Cerebrospinal fluid from morphine-dependent rats precipitates opiate abstinence syndrome

Life Sciences, Vol. 41, pp. 377-383 Printed in the U.S.A.

CEREBROSPINAL

Pergamon Journal

FLUID FROM MORPHINE-DEPENDENT OPIATE ABSTINENCE SYNDROME

RATS PRECIPITATES

David H. Malin, Leah Hatter, Phillip D. Jenkins, Ralph D. Monfort, Patricia D. Bruce, Patricia A. Farley, Robert Ferebee, Ken L. Thrasher, Daniel S. Marullo. Programs in Behavioral and Biological Sciences, Univ. of Houston - Clear Lake, Houston, TX. 77058 (Received in final form May 15, 1987) Summary Cerebrospinal fluid (CSF) was withdrawn from opiatedependent rats following six hours of abstinence. It was infused into the third ventricle of opiatedependent rats, precipitating immediate abstinence signs. The effect was similar to that of infusing the opiate antagonist naloxone, suggesting that opiate-dependent organisms may secrete an endogenous opiate antagonist substance. CSF withdrawn from nondependent rats failed to precipitate an abstinence syndrome in morphine-dependent recipients. Conversely, CSF withdrawn from opiate-dependent rats following six hours of abstinence failed to precipitate an abstinence syndrome in non-dependent recipients. The active factor in the CSF is probably a peptide since it is filterable through a 10,000 MW filter and its activity is destroyed by three different proteolytic enzymes.

The first reports of a m o r p h i n e - t o l e r a n c e - i n d u c i n g peptide factor in brain homogenate of morphine-tolerant rats predated the discovery of the enkephalins by nearly a decade (i). Later studies (2) suggested that extracts of such homogenates, when injected i.p. into recipient animals, induced accelerated dependence formation and tolerance to morphine analgesia (tail pinch test). The extracts also had a naloxone-like morphine antagonist effect on the in vitro vas deferens assay (2). Han et al (3) found that intracerebral injection of brain extract from morphine-tolerant rats or from acupuncture-tolerant rats induced immediate tolerance to morphine analgesia. They characterized the active factor as a peptide. Lu, Johannessen and Mayer (4) reported that intrathecal injection of cerebrospinal fluid (CSF) from tolerant rats induced tolerance to opiate analgesia. Wahlstrom and Terenius (5) found that CSF from morphine-tolerant rats and human heroin addicts contained a factor capable of inducing morphine tolerance in recipient rats. Recently, it has been hypothesized that the neuropeptide CCK might act as an endogenous opiate antagonist (6). Tang, Chou, 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

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CSF Precipitates Opiate Abstinence

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Iadorla, Yang, and Costa (7) have shown that spinal morphine injection causes marked release of CCK-octapeptide. In turn, CCK-8 induces apparent tolerance to the spinal analgesic effects of morphine (8). A striking property of opiate antagonist drugs like naloxone is their ability to precipitate an immediate abstinence syndrome in opiate-dependent organisms. It was therefore of interest to determine whether CSF from abstinent morphine-dependent rats could, like naloxone, precipitate withdrawal in morphine treated rats. The third ventricle was chosen as a target for CSF injection, based on preliminary studies (9) showing that intraventricular naloxone at that site precipitated the most rapid onset of abstinence signs. Method The subjects were thirty 300 g male Sprague-Dawley rats maintained on ad lib food and water and a 12 hour light/dark cycle. Fifteen recipient rats were cannulated in the third ventricle and implanted subcutaneously with two Alzet 2001 osmotic minipumps filled with either 50 mg/ml morphine sulfate in saline or with saline alone. I The continuous morphine infusion rate was 0.33 mg/kg/hr. Cannula placements were subsequently confirmed by examination of brain sections following cannula injection of methylene blue dye. Fifteen donor rats were implanted with either one larger Alzet 2MLI minipump, infusing them with 1.67 mg/kg/hr morphine sulfate or with saline alone. The combination of donors infused at a high rate with recipients infused at a lower rate was the most effective procedure in pilot experiments. The aim was to induce a large amount of endogenous opiate antagonist in the donor CSF relative to the amount of morphine in the recipient. After 7 days of infusion, the donors' pumps were removed and 6 hours was allowed for clearance of morphine from their CSF. It has been reported that 6 hours is sufficient for 96 percent clearance of labeled morphine from rat brain (i0). CSF (80 ~i) was then withdrawn under ether anesthesia from the cisterna magna of each donor and was gradually injected by motorized syringe (i0 ~i/min) into the third ventricle of a conscious recipient rat. CSF samples were coded so that recipients were observed on a blind basis. Recipients were placed in a 45 cm square clear plastic observation chamber and observed for abstinence signs during the 30 minute period following the onset of CSF injection. A standard tally sheet based primarily on Gianutsos et al (II) was employed. Behavioral signs included vigorous wet-dog shakes, abdominal writhes, hindfoot scratches, dyspnea, chewing, seminal ejaculation, genital licking, ptosis and escape jumping. The overall abstinence score for each rat was the total frequency of observed signs across all categories. iIn previous studies in our laboratory, continuous subcutaneous morphine infusion via Alzet 2001 minipump (0.33 mg/kg/hr) was so gradual that the morphine failed to have any analgesic or respiratory depressing effects. Yet, after seven days of infusion, rats showed significant morphine tolerance and exhibited an abstinence syndrome upon naloxone challenge or following abrupt withdrawal (12).

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A d d i t i o n a l e x p e r i m e n t s w e r e c a r r i e d out to p r o v i d e a p r e l i m i n a r y c h a r a c t e r i z a t i o n of the active s u b s t a n c e in CSF from d e p e n d e n t rats. CSF was w i t h d r a w n from t w e n t y - f o u r h i g h l y morphine-dependent rats p r e p a r e d as d e s c r i b e d above. CSF was v a c u u m f i l t e r e d at 4 d e g r e e s C t h r o u g h a c o l l o d i o n filter w i t h a i0,000 mol. wt. cut-off. E i g h t y ~I a l i q u o t s of f i l t r a t e and s a l i n e - r e c o n s t i t u t e d r e t e n t a t e w e r e infused, as d e s c r i b e d above, into the third v e n t r i c l e of m o r p h i n e - d e p e n d e n t r e c i p i e n t rats. As before, rats w e r e o b s e r v e d for 30 m i n u t e s on a s t a n d a r d c h e c k l i s t of o p i a t e a b s t i n e n c e signs. F u r t h e r c h a r a c t e r i z a t i o n p r o c e d u r e s w e r e c a r r i e d out u p o n the f i l t r a t e alone. Four m o r p h i n e - d e p e n d e n t r e c i p i e n t rats w e r e i n f u s e d w i t h CSF f i l t r a t e that had b e e n heat t r e a t e d (I00°C for 150 secs.). Four a d d i t i o n a l groups of m o r p h i n e - d e p e n d e n t r e c i p i e n t rats were i n f u s e d w i t h CSF f i l t r a t e w h i c h had b e e n i n c u b a t e d w i t h v a r i o u s e n z y m e s for 30 mins. at 30 d e g r e e s C. Samples of CSF f i l t r a t e w e r e i n c u b a t e d w i t h o u t e n z y m e or w i t h an excess of c a r b o x y p e p t i d a s e A, trypsin, or c h y m o t r y p s i n . In e a c h case, the i n c u b a t i o n was c a r r i e d out w i t h 6 units of Sigma I n s o l u b l e E n z y m e s per ml of CSF. These enzymes, b o u n d to A g a r o s e beads, w e r e r e m o v e d by c e n t r i f u g a t i o n f o l l o w i n g incubation. A further e x p e r i m e n t t e s t e d w h e t h e r CCK-8 m i g h t c o n s t i t u t e the a c t i v e p e p t i d e in the CSF. CSF was w i t h d r a w n from 12 m o r p h i n e - d e p e n d e n t rats in m o r p h i n e w i t h d r a w a l and from 12 nond e p e n d e n t rats. As w i t h the d o n o r rats above, A l z e t 2MLI o s m o t i c m i n i p u m p s w e r e r e m o v e d six hours prior to CSF s a m p l i n g s u b s e q u e n t to seven days of i n f u s i o n (1.67 m g / k g / h r m o r p h i n e s u l f a t e or saline alone). E i g h t y ~i a l i q u o t s from e a c h rat w e r e c o m b i n e d into two p o o l e d samples (morphine d e p e n d e n t and nondependent) for d e t e r m i n a t i o n of CCK-8 c o n c e n t r a t i o n s by r a d i o i m m u n o a s s a y as d e s c r i b e d by Fried et al (13). The a s s a y was p e r f o r m e d by G.H. Greeley, S u r g i c a l B i o c h e m i s t r y Laboratory, Univ. of Texas M e d i c a l Branch, Galveston. Results In the 5 cases w h e r e the donor and the r e c i p i e n t had b o t h b e e n m o r p h i n e infused, CSF r e c i p i e n t s e x h i b i t e d 42.0 ± 8.6 a b s t i n e n c e signs (M ± SEM), b e g i n n i n g in several rats w i t h i n 5 seconds of the onset of CSF injection. This was not s i g n i f i c a n t l y d i f f e r e n t from the 41.3 • 4.3 signs p r e c i p i t a t e d by 80 ~i of 3 m g / m l n a l o x o n e i n j e c t e d in the same site in three other m o r p h i n e - d e p e n d e n t rats, t(6)= 0.056, NS. The time c o u r s e of these e f f e c t s is i l l u s t r a t e d in Fig. i. As shown in Fig. 2, the p r e d o m i n a n t signs p r e c i p i t a t e d by c e r e b r o s p i n a l fluid from opiate a b s t i n e n t d o n o r s w e r e a b d o m i n a l writhes, w e t - d o g shakes and ptosis. T e e t h chattering, scratching, h e a d shakes, e s c a p e j u m p i n g and seminal e j a c u l a t i o n w e r e also observed. In the 5 c o n t r o l cases w h e r e the d o n o r had b e e n saline i n f u s e d and the r e c i p i e n t had b e e n m o r p h i n e infused, there w e r e o n l y 3.8 • 1.9 a b s t i n e n c e signs. This suggests that n e i t h e r n o r m a l CSF nor the i n j e c t i o n p r o c e d u r e s in t h e m s e l v e s c o u l d p r e c i p i t a t e an a b s t i n e n c e syndrome. In the 5 c o n t r o l cases w h e r e the d o n o r had b e e n m o r p h i n e i n f u s e d and the r e c i p i e n t had b e e n saline infused, there were o n l y 4.4 ± 1.8 a b s t i n e n c e signs.

380

CSF Precipitates Opiate Abstinence

Vol. 41, No. 3, 1987

THIRO VENTRICLE INFUSION OF:

--

/ \ *Q

~

--

NALOXONE n=3

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,,=,

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3

(

I

i

B

g

~2

INFUSION BEGINS

I

I

I

I

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2t

24

27

30

MINUTES

Fig. i. Time course of opiate abstinence signs (cumulated over 3 min. intervals) precipitated by gradual injection (i0 ~i/min.) into the third ventricle of 80 ~i of 3 mg/ml naloxone (solid line) or of CSF (dashed line) drawn from the cisterna magna of donor rats 6 hours after termination of 7 days infusion of morphine sulfate (1.67 mg/kg/hr). BO] J 55 ~I 50 x

MORPHINE INFUSED DONOR/ MORPHINE INFUSED RECIPIENT n=5

-30

SALINE INFUSED DONOR/ MORPHINE INFUSED RECIPIENT n-5

425

45

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'40

MORPHINE INFUSED DONOR/ ~ SALINE INFUSED RECIPIENT n=5

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SEMINAL EJACULATION

=~

OTHER

Fig. 2. Frequency of several types of abstinence signs precipitated by third ventricle injection of 80 ~i CSF drawn from donor rats 6 hours after termination of 7 days infusion with either 1.67 mg/kg/hr morphine sulfate or saline alone. Recipient rats had been infused for 7 days with either 0.33 mg/kg/hr morphine sulfate or saline alone. Recipient rats were observed for 30 min. after initiation of I0 ~i/min. injection.

Vol. 41, No. 3, 1987

CSF Precipitates Opiate Abstinence

38]

This s u g g e s t s that the e n d o g e n o u s a n t a g o n i s t in the CSF, like n a l o x o n e and other e x o g e n o u s antagonists, p r e c i p i t a t e s an a b s t i n e n c e s y n d r o m e w h e n a c u t e l y a d m i n i s t e r e d to o p i a t e d e p e n d e n t o r g a n i s m s only. One w a y a n a l y s i s of v a r i a n c e i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e a m o n g groups, F ( 2 , 1 2 ) = 17.8, p< .01. Post hoc a n a l y s i s by T u k e y ' s H o n e s t l y S i g n i f i c a n t D i f f e r e n c e Test i n d i c a t e d s i g n i f i c a n t l y (p< .01) m o r e s y m p t o m s in the g r o u p w h e r e b o t h donors and r e c i p i e n t s had b e e n m o r p h i n e i n f u s e d than in either of the two c o n t r o l conditions. The d i f f e r e n c e b e t w e e n the two c o n t r o l c o n d i t i o n s was not significant. As shown in Table I, the rats r e c e i v i n g CSF f i l t r a t e (<10,000 MW) e x h i b i t e d far m o r e a b s t i n e n c e signs than rats i n f u s e d w i t h r e t e n t a t e (>10,000 MW). This d i f f e r e n c e was s i g n i f i c a n t a c c o r d i n g to S t u d e n t ' s t test, t(6) = 4.22, p< .01. The rats r e c e i v i n g h e a t - t r e a t e d CSF f i l t r a t e e x h i b i t e d 44.0 5.2 a b s t i n e n c e signs, not s i g n i f i c a n t l y d i f f e r e n t from the g r o u p r e c e i v i n g u n h e a t e d filtrate, t(5) = 1.53, NS. Rats r e c e i v i n g CSF f i l t r a t e i n c u b a t e d w i t h o u t enzyme e x h i b i t e d far m o r e a b s t i n e n c e signs than rats r e c e i v i n g CSF f i l t r a t e i n c u b a t e d w i t h any of the three p r o t e o l y t i c enzymes, as i l l u s t r a t e d in the b o t t o m s e c t i o n of Table I. One-way analysis of v a r i a n c e i n d i c a t e d a s i g n i f i c a n t o v e r a l l d i f f e r e n c e among the four groups r e c e i v i n g v a r i o u s l y i n c u b a t e d CSF, F(3,10) = 4.83, p < .05 A c c o r d i n g to D u n n e t t ' s p r o c e d u r e for c o m p a r i n g a single c o n t r o l g r o u p w i t h m u l t i p l e groups, the rats r e c e i v i n g CSF i n c u b a t e d w i t h o u t e n z y m e showed s i g n i f i c a n t l y m o r e a b s t i n e n c e signs than any other group, p < .05.

TABLE

I

A b s t i n e n t - L i k e Signs P r e c i p i t a t e d in M o r p h i n e - I n f u s e d Rats mg/ml/hr) by ICV I n j e c t i o n of V a r i o u s l y T r e a t e d CSF F r o m M o r p h i n e - D e p e n d e n t Rats. MEAN .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

SEM .

.

.

.

.

.

.

.

.

.

.

n .

.

.

.

.

.

.

.

.

Retentate

(> i0,000

MW)

13.8"*

4.4

5

Filtrate

(< 10,000

MW)

66.0

9.8

4

Boiled .

.

.

.

.

.

Filtrate .

.

.

.

.

.

.

CSF F i l t r a t e

.

.

.

44.0 .

.

.

.

.

.

.

.

Incubated

No E n z y m e

.

.

.

.

.

.

.

.

.

.

.

5.3 .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

15.7

4

6.8*

1.2

4

Trypsin

6.0*

1.2

3

Chymotrypsin

6.3*

2.0

3

Carboxypeptidase

** p < .01 vs. * p < .05 vs.

A

F i l t r a t e Group No Enzyme Group

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

3 .

With: 53.8

.

.

(0.33

382

CSF Precipitates Opiate Abstinence

Vol. 41, No. 3, 1987

Radioimmunoassay of CSF from saline-infused control rats indicated a concentration of 9 picogram/ml CCK-8, while CSF from morphine-abstinent rats exhibited a similar concentration of 10 picogram/ml. Discussion The present results suggest that prolonged opiate exposure induces the appearance in the CSF of an endogenous opiate antagonist capable of contributing to opiate abstinence syndrome. This antagonist activity is present 6 hours after termination of morphine exposure. The continuous presence of such an antagonist might plausibly lead to opiate abstinence syndrome, since chronic exposure to the artificial opiate antagonist naloxone was, unlike acute administration, sufficient to produce a quasi-abstinence syndrome in rats never exposed to opiates. A week of twice daily injections (14) or a day of continuous subcutaneous infusion (15) result in standard behavioral signs (wet-dog shakes etc.), interruption of weight gain, and greatly increased oxygen consumption. As with genuine opiate abstinence syndrome, these signs are potently reversed by morphine and clonidine and exacerbated by phosphodiesterase inhibitor IBMX (16). It seems probable that the active substance in CSF from opiate-dependent rats is a peptide since its bioactivity is destroyed by three different proteolytic enzymes and it appears to be less than I0,000 MW. However, it is unlikely that CCK-8 is the active peptide since active CSF from morphine-dependent rats contained approximately the same amount of CCK-8 as inactive CSF from non-dependent sham control rats. Further experimentation to identify the putative endogenous antagonist may be facilitated by the apparently stable abstinence-precipitating activity of CSF from morphine-dependent rats at room temperature, after boiling, and after freezing and thawing. The survival of activity after boiling is consistant with the hypothesis that the active factor is a small peptide; heat treatment at physiological pH has a far greater denaturing effect on the tertiary structure of large globular proteins than on the primary structure of small unfolded peptides (17). It should be noted that there is no evidence as yet that the endogenous antagonist need be a classic competitive inhibitor at opioid receptors. It might alternatively be a "physiological antagonist", affecting target neurons via non-opioid receptors in a direction opposite to the actions of opiates and endorphins. Acknowledgments Supported by NIDA grant DAO-3966 to D. H. Malin and grants from the Organized Research Program and the Alumni Association of Univ. of Houston - Clear Lake. Naloxone was generously donated by Du Pont Corporation. We thank W. Ronald Mills, Maria Blair, Valli Balasubramanyam and Jane T. Malin for experimental and editorial assistance.

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References i. G.A. UNGAR and M. COHEN, Int. J. Neuropharmacol. 5, 183 (1966). 2. G.A. UNGAR, A. UNGAR, D.H. MALIN, in Opiates and Endogenous Opioid Peptides, H. Kosterlitz, Ed. (Elsevier, Amsterdam, 1976); G.A. UNGAR, A. UNGAR, D.H. MALIN, G. SARANTAKIS, Psychoneuroendocrinol. 2, 1 (1977); D.H. MALIN and G. RADCLIFFE, Soc. Neurosci. Abstr. 5, 290 (1975). 3. C.S. HAN, J. TANG, B.S. HUANG, X.N. LIANG, N.H. ZHANG, in Endogenous and Exogenous Opiate Agonists and Antagonists, E. L. Way, Ed. (Pergamon Press, New York, 1980). 4. G.Q. LU, J.N. JOHANNESSEN, D.J. MAYER, Soc. Neurosci. Abstr. 8, 778 (1982). 5. A. WAHLSTROM and L. TERENIUS, Acta Physiol. Scandia II0, 427 (1980). 6. L.R. WATKINS, I.B. KINSCHECK, D.J. MAYER, Science 224, 395 (1984). 7. J. TANG, J. CHOU, M. IADORLA, H. YANG, E. COSTA, Neuropharmacol. 23, 715 (1984). 8. P.L. FARIS, B.R. KOMISARUK, L.R. WATKINS, D.L. MAYER, Science 219, 310 (1983). 9. D.H. MALIN, L. DUSSACK, P. JENKINS, J. ZOGRAFOS, P.D. BRUCE, L. HARTER and R. MONFORT. Soc. Neurosci. Abstr. ii, 468 (1985). 10. A.L. MISRA in Tissue Responses to Addictive Drugs, D.H. Ford and D.H. Clouet, Ed. pp 1-14, Spectrum Publications, New York, (1976). Ii. G. GIANUTSOS, R. DRAWBAUGH, M. HYNES, H. LAL, in Methods in Narcotic Research, S. Ehrenpreis and A. Neidle, Eds. (Marcel Dekker, New York, 1975), pp. 293-309. 12. D.H. MALIN, K.E. PEAK, K. FREEMAN, W.R. MILLS, L. NEAL, Soc. Neurosci. Abstr. 8, 162 (1982). 13. G.H. FRIED, W.D OGDEN, J.SWIERCZEK, G.H. GREELEY JR., P.L. RAYFORD, J.C. THOMPSON, Gastroenterology 85, 1113-1119 (1983). 14. D.H. MALIN, M.P. LAYNG, P. SWANK, M.J. BAKER, J.L. HOOD, Pharmacol. Biochem. Behav. 17, 389-392 (1982). 15. D.H. MALIN, J.G. LEAVELL, K. FREEMAN, W.C. KINZLER, M.A. REAGAN, Pharmacol. Biochem. Behav. 22, 791-795 (1985). 16. D.H. MALIN, A.G. HEMPEL, R.J. EXLEY, S. ADDINGTON, Pharmacol. Biochem. Behav. 25, 989-993 (1986). 17. A.L. LEHNINGER, Principles of Biochemistry, pp 113 and 140, Worth Publishers, New York (1982).