Psychopharmacology(1991) 104:62-66 0033315891000796
Psychopharmacology © Springer-Verlag 199t
Diazepam withdrawal responses measured in the social interaction test of anxiety and their reversal by baclofen Sandra E. File, P . S . Mabbutt, and N i c k Andrews
PsychopharmacologyResearch Unit, UMDS Divisionof Pharmacology, London University,Guy's Hospital, London SE1 9RT, UK Received August23, 1990 / Finai version October 29, 1990
Abstract. After 21 days of treatment with diazepam (0.5
or 2 mg/kg/day) rats were tolerant to the effects of diazepare to increase social interaction in the low light unfamiliar test condition of the social interaction test of anxiety. When they were tested 24 h after the last of 21 injections they showed significant decreases in social interaction, indicating an anxiogenic withdrawal response. However, the social interaction scores of rats tested 48 h after withdrawal from diazepam treatment were no longer different from those of the control group. The decreased social interaction, indicating increased anxiety, detected 24 h after withdrawal of diazepam (21 daily injections of 0.5 or 2 mg/kg), could be reversed by the usual daily diazepam dose (0.5 or 2 mg/kg, respectively) or by baclofen (0.5 or 1 mg/kg). Baclofen (2 mg/kg) was sedative in both control treated and diazepam-dependent rats, but was ineffective at reversing the decrease in social interaction seen after diazepam withdrawal. Possible sites of action mediating these effects of baclofen are discussed, and it is suggested that either postsynaptic GABAB sites in the hippocampus are involved or that the reversal of the decreased social interaction detected on withdrawal of diazepam treatment is due to a baclofen-mediated inhibition of 5-HT release in the hippocampus. Key words: B e n z o d i a z e p i n e - Tolerance - Withdrawal
- Anxiety - GABAB - 5-HT
There have by now been several demonstrations that tolerance does develop to the effects of the benzodiazepines in animal tests of anxiety and that on drug withdrawal behavioural indications of increased anxiety can be observed (see File 1990 for review). From experiments with chlordiazepoxide it seems that when rats are withdrawn from 3 weeks of treatment it is even possible to detect changes indicating increased anxiety when the treatment was with the minimally effective anxiolytic Offprint requests to: S.E. File
dose (File et al. 1987). However, most of the studies with diazepam have used very high doses, e.g. 40 mg/kg (Oakley et al. 1988) and 240 mg/kg (Emmett-Oglesby et al. ] 987) and therefore one purpose of the current investigation was to determine whether, after 3 weeks of daily injections, tolerance develops to the effects of low doses of diazepam in the social interaction test and whether anxiogenic effects could be detected on drug withdrawal. From previous experiments in which rats were withdrawn from chronic treatment with chlordiazepoxide (File and Baldwin 1989) it seemed that responses indicating increased anxiety could be detected in the social interaction test, whether or not the rats had been previously tested for tolerance. However, this has not yet been explicitly compared in a single experiment, and therefore a second purpose of the current study was to determine whether previous testing in the social interaction test modified the changes detected when they were withdrawn from diazepam treatment. The low light, unfamiliar test condition was used to test whether tolerance had developed to the anxiolytic effects of diazepam, since this test condition is sensitive to the increases in social interaction caused by the benzodiazepines (File 1980). Experiment 1 also investigated whether the occurrence of decreased social interaction after withdrawal from chronic diazepam treatment was determined by whether or not the rats received a vehicle injection before the social interaction test; thus one group of rats w a s tested 24 h after their last injection (i.e. without an injection prior to the social interaction test), whereas a second group of rats was tested 30 min after an IP injection. Finally, in experiment 1 the duration of the decrease in social interaction detected after withdrawal of diazepam treatment was determined, and whether this decreased social interaction could be reversed by the normal daily dose of diazepam. The low light, familiar test condition was selected for the tests after withdrawal of diazepam treatment, since this is the test condition most sensitive to decreases in social interaction, which indicate anxiogenic effects, as the control scores are high. As in previous experiments (File and Baldwin 1989), for all the rats the familiarisation sessions took place with the animals undrugged.
63 T h e b e n z o d i a z e p i n e a n t a g o n i s t f l u m a z e n i l is able to reverse b e h a v i o u r a l c h a n g e s i n d i c a t i n g i n c r e a s e d a n x i e t y on withdrawal from both benzodiazepines (Baldwin and File 1988) a n d a l c o h o l (File et al. 1989). It h a s been r e p o r t e d r e c e n t l y t h a t the G A B A B a g o n i s t b a c l o f e n c a n reverse the b e h a v i o u r a l c h a n g e s seen d u r i n g a l c o h o l w i t h d r a w a l , i n c l u d i n g the d e c r e a s e in social i n v e s t i g a t i o n (File et ai. 1991). T h e p u r p o s e o f e x p e r i m e n t 2 w a s to i n v e s t i g a t e w h e t h e r b a c l o f e n c o u l d reverse the decreases in social i n t e r a c t i o n d e t e c t e d after w i t h d r a w a l f r o m 3 weeks o f d i a z e p a m t r e a t m e n t . T h e d o s e s o f b a c t o f e n were c h o s e n o n t h e basis o f the p r e v i o u s s t u d y o f a l c o h o l w i t h d r a w a l (File e t a l . 1991). In t h a t s t u d y b a c l o f e n (5 m g / k g ) was f o u n d to be e x t r e m e l y s e d a t i v e in b o t h c o n t r o l a n d e t h a n o l - w i t h d r a w n rats, b u t b a c l o f e n (1.25 a n d 2.5 m g / k g ) reversed e t h a n o l w i t h d r a w a l r e s p o n s e s w i t h o u t h a v i n g a n y significant effects in c o n t r o l a n i m a l s ; the r a n g e o f 0 . 5 - 2 m g / k g was t h e r e f o r e selected for the p r e s e n t study.
Materials and methods
Animals Male hooded Lister rats (Olac Ltd, Bicester) weighing approximately 200 g at the start of treatment were housed with food and water freely available in a room with lights on from 0600 to 1700 hours. They were housed in groups of five until 5 days before testing, when they were singly housed. Rats were allocated to test partners on the basis of body weight, such that members of a pair did not differ by more than 5 g. Both members of a test pair always received the same chronic treatment and the same drug treatment on the test day.
Drugs Diazepam (courtesy of Roche Products Ltd) was suspended in distilled water to which a drop of Tween-20 had been added and ultrasonically dispersed for I h before injection; various concentrations were used to give a constant injection volume of 2 ml/kg. All animals received 21 days of handling and injection before testing; thus those that were tested after 5 days of diazepam treatment had previously received 16 days of control injections. Control animals received equi-volume injections of distilled water and Tween for 21 days before the social interaction testing. Baclofen (courtesy of Ciba Geigy) was dissolved in warm distilled water and injected IP in a volume of 2 ml/kg. The vehicle control animals in the baclofen reversal experiment recieved equivolume injection of distilled water. On the test day all injections were IP 30 min before the social interaction test.
Statistics Unless otherwise specified, the data were analysed by analysis of variance, followed by Duncan's tests for significance between individual groups (Kirk 1968). It is the significance of these tests that is cited in this paper.
Apparatus The social interaction test arena was a wooden box 60 x 60 cm, with 35 cm high walls and was lit by dim light (35 radiometric
lux). A camera was mounted vertically above the arena and the rats were observed from a video monitor in the adjacent room. The time spent in active social interaction was scored by an observer blind to the drug treatment. Infrared photocells were mounted in the walls, 4.5 and 12.5 cm from the floor, and the interruption of these beams provided automated measures of locomotor activity and rearing, respectively. The output from the photocells and the scores of the observer were entered into a microcomputer. For further details of this test, see File (]980).
Experiment 1 Group A. Rats were randomly allocated (eight pairs/group) to the following groups: day 5 - control, diazepam (0.5 and 2 mg/kg); day 21 - control, diazepam (0.5 and 2 mg/kg). All rats received 21 days of handling and injections, those in the diazepam day 5 groups received 16 days of vehicle injections prior to their 5 days of diazepam injections; those in the diazepam day 21 groups received 21 days of diazepam treatment. On the test day (day 5 or 21 of diazepam treatment, as appropriate) they were given their usual daily injection and 30 rain later were given a 5-rain social interaction test in a low light, unfamiliar test arena. The test order was randomised for drug treatment and the rats were tested between 0730 and 1200 hours. The following day the rats in the day 21 groups were given a further 7.5 rain familiarisation with the test arena before receiving their usual dose of diazepam. For this familiarisation session rats were placed singly into the arena. They were then retested undrugged (and uninjected) in the by now familiar arena, 24 h after their last injection. The test order was randomised for prior chronic drug treatment. The rats were again tested (drug free) the next day, 48 h after the last diazepam injection. Group B. Rats in this group were randomly allocated (18 pairs/ group) to control or diazepam (0.5 and 2 mg/kg) groups. All rats received 21 daily injections and on days 20 and 21 the rats were familiarised singly with the test arena for 7.5 min. As was the case for the group A rats, the rats were undrugged when they were given their familiarisation sessions. Immediately after the familiarisation session each rat received its usual daily injection. On day 22 half of each group was tested after a vehicle injection and half was tested after the usual diazepam injection. Rats were tested in an order randomised for drug treatment, between 0730 and 1200 hours. Experiment 2 Rats were randomly allocated to control (21 days of vehicle, n = 50 rats) or diazepam (21 days of 2 mg/kg, n=48 rats) pretreatment. On days 20 and 21 the rats were familiarised singly with the test arena for 7.5 rain, immediately before their usual daily injection. They were allocated to test partners of the same pretreatment and within each pretreatment, pairs of rats were randomly allocated among the drug test conditions: control; baclofen (0.5, 1 or 2 mg/ kg). The rats were tested in an order randomised for drug treatment between 0730 and 1200 horn's.
Results Table 1 s h o w s t h a t c o m p a r e d with t h e c o n t r o l g r o u p , rats t r e a t e d for 5 d a y s w i t h d i a z e p a m (0.5 a n d 2 m g / k g ) h a d a significantly i n c r e a s e d time s p e n t in social interact i o n in the low light, u n f a m i l i a r test c o n d i t i o n , i n d i c a t i n g an a n x i o l y t i c action. H o w e v e r , the r a t s tested a f t e r 21 d a y s o f d i a z e p a m t r e a t m e n t (0.5 a n d 2 m g / k g ) h a d social
64 Table 1. Mean (_+SEM) time spent in active social interaction by pairs of rats tested in a low light, unfamiliar arena 30 rain after their final injection of vehicle or diazepam (0.5 or 2 mg/kg) Day 5 Vehicle Diazepam 0.5 mg/kg 2 mg/kg
81.0 ± 9.5 114.9" +_10.6 114.8"_+11.7
I
200I
Day 21 150
78.4_+6.7 77.3 +_6.9 77.8___4.0
Prior to the test day, all rats had received 21 days of handling and injection: those in the day 5 diazepam groups thus received 16 days of vehicle injections before their 5 days of diazepam injections; those in the day 21 diazepam groups received 21 daily diazepare injections * P
Psychopharmacology © Springer-Verlag 199t
Diazepam withdrawal responses measured in the social interaction test of anxiety and their reversal by baclofen Sandra E. File, P . S . Mabbutt, and N i c k Andrews
PsychopharmacologyResearch Unit, UMDS Divisionof Pharmacology, London University,Guy's Hospital, London SE1 9RT, UK Received August23, 1990 / Finai version October 29, 1990
Abstract. After 21 days of treatment with diazepam (0.5
or 2 mg/kg/day) rats were tolerant to the effects of diazepare to increase social interaction in the low light unfamiliar test condition of the social interaction test of anxiety. When they were tested 24 h after the last of 21 injections they showed significant decreases in social interaction, indicating an anxiogenic withdrawal response. However, the social interaction scores of rats tested 48 h after withdrawal from diazepam treatment were no longer different from those of the control group. The decreased social interaction, indicating increased anxiety, detected 24 h after withdrawal of diazepam (21 daily injections of 0.5 or 2 mg/kg), could be reversed by the usual daily diazepam dose (0.5 or 2 mg/kg, respectively) or by baclofen (0.5 or 1 mg/kg). Baclofen (2 mg/kg) was sedative in both control treated and diazepam-dependent rats, but was ineffective at reversing the decrease in social interaction seen after diazepam withdrawal. Possible sites of action mediating these effects of baclofen are discussed, and it is suggested that either postsynaptic GABAB sites in the hippocampus are involved or that the reversal of the decreased social interaction detected on withdrawal of diazepam treatment is due to a baclofen-mediated inhibition of 5-HT release in the hippocampus. Key words: B e n z o d i a z e p i n e - Tolerance - Withdrawal
- Anxiety - GABAB - 5-HT
There have by now been several demonstrations that tolerance does develop to the effects of the benzodiazepines in animal tests of anxiety and that on drug withdrawal behavioural indications of increased anxiety can be observed (see File 1990 for review). From experiments with chlordiazepoxide it seems that when rats are withdrawn from 3 weeks of treatment it is even possible to detect changes indicating increased anxiety when the treatment was with the minimally effective anxiolytic Offprint requests to: S.E. File
dose (File et al. 1987). However, most of the studies with diazepam have used very high doses, e.g. 40 mg/kg (Oakley et al. 1988) and 240 mg/kg (Emmett-Oglesby et al. ] 987) and therefore one purpose of the current investigation was to determine whether, after 3 weeks of daily injections, tolerance develops to the effects of low doses of diazepam in the social interaction test and whether anxiogenic effects could be detected on drug withdrawal. From previous experiments in which rats were withdrawn from chronic treatment with chlordiazepoxide (File and Baldwin 1989) it seemed that responses indicating increased anxiety could be detected in the social interaction test, whether or not the rats had been previously tested for tolerance. However, this has not yet been explicitly compared in a single experiment, and therefore a second purpose of the current study was to determine whether previous testing in the social interaction test modified the changes detected when they were withdrawn from diazepam treatment. The low light, unfamiliar test condition was used to test whether tolerance had developed to the anxiolytic effects of diazepam, since this test condition is sensitive to the increases in social interaction caused by the benzodiazepines (File 1980). Experiment 1 also investigated whether the occurrence of decreased social interaction after withdrawal from chronic diazepam treatment was determined by whether or not the rats received a vehicle injection before the social interaction test; thus one group of rats w a s tested 24 h after their last injection (i.e. without an injection prior to the social interaction test), whereas a second group of rats was tested 30 min after an IP injection. Finally, in experiment 1 the duration of the decrease in social interaction detected after withdrawal of diazepam treatment was determined, and whether this decreased social interaction could be reversed by the normal daily dose of diazepam. The low light, familiar test condition was selected for the tests after withdrawal of diazepam treatment, since this is the test condition most sensitive to decreases in social interaction, which indicate anxiogenic effects, as the control scores are high. As in previous experiments (File and Baldwin 1989), for all the rats the familiarisation sessions took place with the animals undrugged.
63 T h e b e n z o d i a z e p i n e a n t a g o n i s t f l u m a z e n i l is able to reverse b e h a v i o u r a l c h a n g e s i n d i c a t i n g i n c r e a s e d a n x i e t y on withdrawal from both benzodiazepines (Baldwin and File 1988) a n d a l c o h o l (File et al. 1989). It h a s been r e p o r t e d r e c e n t l y t h a t the G A B A B a g o n i s t b a c l o f e n c a n reverse the b e h a v i o u r a l c h a n g e s seen d u r i n g a l c o h o l w i t h d r a w a l , i n c l u d i n g the d e c r e a s e in social i n v e s t i g a t i o n (File et ai. 1991). T h e p u r p o s e o f e x p e r i m e n t 2 w a s to i n v e s t i g a t e w h e t h e r b a c l o f e n c o u l d reverse the decreases in social i n t e r a c t i o n d e t e c t e d after w i t h d r a w a l f r o m 3 weeks o f d i a z e p a m t r e a t m e n t . T h e d o s e s o f b a c t o f e n were c h o s e n o n t h e basis o f the p r e v i o u s s t u d y o f a l c o h o l w i t h d r a w a l (File e t a l . 1991). In t h a t s t u d y b a c l o f e n (5 m g / k g ) was f o u n d to be e x t r e m e l y s e d a t i v e in b o t h c o n t r o l a n d e t h a n o l - w i t h d r a w n rats, b u t b a c l o f e n (1.25 a n d 2.5 m g / k g ) reversed e t h a n o l w i t h d r a w a l r e s p o n s e s w i t h o u t h a v i n g a n y significant effects in c o n t r o l a n i m a l s ; the r a n g e o f 0 . 5 - 2 m g / k g was t h e r e f o r e selected for the p r e s e n t study.
Materials and methods
Animals Male hooded Lister rats (Olac Ltd, Bicester) weighing approximately 200 g at the start of treatment were housed with food and water freely available in a room with lights on from 0600 to 1700 hours. They were housed in groups of five until 5 days before testing, when they were singly housed. Rats were allocated to test partners on the basis of body weight, such that members of a pair did not differ by more than 5 g. Both members of a test pair always received the same chronic treatment and the same drug treatment on the test day.
Drugs Diazepam (courtesy of Roche Products Ltd) was suspended in distilled water to which a drop of Tween-20 had been added and ultrasonically dispersed for I h before injection; various concentrations were used to give a constant injection volume of 2 ml/kg. All animals received 21 days of handling and injection before testing; thus those that were tested after 5 days of diazepam treatment had previously received 16 days of control injections. Control animals received equi-volume injections of distilled water and Tween for 21 days before the social interaction testing. Baclofen (courtesy of Ciba Geigy) was dissolved in warm distilled water and injected IP in a volume of 2 ml/kg. The vehicle control animals in the baclofen reversal experiment recieved equivolume injection of distilled water. On the test day all injections were IP 30 min before the social interaction test.
Statistics Unless otherwise specified, the data were analysed by analysis of variance, followed by Duncan's tests for significance between individual groups (Kirk 1968). It is the significance of these tests that is cited in this paper.
Apparatus The social interaction test arena was a wooden box 60 x 60 cm, with 35 cm high walls and was lit by dim light (35 radiometric
lux). A camera was mounted vertically above the arena and the rats were observed from a video monitor in the adjacent room. The time spent in active social interaction was scored by an observer blind to the drug treatment. Infrared photocells were mounted in the walls, 4.5 and 12.5 cm from the floor, and the interruption of these beams provided automated measures of locomotor activity and rearing, respectively. The output from the photocells and the scores of the observer were entered into a microcomputer. For further details of this test, see File (]980).
Experiment 1 Group A. Rats were randomly allocated (eight pairs/group) to the following groups: day 5 - control, diazepam (0.5 and 2 mg/kg); day 21 - control, diazepam (0.5 and 2 mg/kg). All rats received 21 days of handling and injections, those in the diazepam day 5 groups received 16 days of vehicle injections prior to their 5 days of diazepam injections; those in the diazepam day 21 groups received 21 days of diazepam treatment. On the test day (day 5 or 21 of diazepam treatment, as appropriate) they were given their usual daily injection and 30 rain later were given a 5-rain social interaction test in a low light, unfamiliar test arena. The test order was randomised for drug treatment and the rats were tested between 0730 and 1200 hours. The following day the rats in the day 21 groups were given a further 7.5 rain familiarisation with the test arena before receiving their usual dose of diazepam. For this familiarisation session rats were placed singly into the arena. They were then retested undrugged (and uninjected) in the by now familiar arena, 24 h after their last injection. The test order was randomised for prior chronic drug treatment. The rats were again tested (drug free) the next day, 48 h after the last diazepam injection. Group B. Rats in this group were randomly allocated (18 pairs/ group) to control or diazepam (0.5 and 2 mg/kg) groups. All rats received 21 daily injections and on days 20 and 21 the rats were familiarised singly with the test arena for 7.5 min. As was the case for the group A rats, the rats were undrugged when they were given their familiarisation sessions. Immediately after the familiarisation session each rat received its usual daily injection. On day 22 half of each group was tested after a vehicle injection and half was tested after the usual diazepam injection. Rats were tested in an order randomised for drug treatment, between 0730 and 1200 hours. Experiment 2 Rats were randomly allocated to control (21 days of vehicle, n = 50 rats) or diazepam (21 days of 2 mg/kg, n=48 rats) pretreatment. On days 20 and 21 the rats were familiarised singly with the test arena for 7.5 rain, immediately before their usual daily injection. They were allocated to test partners of the same pretreatment and within each pretreatment, pairs of rats were randomly allocated among the drug test conditions: control; baclofen (0.5, 1 or 2 mg/ kg). The rats were tested in an order randomised for drug treatment between 0730 and 1200 horn's.
Results Table 1 s h o w s t h a t c o m p a r e d with t h e c o n t r o l g r o u p , rats t r e a t e d for 5 d a y s w i t h d i a z e p a m (0.5 a n d 2 m g / k g ) h a d a significantly i n c r e a s e d time s p e n t in social interact i o n in the low light, u n f a m i l i a r test c o n d i t i o n , i n d i c a t i n g an a n x i o l y t i c action. H o w e v e r , the r a t s tested a f t e r 21 d a y s o f d i a z e p a m t r e a t m e n t (0.5 a n d 2 m g / k g ) h a d social
64 Table 1. Mean (_+SEM) time spent in active social interaction by pairs of rats tested in a low light, unfamiliar arena 30 rain after their final injection of vehicle or diazepam (0.5 or 2 mg/kg) Day 5 Vehicle Diazepam 0.5 mg/kg 2 mg/kg
81.0 ± 9.5 114.9" +_10.6 114.8"_+11.7
I
200I
Day 21 150
78.4_+6.7 77.3 +_6.9 77.8___4.0
Prior to the test day, all rats had received 21 days of handling and injection: those in the day 5 diazepam groups thus received 16 days of vehicle injections before their 5 days of diazepam injections; those in the day 21 diazepam groups received 21 daily diazepare injections * P
