interaction in diazepam withdrawn rats and in the plus- maze also this dose significantly reversed the anxiogenic effects of diazepam withdrawal. Buspirone (400 ...
Psychopharmacology (1991) 105:578 582
Psychopharmacology © Springer-Verlag 1991
Low but not high doses of buspirone reduce the anxiogenic effects of diazepam withdrawal Sandra E. File and N i c k Andrews
Psychopharmacology Research Unit, UMDS Division of Pharmacology, London University, Guy's Hospital, London SE1 9RT, UK Received April 4, 1991 / Final version May 7, 1991 Abstract. After 21 days of treatment with diazepam
(2 mg/kg/day IP) rats were tested 24 h after the last injection in the social interaction and elevated plus-maze tests of anxiety. Compared with control-treated rats, they showed significant decreases in social interaction, in the % numbers of entries onto open arms of the plus-maze and in the % of time spent on the open arms, indicating an anxiogenic response on withdrawal from diazepam. Buspirone (200 gg/kg SC) significantly increased social interaction in diazepam withdrawn rats and in the plusmaze also this dose significantly reversed the anxiogenic effects of diazepam withdrawal. Buspirone (400 ~tg/kg SC) was without effect in the plus-maze, but buspirone (800 gg/kg SC) significantly decreased the % of time spent on open arms in control-treated rats, indicating an anxiogenic effect. In the social interaction test buspirone (800 gg/kg SC) was without significant effect. The contrasting effects of the 200 and 800 gg/kg doses are discussed in terms of the pre- and post-synaptic actions of buspirone. The findings are consistent with earlier proposals that the increased anxiety during benzodiazepine withdrawal is at least partly caused by an increased release of hippocampal 5-HT. Key words: B e n z o d i a z e p i n e - W i t h d r a w a l - A n x i e t y -
5-HT1A receptors
The detailed neurochemical changes that underlie benzodiazepine dependence are still not fully delineated, but there is increasing evidence for a change in the functioning of the GABA-benzodiazepine receptor complex. The benzodiazepine antagonist, flumazenil, can reverse the increased anxiety and the decreased seizure threshold that can be detected on withdrawal from chronic benzodiazepine treatment (Baldwin and File t988; Hitchcott et al. 1989) and the GABA subsensitivity of dorsal raph~ neurons that occurs after chronic benzodiazepine treatOffprint requests to ." S.E. File
ment (Gonsalves and Gallager 1985, 1988). When flumazenil is administered during the period of benzodiazepine treatment, it can prevent the development of dependence, as evidenced by increased anxiety or physical signs of withdrawal (Gallager et al. 1986; Baldwin and File 1989; Baldwin et al. 1990a). It has been proposed that flumazenil is able to reset the GABA-benzodiazepine receptor complex to a drug naive state, thus reversing the process of dependence (Gallager et al. 1986; File and Hitchcott 1990). The changes at the GABA-benzodiazepine complex that occur during benzodiazepine dependence are likely to result in changes in other neurotransmitter pathways. There is increasing evidence that changes in different neurotransmitters underlie different withdrawal responses. For example, clonidine reverses the hyperactivity and diarrhoea (Kunchandy and Kulkarni 1986), but not the increased anxiety (Baldwin et al. 1989) seen on benzodiazepine withdrawal. There is growing evidence that changes in 5-hydroxytryptamine (5 HT) may underlie the increased anxiety detected during benzodiazepine withdrawal. Hitchcott et al. (1990) found increased release of 5-HT in hippocampal slices taken from rats withdrawn from chronic diazepam, and Costall et al. (1989) reported that the 5-HT3 receptor antagonist, ondansetron, reversed the anxiogenic effects of diazepam withdrawal. The GABAB agonist, baclofen, is also able to reverse the anxiogenic effects of diazepam and ethanol withdrawal (File et al. 1991a, b). Whilst the mechanism of these reversals by baclofen is unknown, one possibility is that baclofen was active by reducing 5-HT release in the hippocampus. Baclofen has an inhibitory effect on the release of T-aminobutyric acid (GABA), and through an action at pre-synaptic hetero-receptors it also decreases the release of 5-HT and noradrenaline (Gray and Green 1987; Bowery et al. 1980). In order to explore further the role of 5-HT in mediating the anxiogenic responses detected on withdrawal from benzodiazepines, we examined the effects of buspirone, which acts at 5-HT1A receptors. Buspirone was selected because of its known clinical effects. Buspirone
579 has been r e p o r t e d to have clinical efficacy as a n anxiolytic ( L a d e r 1988) a n d to reduce the increased anxiety experienced d u r i n g alcohol w i t h d r a w a l ( B r u n o 1988). H o w e v e r , b u s p i r o n e was n o t f o u n d to be effective d u r i n g b e n z o d i a z e p i n e w i t h d r a w a l and, indeed, h a d the tend e n c y to increase the s y m p t o m s o f anxiety ( A s h t o n et al. 1990). T h e results o f the p r e s e n t s t u d y suggest that busp i r o n e m a y have b i p h a s i c effects o n b e n z o d i a z e p i n e withd r a w a l responses, with a low dose alleviating, a n d a high dose t e n d i n g to exacerbate, anxiety.
Materials and methods Animals. Male hooded Lister rats (Olac Ltd; Bicester) weighing approximately 180 g at the start of treatment were housed with food and water freely available in a room with lights on from 0600 to 1800 hours. They were housed in groups of five until 5 days before testing, when they were singly housed. 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 1 h before injection; the injection volume was 2 ml/kg. All animals received 21 days of handling and an intraperitoneal (IP) injection (diazepam 2 mg/kg/day or water/Tween, as appropriate) before testing. On the test day each animal received a subcutaneous injection of water or buspirone (100-800 lag/kg) 15 min before the social interaction or plus-maze test. Statistics. The scores from the control group and the diazepam withdrawal group were first analysed by a one-way analysis of variance in order to establish whether there was a significant anxiogenic effect of diazepam withdrawal. The data for each dose of buspirone were then analysed by two-way analyses of variance with the chronic treatment (control or diazepam) as one factor and the acute treatment (control or buspirone) as the second. A significant buspirone factor would indicate that buspirone had an overall anxiolytic or anxiogenic effect in both control and withdrawn animals; whereas a significant diazepam x buspirone interaction factor would indicate that buspirone was having a significant effect in one group (e.g. the withdrawal group) only. In cases where the buspirone factor was significant, Duncan's tests were used to establish the significance of differences between individual groups. 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 (1980). The elevated plus-maze was wooden, with two opposite open arms, 50 x 10 cm, and two opposite enclosed arms of the same size, but with walls 40 em high. The arms were connected by a central square and thus the maze formed a plus-sign. It was elevated 50 cm above the floor. The rats were observed on a TV monitor from an adjacent room. Procedure Social interaction test. Rats were randomly allocated to control or diazepam (2 mg/kg/day) chronic treatment. 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; they were undrugged when they were given their familiarisation sessions. Immediately after the familiarisation session each rat received its usual daily injection. 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. On day 22 (i.e. 24 h after the last injection) rats in each chronic treatment condition were randomly allocated to the acute drug groups: control (water); buspirone (200 or 800 gg/kg). There were eight pairs in each group, except for the untreated diazepam withdrawal group which had ten pairs. Rats were tested in an order randomised for drug treatment, between 0730 and 1200 hours. The social interaction test lasted 5 rain and an observer, blind to the treatment of the rats, scored the time spent in active social interaction (sniffing, following and grooming the partner). Aggressive behaviours (boxing, wrestling and submitting) were excluded from this score since we have found these behaviours to markedly increase during ethanol withdrawal, in contrast to the decrease that is seen in social investigatory behaviours (File et al. 1991b). During benzodiazepine withdrawal there is no overall change in aggressive behaviours, but in some rats we have observed a marked increase.
Elevatedplus-maze. The rats received the same chronic treatments of control (water/Tween) or diazepam (2 mg/kg/day) for 21 days, and were tested on day 22 (24 h after the last injection). They were then randomly allocated among the following acute treatment groups: control (water); huspirone (100, 200, 400 or 800 gg/kg). There were 8 rats in each group, except for the untreated diazepam withdrawal group which had 12. Each rat was placed in the central square of the plus-maze, facing an enclosed arm. The time spent on open arms and the time spent in enclosed arms was scored for a 5-rain test period. An arm entry was defined as all four feet in the arm and the numbers of entries into open and enclosed arms were scored. The rats were scored by an observer who was without knowledge of either the chronic or acute drug treatment and who observed the rats from a TV monitor in an adjacent room. The rats were tested in an order randomised for drug treatment, between 0800 and 1300 hours.
Results A c o m p a r i s o n b e t w e e n the c o n t r o l g r o u p a n d the g r o u p w i t h d r a w n f r o m d i a z e p a m showed t h a t d i a z e p a m withd r a w a l resulted i n a significant decrease in social interaction [F ( 1 , 1 6 ) = 9 . 7 , P < 0 . 0 1 ] , b u t n o c h a n g e i n m o t o r activity or rears [ F < 1.0 in b o t h cases], see Fig. 1 a n d T a b l e 1. B u s p i r o n e (200 gg/kg) significantly increased social i n t e r a c t i o n [F ( 1 , 3 0 ) = 19.2, P = 0 . 0 0 0 1 ) a n d the d i a z e p a m w i t h d r a w a l g r o u p treated with this dose o f b u s p i r o n e h a d significantly ( P < 0.01) higher scores t h a n the u n t r e a t e d w i t h d r a w a l g r o u p ; however, o n c o m p a r i n g the i n d i v i d u a l g r o u p s given c h r o n i c vehicle t r e a t m e n t , b u s p i r o n e (200 gg/kg) was n o t significantly different f r o m the controls, see Fig. 1. B u s p i r o n e (800 gg/kg) was w i t h o u t significant effect ( P < 1.0) a n d a l t h o u g h this dose t e n d e d to reverse the a n x i o g e n i c effects of d i a z e p a m w i t h d r a w a l , the b u s p i r o n e x w i t h d r a w a l i n t e r a c t i o n failed to reach significance [F ( 1 , 3 0 ) = 2.5, P = 0.12). A l t h o u g h there were n o differences in m o t o r activity or rears b e t w e e n the c o n t r o l g r o u p a n d t h a t w i t h d r a w n f r o m d i a z e p a m , b u s p i r o n e (800 gg/kg) significantly red u c e d these measures in b o t h the c o n t r o l - t r e a t e d a n d the d i a z e p a m w i t h d r a w a l groups, see T a b l e 1. The social i n t e r a c t i o n d a t a for this dose were therefore r e - a n a l y s e d
580 CONTROLTREATED
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Fig. 1. Mean (4- SEM) time spent in social interaction by rats treated for 21 days with water (control-treated, clear columns) or diazepam (2 mg/kg/day) and tested 24 h after the last injection (diazepam withdrawal, hatched columns). They were tested 15 rain after subcutaneous injection with water (withdrawal group) or buspirone (200 or 800 pg/kg) + +P < 0.01 compared with control group ; **P
