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Cyclosporine and alpha-interferon do not attenuate morphine withdrawal in rats but do impair thermoregulation
Physiology&Behavior,Vol. 39, pp. 593-598. Copyright©Pergamon Journals Ltd., 1987. Printed in the U.S.A.
0031-9384/87 $3.00 + .00
Cyclosporine and Alpha-Interferon Do Not Attenuate Morphine Withdrawal in Rats but Do Impair Thermoregulation R. D A N T Z E R , *
E. S A T I N O F F t §
AND K. W. KELLEY~:§
*Psychobiologie des Comportements adaptatifs, I N R A - I N S E R M U259 Rue Camille St-Saens, 33077 Bordeaux Cedex, France tDepartment of Psychology, University of Illinois at Urbana-Champaign ~Laboratory of lmmunophysiology, Department of Animal Sciences University of Illinois at Urbana-Champaign §Program in Neural and Behavioral Biology, University o f Illinois at Urbana-Champaign R e c e i v e d 15 O c t o b e r 1986 DANTZER, R., E. SATINOFF AND K. W. KELLEY. Cyclosporine and alpha-interferon do not attenuate morphine withdrawalin rats but do impair thermoregulation. PHYSIOL BEHAV 39(5) 593-598, 1987.--Immunomodulating drugs as diverse as alpha-interferon and cyclosporine have been reported to attenuate physical signs of morphine withdrawal in rats. On the basis of these results, the immune system has been claimed to be involved in opiate addiction. To assess whether this is the case, the effects of alpha-interferon and cyclosporine were studied on objective signs of morphine withdrawal in morphine-dependent rats. Rats made dependent upon morphine by implantation of a 75-mg morphine pellet were challenged three days later by naloxone (1 mg/kg). Pretreatment with alpha-interferon (150 U/g) or cyclosporine (15 mg/kg) did not attenuate the reduction in body weight or the behavioral suppression induced by naloxone in morphine-dependent rats trained to press a lever for food reinforcement on a fixed-ratio 10 schedule. Alpha-interferon pretreatment blocked the capacity of naloxone to decrease body temperature in these rats and actually induced an hyperthermic response. In contrast, cyclosporine tended to enhance the drop in body temperature induced by naloxone. This last effect was more striking when the rats were placed in a cold room at 3.5°C. Cyclosporine by itself induced a drop in body temperature in normal rats exposed to 3.5°C. These results indicate that alpha-interferon and cyclosporine impair thermoregulation but do not directly interfere with morphine withdrawal signs. Morphine Dependence Alpha-interferon
Operant behavior
Body temperature
IN recent years, evidence has accumulated that the central nervous system influences the immune system. Lymphoid organs are innervated by both the sympathetic and parasympathetic branches of the autonomic nervous system [10]. Lymphocytes have receptors for a wide variety o f neurotransmitters and neuropeptides, and most of these substances have been shown to modulate cellular immune responses and natural killer cell activity [20, 22, 24]. These findings have been interpreted to indicate that the immune system is under extrinsic neural and/or neuroendocrine influences. F o r these regulations to be effective, the brain needs to be informed about the functional state of the immune system. There are some suggestions as to which signals may transmit this information. F o r instance, cytokines such as interleukin-1, which are elaborated by lymphocytes, have central and neuroendocrine effects [4,15]. Moreover, lymphocytes can express genes coding for the synthesis of the proopiomelanocortin family of peptides [28], preproenkephalins [29] and prolactin [13], and release them in apparently physiologically effective concentrations [24,25]. Unfortunately, in vivo evidence about immune system
Rats
Cyclosporine
communication with the brain is scarce. The development of the humoral immune response is accompanied by parallel neurohormonal changes affecting plasma corticosterone levels and norepinephrine concentrations in the spleen [3]. Changes in firing rates of hypothalamic neurons and in hypothalamic norepinephrine metabolism have been reported in rats injected with sheep red blood cells [3]. In a recent series of experiments, Dafny et al. [8, 9, 14] observed that manipulations of the immune system by such diverse treatments as alpha-interferon, cyclosporine, or irradiation attenuate signs of morphine withdrawal. These data have been interpreted to indicate that the immune system plays a role in morphine addiction. Alpha-interferon, an immunoenhancing substance produced by leukocytes, has opiate-like properties both in vitro and in vivo [5]. The possibility that the effects of alpha-interferon are mediated by opiate receptors is, however, disputed [21]. In contrast, cyclosporine and irradiation are immunosuppressive treatments that have no known effect on opiate receptors. The effects of these treatments on morphine withdrawal are intriguing for two reasons. First, the changes are dramatic, in
593
594
DANTZER, S A T I N O F F A N D K E L L E Y
contrast to the more subtle reciprocal influences that have been described between the brain and the immune system. Second, the changes might provide proof of a functional influence of the immune system on the brain. Because of the potential importance of these findings for our conception of the relationships between the brain and the immune system, we decided to investigate further the effects of alphainterferon and cyclosporine on morphine withdrawal signs in rats. Dafny et al. [8, 9, 14] scored by direct observation physical symptoms of morphine withdrawal, such as wet dog shakes, teeth chattering, scream-to-touch and changes in activity. These measures, though valid, are sensitive to many environmental and organismic variables. We hypothesized that the disappearance of these signs in interferon- or cyclosporine-treated rats could have been due to a general sickness or malaise produced by these drugs, rather than to a specific antagonism of morphine withdrawal. To test this hypothesis, we chose to use the disruption of schedule-controlled behavior induced by an injection of naloxone in morphine-dependent rats as an objective indicator of withdrawal [2, 11, 26]. Any nonspecific malaise induced by the treatment should enhance rather than decrease behavioral suppression. We report here that neither alphainterferon nor cyclosporine alleviated naloxone-precipitated suppression of operant responding in morphine-dependent rats. Moreover, we demonstrate that these compounds alter normal body temperature and we suggest that disturbances in thermoregulation may be responsible for the reduction in morphine withdrawal symptoms observed by Dafny et al. [8, 9, 141.
METHOD Subjects and Housing
The subjects were 37 male and 20 female hooded rats of the Long Evans strain born and raised in the Psychology Department animal colony. They weighed between 205 and 510g. Rats used in the behavioral experiments were housed four to a cage in polypropylene cages with sawdust litter. The cages were kept in a colony room at an ambient temperature of 23-1°C and a 12:12 light/dark photoperiod (lights on at 7.00 a.m.). The rats were partially food-deprived until they reached 80% of their free-feeding body weight and they were maintained at this weight by being given 12-15 g of rat chow within one hour after a session in the operant chamber. Water was available ad lib. Rats used in the thermoregulation experiments were first anesthetized with ether, and a small (weight 2.3 g) a.m. temperature transmitter (Model M, Minimitter Co., Sun River, OR) was implanted into the peritoneal cavity. Then they were housed in individual Plexiglas cages with sawdust litter. Food and water were available ad lib. Behavioral Experiments
Food-deprived male rats (n=25) were trained to press a lever on a fixed-ratio 10 schedule (FR 10) for a 45 mg Noyes pellet reward in a standard operant chamber. The response rate was stable after about 12 sessions. All testing took place in the early afternoon and ended after the rat had earned 40 pellets, which usually required 6-8 min after stable responding had been achieved. After two weeks of stable F R 10 responding, the rats were implanted between 8.00 and
TABLE 1 EXPERIMENTAL DESIGN FOR THE INVESTIGATIONOF THE EFFECTS OF ALPHA-INTERFERONAND CYCLOSPORINE ON MORPHINE WITHDRAWALSIGNS Pellet Implantation (Day 0)
Treatment (Day 3) 8.00 a.m.
Morphine (n= 15)
Pellet Cyclosporine Removal (n=5)
10.00a.m.
11.00 a.m.
Pellet Removal Pellet Removal Placebo (n= 10)
Naloxone Interferon Naloxone (n=5)
Saline (n=3)
(or) Saline (n=2)
Pellet Cyclosporine Removal (n=5) Pellet Removal
12.00 a.m
Naloxone
Naloxone Interferon Naloxone (n=5)
9.00 a.m. with a pellet containing 75 mg of morphine or with a placebo pellet. Pellets were provided by the National Institute on Drug Abuse (Rockville, MD). While rats were lightly anesthetized with ether, an incision was made on the left side of the abdominal wall and a pellet inserted subcutaneously. The incision was then closed with nylon surgical thread. The rats were then housed individually. The pellets were removed on the third day after surgery, again under ether anesthesia, and between 7:00 and 8:00 a.m. The placebo pellet-implanted rats (n= 10) were randomly divided into 2 groups treated with alpha-interferon or with cyclosporine. The morphine pellet-implanted rats (n=15) were randomly divided into three groups treated with alpha-interferon, cyclosporine or a placebo. Two hours after removal of the pellet, cyclosporine-treated rats were injected intraperitoneally with 15 mg/kg cyclosporine (Sandimmun, Sandoz, injectable form, diluted with saline). Lyophilized recombinant human leukocyte alpha-interferon (Hoffman-La Roche) was reconstituted in saline and injected intraperitoneally to the interferon-treated rats at a dose of 150 U/g 3 hours after removal of the pellet. Three placebotreated animals were injected with saline two hours, and the others three hours, after removal of the morphine pellet. As there were no differences between these last two groups, their data were pooled. All rats were injected with naloxone (1 mg/kg, IP) four hours after pellet removal. Dosages and schedules of drug administration are summarized in Table 1. They were those that were used by Dafny et al. [8,9]. The volume of all injections was 2 ml/kg. Two behavioral sessions were run, one immediately before the naloxone challenge and one 1 hr later. In the interim, the rats were replaced in their home cages. Body weights were measured before each session. Both test sessions were terminated after the rats had earned 30 pellets or after ten min had elapsed. There were two dependent variables: the operant response rates in each session, expressed as the percentage of response rates on the last day before removal of the pellet, and weight loss, calculated as body weight before the first test session minus body weight before the second test session.
IMMUNITY AND MORPHINE DEPENDENCE
595
EFFECTS OF INTERFERON AND CYCLOSPORINE ON DISRUPTION OF FR-IO OPERANT BEHAVIOR INDUCED BY NALOXONE IN MORPHINE-DEPENDENT RATS
Response rate ii~~__o l tT " ~ 100/a~ percen/[°as
r ........
EFFECTS OF INTERFERON AND CYCLOSPORINE ON BODY WEIGHT LOSSES INDUCED BY ADMINISTRATION OF NALOXONE TO M O R P H I N E DEPENDENT RATS Weight loss (g) after challenge by naloxone -*-
I----] before ) naloxone ~ ~ ~,~.^~ I;~,.N.N~after9 . . . . '~"~=~
eline I'
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I
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IL.v_lCyclospo I ne 0
Interferon Placebo Cyclosporine m
PLACEBO
,
PELLET
~
Interferon
MORPHINE
~2.5
J
ym
Interferon Placebo Cyclosporine
InterJferon
Cyclosporine
PELLET
FIG. 1. Effects of alpha-interferon and cyclosporine on response rates in animals implanted with a placebo pellet or a morphine pellet. Each column represents the mean response rate expressed as percent of baseline, during test sessions run before and after injection of naloxone. **p<0.01 compared to response rate before injection of naloxone, (*)p<0.05 compared to baseline values.
Body Temperature (Tb) Experiments One week after implantation of the transmitter, under ether, male rats (n= 12) were implanted with a 75-mg morphine pellet at the dorsal base of the neck. Three days later the pellet was removed, again under ether, and the rats were randomly divided into 3 groups, and treated to the same drugs and dosages according to the same time schedule as described above. Four hr after pellet removal, naloxone (1 mg/kg, IP) was injected in all rats and they were immediately placed, in their home cages, in a chamber whose temperature was maintained at 23-+0.5°C. They remained there for 6 hr. T b ' s were measured through the implanted transmitters, which emit a series of discrete AM signals, or " b e a t s . " The time between beats is proportional to the temperature of the telemeter. At 10 min intervals, the computer sampled the beats from each rat's transmitter, determined the interval between them in msec, translated them into °C, and stored them on diskette for later printout. The body temperature measured at 9.00 a.m. approximately 2 hours after removal of the pellet, served as the control baseline for statistical comparisons and changes in body temperature ~vere calculated by subtracting each value from this baseline value. F o r experiments in the cold, 6 female rats were implanted with the 75-mg morphine pellet which was removed 3 days later. Two hours after pellet removal, the rats were treated with either cyclosporine (15 mg/kg) or the vehicle. Four hours after pellet removal, the rats were injected with naloxone (1 mg/kg) and exposed to an ambient temperature
""-,,~-,'/~ PLACEBO PELLET
nl
/
MORP~HtNE PELLET
FIG. 2. Effects of cyclosporine and alpha-interferon on losses in body weight suffered after injection of naloxone to rats implanted with a placebo or a morphine pellet. Each column represents the mean (-+sere) weight loss. *p<0.05 and **p<0.01 compared to animals implanted with the placebo pellet.
of 3.5+-0.5°C for 90 min. Tb measured immediately after naloxone injection was the baseline Tb, and all changes were calculated by subtracting each value from this baseline. In another experiment, the effects of cyclosporine on Tb of naive rats in the cold was tested. One week after transmitter implantation, female rats ( n = 6 ) w e r e given cyclosporine (25 mg/kg of the oral solution dissolved in olive oil) orally in a volume of 2 ml/250 g. Vehicle-treated animals (n=7) received an equivalent volume of olive oil. Two hr later, the rats were exposed to cold for 1 hr. Tb at the start of the cold test was the baseline for statistical comparisons.
RESULTS
Effects of Alpha-Interferon and Cyclosporine on Naloxone-Induced Suppression of FR 10 Behavior in Morphine-Dependent Rats Two days after implantation of the morphine pellet, the mean rate of response was 0.74+-0.07 responses per second versus 1.0+-0.07 in rats implanted with the placebo pellet, F(1,24)=6.84, p<0.05. Figure 1 represents the mean response rates expressed as percent of these baseline values after injection of interferon or cyclosporine and after injection of naloxone. A 2-way analysis of variance (5 treatments x 2 time periods) with repeated measurements on the time factor, revealed a significant treatment and time factor, F(4,20)=5.74, p<0.01, and F(1,20)=32.5, p<0.001, respectively, and a significant treatment x time interaction,
596
DANTZER, S A T I N O F F AND K E L L E Y TABLE 2 EFFECTS OF MORPHINEON BODYTEMPERATURE1
Treatment Morphine Pellet
(n= 12) Placebo Pellet (n=6) No Pellet (n=6)
Tb Before Surgery
EFFECTS OF INTERFERONAND CYCLOSPORINE ON CHANGES IN BODY TEMPERATURE INDUCED BY NALOXONE IN MORPHINE-DEPENDENT RATS
Tb After Surgery 0-24 hr
24-48 hr
48-72hr
37.05
37.81:~
37.23~
37.16"
(0.06)
(0.10)
(0.08)
(0.09)
37.23 (0.07) 37.30 (0.09)
37.26 (0.10) 37.27 (0.11)
--
--
in body IChanges temperature (°C)
I
+ 1.0ICY e~-IFNNX --
interferon (n= 4)
--
JMean (and sem) of 24 hr recording. *Significantly different at p<0.001 (~), p<0.01 (t) or p<0.05 (*) from Tb before surgery. Note that Tb measured after surgery was also significantly different (p < 0.001) from Tb meas ured 24-48 hr and 48-72 hr after surgery.
1
2~\ ~
F(4,20)=4.46,p<0.01. Post hoc comparisons of group means with the least significant difference test indicated that cyclosporine-treated animals pressed the lever at a significantly lower rate than on the baseline session (p<0.05) and that treatment with naloxone induced a complete cessation of responding that was not modified by either interferon or cyclosporine pretreatment (p <0.01). Figure 2 shows changes in body weight one hour after injection of naloxone. A one way analysis of variance revealed a significant treatment effect, F(4,20)=5.74, p<0.01. Post hoc comparisons of group means indicated that rats implanted with the morphine pellet lost significantly more weight than rats implanted with the placebo pellet, independent of the nature of the pretreatment.
Effects of Alpha-Interferon and Cyclosporine on Changes in Body Temperature Induced by Naloxone in MorphineDependent Rats Implantation of the morphine pellet induced a significant increase in Tb on the day of surgery, F(3,33)=93.3, p<0.001 (Table 2). Tolerance developed gradually to this effect. This hyperthermic response was not due to surgery since implantation of a placebo pellet in a different group of animals did not result in any significant change in mean body temperature (Table 2). Figure 3 illustrates the mean changes in Tb observed after removal of the morphine pellet and injection of naloxone preceded by interferon or cyclosporine. One control animal was removed from the experiment because its transmitter was no longer functioning. A 2-way analysis of variance (3 treatments × 7 time periods) revealed a significant influence of the treatment and time factors, F(2,8) =8.50, p =0.01, and F(6,48)=2.36, p<0.05, respectively, and a significant treatment x time interaction, F(12,48)=2.69, p<0.05. This was because the interferon-treated rats gradually increased their Tb whereas placebo- and cyclosporine-treated rats showed a drop in body temperature in response to naloxone. The decrease in Tb tended to be more pronounced in cyclosporine-treated animals. All rats had diarrhea and lost a considerable amount of weight in the four hours following the injection of naloxone. This loss was not modified by cyclosporine or interferon
-
3 f/,~".. ~
~
time (hr) saline (n=3) , cyclosporine In=4)
1.0
FIG. 3. Effects of cyclosporine and alpha-interferon on mean changes in body temperature observed in rats implanted with a morphine pellet and challenged with naloxone after removal of the pellet. Cyclosporine was injected at time 0, alpha-interferon 1 hr later and naloxone 2 hr after the beginning of the experiment. Each point represents the mean change in Tb (_+sem) from baseline values measured before the first injection.
treatment (placebo: 17.8± 1.65 g, cyclosporine: 14.0_+2.68, interferon: 17.2_+2.06, F(2,9)=0.88).
Effects of Cyclosporine on Changes in Tb Induced by Naloxone in Morphine-Dependent Rats Exposed to Cold The results of the previous experiment suggested that cyclosporine might have intensified the drop in Tb caused by naloxone in morphine-dependent animals. To test this, 6 rats were made dependent on morphine and exposed to cold after challenge with naloxone. Figure 4 represents the changes in body temperature observed in these animals. A 2-way analysis of variance revealed a significant effect of the treatment and time factors, F(1,4)=10.7, p<0.05, and F(13,52)=7.19, p<0.001, respectively, and a significant treatment x time interaction, F(13,52)=10.1, p<0.001. Placebo-treated animals maintained their Tb within 0. I°C of control levels during 90 min in the cold. In contrast, the Tb of cyclosporine-treated animals dropped an average of almost 2°C.
Effects of Cyclosporine on Tb in Rats Exposed to Cold In order to determine whether the changes in Tb seen in cyclosporine-treated rats were due to cyclosporine per se or to a potentiation of the hypothermic response to naloxone normally occurring in morphine-dependent rats, 13 rats were exposed to cold 2 hr after oral administration of cyclosporine (n=6) or the vehicle (n=7). Figure 5 represents the changes
IMMUNITY AND MORPHINE DEPENDENCE
597
EFFECTS OF CYCLOSPORINEON BODY TEMPERATURE DURING COLD EXPOSURE
EFFECTS OF CYCLOSPORINE ON CHANGES IN BODY TEMPERATURE INDUCED BY NALOXONE IN MORPHINE DEPENDENT RATS EXPOSED TO COLD
+
Changes in body temperature ( °C )
1,0
+0.5
+1.0
Changes in body temoerature (°C) m
• m
".
.
"
;o02:0 ° •
~ ~.- " - ~ ' " i
15
.
.
~
placebo (n=7) I
30
,
n
,
I
45
,
,
I
60 min .'--6 )
(n=3)
-0.5 -1.0
-1.0
-
1.5
cyclosporine -
FIG. 5. Effects of cyclosporine on changes in body temperature in response to cold. Animals were placed in the cold room at 3.5°C at time 0, two hours after cyclosporine or placebo injection, and Tb was recorded every 5 min. Each point represents the mean change in Tb (_+sem) from baseline values measured at the start of the cold test.
2.0
FIG. 4. Effects of cyclosporine on mean changes in body temperature observed during morphine withdrawal in animals exposed to cold. Rats made morphine dependent were injected with naloxone after removal of the morphine pellet. Cyclosporine or saline was injected 2 hours before naloxone. After naloxone injection, rats were placed in a cold room at 3.5°C. Each point represents the mean change in Tb (-+sere) from baseline values measured at the start of the cold test.
in Tb for 60 min at 3.5°C. A two-way analysis of variance on changes in Tb revealed a significant treatment effect, F(1,1 I)= 17.9, p =0.001, and a significant treatment x time interaction, F(1,121)=2.98, p=0.001. Placebo-treated animals responded to cold by increasing their Tb whereas cyclosporine-treated animals displayed a significant reduction in Tb. DISCUSSION The present experiments provide no evidence for any attenuation of naloxone-precipitated morphine withdrawal signs by alpha-interferon or cyclosporine. Neither of the two objective indicators of morpine withdrawal, disruption of operant responding and body weight loss, was alleviated by either treatment. Disruption of schedule-controlled behavior after administration of naloxone to morphine-dependent rats is a sensitive indicator of morphine withdrawal and is used routinely in many laboratories for studying the mechanisms of morphine withdrawal and the effects o f potential agonists and antagonists [2, 11, 26]. In a study of the effects of naloxone on morphine-dependent rats, Gellert and Sparber
[11] showed a strong correlation between the severity of behavioral disruption of F R 10 responding and the amount of weight loss. Using the same dependent variables, we found that neither cyclosporine nor alpha-interferon attenuated these morphine withdrawal signs. These results are obviously limited by the fact that only one dose was used for the different treatments under investigation, obviating therefore the possibility of observing graded effects if any. However, the selected doses were the same as those used by Dafny et al. in their studies. The question remains as to why Dafny et al. observed an effect using different dependent variables. Possibly the drugs had toxic side effects which interacted with the physical signs that they used as their behavioral end-points. Alphainterferon, for example, is pyrogenic in laboratory animals [6] and in humans induces hyperthermia, headache, myalgia, nausea, perceptual and conceptual confusion and fatigue (see [17] for review). Cyclosporine administered chronically is toxic and induces hypothermia [16]. Because of the pyrogenic effects of alpha-interferon and the hypothermic effects of cyclosporine on the one hand, and the fact that wet-dog shakes, or shivering, are seen both during morphine withdrawal and cold exposure [27], we examined the effects of alpha-interferon and cyclosporine on Tb. Using freely moving animals exposed to an ambient temperature of 23°C, we confirmed that implantation of a morphine pellet induces a hyperthermic response that shows rapid tolerance [1,7], just as it does when morphine is injected over several days [23] and that injection of naloxone, which by itself causes small declines in Tb [12] results in a greater drop in Tb when it is given to morphine-dependent rats [1, 18, 19]. More to the point, we observed that both alpha-interferon and cyclosporine altered the changes in Tb induced by naloxone in morphine-dependent rats. The former compound caused a gradual increase in Tb consistent with its known pyrogenic activity [6,17]. In contrast, the
598
DANTZER, SATINOFF AND KELLEY
results w i t h c y c l o s p o r i n e s u g g e s t e d it m i g h t e n h a n c e the d r o p in Tb. B e c a u s e o f this possibility, w e p l a c e d the rats in a cold e n v i r o n m e n t , where their thermoregulatory mechanisms would be more taxed, and where, therefore, any effect o f a drug o n t h e r m o r e g u l a t i o n w o u l d b e m o r e e v i d e n t . In fact, in t h e cold c y c l o s p o r i n e e x a g g e r a t e d t h e h y p o t h e r mia. W h e n we t h e n e x p o s e d naive rats to t h e cold, we f o u n d t h a t c y c l o s p o r i n e b y itself h a d a h y p o t h e r m i c effect, w h i c h c o u l d be i n d i c a t i v e o f a l o w e r i n g o f the t h e r m o r e g u l a t o r y s e t p o i n t , but m o r e likely i n d i c a t e s a n i n a d e q u a c y o f o n g o i n g t h e r m o r e g u l a t o r y e f f e c t o r m e c h a n i s m s . H o w e v e r , regardless o f the m e c h a n i s m s o f t h e t h e r m o r e g u l a t o r y d i s t u r b a n c e , t h e fact t h a t b o d y t e m p e r a t u r e is a l t e r e d m a y well b e indica-
tive o f o t h e r physiological c h a n g e s t h a t c o u l d a c c o u n t for the results r e p o r t e d here.
ACKNOWLEDGEMENTS Supported by NSF grant No. BNS 8311466 to E.S. These experiments were carried out while the first author was on sabbatical leave at the Department of Animal Science at the University of Illinois at Urbana-Champaign. Many thanks are due to Dr. G. F. Koob (Scripps Institute, San Diego) and Dr. S. Sparber (Department of Pharmacology, Minneapolis) who helped in many ways. Alphainterferon was kindly provided by Hoffman-La Roche and cyclosporine by Sandoz.
REFERENCES 1. Ary, M. and P. Lomax. Temperature changes during morphine dependence and withdrawal in the rat. In: Drugs, Biogenic Amines and Body Temperature, edited by K. E. Cooper, P. Lomax and E. Schonbaum. Basel: Karger, 1977. 2. Babbini, M., M. Gaiardi and M. Bartoletti. Changes in operant behavior as an index of withdrawal state from morphine in rats. Psychon Sci 29: 142-144, 1972. 3. Besedovsky, H. O., A. E. del Rey and E. Sorkin. Immuneneuroendocrine interactions. J Immunol 135: 750s-754s, 1985. 4. Besedovsky, H. O., A. E. Del Rey, E. Sorkin and C. A. Dinarello. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 233: 652-654, 1986. 5. Blalock, J. E. and E. M. Smith. Human leukocyte interferon: potent endorphin-like opioid activity. Biochem Biophys Res Commun 101: 472-478, 1981. 6. Clark, W. G. and Y. L. Clark. Changes in body temperature after administration of antipyretics, LSD, delta9-THC, CNS depressants and stimulants, hormones, inorganic ions, gases, 2,4DNP and miscellaneous agents. Neurosci Biobehav Rev 5: 1-136, 1981. 7. Clark, W. G. and J. M. Lipton. Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins and related agents: II. Neurosci Biobehav Rev 9: 47%552, 1985. 8. Dafny, N., B. Prieto-Gomez and C. Reyes-Vasquez. Does the immune system communicate with the central nervous system? Interferon modifies central nervous activity. J Neuroimmunol 9: 1-12, 1985. 9. Dafny, N., V. G. Wagle and D. B. Brath. Cyclosporine alters opiate withdrawal in rodents. Life Sci 36: 1721-1726, 1985. 10. Felten, D. L., S. Y. Felten, S. L. Carlson, J. A. Olschowka and S. Livnat. Noradrenergic and peptidergic innervation of lymphoid tissue. J lmmunol 135: 755s-764s, 1985. 11. Gellert, V. F. and S. B. Sparber. A comparison of the effects of naloxone upon body weight loss and suppression of fixed-ratio operant behavior in morphine-dependent rats. J Pharmacol Exp Ther 201: 44-54, 1977. 12. Goidstein, A. and P. J. Lowry. Effect of the opiate antagonist naloxone on body temperature in rats. Life Sci 17: 927-932, 1975. 13. Hiestand, P. C., P. Mekler, R. Nordmann, A. Grieder and C. Permmongkol. Prolactin as a modulator of lymphocyte responsiveness provides a possible mechanism of action for cyclosporine. Proc Natl Acad Sci USA 83: 2599-2603, 1986. 14. Kletzly, N. E., P. M. Dougherty, J. Aronowski, N. R. Pellis and N. Dafny. Evidence that opiate addiction involves an immune response. Soc Neurosci Abstr 11: 1201, 1985. 15. Krueger, J., C. Dinarello, M. Wolff, L. Chedid and J. Walter. Sleep-promoting effects of endogenous pyrogen (intedeukin l). Am J Physiol 246: R994-R1001, 1984.
16. Magnus, G., M. CavaUini, F. Halberg, G. Cornelissen, D. E. R. Sutherland, J. A. Najarian and W. J. M. Hrushesky. Circadian toxicology of cyclosporin. Toxicol Appl Pharmaco177: 181-185, 1985. 17. Mannering, G. J. and L. B. Deloria. The pharmacology and toxicology of the interferons: An overview. Annu Rev Pharmacol Toxicol 26: 455-515, 1986. 18. Mucha, R. F., H. Kalant and M. A. Linsemann. Quantitative relationships among measures of morphine tolerance and physical dependence in the rat. Pharmacol Biochem Behav 10: 397-405, 1979. 19. Nielsen, J. A. and S. B. Sparber. Indomethacin facilitates acute tolerance to and dependence upon morphine as measured by changes in fixed-ratio behavior and rectal temperature in rats. Pharmacol Biochem Behav 22: 921-931, 1985. 20. O'Dorisio, M. S., C. L. Wood and T. M. O'Dorisio. Vasoactive intestinal peptide and neuropeptide modulation of the immune response. J lmmunol 135: 792s-796s, 1985. 21. Reyes-Vasquez, C., N. Weisbrodt and N. Dafny. Does interferon exert its actions through opiate receptors? Life Sci 35: 1015-1021, 1984. 22. Shavit, Y., G. W. Terman, F. C. Martin, J. W. Lewis, J. C. Liebeskind and R. P. Gale. Stress, opioid peptides, the immune system and cancer. J lmmuno! 135: 834s-837s, 1985. 23. Siegel, S. Tolerance to the hyperthermic effect of morphine in the rat is a learned response. J Comp Physiol Psychol 92: 1137-1149, 1978. 24. Smith, E. M., D. Harbour-McMenamin and J. E. Blalock. Lymphocyte production of endorphin and endorphin-mediated immunoregulatory activity. J lmmunol 135: 779s-782s, 1985. 25. Smith, E. M., W. J. Meyer and J. E. Blalock. Virus-induced increases in corticosterone in hypophysectomized mice: a possible lymphoid-adrenal axis. Science 218:1311-1313, 1982. 26. Sparber, S. B., V. F. Gellert, L. Lichtblau and R. Eisenberg. The use of operant methods to study aggression and effects of acute and chronic morphine administration in rats. In: Factors Affecting the Action o f Narcotics, edited by M. W. Adler, L. Manara and R. Samanin. New York: Raven Press, 1978. 27. Wei, E., L. F. Tseng, H. Loh and E. L. Way. Similarity of morphine abstinence signs to thermoregulatory behavior. Nature 247: 398-399, 1974. 28. Westly, H. J., A. J. Kleiss, K, W. Kelley, P. K. Y. Wong and P. H. Yuen. Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J Exp Med 163: 158%1594, 1986. 29. Zurawski, G., M. Benedik, B. J. Kamb, J. S. Abrams, S. M. Zurawski and F. D. Lee. Activation of mouse T-helper cells induce abundant preproenkephalin mRNA synthesis. Science 232: 772-775, 1986.
0031-9384/87 $3.00 + .00
Cyclosporine and Alpha-Interferon Do Not Attenuate Morphine Withdrawal in Rats but Do Impair Thermoregulation R. D A N T Z E R , *
E. S A T I N O F F t §
AND K. W. KELLEY~:§
*Psychobiologie des Comportements adaptatifs, I N R A - I N S E R M U259 Rue Camille St-Saens, 33077 Bordeaux Cedex, France tDepartment of Psychology, University of Illinois at Urbana-Champaign ~Laboratory of lmmunophysiology, Department of Animal Sciences University of Illinois at Urbana-Champaign §Program in Neural and Behavioral Biology, University o f Illinois at Urbana-Champaign R e c e i v e d 15 O c t o b e r 1986 DANTZER, R., E. SATINOFF AND K. W. KELLEY. Cyclosporine and alpha-interferon do not attenuate morphine withdrawalin rats but do impair thermoregulation. PHYSIOL BEHAV 39(5) 593-598, 1987.--Immunomodulating drugs as diverse as alpha-interferon and cyclosporine have been reported to attenuate physical signs of morphine withdrawal in rats. On the basis of these results, the immune system has been claimed to be involved in opiate addiction. To assess whether this is the case, the effects of alpha-interferon and cyclosporine were studied on objective signs of morphine withdrawal in morphine-dependent rats. Rats made dependent upon morphine by implantation of a 75-mg morphine pellet were challenged three days later by naloxone (1 mg/kg). Pretreatment with alpha-interferon (150 U/g) or cyclosporine (15 mg/kg) did not attenuate the reduction in body weight or the behavioral suppression induced by naloxone in morphine-dependent rats trained to press a lever for food reinforcement on a fixed-ratio 10 schedule. Alpha-interferon pretreatment blocked the capacity of naloxone to decrease body temperature in these rats and actually induced an hyperthermic response. In contrast, cyclosporine tended to enhance the drop in body temperature induced by naloxone. This last effect was more striking when the rats were placed in a cold room at 3.5°C. Cyclosporine by itself induced a drop in body temperature in normal rats exposed to 3.5°C. These results indicate that alpha-interferon and cyclosporine impair thermoregulation but do not directly interfere with morphine withdrawal signs. Morphine Dependence Alpha-interferon
Operant behavior
Body temperature
IN recent years, evidence has accumulated that the central nervous system influences the immune system. Lymphoid organs are innervated by both the sympathetic and parasympathetic branches of the autonomic nervous system [10]. Lymphocytes have receptors for a wide variety o f neurotransmitters and neuropeptides, and most of these substances have been shown to modulate cellular immune responses and natural killer cell activity [20, 22, 24]. These findings have been interpreted to indicate that the immune system is under extrinsic neural and/or neuroendocrine influences. F o r these regulations to be effective, the brain needs to be informed about the functional state of the immune system. There are some suggestions as to which signals may transmit this information. F o r instance, cytokines such as interleukin-1, which are elaborated by lymphocytes, have central and neuroendocrine effects [4,15]. Moreover, lymphocytes can express genes coding for the synthesis of the proopiomelanocortin family of peptides [28], preproenkephalins [29] and prolactin [13], and release them in apparently physiologically effective concentrations [24,25]. Unfortunately, in vivo evidence about immune system
Rats
Cyclosporine
communication with the brain is scarce. The development of the humoral immune response is accompanied by parallel neurohormonal changes affecting plasma corticosterone levels and norepinephrine concentrations in the spleen [3]. Changes in firing rates of hypothalamic neurons and in hypothalamic norepinephrine metabolism have been reported in rats injected with sheep red blood cells [3]. In a recent series of experiments, Dafny et al. [8, 9, 14] observed that manipulations of the immune system by such diverse treatments as alpha-interferon, cyclosporine, or irradiation attenuate signs of morphine withdrawal. These data have been interpreted to indicate that the immune system plays a role in morphine addiction. Alpha-interferon, an immunoenhancing substance produced by leukocytes, has opiate-like properties both in vitro and in vivo [5]. The possibility that the effects of alpha-interferon are mediated by opiate receptors is, however, disputed [21]. In contrast, cyclosporine and irradiation are immunosuppressive treatments that have no known effect on opiate receptors. The effects of these treatments on morphine withdrawal are intriguing for two reasons. First, the changes are dramatic, in
593
594
DANTZER, S A T I N O F F A N D K E L L E Y
contrast to the more subtle reciprocal influences that have been described between the brain and the immune system. Second, the changes might provide proof of a functional influence of the immune system on the brain. Because of the potential importance of these findings for our conception of the relationships between the brain and the immune system, we decided to investigate further the effects of alphainterferon and cyclosporine on morphine withdrawal signs in rats. Dafny et al. [8, 9, 14] scored by direct observation physical symptoms of morphine withdrawal, such as wet dog shakes, teeth chattering, scream-to-touch and changes in activity. These measures, though valid, are sensitive to many environmental and organismic variables. We hypothesized that the disappearance of these signs in interferon- or cyclosporine-treated rats could have been due to a general sickness or malaise produced by these drugs, rather than to a specific antagonism of morphine withdrawal. To test this hypothesis, we chose to use the disruption of schedule-controlled behavior induced by an injection of naloxone in morphine-dependent rats as an objective indicator of withdrawal [2, 11, 26]. Any nonspecific malaise induced by the treatment should enhance rather than decrease behavioral suppression. We report here that neither alphainterferon nor cyclosporine alleviated naloxone-precipitated suppression of operant responding in morphine-dependent rats. Moreover, we demonstrate that these compounds alter normal body temperature and we suggest that disturbances in thermoregulation may be responsible for the reduction in morphine withdrawal symptoms observed by Dafny et al. [8, 9, 141.
METHOD Subjects and Housing
The subjects were 37 male and 20 female hooded rats of the Long Evans strain born and raised in the Psychology Department animal colony. They weighed between 205 and 510g. Rats used in the behavioral experiments were housed four to a cage in polypropylene cages with sawdust litter. The cages were kept in a colony room at an ambient temperature of 23-1°C and a 12:12 light/dark photoperiod (lights on at 7.00 a.m.). The rats were partially food-deprived until they reached 80% of their free-feeding body weight and they were maintained at this weight by being given 12-15 g of rat chow within one hour after a session in the operant chamber. Water was available ad lib. Rats used in the thermoregulation experiments were first anesthetized with ether, and a small (weight 2.3 g) a.m. temperature transmitter (Model M, Minimitter Co., Sun River, OR) was implanted into the peritoneal cavity. Then they were housed in individual Plexiglas cages with sawdust litter. Food and water were available ad lib. Behavioral Experiments
Food-deprived male rats (n=25) were trained to press a lever on a fixed-ratio 10 schedule (FR 10) for a 45 mg Noyes pellet reward in a standard operant chamber. The response rate was stable after about 12 sessions. All testing took place in the early afternoon and ended after the rat had earned 40 pellets, which usually required 6-8 min after stable responding had been achieved. After two weeks of stable F R 10 responding, the rats were implanted between 8.00 and
TABLE 1 EXPERIMENTAL DESIGN FOR THE INVESTIGATIONOF THE EFFECTS OF ALPHA-INTERFERONAND CYCLOSPORINE ON MORPHINE WITHDRAWALSIGNS Pellet Implantation (Day 0)
Treatment (Day 3) 8.00 a.m.
Morphine (n= 15)
Pellet Cyclosporine Removal (n=5)
10.00a.m.
11.00 a.m.
Pellet Removal Pellet Removal Placebo (n= 10)
Naloxone Interferon Naloxone (n=5)
Saline (n=3)
(or) Saline (n=2)
Pellet Cyclosporine Removal (n=5) Pellet Removal
12.00 a.m
Naloxone
Naloxone Interferon Naloxone (n=5)
9.00 a.m. with a pellet containing 75 mg of morphine or with a placebo pellet. Pellets were provided by the National Institute on Drug Abuse (Rockville, MD). While rats were lightly anesthetized with ether, an incision was made on the left side of the abdominal wall and a pellet inserted subcutaneously. The incision was then closed with nylon surgical thread. The rats were then housed individually. The pellets were removed on the third day after surgery, again under ether anesthesia, and between 7:00 and 8:00 a.m. The placebo pellet-implanted rats (n= 10) were randomly divided into 2 groups treated with alpha-interferon or with cyclosporine. The morphine pellet-implanted rats (n=15) were randomly divided into three groups treated with alpha-interferon, cyclosporine or a placebo. Two hours after removal of the pellet, cyclosporine-treated rats were injected intraperitoneally with 15 mg/kg cyclosporine (Sandimmun, Sandoz, injectable form, diluted with saline). Lyophilized recombinant human leukocyte alpha-interferon (Hoffman-La Roche) was reconstituted in saline and injected intraperitoneally to the interferon-treated rats at a dose of 150 U/g 3 hours after removal of the pellet. Three placebotreated animals were injected with saline two hours, and the others three hours, after removal of the morphine pellet. As there were no differences between these last two groups, their data were pooled. All rats were injected with naloxone (1 mg/kg, IP) four hours after pellet removal. Dosages and schedules of drug administration are summarized in Table 1. They were those that were used by Dafny et al. [8,9]. The volume of all injections was 2 ml/kg. Two behavioral sessions were run, one immediately before the naloxone challenge and one 1 hr later. In the interim, the rats were replaced in their home cages. Body weights were measured before each session. Both test sessions were terminated after the rats had earned 30 pellets or after ten min had elapsed. There were two dependent variables: the operant response rates in each session, expressed as the percentage of response rates on the last day before removal of the pellet, and weight loss, calculated as body weight before the first test session minus body weight before the second test session.
IMMUNITY AND MORPHINE DEPENDENCE
595
EFFECTS OF INTERFERON AND CYCLOSPORINE ON DISRUPTION OF FR-IO OPERANT BEHAVIOR INDUCED BY NALOXONE IN MORPHINE-DEPENDENT RATS
Response rate ii~~__o l tT " ~ 100/a~ percen/[°as
r ........
EFFECTS OF INTERFERON AND CYCLOSPORINE ON BODY WEIGHT LOSSES INDUCED BY ADMINISTRATION OF NALOXONE TO M O R P H I N E DEPENDENT RATS Weight loss (g) after challenge by naloxone -*-
I----] before ) naloxone ~ ~ ~,~.^~ I;~,.N.N~after9 . . . . '~"~=~
eline I'
1
T
..................
,T ............ I
]
. . . . . . . . . . . . . . .
(.)
(.)
I
+5.0
1
50 +2.5
IL.v_lCyclospo I ne 0
Interferon Placebo Cyclosporine m
PLACEBO
,
PELLET
~
Interferon
MORPHINE
~2.5
J
ym
Interferon Placebo Cyclosporine
InterJferon
Cyclosporine
PELLET
FIG. 1. Effects of alpha-interferon and cyclosporine on response rates in animals implanted with a placebo pellet or a morphine pellet. Each column represents the mean response rate expressed as percent of baseline, during test sessions run before and after injection of naloxone. **p<0.01 compared to response rate before injection of naloxone, (*)p<0.05 compared to baseline values.
Body Temperature (Tb) Experiments One week after implantation of the transmitter, under ether, male rats (n= 12) were implanted with a 75-mg morphine pellet at the dorsal base of the neck. Three days later the pellet was removed, again under ether, and the rats were randomly divided into 3 groups, and treated to the same drugs and dosages according to the same time schedule as described above. Four hr after pellet removal, naloxone (1 mg/kg, IP) was injected in all rats and they were immediately placed, in their home cages, in a chamber whose temperature was maintained at 23-+0.5°C. They remained there for 6 hr. T b ' s were measured through the implanted transmitters, which emit a series of discrete AM signals, or " b e a t s . " The time between beats is proportional to the temperature of the telemeter. At 10 min intervals, the computer sampled the beats from each rat's transmitter, determined the interval between them in msec, translated them into °C, and stored them on diskette for later printout. The body temperature measured at 9.00 a.m. approximately 2 hours after removal of the pellet, served as the control baseline for statistical comparisons and changes in body temperature ~vere calculated by subtracting each value from this baseline value. F o r experiments in the cold, 6 female rats were implanted with the 75-mg morphine pellet which was removed 3 days later. Two hours after pellet removal, the rats were treated with either cyclosporine (15 mg/kg) or the vehicle. Four hours after pellet removal, the rats were injected with naloxone (1 mg/kg) and exposed to an ambient temperature
""-,,~-,'/~ PLACEBO PELLET
nl
/
MORP~HtNE PELLET
FIG. 2. Effects of cyclosporine and alpha-interferon on losses in body weight suffered after injection of naloxone to rats implanted with a placebo or a morphine pellet. Each column represents the mean (-+sere) weight loss. *p<0.05 and **p<0.01 compared to animals implanted with the placebo pellet.
of 3.5+-0.5°C for 90 min. Tb measured immediately after naloxone injection was the baseline Tb, and all changes were calculated by subtracting each value from this baseline. In another experiment, the effects of cyclosporine on Tb of naive rats in the cold was tested. One week after transmitter implantation, female rats ( n = 6 ) w e r e given cyclosporine (25 mg/kg of the oral solution dissolved in olive oil) orally in a volume of 2 ml/250 g. Vehicle-treated animals (n=7) received an equivalent volume of olive oil. Two hr later, the rats were exposed to cold for 1 hr. Tb at the start of the cold test was the baseline for statistical comparisons.
RESULTS
Effects of Alpha-Interferon and Cyclosporine on Naloxone-Induced Suppression of FR 10 Behavior in Morphine-Dependent Rats Two days after implantation of the morphine pellet, the mean rate of response was 0.74+-0.07 responses per second versus 1.0+-0.07 in rats implanted with the placebo pellet, F(1,24)=6.84, p<0.05. Figure 1 represents the mean response rates expressed as percent of these baseline values after injection of interferon or cyclosporine and after injection of naloxone. A 2-way analysis of variance (5 treatments x 2 time periods) with repeated measurements on the time factor, revealed a significant treatment and time factor, F(4,20)=5.74, p<0.01, and F(1,20)=32.5, p<0.001, respectively, and a significant treatment x time interaction,
596
DANTZER, S A T I N O F F AND K E L L E Y TABLE 2 EFFECTS OF MORPHINEON BODYTEMPERATURE1
Treatment Morphine Pellet
(n= 12) Placebo Pellet (n=6) No Pellet (n=6)
Tb Before Surgery
EFFECTS OF INTERFERONAND CYCLOSPORINE ON CHANGES IN BODY TEMPERATURE INDUCED BY NALOXONE IN MORPHINE-DEPENDENT RATS
Tb After Surgery 0-24 hr
24-48 hr
48-72hr
37.05
37.81:~
37.23~
37.16"
(0.06)
(0.10)
(0.08)
(0.09)
37.23 (0.07) 37.30 (0.09)
37.26 (0.10) 37.27 (0.11)
--
--
in body IChanges temperature (°C)
I
+ 1.0ICY e~-IFNNX --
interferon (n= 4)
--
JMean (and sem) of 24 hr recording. *Significantly different at p<0.001 (~), p<0.01 (t) or p<0.05 (*) from Tb before surgery. Note that Tb measured after surgery was also significantly different (p < 0.001) from Tb meas ured 24-48 hr and 48-72 hr after surgery.
1
2~\ ~
F(4,20)=4.46,p<0.01. Post hoc comparisons of group means with the least significant difference test indicated that cyclosporine-treated animals pressed the lever at a significantly lower rate than on the baseline session (p<0.05) and that treatment with naloxone induced a complete cessation of responding that was not modified by either interferon or cyclosporine pretreatment (p <0.01). Figure 2 shows changes in body weight one hour after injection of naloxone. A one way analysis of variance revealed a significant treatment effect, F(4,20)=5.74, p<0.01. Post hoc comparisons of group means indicated that rats implanted with the morphine pellet lost significantly more weight than rats implanted with the placebo pellet, independent of the nature of the pretreatment.
Effects of Alpha-Interferon and Cyclosporine on Changes in Body Temperature Induced by Naloxone in MorphineDependent Rats Implantation of the morphine pellet induced a significant increase in Tb on the day of surgery, F(3,33)=93.3, p<0.001 (Table 2). Tolerance developed gradually to this effect. This hyperthermic response was not due to surgery since implantation of a placebo pellet in a different group of animals did not result in any significant change in mean body temperature (Table 2). Figure 3 illustrates the mean changes in Tb observed after removal of the morphine pellet and injection of naloxone preceded by interferon or cyclosporine. One control animal was removed from the experiment because its transmitter was no longer functioning. A 2-way analysis of variance (3 treatments × 7 time periods) revealed a significant influence of the treatment and time factors, F(2,8) =8.50, p =0.01, and F(6,48)=2.36, p<0.05, respectively, and a significant treatment x time interaction, F(12,48)=2.69, p<0.05. This was because the interferon-treated rats gradually increased their Tb whereas placebo- and cyclosporine-treated rats showed a drop in body temperature in response to naloxone. The decrease in Tb tended to be more pronounced in cyclosporine-treated animals. All rats had diarrhea and lost a considerable amount of weight in the four hours following the injection of naloxone. This loss was not modified by cyclosporine or interferon
-
3 f/,~".. ~
~
time (hr) saline (n=3) , cyclosporine In=4)
1.0
FIG. 3. Effects of cyclosporine and alpha-interferon on mean changes in body temperature observed in rats implanted with a morphine pellet and challenged with naloxone after removal of the pellet. Cyclosporine was injected at time 0, alpha-interferon 1 hr later and naloxone 2 hr after the beginning of the experiment. Each point represents the mean change in Tb (_+sem) from baseline values measured before the first injection.
treatment (placebo: 17.8± 1.65 g, cyclosporine: 14.0_+2.68, interferon: 17.2_+2.06, F(2,9)=0.88).
Effects of Cyclosporine on Changes in Tb Induced by Naloxone in Morphine-Dependent Rats Exposed to Cold The results of the previous experiment suggested that cyclosporine might have intensified the drop in Tb caused by naloxone in morphine-dependent animals. To test this, 6 rats were made dependent on morphine and exposed to cold after challenge with naloxone. Figure 4 represents the changes in body temperature observed in these animals. A 2-way analysis of variance revealed a significant effect of the treatment and time factors, F(1,4)=10.7, p<0.05, and F(13,52)=7.19, p<0.001, respectively, and a significant treatment x time interaction, F(13,52)=10.1, p<0.001. Placebo-treated animals maintained their Tb within 0. I°C of control levels during 90 min in the cold. In contrast, the Tb of cyclosporine-treated animals dropped an average of almost 2°C.
Effects of Cyclosporine on Tb in Rats Exposed to Cold In order to determine whether the changes in Tb seen in cyclosporine-treated rats were due to cyclosporine per se or to a potentiation of the hypothermic response to naloxone normally occurring in morphine-dependent rats, 13 rats were exposed to cold 2 hr after oral administration of cyclosporine (n=6) or the vehicle (n=7). Figure 5 represents the changes
IMMUNITY AND MORPHINE DEPENDENCE
597
EFFECTS OF CYCLOSPORINEON BODY TEMPERATURE DURING COLD EXPOSURE
EFFECTS OF CYCLOSPORINE ON CHANGES IN BODY TEMPERATURE INDUCED BY NALOXONE IN MORPHINE DEPENDENT RATS EXPOSED TO COLD
+
Changes in body temperature ( °C )
1,0
+0.5
+1.0
Changes in body temoerature (°C) m
• m
".
.
"
;o02:0 ° •
~ ~.- " - ~ ' " i
15
.
.
~
placebo (n=7) I
30
,
n
,
I
45
,
,
I
60 min .'--6 )
(n=3)
-0.5 -1.0
-1.0
-
1.5
cyclosporine -
FIG. 5. Effects of cyclosporine on changes in body temperature in response to cold. Animals were placed in the cold room at 3.5°C at time 0, two hours after cyclosporine or placebo injection, and Tb was recorded every 5 min. Each point represents the mean change in Tb (_+sem) from baseline values measured at the start of the cold test.
2.0
FIG. 4. Effects of cyclosporine on mean changes in body temperature observed during morphine withdrawal in animals exposed to cold. Rats made morphine dependent were injected with naloxone after removal of the morphine pellet. Cyclosporine or saline was injected 2 hours before naloxone. After naloxone injection, rats were placed in a cold room at 3.5°C. Each point represents the mean change in Tb (-+sere) from baseline values measured at the start of the cold test.
in Tb for 60 min at 3.5°C. A two-way analysis of variance on changes in Tb revealed a significant treatment effect, F(1,1 I)= 17.9, p =0.001, and a significant treatment x time interaction, F(1,121)=2.98, p=0.001. Placebo-treated animals responded to cold by increasing their Tb whereas cyclosporine-treated animals displayed a significant reduction in Tb. DISCUSSION The present experiments provide no evidence for any attenuation of naloxone-precipitated morphine withdrawal signs by alpha-interferon or cyclosporine. Neither of the two objective indicators of morpine withdrawal, disruption of operant responding and body weight loss, was alleviated by either treatment. Disruption of schedule-controlled behavior after administration of naloxone to morphine-dependent rats is a sensitive indicator of morphine withdrawal and is used routinely in many laboratories for studying the mechanisms of morphine withdrawal and the effects o f potential agonists and antagonists [2, 11, 26]. In a study of the effects of naloxone on morphine-dependent rats, Gellert and Sparber
[11] showed a strong correlation between the severity of behavioral disruption of F R 10 responding and the amount of weight loss. Using the same dependent variables, we found that neither cyclosporine nor alpha-interferon attenuated these morphine withdrawal signs. These results are obviously limited by the fact that only one dose was used for the different treatments under investigation, obviating therefore the possibility of observing graded effects if any. However, the selected doses were the same as those used by Dafny et al. in their studies. The question remains as to why Dafny et al. observed an effect using different dependent variables. Possibly the drugs had toxic side effects which interacted with the physical signs that they used as their behavioral end-points. Alphainterferon, for example, is pyrogenic in laboratory animals [6] and in humans induces hyperthermia, headache, myalgia, nausea, perceptual and conceptual confusion and fatigue (see [17] for review). Cyclosporine administered chronically is toxic and induces hypothermia [16]. Because of the pyrogenic effects of alpha-interferon and the hypothermic effects of cyclosporine on the one hand, and the fact that wet-dog shakes, or shivering, are seen both during morphine withdrawal and cold exposure [27], we examined the effects of alpha-interferon and cyclosporine on Tb. Using freely moving animals exposed to an ambient temperature of 23°C, we confirmed that implantation of a morphine pellet induces a hyperthermic response that shows rapid tolerance [1,7], just as it does when morphine is injected over several days [23] and that injection of naloxone, which by itself causes small declines in Tb [12] results in a greater drop in Tb when it is given to morphine-dependent rats [1, 18, 19]. More to the point, we observed that both alpha-interferon and cyclosporine altered the changes in Tb induced by naloxone in morphine-dependent rats. The former compound caused a gradual increase in Tb consistent with its known pyrogenic activity [6,17]. In contrast, the
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DANTZER, SATINOFF AND KELLEY
results w i t h c y c l o s p o r i n e s u g g e s t e d it m i g h t e n h a n c e the d r o p in Tb. B e c a u s e o f this possibility, w e p l a c e d the rats in a cold e n v i r o n m e n t , where their thermoregulatory mechanisms would be more taxed, and where, therefore, any effect o f a drug o n t h e r m o r e g u l a t i o n w o u l d b e m o r e e v i d e n t . In fact, in t h e cold c y c l o s p o r i n e e x a g g e r a t e d t h e h y p o t h e r mia. W h e n we t h e n e x p o s e d naive rats to t h e cold, we f o u n d t h a t c y c l o s p o r i n e b y itself h a d a h y p o t h e r m i c effect, w h i c h c o u l d be i n d i c a t i v e o f a l o w e r i n g o f the t h e r m o r e g u l a t o r y s e t p o i n t , but m o r e likely i n d i c a t e s a n i n a d e q u a c y o f o n g o i n g t h e r m o r e g u l a t o r y e f f e c t o r m e c h a n i s m s . H o w e v e r , regardless o f the m e c h a n i s m s o f t h e t h e r m o r e g u l a t o r y d i s t u r b a n c e , t h e fact t h a t b o d y t e m p e r a t u r e is a l t e r e d m a y well b e indica-
tive o f o t h e r physiological c h a n g e s t h a t c o u l d a c c o u n t for the results r e p o r t e d here.
ACKNOWLEDGEMENTS Supported by NSF grant No. BNS 8311466 to E.S. These experiments were carried out while the first author was on sabbatical leave at the Department of Animal Science at the University of Illinois at Urbana-Champaign. Many thanks are due to Dr. G. F. Koob (Scripps Institute, San Diego) and Dr. S. Sparber (Department of Pharmacology, Minneapolis) who helped in many ways. Alphainterferon was kindly provided by Hoffman-La Roche and cyclosporine by Sandoz.
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