g vs 6.14 g; P ~< 0.05).
0
R C G U analysis
/J,/
/" S
ICV N
A
L
O
X
,
,
n o
e
o
-
y e s
FJ8. 3. Correlation of OD ratios for i.c.v, versus s.c. dependent rats. O, dependent rats in withdrawal (r = 0.958); O, dependent rats not in withdrawal (r = 0.97]).
RESULTS Behavioral analysis
Rats in withdrawal that were made dependent by either i.c.v, or s.c. administration of morphine had an increase in weight loss, A N S score, and incidence of diarrhea compared to all controls (Fig. 1). Jumps occurred only in morphine-dependent rats that received naloxone. Rats made dependent by s.c. morphine pellets had an increased incidence of jumping compared to their controls not in withdrawal, whereas rats made dependent by i.c.v. MS infusion did not. In comparing the two groups of rats in withdrawal (i.c.v. and s.c. morphinetreated), there were no differences in incidence of jumps, A N S score, or incidence of diarrhea. There was, however, greater weight loss during withdrawal in s.c. morphine-treated rats compared to i.c.v. MS-treated
In Fig. 2 A - C , brain structures have been arbitrarily divided into 3 categories based on R C G U changes. Structures in Fig. 2A have increased R C G U in both groups of rats in withdrawal compared to their respective dependent controls not in withdrawal. Structures in Fig. 2B have increased R C G U in one of the groups in withdrawal (either i.c.v. MS or s.c. morphine pellettreated) compared to its controls, but not the other group in withdrawal compared to its controls. Structures in Fig. 2C show no difference between groups in withdrawal and controls. With the exception of the lateral septum, there were no differences in O D ratios between the two groups in withdrawal. The correlation between O D ratios for i.c.v, vs s.c. morphine administration for rats in withdrawal and controls is shown in Fig. 3. The correlation is high with an r value of 0.971 for i.c.v, vs s.c. morphinetreated controls (i.e. receiving saline on experimental day); while the r value for the correlation between i.c.v. MS- and s.c. morphine pellet-treated rats receiving naloxone on the experimental day was 0.958. DISCUSSION The chronic administration of morphine by either the i.c.v, or s.c. route results in robust withdrawal behavior upon treatment with naloxone. Rats made dependent by i.c.v, infusions received a total of 2.9 mg of morphine over 3 days, while rats made dependent by s.c. pellet
TABLE I Summary of behavioral data for ANS, jumps, and incidence of diarrhea
Diarrhea is defined as increased frequency and/or water content of stools over the 50 min observation period. Range
No. of animals responding
ANS score/>1 i.c.v. MS s.c. Pellets Jumps i.c.v. MS s.c. Pellets Diarrhea i.c.v. MS s.c. Pellets
Mean
NALOX
n.s.
P
NALOX
n.s.
NALOX
n.s.
7/7 5/5
0/4 0/4
O.003 0.008
3.5-5.5 5.0-6.5
0-1 0-1
4.5 5.4
0.5 (1.5
4/7 4/5
0/4 0/4
0.106 0.0397
0-4 0-10
0 0
1.14 5.2
0 0
7/7 5/5
1/4 1/4
0.024 0.048
implantation received 675-825 mg of morphine over 5 days. The amount of morphine released from these pellets was not directly measured in this study but has been measured by others to be 25-50% in the first 2 days after implantation 8. Even if a small amount of the i.c.v.-administered morphine entered the systemic circulation, the concentration would be too low to produce any significant pharmacological effect in the periphery. Therefore, RCGU and behavioral signs of morphine withdrawal in rats made dependent by the i.c.v, route could only result from the chronic effects of morphine at sites of action in the central nervous system (CNS). Behaviorally, there was little difference between the two groups in withdrawal with the notable exception of the much greater weight loss during withdrawal in rats made dependent by s.c. pellet implantation. The 100% incidence of diarrhea in rats made dependent by the i.c.v. MS route indicates that there is a purely central component to withdrawal diarrhea as suggested by others 9'1t'12. This is likely a part of the generalized autonomic release manifested by both dependent groups in withdrawal. The magnitude of diarrhea as estimated by weight loss, however, is higher in rats made dependent by s.c. morphine pellets, suggesting a significant peripheral or spinal component to withdrawal diarrhea in that group. Due to limitations in our model for central dependence, our study cannot address effects at the spinal level. With our i.c.v, infusions we had no means of determining whether infused MS gained access to the spinal cord via the subarachnoid space. We therefore cannot exclude a spinal influence on gut motility through the action of i.c.v, morphine on spinal cord receptors. Thus, we cannot say for certain whether the increased gut motility is generated at the spinal level, higher in the CNS, or perhaps some combination. In concordance with work from other groups it appears likely that there is a significant contribution by enteric opiate receptors to the increased diarrheal weight loss of rats made dependent on systemically administered morphine 2'4'1°. RCGU analysis showed that, with the exception of the lateral septum, changes in cerebral metabolism during withdrawal is similar whether dependence is induced by systemic or central administration of morphine. The area of the lateral septum where measurements of OD were made is proximal to the site of MS infusion; thus, the lack
of increased RCGU may be an artifact of the infusion. Furthermore, while the mean OD ratios of lateral septum for the two groups in withdrawal are different, their relative increases over their controls not in withdrawal are quite similar (Fig. 2A). Artifacts of i.c.v. MS infusion can be controlled by comparison of rats in withdrawal and those not in withdrawal. One limitation to the use of i.c.v, infusions for the production of dependence is the production of ventriculitis with prolonged, voluminous i.c.v, infusions. For this reason, i.c.v, infusions were limited to 3 days at a rate of 0.5 pl/h. Above this rate and duration, the ventricle became progressively enlarged and the ependymal lining inflamed, obscuring the RCGU pattern of periventricular structures. Lack of solubility limited the usable MS concentration to 80 mg/ml. Above this concentration, MS would precipitate in the infusion apparatus or lateral ventricle. Thus, technical restrictions limited the degree of dependence that could be produced by i.c.v, morphine infusion. It is therefore possible, but unlikely5'6, that a qualitatively different RCGU pattern during withdrawal would emerge if greater dependence could be produced by the i.c.v, route. A more potent opiate agonist infused intracerebroventricularly at a lower rate might produce greater dependence and more robust withdrawal. Nevertheless, rats in withdrawal after production of dependence by the i.c.v, or s.c. route of morphine administration have an essentially identical profile of RCGU changes. Withdrawal behaviors were also essentially identical with the single exception of greater weight loss with subcutaneous administration, suggesting withdrawal at visceral sites in addition to or possibly synergistic with withdrawal in the CNS. Thus, the unique pattern of changes in RCGU seen during morphine withdrawal appears to result exclusively from chronic effects of morphine in the brain. There appear to be no significant contributions of peripheral effects of chronic morphine administration on the pattern of RCGU during precipitated morphine withdrawal.
ABBREVIATIONS (USED IN FIG. 2)
IPN DBB MSEPT VMN H DMN PT CENTR VMN TH STR
MPOA LPOA LSEPT CNA PVN LHypo LHB
medial preoptic area lateral preoptic area lateral septum central nucleus of amygdala periventricular nucleus of hypothalamus lateral hypothalamus lateral habenula
Acknowledgements. This work was supported by NIH-NIDA DA03787 and by the Mary Anderson Harrison Endowment of the University of Virginia. We wish to thank Rose Powell and Pamela Norem for their efforts in the preparation of this manuscript and Dr. Don Kaiser for statistical assistance. interpeduncular nucleus nucleus diagonal band of broca medial septum ventromedial nucleus of hypothalamus dorsomedial nucleus of hypothalamus paratenial nucleus thalamus centromedian nucleus thalamus ventromedial nucleus thalamus striatum
10 GP ACC MMam LMam
globus pallidus nucleus accumbens medial mammillary nucleus lateral mammillary nucleus
REFERENCES 1 Armitage, P., Statistical Methods in Medical Research, Wiley, New York, 1971, 504 pp. 2 Bianchetti, A., Giudice, A., Nava, E and Manara, L., Dissociation of morphine withdrawal diarrhea and jumping in mice by the peripherally selective opioid antagonist SR 58002 C, Life Sci., 39 (1986) 2297-2303. 3 Duncan, D.B., Multiple range and multiple F-tests, Biometrics, 11 (1955) 171-189. 4 Ehrenpreis, S., Greenberg, J. and Comaty, J.E., Mechanism of development of tolerance to injected morphine by guinea pig ileum, Life Sci., 17 (1975) 49-54. 5 Geary II, W.A. and Wooten, G.F., A behavioral and 2deoxyglucose autoradiographic study of the effects of the cumulative morphine dose on naloxone precipitated withdrawal in the rat, Brain Research, 275 (1983) 117-126. 6 Geary II, W.A. and Wooten, G.F., Dose effects of naloxone on fixed morphine dependence: simultaneous behavioral and 2deoxyglucose study in the rat, Brain Research, 332 (1985) 69-78. 7 Geary II, W.A. and Wooten, G.F., Similar functional anatomy of spontaneous and precipitated morphine withdrawal, Brain Research, 334 (1985) 183-186. 8 Gibson, R.D. and Tingstad, J.E., Formulation of a morphine implantation pellet suitable for tolerance-physical dependence
AVAM Z1 RAPHE SCSG
anteroventral/anteromedial nucleus thalamus zona incerta median raphe superior colliculus superficial grey
studies in mice, J. Pharmacol. Sci., 59 (1970) 426-427. 9 Huffman, R.D., Simmons, K.E. and Lum, J.T., An intraventricular infusion model for inducing morphine dependence in rats: quantitative assessment of precipitated withdrawal, Behav. Neurosci., 99 (1985) 861-880. 10 Kromer, W. and Woinoff, R., Peristalsis in the isolated guineapig ileum during opiate withdrawal, Arch. Pharmacol., 314 (1980) 191-193. 11 Laschka, E., Herz, A. and Blasig, J., Sites of action of morphine involved in the development of physical dependence in rats. I. Comparison of precipitated morphine withdrawal after intraperitoneal and intraventricular injection of morphine antagonists, Psychopharmacologia, 46 (1976) 133-139. 12 Laschka, E., Teschemacher, H., Mehraein, P. and Herz, A., Sites of action of morphine involved in the development of physical dependence in rats. II. Morphine withdrawal precipitated by application of morphine antagonists into restricted parts of the ventricular system and by microinjection into various brain areas, Psychopharmacologia, 46 (1976) 141-147. 13 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1986, 119 plates. 14 Wooten, G.F., DiStefano, P. and Collins, R.C., Regional cerebral glucose utilization during morphine withdrawal in the rat, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3360-3364.
Brain Research, 518 (1990) 6-10 Elsevier
BRES 15509
Dependence and withdrawal following intracerebroventricular and systemic morphine administration" functional anatomy and behavior Robert E. Adams and G. Frederick Wooten Department of Neurology, University of Virginia School of Medicine, Charlottesville, VA 22908 (U.S.A.) (Accepted 7 November 1989) Key words: Morphine; Intracerebroventricular; 2-Deoxy-o-glucose; Autoradiography; Naloxone; Minipump; Regional cerebral glucose utilization
Regional cerebral glucose utilization (RCGU) and behavior during precipitated morphine withdrawal were studied in rats made dependent by either intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of morphine. [14C]2-deoxy-D-glucoseautoradiography revealed that RCGU increased in an anatomically related group of limbic and brainstem structures in rats that were in morphine withdrawal precipitated by naloxone administration compared to morphine-dependent controls that were not in precipitated withdrawal. Correlation of RCGU for 24 brain structures comparing i.c.v, vs s.c. morphine-treated rats was highly significant for groups in withdrawal and for controls (r values, 0.958 and 0.971, respectively). Withdrawal behaviors including autonomic signs of withdrawal, withdrawal jumping, and incidence of diarrhea were not different between the two groups in withdrawal (i.c.v. and s.c.), Weight loss during withdrawal increased (P < 0.05) in rats made dependent by s.c. morphine administration compared to rats that received morphine by the i.c.v, route. Taken together, these results indicate that RCGU changes during morphine withdrawal result solely from effects of chronic morphine in the central nervous system, not in peripheral sites. The increased weight loss of s.c.-treated, morphine-dependent rats in withdrawal suggests an independent peripheral effect perhaps mediated by visceral opiate receptors. INTRODUCTION Increases in
MATERIALS AND METHODS regional cerebral
glucose
utilization
( R C G U ) occur in an anatomically related group of limbic and brainstem structures during morphine withdrawal in the rat 5-7A4. The magnitude of these regionally selective increases in R C G U is directly related to both the total morphine dose administered to produce dependence and the naloxone dose used to precipitate withdrawal 5'6. The same structures are metabolically active whether withdrawal is precipitated by naloxone or occurs through abstinence 7. Behavioral and physiological (in vivo and in vitro) studies have suggested that at least some manifestations of opiate withdrawal may result in part from the effects of opiates at peripheral sites 2'4'~°-1z. For example, diarrhea during opiate withdrawal may result from not only central effects but also systemic effects of opiates on the gut 2"9"11,12. Previously, R C G U during withdrawal was only studied after systemic administration of morphine. To address the relative roles of central vs peripheral effects of chronic morphine administration on R C G U during withdrawal, we have also studied R C G U and behavior during naloxone-precipitated morphine withdrawal in rats made d e p e n d e n t upon morphine administered by the intracerebroventricular (i.c.v.) route.
Male Sprague-Dawley rats (250-300 g, Dominion Labs, Dublin, VA) were given food and water ad libitum and kept in a 12 h light-dark cycle throughout the study. Morphine dependence was produced in some groups by i.c.v, infusion of morphine sulfate (MS) (Mallinckrodt, St. Louis, MO), and in other groups by subcutaneous (s.c.) implantation of sustained-release, morphine-base pellets (NIDA, Rockville, MD). An effort was made to produce similar degrees of dependence (i.e. comparable degrees of behavioral withdrawal) in all dependent groups whether morphine was administered by the i.c.v, or s.c. route. 1.c. v. infusions Rats were anesthetized with a 1 ml/kg intraperitoneal injection of 1:1 ketamine/xylozine cocktail (100 mg/ml to 20 mg/ml) and placed in a Kopf stereotactic frame (incisor bar, -3.3 to -3.5 ram). The skull was exposed by making a mid-sagittal scalp incision, and a burr hole was made over the right lateral ventricle (bregma -0.9 mm, right -1.5 mm, ventrally 3.5 mm from brain surface) 13. The infusion apparatus consisted of an Alzet osmotic minipump (Model 2002) connected to a 26 gauge stainless steel blunt tube via PE 60 tubing. The entire apparatus, primed and filled with MS (80 mg/ml distilled water), was implanted subcutaneously between the scapulae with the tip of the blunt tube in the right lateral ventricle held in place by skull screws and dental acrylic. Morphine sulfate was then infused at the rate of 40/~g.0.5/d 1.h-1 for 3 days by the apparatus. A concentration of 40 ktg/0.5/~1 has proven to be well within the limits of solubility at in vivo temperatures. In addition, no precipitation of MS was found in any rats upon sectioning of infusion apparatus, and histologic examination of tissue. Infusions of higher concentrations of morphine sulfate (over 50 ~g.#l-l.h-~) resulted in definite precipitation and ventriculitis.
Correspondence: G.F. Wooten, Department of Neurology, Box 394, University of Virginia Medical Center, Charlottesville, VA 22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Morphine pellet implantation Under light halothane anesthesia (2%), a 1 cm midline incision dorsal to the mid thoracic spine was produced in the rat's trunk. An s.c. pocket was made, into which sustained-release, morphine-base pellets containing 75 mg of morphine per pellet were implanted (1 pellet/45 g body weight). Three days later, using the same technique, pellets were implanted into a second s.c. pocket (l pellet/70 g) and left in place 2 additional days for a total of 5 days of morphine pellet exposure.
measurements of each lateral structure or 4 total measurements of each midline structure were taken and expressed as an average for each brain.
Statistics Comparisons of OD values and weight loss among the 5 groups were made using Duncan's Multiple Range TesP. Analysis of jumping behavior, ANS, and presence of diarrhea was performed using Fisher's Exact Test 1.
Experimental groups Groups of rats in withdrawal consisted of rats made dependent by i.c.v, or s.c. morphine administration and given an intravenous (i.v.) injection of naloxone HCI (Dupont, Wilmington, DE) on the day of experimentation. Controls consisted of morphine-dependent rats (i.c.v. or s.c.) given i.v. normal saline on experimental day and rats that received a 3-day, chronic, i.c.v, infusion of sterile distilled water (dHzO) (using the same technique described for the infusion of MS above) and i.v. naloxone on the experimental day.
[~ I ~ [z23
= °e
~
A
SC PELLETS - NALOX SC PELLETS - NS ICV MS % NALOX ICY MS - NS
6
o L
Experimentation On the day of experimentation (day 3 for i.c.v.-administered groups, day 5 for s.c.-administered groups) under 2% halothane anesthesia, the right jugular vein was cannulated with PE 50 tubing and the other end was tunneled subcutaneously and exposed through a stab wound in the back. Rats were then placed in separate cages to recover for 2-4 h prior to experimentation. After recovery from general anesthesia, rats were placed in a cylindrical glass container (30 cm diameter, 37 cm height) and given a 1 mg/kg injection of either naloxone HCI 5 mg/ml in normal saline or normal saline alone, and, 1 min later, all groups were injected with 10 ItCi/lO0 g of [x4C]2-deoxy-D-glucose (American Radiolabelled Chemicals, St. Louis, MO). The following behavioral signs of withdrawal were then observed and recorded: jumps/20 min, weight loss/50 min, and the presence of autonomic signs (ANS) for 50 min. Autonomic signs recorded included tachypnea, teeth chatter, vocalization to touch, irritability, ptosis, writhing, forepaw tremor, diarrhea, urination, lacrimation, salivation, rhinorrhea, erection and ejaculation. Each ANS was assigned l/z point for a total potential ANS score of 7. After the 50 min observation period, rats were killed by decapitation, and the brains were removed, frozen and embedded. Twenty/tm coronal sections were taken and exposed to Kodak SB-5 X-ray film for 5 days to produce an autoradiogram. Optical densities of various structures were measured using a Leitz Ergolux variable aperture microdensitometer and expressed as a ratio of the optical density (OD) of that structure to the OD of corpus callosum (representing pure white matter). No fewer than 4 bilateral
g o P,
[EEE] S C P E L L E T S - N A L O X I SO PELLETS - NS ICY M S - N A L O X
"z
EZZ] ICY MS - NS I ~ ICY dH20 - NALOX
15-
10-
LSEPT
CNA
PVN
LHYPO
LHB
IPN
B
10~
w,
4
0 DBB
10
WSEPT
)
VMN H
DMN
1i11
PT
CENTR VMN TH
6
20
I
i
~ STR
I
S
W w
0i
LPOA
4
20.
E
0 MPOA
or--
o
2
r-
i
c
Fig. 1. Weight loss, mean + S.D., and statistical group assignment (A,B,C) according to the Duncan Multiple Range test. A, B, and C are defined by Duncan Multiple Range analysis to be groups of numbers (in this case, g of weight loss/rat) that are statistically unrelated to groups of different letter, and related to groups of same letter (P ~ 0.05).
GP
ACC
MMAM LIWAM AVAM
Zl
RAPHE SCSG
Fig. 2. Mean O D ratios (OD CNS structure/OD corpus callosum) and S.D. for 24 CMS structures measured. * Indicate significantly increased values (P ~< 0.05) for rats in withdrawal (i.c.v and/or s.c.) compared to their respective dependent controls not in withdrawal.
A: CNS structures in which OD ratios are significantly increased for rats of both groups in withdrawal compared to non-withdrawing controls. B: CNS structures in which OD ratios are significantly increased for rats of only one group in withdrawal (i.c.v. or s.c.) compared to their controls. C: CNS structures in which OD ratios are not significantly different between groups in withdrawal and those not in withdrawal. See Abbreviation List at end of article for abbreviations.
rats. In another study, 4 rats (300 g), implanted with 3 pellets (225 mg of morphine) and given naloxone 3 days later, manifested less pronounced withdrawal (data not shown) than the s.c. pellet or i.c.v. MS-treated rats used in this study; yet the amount of weight loss was still higher than in rats made dependent by i.c.v. MS infusion (11.0 g vs 6.14 g; P ~< 0.05).
0
R C G U analysis
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FJ8. 3. Correlation of OD ratios for i.c.v, versus s.c. dependent rats. O, dependent rats in withdrawal (r = 0.958); O, dependent rats not in withdrawal (r = 0.97]).
RESULTS Behavioral analysis
Rats in withdrawal that were made dependent by either i.c.v, or s.c. administration of morphine had an increase in weight loss, A N S score, and incidence of diarrhea compared to all controls (Fig. 1). Jumps occurred only in morphine-dependent rats that received naloxone. Rats made dependent by s.c. morphine pellets had an increased incidence of jumping compared to their controls not in withdrawal, whereas rats made dependent by i.c.v. MS infusion did not. In comparing the two groups of rats in withdrawal (i.c.v. and s.c. morphinetreated), there were no differences in incidence of jumps, A N S score, or incidence of diarrhea. There was, however, greater weight loss during withdrawal in s.c. morphine-treated rats compared to i.c.v. MS-treated
In Fig. 2 A - C , brain structures have been arbitrarily divided into 3 categories based on R C G U changes. Structures in Fig. 2A have increased R C G U in both groups of rats in withdrawal compared to their respective dependent controls not in withdrawal. Structures in Fig. 2B have increased R C G U in one of the groups in withdrawal (either i.c.v. MS or s.c. morphine pellettreated) compared to its controls, but not the other group in withdrawal compared to its controls. Structures in Fig. 2C show no difference between groups in withdrawal and controls. With the exception of the lateral septum, there were no differences in O D ratios between the two groups in withdrawal. The correlation between O D ratios for i.c.v, vs s.c. morphine administration for rats in withdrawal and controls is shown in Fig. 3. The correlation is high with an r value of 0.971 for i.c.v, vs s.c. morphinetreated controls (i.e. receiving saline on experimental day); while the r value for the correlation between i.c.v. MS- and s.c. morphine pellet-treated rats receiving naloxone on the experimental day was 0.958. DISCUSSION The chronic administration of morphine by either the i.c.v, or s.c. route results in robust withdrawal behavior upon treatment with naloxone. Rats made dependent by i.c.v, infusions received a total of 2.9 mg of morphine over 3 days, while rats made dependent by s.c. pellet
TABLE I Summary of behavioral data for ANS, jumps, and incidence of diarrhea
Diarrhea is defined as increased frequency and/or water content of stools over the 50 min observation period. Range
No. of animals responding
ANS score/>1 i.c.v. MS s.c. Pellets Jumps i.c.v. MS s.c. Pellets Diarrhea i.c.v. MS s.c. Pellets
Mean
NALOX
n.s.
P
NALOX
n.s.
NALOX
n.s.
7/7 5/5
0/4 0/4
O.003 0.008
3.5-5.5 5.0-6.5
0-1 0-1
4.5 5.4
0.5 (1.5
4/7 4/5
0/4 0/4
0.106 0.0397
0-4 0-10
0 0
1.14 5.2
0 0
7/7 5/5
1/4 1/4
0.024 0.048
implantation received 675-825 mg of morphine over 5 days. The amount of morphine released from these pellets was not directly measured in this study but has been measured by others to be 25-50% in the first 2 days after implantation 8. Even if a small amount of the i.c.v.-administered morphine entered the systemic circulation, the concentration would be too low to produce any significant pharmacological effect in the periphery. Therefore, RCGU and behavioral signs of morphine withdrawal in rats made dependent by the i.c.v, route could only result from the chronic effects of morphine at sites of action in the central nervous system (CNS). Behaviorally, there was little difference between the two groups in withdrawal with the notable exception of the much greater weight loss during withdrawal in rats made dependent by s.c. pellet implantation. The 100% incidence of diarrhea in rats made dependent by the i.c.v. MS route indicates that there is a purely central component to withdrawal diarrhea as suggested by others 9'1t'12. This is likely a part of the generalized autonomic release manifested by both dependent groups in withdrawal. The magnitude of diarrhea as estimated by weight loss, however, is higher in rats made dependent by s.c. morphine pellets, suggesting a significant peripheral or spinal component to withdrawal diarrhea in that group. Due to limitations in our model for central dependence, our study cannot address effects at the spinal level. With our i.c.v, infusions we had no means of determining whether infused MS gained access to the spinal cord via the subarachnoid space. We therefore cannot exclude a spinal influence on gut motility through the action of i.c.v, morphine on spinal cord receptors. Thus, we cannot say for certain whether the increased gut motility is generated at the spinal level, higher in the CNS, or perhaps some combination. In concordance with work from other groups it appears likely that there is a significant contribution by enteric opiate receptors to the increased diarrheal weight loss of rats made dependent on systemically administered morphine 2'4'1°. RCGU analysis showed that, with the exception of the lateral septum, changes in cerebral metabolism during withdrawal is similar whether dependence is induced by systemic or central administration of morphine. The area of the lateral septum where measurements of OD were made is proximal to the site of MS infusion; thus, the lack
of increased RCGU may be an artifact of the infusion. Furthermore, while the mean OD ratios of lateral septum for the two groups in withdrawal are different, their relative increases over their controls not in withdrawal are quite similar (Fig. 2A). Artifacts of i.c.v. MS infusion can be controlled by comparison of rats in withdrawal and those not in withdrawal. One limitation to the use of i.c.v, infusions for the production of dependence is the production of ventriculitis with prolonged, voluminous i.c.v, infusions. For this reason, i.c.v, infusions were limited to 3 days at a rate of 0.5 pl/h. Above this rate and duration, the ventricle became progressively enlarged and the ependymal lining inflamed, obscuring the RCGU pattern of periventricular structures. Lack of solubility limited the usable MS concentration to 80 mg/ml. Above this concentration, MS would precipitate in the infusion apparatus or lateral ventricle. Thus, technical restrictions limited the degree of dependence that could be produced by i.c.v, morphine infusion. It is therefore possible, but unlikely5'6, that a qualitatively different RCGU pattern during withdrawal would emerge if greater dependence could be produced by the i.c.v, route. A more potent opiate agonist infused intracerebroventricularly at a lower rate might produce greater dependence and more robust withdrawal. Nevertheless, rats in withdrawal after production of dependence by the i.c.v, or s.c. route of morphine administration have an essentially identical profile of RCGU changes. Withdrawal behaviors were also essentially identical with the single exception of greater weight loss with subcutaneous administration, suggesting withdrawal at visceral sites in addition to or possibly synergistic with withdrawal in the CNS. Thus, the unique pattern of changes in RCGU seen during morphine withdrawal appears to result exclusively from chronic effects of morphine in the brain. There appear to be no significant contributions of peripheral effects of chronic morphine administration on the pattern of RCGU during precipitated morphine withdrawal.
ABBREVIATIONS (USED IN FIG. 2)
IPN DBB MSEPT VMN H DMN PT CENTR VMN TH STR
MPOA LPOA LSEPT CNA PVN LHypo LHB
medial preoptic area lateral preoptic area lateral septum central nucleus of amygdala periventricular nucleus of hypothalamus lateral hypothalamus lateral habenula
Acknowledgements. This work was supported by NIH-NIDA DA03787 and by the Mary Anderson Harrison Endowment of the University of Virginia. We wish to thank Rose Powell and Pamela Norem for their efforts in the preparation of this manuscript and Dr. Don Kaiser for statistical assistance. interpeduncular nucleus nucleus diagonal band of broca medial septum ventromedial nucleus of hypothalamus dorsomedial nucleus of hypothalamus paratenial nucleus thalamus centromedian nucleus thalamus ventromedial nucleus thalamus striatum
10 GP ACC MMam LMam
globus pallidus nucleus accumbens medial mammillary nucleus lateral mammillary nucleus
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AVAM Z1 RAPHE SCSG
anteroventral/anteromedial nucleus thalamus zona incerta median raphe superior colliculus superficial grey
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