Psychopharmacology
Psychopharmacology 64, 125-126 (1979)
9
by Springer-Verlag 1979
Benzodiazepines Alter Acquisition and Retention of an Inhibitory Avoidance Response in Mice R o b e r t A. Jensen, Joe L. M a r t i n e z , Jr., Beatriz J. Vasquez, a n d J a m e s L. M c G a u g h Department of Psychobiology, School of Biological Sciences, University of California, Irvine, California 92717, U.S,A.
Abstract. These e x p e r i m e n t s were p e r f o r m e d to exa m i n e the effects of g r a d e d doses of d i a z e p a m , fluraz e p a m , o r l o r a z e p a m given to S w i s s - W e b s t e r mice either 30 rain p r i o r to t r a i n i n g o r i m m e d i a t e l y after t r a i n i n g in a o n e - t r i a l i n h i b i t o r y (passive) a v o i d a n c e task. A 350 g A f o o t s h o c k was a d m i n i s t e r e d following entry into a d a r k e n e d c o m p a r t m e n t a n d r e t e n t i o n was tested three d a y s later. D o s e s of 10.0 m g / k g d i a z e p a m a n d 20.0 m g / k g l o r a z e p a m given before training significantly i m p a i r e d acquisition, while 1.0 m g / k g fluraz e p a m , given i m m e d i a t e l y after training, p r o d u c e d r e t r o g r a d e amnesia. These results indicate that benzodiazepines affect m e m o r y processes a n d t h a t various drugs of the b e n z o d i a z e p i n e family differentially affect acquisition a n d m e m o r y c o n s o l i d a t i o n .
Key words: Benzodiazepine,
Memory, Diazepam, Flurazepam, Lorazepam, Inhibitory Avoidance, Mice
B e n z o d i a z e p i n e s often p r o d u c e a n t e r o g r a d e a m n e s i a in humans. I n t r a v e n o u s a d m i n i s t r a t i o n of 1 0 - 1 5 m g d i a z e p a m to h u m a n s induces a m e m o r y deficit for at least I/2 h after the injection ( D u n d e e a n d Pandit, 1972), while l o r a z e p a m , given in doses of 3 - 5 m g is r e p o r t e d to have a s o m e w h a t m o r e p r o l o n g e d amnestic effect (Wilson a n d Ellis, 1973). H o w e v e r , few investigations of the a m n e s t i c effects of b e n z o d i a z e p i n e s have been c a r r i e d o u t in c o n t r o l l e d e x p e r i m e n t s on l a b o r a t o r y animals. This study investigated the effect on r e t e n t i o n of three different b e n z o d i a z e p i n e s given before o r after t r a i n i n g in a o n e - t r i a l i n h i b i t o r y a v o i d a n c e situation. This t a s k is p a r t i c u l a r l y well-suited to the s t u d y of learning a n d m e m o r y processes since the time t h a t learning occurs is well defined ( M c G a u g h a n d Herz, 1972).
Materials and Methods The subjects were 732 male Swiss-Webster mice 50-60 days of age. They were housed eight to a cage and were mail3tained with food and water freely available at all times. A 12 : 12 light-dark cycle (7 : 00 am, on) was in effect throughout the study. All behavioral manipulations were performed between 1: 00 and 5 : 00 pro. The mice were trained in one of three identical two-compartment runways (Jarvik and Kopp, 1967). On the training day, each mouse was placed in the brightly-lit smaller compartment facing the door. Five seconds later, the door was opened and when the mouse stepped through and bridged the rear set of plates (8.3 cm from the door), a timer recording entrance latency was automatically stopped, and a 350 pA footshock (700 laAsquare wave, on 50% of the time) was given. Shock was terminated when the mouse escaped back into the smaller compartment, typically within 1.0 - 1.5 s. Retention was tested 3 days later and the procedure was the same as training except that no shock was given upon entry into the darker compartment. Three benzodiazepines were used in this study: diazepam, flurazepam, and lorazepam. In the early stages of this research, we noticed that the commonly employed injection vehicle for diazepam, propylene glycol, acted synergistically with diazepam to produce amnesia, and under some conditions, propylene glycol alone produced a retention deficit (Vasquez et al., 1977). For this reason, the diazepam used in this study was dissolved in 1.0 N HC1 and then taken to pH 2.7 - 2.9 with 1.0 N NaOH. The doses used were 0.01,0.1, 1.0, and 10.0 mg/kg. The flurazepam was dissolved in saline, and given in doses of 0.025, 0.25, 1.0, and 2.5 mg/kg. The lorazepam was suspended in peanut oil and given in doses of 0.2, 2.0, 5.0, and 20.0 mg/kg. Control injections were of the appropriate injection vehicle and all injection volumes were 1 ml/100 g body weight, given I.P. The dose ranges we employed were determined on the basis of pilot work and the high doses were those that did not seriously disrupt locomotion. All injections were given either 30 rain prior to training, or immediately after training. The number of animals in each dose and training condition was between 21 and 26. Animals from any one shipment were randomly distributed among different regimes drug within practical constraints.
Results T h e percentage of a n i m a l s showing r e t e n t i o n latencies below 60 s in the d i a z e p a m p r e t r a i n i n g g r o u p is shown in F i g 1. Because m o s t a n i m a l s showed either very high
0033-3158/79/0064/0125/$01.00
126
Psychopharmacology 64 (1979)
Discussion
I00 o cO .~ ~ rr
80.
c O t-
=~ 1/~ .J
60. 40-
HH
O
E~ ~c g o
g3
20"
II !
i
Control
0.01
Diazepam
i
0.10
1.0
I i
IO.O
(mg/kg)
Fig. 1. The percent of animals showing entrance latencies on test day of less than 60 s that receivedeither diazepam or a control injection 30 min before training. Only the 10.0 mg/kg dose produced a significant impairment (p = 0.03)
These data indicate that diazepam and lorazepam affect acquisition of the task, while flurazepam m a y affect m e m o r y storage processes since its effect was seen only with post-trial administration. It is possible that the pretraining effects of diazepam and lorazepam m a y be due to drug dissociation (Hendriksson and J~irbe, 1971) or state dependency (Colpaert et al., 1976). These hypotheses were not specifically tested in this experiment. The mechanism by which benzodiazepines act is u n k n o w n , although a n u m b e r of neurotransmitter systems have been suggested as mediating their effects (see C o o k and Sepinwall, 1975).
Acknowledgements. We
thank Dr. Louis Gottschalk of the Department of Psychiatry, California Collegeof Medicine, University of California, Irvine, for the donation of the diazepam and flurazepam, and Wyeth Laboratories, Inc., for the lorazepam. This research was supported by Research grants MH 12526 (J. L. McG), Postdoctoral Fellowships MH 05358 (R.A.J.)and MH 05429 (J.L.M., Jr.).
References or very low scores, and the data were dichotomous, p r o p o r t i o n a l representation, as in Fig 1, is the m o s t appropriate m e t h o d of presenting the findings. To eliminate the possibility that some differences might be due to chance, overall goodness-of-fit Chi square tests (Hays, 1963) were performed. Only the diazepam pretraining administration g r o u p (Zz=35.47, df=3, p < 0.001), the lorazepam pretraining group (Z2 = 16.71, df= 3, p < 0.001), and the flurazepam posttraining g r o u p (Z z = 12.55, df= 3, p < 0.01) showed significant overall differences from their respective control groups. Following this, individual comparisons were then m a d e in these groups using M a n n - W h i t n e y U-tests. A dose of 10.0 m g / k g diazepam, given 30 min before training, p r o d u c e d a significant retention deficit ( U = 1 9 1 . 5 , z = 2 . 1 7 , p = 0 . 0 3 , two-tailed test), as did lorazepam given before training at a dose of 20.0 m g / k g (U = 160.5, z = 2.95, p =0.003). Flurazepam, 1.0 mg/kg, given immediately after training produced a significant retrograde amnesia ( U = 1 9 4 , z = 2 . 1 2 , p=0.034). N o other experimental condition p r o d u c e d significant effects on retention.
Colpaert, F. C., Desmedt, L. K. C., Janssen, P. A. J.: Discriminative stimulus properties of benzodiazepines, barbiturates and pharmacologically related drugs; relation to some intrinsic and anticonvulsant effects. Eur. J. Pharmacol. 37, 113-123 (1976) Cook, L., Sepinwall, J.: Behavioral analysis of the effects and mechanisms of action of benzodiazepines. In: Mechanisms of Action of Benzodiazepines. E. Costa and P. Greengard, eds., pp. 1-28. New York: Raven Press 1975 Dundee, J. W., Pandit, S. K.: Anterograde amnesic effectsof pethidine, hyoscine, and diazepam in adults. Br. J. Pharmacol. 44, 140 - 144 (1972) Hays, W. L.: Statistics for Psychologists, pp. 582-589, New York: Holt, Reinhart & Winston 1963 Henriksson, B. G., J~irbe, T.: Effects of diazepam on conditioned avoidance learning in rats and its transfer to normal state conditions. Psychopharmacologia (Berl.) 20), 186-190 (1971) Jarvik, M. E., Kopp, A.: An improved one-trial passive avoidance learning situation. Psychol. Rep. 21, 221-224 (1967) McGaugh, J. L., Herz, M. J.: Memory Consolidation. San Francisco: Albion Publishing Co. 1972 Vasquez, B. J., Martinez, J. L., Jr., Jensen, R. A., McGaugh, J. L.: Amnestic effects of propylene glycol in mice. Proc. West Pharmacol. Soc. 20, 179-183 (1977) Wilson, H., Ellis, F. R.: Oral premedication with lorazepam (Ativan): a comparison with heptabarbitone (Medomin) and diazepam (Valium). Br. J. Anaest. 45, 738-744 (1973) Received May 31, 1978; Final Version January 19, 1979
Psychopharmacology 64, 125-126 (1979)
9
by Springer-Verlag 1979
Benzodiazepines Alter Acquisition and Retention of an Inhibitory Avoidance Response in Mice R o b e r t A. Jensen, Joe L. M a r t i n e z , Jr., Beatriz J. Vasquez, a n d J a m e s L. M c G a u g h Department of Psychobiology, School of Biological Sciences, University of California, Irvine, California 92717, U.S,A.
Abstract. These e x p e r i m e n t s were p e r f o r m e d to exa m i n e the effects of g r a d e d doses of d i a z e p a m , fluraz e p a m , o r l o r a z e p a m given to S w i s s - W e b s t e r mice either 30 rain p r i o r to t r a i n i n g o r i m m e d i a t e l y after t r a i n i n g in a o n e - t r i a l i n h i b i t o r y (passive) a v o i d a n c e task. A 350 g A f o o t s h o c k was a d m i n i s t e r e d following entry into a d a r k e n e d c o m p a r t m e n t a n d r e t e n t i o n was tested three d a y s later. D o s e s of 10.0 m g / k g d i a z e p a m a n d 20.0 m g / k g l o r a z e p a m given before training significantly i m p a i r e d acquisition, while 1.0 m g / k g fluraz e p a m , given i m m e d i a t e l y after training, p r o d u c e d r e t r o g r a d e amnesia. These results indicate that benzodiazepines affect m e m o r y processes a n d t h a t various drugs of the b e n z o d i a z e p i n e family differentially affect acquisition a n d m e m o r y c o n s o l i d a t i o n .
Key words: Benzodiazepine,
Memory, Diazepam, Flurazepam, Lorazepam, Inhibitory Avoidance, Mice
B e n z o d i a z e p i n e s often p r o d u c e a n t e r o g r a d e a m n e s i a in humans. I n t r a v e n o u s a d m i n i s t r a t i o n of 1 0 - 1 5 m g d i a z e p a m to h u m a n s induces a m e m o r y deficit for at least I/2 h after the injection ( D u n d e e a n d Pandit, 1972), while l o r a z e p a m , given in doses of 3 - 5 m g is r e p o r t e d to have a s o m e w h a t m o r e p r o l o n g e d amnestic effect (Wilson a n d Ellis, 1973). H o w e v e r , few investigations of the a m n e s t i c effects of b e n z o d i a z e p i n e s have been c a r r i e d o u t in c o n t r o l l e d e x p e r i m e n t s on l a b o r a t o r y animals. This study investigated the effect on r e t e n t i o n of three different b e n z o d i a z e p i n e s given before o r after t r a i n i n g in a o n e - t r i a l i n h i b i t o r y a v o i d a n c e situation. This t a s k is p a r t i c u l a r l y well-suited to the s t u d y of learning a n d m e m o r y processes since the time t h a t learning occurs is well defined ( M c G a u g h a n d Herz, 1972).
Materials and Methods The subjects were 732 male Swiss-Webster mice 50-60 days of age. They were housed eight to a cage and were mail3tained with food and water freely available at all times. A 12 : 12 light-dark cycle (7 : 00 am, on) was in effect throughout the study. All behavioral manipulations were performed between 1: 00 and 5 : 00 pro. The mice were trained in one of three identical two-compartment runways (Jarvik and Kopp, 1967). On the training day, each mouse was placed in the brightly-lit smaller compartment facing the door. Five seconds later, the door was opened and when the mouse stepped through and bridged the rear set of plates (8.3 cm from the door), a timer recording entrance latency was automatically stopped, and a 350 pA footshock (700 laAsquare wave, on 50% of the time) was given. Shock was terminated when the mouse escaped back into the smaller compartment, typically within 1.0 - 1.5 s. Retention was tested 3 days later and the procedure was the same as training except that no shock was given upon entry into the darker compartment. Three benzodiazepines were used in this study: diazepam, flurazepam, and lorazepam. In the early stages of this research, we noticed that the commonly employed injection vehicle for diazepam, propylene glycol, acted synergistically with diazepam to produce amnesia, and under some conditions, propylene glycol alone produced a retention deficit (Vasquez et al., 1977). For this reason, the diazepam used in this study was dissolved in 1.0 N HC1 and then taken to pH 2.7 - 2.9 with 1.0 N NaOH. The doses used were 0.01,0.1, 1.0, and 10.0 mg/kg. The flurazepam was dissolved in saline, and given in doses of 0.025, 0.25, 1.0, and 2.5 mg/kg. The lorazepam was suspended in peanut oil and given in doses of 0.2, 2.0, 5.0, and 20.0 mg/kg. Control injections were of the appropriate injection vehicle and all injection volumes were 1 ml/100 g body weight, given I.P. The dose ranges we employed were determined on the basis of pilot work and the high doses were those that did not seriously disrupt locomotion. All injections were given either 30 rain prior to training, or immediately after training. The number of animals in each dose and training condition was between 21 and 26. Animals from any one shipment were randomly distributed among different regimes drug within practical constraints.
Results T h e percentage of a n i m a l s showing r e t e n t i o n latencies below 60 s in the d i a z e p a m p r e t r a i n i n g g r o u p is shown in F i g 1. Because m o s t a n i m a l s showed either very high
0033-3158/79/0064/0125/$01.00
126
Psychopharmacology 64 (1979)
Discussion
I00 o cO .~ ~ rr
80.
c O t-
=~ 1/~ .J
60. 40-
HH
O
E~ ~c g o
g3
20"
II !
i
Control
0.01
Diazepam
i
0.10
1.0
I i
IO.O
(mg/kg)
Fig. 1. The percent of animals showing entrance latencies on test day of less than 60 s that receivedeither diazepam or a control injection 30 min before training. Only the 10.0 mg/kg dose produced a significant impairment (p = 0.03)
These data indicate that diazepam and lorazepam affect acquisition of the task, while flurazepam m a y affect m e m o r y storage processes since its effect was seen only with post-trial administration. It is possible that the pretraining effects of diazepam and lorazepam m a y be due to drug dissociation (Hendriksson and J~irbe, 1971) or state dependency (Colpaert et al., 1976). These hypotheses were not specifically tested in this experiment. The mechanism by which benzodiazepines act is u n k n o w n , although a n u m b e r of neurotransmitter systems have been suggested as mediating their effects (see C o o k and Sepinwall, 1975).
Acknowledgements. We
thank Dr. Louis Gottschalk of the Department of Psychiatry, California Collegeof Medicine, University of California, Irvine, for the donation of the diazepam and flurazepam, and Wyeth Laboratories, Inc., for the lorazepam. This research was supported by Research grants MH 12526 (J. L. McG), Postdoctoral Fellowships MH 05358 (R.A.J.)and MH 05429 (J.L.M., Jr.).
References or very low scores, and the data were dichotomous, p r o p o r t i o n a l representation, as in Fig 1, is the m o s t appropriate m e t h o d of presenting the findings. To eliminate the possibility that some differences might be due to chance, overall goodness-of-fit Chi square tests (Hays, 1963) were performed. Only the diazepam pretraining administration g r o u p (Zz=35.47, df=3, p < 0.001), the lorazepam pretraining group (Z2 = 16.71, df= 3, p < 0.001), and the flurazepam posttraining g r o u p (Z z = 12.55, df= 3, p < 0.01) showed significant overall differences from their respective control groups. Following this, individual comparisons were then m a d e in these groups using M a n n - W h i t n e y U-tests. A dose of 10.0 m g / k g diazepam, given 30 min before training, p r o d u c e d a significant retention deficit ( U = 1 9 1 . 5 , z = 2 . 1 7 , p = 0 . 0 3 , two-tailed test), as did lorazepam given before training at a dose of 20.0 m g / k g (U = 160.5, z = 2.95, p =0.003). Flurazepam, 1.0 mg/kg, given immediately after training produced a significant retrograde amnesia ( U = 1 9 4 , z = 2 . 1 2 , p=0.034). N o other experimental condition p r o d u c e d significant effects on retention.
Colpaert, F. C., Desmedt, L. K. C., Janssen, P. A. J.: Discriminative stimulus properties of benzodiazepines, barbiturates and pharmacologically related drugs; relation to some intrinsic and anticonvulsant effects. Eur. J. Pharmacol. 37, 113-123 (1976) Cook, L., Sepinwall, J.: Behavioral analysis of the effects and mechanisms of action of benzodiazepines. In: Mechanisms of Action of Benzodiazepines. E. Costa and P. Greengard, eds., pp. 1-28. New York: Raven Press 1975 Dundee, J. W., Pandit, S. K.: Anterograde amnesic effectsof pethidine, hyoscine, and diazepam in adults. Br. J. Pharmacol. 44, 140 - 144 (1972) Hays, W. L.: Statistics for Psychologists, pp. 582-589, New York: Holt, Reinhart & Winston 1963 Henriksson, B. G., J~irbe, T.: Effects of diazepam on conditioned avoidance learning in rats and its transfer to normal state conditions. Psychopharmacologia (Berl.) 20), 186-190 (1971) Jarvik, M. E., Kopp, A.: An improved one-trial passive avoidance learning situation. Psychol. Rep. 21, 221-224 (1967) McGaugh, J. L., Herz, M. J.: Memory Consolidation. San Francisco: Albion Publishing Co. 1972 Vasquez, B. J., Martinez, J. L., Jr., Jensen, R. A., McGaugh, J. L.: Amnestic effects of propylene glycol in mice. Proc. West Pharmacol. Soc. 20, 179-183 (1977) Wilson, H., Ellis, F. R.: Oral premedication with lorazepam (Ativan): a comparison with heptabarbitone (Medomin) and diazepam (Valium). Br. J. Anaest. 45, 738-744 (1973) Received May 31, 1978; Final Version January 19, 1979
