P sycho pharmacology
Psychopharmacology 54, 197-201 (i977)
9 by Springer-Verlag 1977
The Development of Tolerance to Morphine in the Rat M. FERNANDES, S. KLUWE, and H. COPER Institute for Neuropsychopharmacology, Free University Berlin, Ulmenallee 30, D-1000 Berlin 19
Abstract. T o l e r a n c e to v a r i o u s effects o f m o r p h i n e in the r a t c a n be q u a n t i f i e d b y m e a n s o f a shift o f semil o g a r i t h m i c d o s e - r e s p o n s e curves. T o l e r a n c e to analgesia (hot plate, acetic a c i d writhing), catalepsy, a n d the tilted p l a n e d e v e l o p s in a closely similar m a n n e r . A l s o , the s t i m u l a t i n g effects o f a b o u t 1 m g / k g m o r p h i n e - H C 1 tested in a n open-field p r o c e d u r e are s o m e w h a t less p r o n o u n c e d in c h r o n i c a l l y t r e a t e d rats t h a n in naive ones. T h e r e is no c o r r e l a t i o n b e t w e e n t o l e r a n c e d e v e l o p m e n t a n d the acute EDso o f different tests.
can be quantified b y the d e l t a - l o g p r o c e d u r e , too. A d d i t i o n a l l y , the effect o f r e p e a t e d m o r p h i n e t r e a t m e n t on v a r i o u s s y m p t o m s o c c u r i n g after low doses o f m o r p h i n e o f a b o u t 1 m g / k g , in m o s t cases signs o f b e h a v i o r a l s t i m u l a t i o n ( A y h a m a n d R a n d r u p , 1973; F o g , 1970), was investigated. T h e r e l a t i o n s h i p between t o l e r a n c e a n d d e p e n d e n c e will be discussed in a separate paper.
MATERIALS AND METHODS
Key words: M o r p h i n e - T o l e r a n c e - R a t s Animals, Drug, and Tolerance Induction
It is well e s t a b l i s h e d t h a t r e p e a t e d a d m i n i s t r a t i o n o f m o r p h i n e i n t h e rat causes the d e v e l o p m e n t o f t o l e r a n c e to several d e p r e s s a n t effects o f the o p i a t e ( M a r t i n et al., 1963; Cicero a n d M e y e r , 1973; C l o u e t a n d I w a t s u b o , 1975; G r e c k s c h et al., 1974; A y h a m a n d R a n d r u p , 1973; P u r i a n d Lal, 1974; K u m a r et al., 1971). F r o m these p a p e r s it can be suggested t h a t the degree o f t o l e r a n c e varies with the m e t h o d o f investigation. A s K a l a n t et al. (1971) p o i n t e d out, t o l e r a n c e can be quantified o n l y if c h r o n i c d r u g t r e a t m e n t causes a p a r a l l e l shift o f s e m i l o g a r i t h m i c d o s e - r e s p o n s e curves. T h e degree o f this shift is used as a m e a s u r e o f t o l e r a n c e ( = d e l t a - l o g p r o c e d u r e ) . This p r o c e d u r e has n o t yet been used in the rat. I n mice we have e s t a b l i s h e d t h a t this m e t h o d can be a p p l i e d to the d e v e l o p m e n t o f t o l e r a n c e to analg e s i a - h o t p l a t e a n d acetic a c i d w r i t h i n g - h y p o t h e r m i a , a n d l e t h a l i t y b u t n o t to the s t i m u l a t i o n o f m o t i l i t y ( F e r n a n d e s et al., 1977). S p e e d a n d degree o f t o l e r a n c e d e v e l o p m e n t were f o u n d to v a r y with the test p r o c e dure. T h e p r e s e n t s t u d y has been u n d e r t a k e n to s h o w t h a t in the rat t o l e r a n c e to several effects o f m o r p h i n e
Male adult Wistar rats (breeder: Hagemann, Hannover), weighing 200-250 g, were used. In order to avoid the effects of repeated testing on tolerance development, all animals were used only once. Morphine-HC1 was obtained from Merck, Darmstadt and injected subcutaneously one or twice daily for up to 20 days in doses of 16 or 32 mg/kg. Injection time was 9 a.m. and, in the case of a second daily injection, 5 p.m. All experiments on test days were carried out between 9 and 11 a.m. The doses given in the paper refer to morphine-HCI.
Test Procedures In order to minimize the influence of altered pharmacokinetic properties of morphine during chronic treatment, peak effects were determined if possible.
Lethality. Rats were kept in groups of ten in plastic cages (26 x 42 x 15 cm). Incidence of death was checked 24 h after the test dose of morphine.
Catalepsy. The front paws of the rats were placed on a wooden bar of 7 cm height 60 and 90 rain after the test dose of morphine. Orientating experiments showed a maximum cataleptic reaction at both times. Holding time in seconds was determined by means of a stopwatch for up to i20 s. The higher value was taken as response. Hot Plate. The hot plate test of Eddy and Leimbach (1953) was adapted to rats. The plate temperature was 58~C, the latency times until the rats licked their paws or jumped determined by means of a stop-watch before, as well as 20 and 40 rain after, morphine administration. The greatest difference in latency time (s) was taken as the response. To avoid burning, rats that did not lick their
198 paws or jump within 20 s were removed from the plate and a latency time of 20 s was recorded.
Acetic Acid Writhing. Acetic acid, 0.3 ml, 10 ~, was injected intraperitoneally 40 min after the morphine test dose. The number of writhing movements were counted from the 5th to 20th min after acid injection.
Body Temperature. The probe of an electric thermometer (Atmos) was inserted 6 cm into the rectum. The greatest increase in body temperature, 60 or 90 min after morphine administration, was taken as the effect. Ambient temperature was 22-23~ Under these conditions morphine-HC1 did not produce any significant hypothermia. Tilted Plane. The rats were placed on a wooden plane that con-
Psychopharmacology 54 (1977) linearity were performed. In ai1 cases given in Table 1, the analysis was found to be valid. The degree of tolerance induced by all other morphine administration schedules used in the present investigation was found to be located within both dose-responserelationships which were tested for parallelism. These results were also assumed to be on parallel curves. With the exception of the shifts of the semilogarithmic dose-response curves presented in Table 1, the degree of all others was in general determined graphically. Occasional checking by probit analysis or by other methods based on the parallelism of straight lines (see CavaIli-Sforza, 1969) revealed an
~
tinuously moved into upright position within 8 s. Untreated animals started to slide at an angel of 73 + 3 degrees. The sliding angel was determined before as well as 60 and 90 min after morphine. The greatest decrease after the test dose of the drug was taken as the response on the tilted plane.
Open-Fieid Procedure. Pairs of rats were placed overnight into plastic cages (26• 15 cm) to allow acclimatization. In the morning food and water were removed, the rats injected and immediately replaced into the cage. From the 40th to the 60th rain after morphine administration the following synrptoms were counted: 1. crossing of lines at the floor deviding the cage into four equal parts as expression of motility, 2. rearing, 3. grooming by paws, 4. grooming by mouth 5. chewing of sawdust, 6. chewing of the paws, 7, licking of the paws, 8. touching the partner, 9. time interaction, i.e., the proportion of the time that the two animals spent in the same quarter of the cage. Symptoms 2 to 8 were counted as a new event when they lasted more than 10 s. Symptoms such as chewing without object, gnawing on the cage, licking of the cage, shaking of the body, and negative interactions such as fear, flight, fright, or kicking of the hind legs, and aggression (such as biting) occurred at a frequency of less than 3/20 min and are not presented here. In chronically treated animals attention has been focused also on new symptoms.
e-
e---~// 010
i e-
!t
I:0
210
Complete dose-response relationships were determined acutely and after 20 days of treatment with 32 mg/kg morphine-HCl twice daily, with the exception of the lethality. The low watersolubility of morphine-HCl did not allow the administration of the amounts of the opioid necessary to obtain a complete doseresponse relationship after this dosage schedule. Therefore, in lethality the relationship obtained after 2 • 16 mg/kg for 20 days was used further. Parallelism of these semilogarithmic dose-response curves was examined by analysis of variance or, in the case of all or no responses, by probit analysis (Cavalli-Sforza, 1969) as has been pointed out earlier (Fernandes et al., 1977). Nonsignificance indicates parallelism. In analysis of variance only those points lying on the linear parts of the semilogarithmic relationships were used. Tests of the validity of the procedure and an examination of regression and
catalepsy 3.0
/
/o/1/
J
hot plate
e., 9
0:0
0
110 9
1~
/
J~ A/~/
l
2.0
3:0
~-
?
4
./ 0[0
acetic acid test 1[0
.,o
2.'0
i,o
3~
hyperthermia olo
~Io
2:0 o,. e
/ / .t /
4/
tilted plane
.d olo
2.0
1.10
/
50
Statistical Analysis
9
soq
Suppression of Stress-Induced Defecation. Rats were stressed by placing them into a plastic cylinder [5.8 (i.d.)x I8 cm]. Eightytwo percent (18/22) of saline-treated rats defecated within 10 min after imprisonment. Forty minutes after morphine injection the percentage of animals that showed defecation within 10 min was recorded.
120~ 100~
25
lethality
/
/
o
./
olo
ot /-. i =t. /
310
2~o
31o
A
2O
,o 1
olo
/
suppressionof stress -induced defecation ~o
210
log morphine dose
3~o
Fig. 1. Dose-response curves to morphine-HCl in different test situations. 9 acute, O 1 x 16 mg/kg 20 days, [] 2 x 16 mg/kg 20 days, A 2 x 32 mg/kg 20 days. Each point represents mean results from at least 10 animals
199
M. Fernandes et al. : Tolerance to Morphine in the Rat Table 1.
Tests on parallelism acute vs. 2 x 32 mg/kg morphine-HC1 for 20 days
Test
F
df
P
Shift
95 ~ Confidence limits
Catalepsy Hot plate Acetic acid writhing Hyperthermia Tilted plane Time interaction
2.82 1.72 0.002 3.27 0.005 0.24
1/68 1/146 1/96 1/89 1/97 I/50
NS NS NS NS NS NS
1.32 1.20 1.29 0.21 0.76 0.84
1.09- 1.38 1.08 - I. 28 0.98-1.30 0.13 - 0 . 3 0 0 . 6 6 - 0.83 0.27 - 0.92
Test
)~2
df
P
shift
95 ~ confid, limits
Dosis lethalis" Stress-induced defecation
0.43 0.24
1 1
NS NS
1.48 1.18
1.32-1.63 a i .01 - 1.32
NS = not significant, which indicates parallelity df = degrees of freedom F = variance ratio shift = semilogarithmic shift of dose-response curves caused by chronic treatment /2 = dispersion index resulting from probit analysis a In dosis lethalis the dose-response relationships obtained acute and after 16 mg/kg morphine-HC1 twice daily for 20 days were tested on parallelism because of the water-insolubility of morphine
/
error of the graphic method below log. 0.12. in view of the few points lying on the ascending or descending branches of the bellshaped dose-response relationships obtained in the open-field experiment, statistical analysis was not perforraed.
hyperthermia
4
1.0
1.0
dosislethalis ~.~-~. ~ L
i
RESULTS
--ex
aceticacidtest 4--
,o
E 0
i
==
/:i i
lo
1.0
oj.~....
i
I catalepsy
0 "0
2
--4
i
I tiltedplans 1.0
Psychopharmacology 54, 197-201 (i977)
9 by Springer-Verlag 1977
The Development of Tolerance to Morphine in the Rat M. FERNANDES, S. KLUWE, and H. COPER Institute for Neuropsychopharmacology, Free University Berlin, Ulmenallee 30, D-1000 Berlin 19
Abstract. T o l e r a n c e to v a r i o u s effects o f m o r p h i n e in the r a t c a n be q u a n t i f i e d b y m e a n s o f a shift o f semil o g a r i t h m i c d o s e - r e s p o n s e curves. T o l e r a n c e to analgesia (hot plate, acetic a c i d writhing), catalepsy, a n d the tilted p l a n e d e v e l o p s in a closely similar m a n n e r . A l s o , the s t i m u l a t i n g effects o f a b o u t 1 m g / k g m o r p h i n e - H C 1 tested in a n open-field p r o c e d u r e are s o m e w h a t less p r o n o u n c e d in c h r o n i c a l l y t r e a t e d rats t h a n in naive ones. T h e r e is no c o r r e l a t i o n b e t w e e n t o l e r a n c e d e v e l o p m e n t a n d the acute EDso o f different tests.
can be quantified b y the d e l t a - l o g p r o c e d u r e , too. A d d i t i o n a l l y , the effect o f r e p e a t e d m o r p h i n e t r e a t m e n t on v a r i o u s s y m p t o m s o c c u r i n g after low doses o f m o r p h i n e o f a b o u t 1 m g / k g , in m o s t cases signs o f b e h a v i o r a l s t i m u l a t i o n ( A y h a m a n d R a n d r u p , 1973; F o g , 1970), was investigated. T h e r e l a t i o n s h i p between t o l e r a n c e a n d d e p e n d e n c e will be discussed in a separate paper.
MATERIALS AND METHODS
Key words: M o r p h i n e - T o l e r a n c e - R a t s Animals, Drug, and Tolerance Induction
It is well e s t a b l i s h e d t h a t r e p e a t e d a d m i n i s t r a t i o n o f m o r p h i n e i n t h e rat causes the d e v e l o p m e n t o f t o l e r a n c e to several d e p r e s s a n t effects o f the o p i a t e ( M a r t i n et al., 1963; Cicero a n d M e y e r , 1973; C l o u e t a n d I w a t s u b o , 1975; G r e c k s c h et al., 1974; A y h a m a n d R a n d r u p , 1973; P u r i a n d Lal, 1974; K u m a r et al., 1971). F r o m these p a p e r s it can be suggested t h a t the degree o f t o l e r a n c e varies with the m e t h o d o f investigation. A s K a l a n t et al. (1971) p o i n t e d out, t o l e r a n c e can be quantified o n l y if c h r o n i c d r u g t r e a t m e n t causes a p a r a l l e l shift o f s e m i l o g a r i t h m i c d o s e - r e s p o n s e curves. T h e degree o f this shift is used as a m e a s u r e o f t o l e r a n c e ( = d e l t a - l o g p r o c e d u r e ) . This p r o c e d u r e has n o t yet been used in the rat. I n mice we have e s t a b l i s h e d t h a t this m e t h o d can be a p p l i e d to the d e v e l o p m e n t o f t o l e r a n c e to analg e s i a - h o t p l a t e a n d acetic a c i d w r i t h i n g - h y p o t h e r m i a , a n d l e t h a l i t y b u t n o t to the s t i m u l a t i o n o f m o t i l i t y ( F e r n a n d e s et al., 1977). S p e e d a n d degree o f t o l e r a n c e d e v e l o p m e n t were f o u n d to v a r y with the test p r o c e dure. T h e p r e s e n t s t u d y has been u n d e r t a k e n to s h o w t h a t in the rat t o l e r a n c e to several effects o f m o r p h i n e
Male adult Wistar rats (breeder: Hagemann, Hannover), weighing 200-250 g, were used. In order to avoid the effects of repeated testing on tolerance development, all animals were used only once. Morphine-HC1 was obtained from Merck, Darmstadt and injected subcutaneously one or twice daily for up to 20 days in doses of 16 or 32 mg/kg. Injection time was 9 a.m. and, in the case of a second daily injection, 5 p.m. All experiments on test days were carried out between 9 and 11 a.m. The doses given in the paper refer to morphine-HCI.
Test Procedures In order to minimize the influence of altered pharmacokinetic properties of morphine during chronic treatment, peak effects were determined if possible.
Lethality. Rats were kept in groups of ten in plastic cages (26 x 42 x 15 cm). Incidence of death was checked 24 h after the test dose of morphine.
Catalepsy. The front paws of the rats were placed on a wooden bar of 7 cm height 60 and 90 rain after the test dose of morphine. Orientating experiments showed a maximum cataleptic reaction at both times. Holding time in seconds was determined by means of a stopwatch for up to i20 s. The higher value was taken as response. Hot Plate. The hot plate test of Eddy and Leimbach (1953) was adapted to rats. The plate temperature was 58~C, the latency times until the rats licked their paws or jumped determined by means of a stop-watch before, as well as 20 and 40 rain after, morphine administration. The greatest difference in latency time (s) was taken as the response. To avoid burning, rats that did not lick their
198 paws or jump within 20 s were removed from the plate and a latency time of 20 s was recorded.
Acetic Acid Writhing. Acetic acid, 0.3 ml, 10 ~, was injected intraperitoneally 40 min after the morphine test dose. The number of writhing movements were counted from the 5th to 20th min after acid injection.
Body Temperature. The probe of an electric thermometer (Atmos) was inserted 6 cm into the rectum. The greatest increase in body temperature, 60 or 90 min after morphine administration, was taken as the effect. Ambient temperature was 22-23~ Under these conditions morphine-HC1 did not produce any significant hypothermia. Tilted Plane. The rats were placed on a wooden plane that con-
Psychopharmacology 54 (1977) linearity were performed. In ai1 cases given in Table 1, the analysis was found to be valid. The degree of tolerance induced by all other morphine administration schedules used in the present investigation was found to be located within both dose-responserelationships which were tested for parallelism. These results were also assumed to be on parallel curves. With the exception of the shifts of the semilogarithmic dose-response curves presented in Table 1, the degree of all others was in general determined graphically. Occasional checking by probit analysis or by other methods based on the parallelism of straight lines (see CavaIli-Sforza, 1969) revealed an
~
tinuously moved into upright position within 8 s. Untreated animals started to slide at an angel of 73 + 3 degrees. The sliding angel was determined before as well as 60 and 90 min after morphine. The greatest decrease after the test dose of the drug was taken as the response on the tilted plane.
Open-Fieid Procedure. Pairs of rats were placed overnight into plastic cages (26• 15 cm) to allow acclimatization. In the morning food and water were removed, the rats injected and immediately replaced into the cage. From the 40th to the 60th rain after morphine administration the following synrptoms were counted: 1. crossing of lines at the floor deviding the cage into four equal parts as expression of motility, 2. rearing, 3. grooming by paws, 4. grooming by mouth 5. chewing of sawdust, 6. chewing of the paws, 7, licking of the paws, 8. touching the partner, 9. time interaction, i.e., the proportion of the time that the two animals spent in the same quarter of the cage. Symptoms 2 to 8 were counted as a new event when they lasted more than 10 s. Symptoms such as chewing without object, gnawing on the cage, licking of the cage, shaking of the body, and negative interactions such as fear, flight, fright, or kicking of the hind legs, and aggression (such as biting) occurred at a frequency of less than 3/20 min and are not presented here. In chronically treated animals attention has been focused also on new symptoms.
e-
e---~// 010
i e-
!t
I:0
210
Complete dose-response relationships were determined acutely and after 20 days of treatment with 32 mg/kg morphine-HCl twice daily, with the exception of the lethality. The low watersolubility of morphine-HCl did not allow the administration of the amounts of the opioid necessary to obtain a complete doseresponse relationship after this dosage schedule. Therefore, in lethality the relationship obtained after 2 • 16 mg/kg for 20 days was used further. Parallelism of these semilogarithmic dose-response curves was examined by analysis of variance or, in the case of all or no responses, by probit analysis (Cavalli-Sforza, 1969) as has been pointed out earlier (Fernandes et al., 1977). Nonsignificance indicates parallelism. In analysis of variance only those points lying on the linear parts of the semilogarithmic relationships were used. Tests of the validity of the procedure and an examination of regression and
catalepsy 3.0
/
/o/1/
J
hot plate
e., 9
0:0
0
110 9
1~
/
J~ A/~/
l
2.0
3:0
~-
?
4
./ 0[0
acetic acid test 1[0
.,o
2.'0
i,o
3~
hyperthermia olo
~Io
2:0 o,. e
/ / .t /
4/
tilted plane
.d olo
2.0
1.10
/
50
Statistical Analysis
9
soq
Suppression of Stress-Induced Defecation. Rats were stressed by placing them into a plastic cylinder [5.8 (i.d.)x I8 cm]. Eightytwo percent (18/22) of saline-treated rats defecated within 10 min after imprisonment. Forty minutes after morphine injection the percentage of animals that showed defecation within 10 min was recorded.
120~ 100~
25
lethality
/
/
o
./
olo
ot /-. i =t. /
310
2~o
31o
A
2O
,o 1
olo
/
suppressionof stress -induced defecation ~o
210
log morphine dose
3~o
Fig. 1. Dose-response curves to morphine-HCl in different test situations. 9 acute, O 1 x 16 mg/kg 20 days, [] 2 x 16 mg/kg 20 days, A 2 x 32 mg/kg 20 days. Each point represents mean results from at least 10 animals
199
M. Fernandes et al. : Tolerance to Morphine in the Rat Table 1.
Tests on parallelism acute vs. 2 x 32 mg/kg morphine-HC1 for 20 days
Test
F
df
P
Shift
95 ~ Confidence limits
Catalepsy Hot plate Acetic acid writhing Hyperthermia Tilted plane Time interaction
2.82 1.72 0.002 3.27 0.005 0.24
1/68 1/146 1/96 1/89 1/97 I/50
NS NS NS NS NS NS
1.32 1.20 1.29 0.21 0.76 0.84
1.09- 1.38 1.08 - I. 28 0.98-1.30 0.13 - 0 . 3 0 0 . 6 6 - 0.83 0.27 - 0.92
Test
)~2
df
P
shift
95 ~ confid, limits
Dosis lethalis" Stress-induced defecation
0.43 0.24
1 1
NS NS
1.48 1.18
1.32-1.63 a i .01 - 1.32
NS = not significant, which indicates parallelity df = degrees of freedom F = variance ratio shift = semilogarithmic shift of dose-response curves caused by chronic treatment /2 = dispersion index resulting from probit analysis a In dosis lethalis the dose-response relationships obtained acute and after 16 mg/kg morphine-HC1 twice daily for 20 days were tested on parallelism because of the water-insolubility of morphine
/
error of the graphic method below log. 0.12. in view of the few points lying on the ascending or descending branches of the bellshaped dose-response relationships obtained in the open-field experiment, statistical analysis was not perforraed.
hyperthermia
4
1.0
1.0
dosislethalis ~.~-~. ~ L
i
RESULTS
--ex
aceticacidtest 4--
,o
E 0
i
==
/:i i
lo
1.0
oj.~....
i
I catalepsy
0 "0
2
--4
i
I tiltedplans 1.0
