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Attenuation of amnesia by hydrocortisone in the mouse
Physiology & Behavior, Vol. 20, pp. 607-611. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A
Attenuation of Amnesia by Hydrocortisone in the M o u s e I SHINSHU NAKAJIMA
Department o f Psychology, Dalhousie Universi~, Halifax, Nova Scotia, Canada ( R e c e i v e d 10 O c t o b e r 1 9 7 7 ) NAKAJIMA, S. Attenuation of amnesia by hydrocortisone in the mouse. PHYSIOL. BEHAV. 20(5) 6 0 7 - 6 1 1 , 1978. Effects of hydrocortisone on the retrograde amnesia produced by electroconvulsive shock (ECS) was studied in 228 mice. The animals were given ECS after one-trial training in a step-through apparatus, and tested for passive avoidance behavior 7 days later. The amnesic effect of ECS was absent in the animals that were injected with hydrocortisone SC prior to the administration of ECS. Hydrocortisone alone, without ECS, had no significant effect on passive avoidance. There was no indication that the hormone suppressed the ECS-induced seizure activity in the brain. The results suggest that hydrocortisone protected the animals from amnesia by acting on the brain before the onset of the disruptive action of ECS. Amnesia
Corticosteroids
ECS
Hydrocortisone
Memory
Passive avoidance
They were h o u s e d in plastic cages, 5 - 8 t o a cage, a n d given free access to f o o d a n d water. The a n i m a l c o l o n y was i l l u m i n a t e d from 0 8 : 0 0 - 2 0 : 0 0 hr, a n d the b e h a v i o r a l tests were c o n d u c t e d b e t w e e n 0 9 : 3 0 - 1 2 : 3 0 h r to m i n i m i z e t h e effects of circadian f l u c t u a t i o n of h y p o p h y s e a l - a d r e n a l functions. Details of the s t e p - t h r o u g h passive a v o i d a n c e task h a v e been given in a previous p a p e r [ 12] ; a b r i e f o u t l i n e o f t h e p r o c e d u r e is as follows. Each m o u s e was p l a c e d in a small, well-illuminated c o m p a r t m e n t , facing away f r o m a d o o r way. When the animal t u r n e d a r o u n d t o w a r d t h e d o o r , it was o p e n e d a n d a t i m e r started. When the a n i m a l s t e p p e d t h r o u g h the d o o r w a y i n t o a relatively large, d a r k c o m p a r t m e n t , the t i m e r was s t o p p e d , a n d f o o t s h o c k (0.35 m A , AC) was delivered t h r o u g h m e t a l plates c o v e r i n g t h e f l o o r a n d the walls. The animal was allowed t o r e t r e a t to t h e small c o m p a r t m e n t , where it was p i c k e d u p a n d given ECS i m m e d i a t e l y , or r e t u r n e d to the h o m e cage w i t h o u t ECS. The ECS was 60 Hz AC, 10 m A in i n t e n s i t y , lasting for 0.2 sec, a n d was applied to the f o r e h e a d j u s t a b o v e the eyes, using r o u n d e d tips of stainless-steel r o d s (1.5 m m dia., 10 m m a p a r t ) as electrodes. Twelve groups of mice (10 per g r o u p ) were given one trial of f o o t s h o c k training: 6 groups receiving ECS a n d the o t h e r 6 w i t h o u t ECS. H y d r o c o r t i s o n e - 2 1 - s o d i u m s u c c i n a t e (Sigma Chemical Co.) was dissolved in p h y s i o l o g i c a l saline and i n j e c t e d s u b c u t a n e o u s l y 42 m g / k g e i t h e r 15, 5 or 1 m i n before, or 1 rain or 24 h r a f t e r the t r a i n i n g trial. One ECS group a n d one No-ECS group were assigned to e a c h of the 5 training-injection intervals. The r e m a i n i n g 2 groups, one ECS and one No-ECS, did n o t receive a n y i n j e c t i o n a n d served as No-Injection c o n t r o l groups. Passive avoidance
T H E R E are several drugs t h a t a t t e n u a t e the amnesic e f f e c t of e l e c t r o c o n v u l s i v e s h o c k (ECS). Neural s t i m u l a n t s such as s t r y c h n i n e and p e n t y l e n e t e t r a z o l , at relatively low dosage levels, reduce the a m n e s i c e f f e c t if t h e y are i n j e c t e d before the a d m i n i s t r a t i o n o f ECS [ 1 5 ] . Similarly, t h e a m n e s i c effect o f ECS is a t t e n u a t e d b y t r i c y a n o a m i n o p r o p e n e [ 6 ] , w h i c h facilitates b i o s y n t h e s i s of r i b o n u c l e i c acid, a n d by p h y s o s t i g m i n e [ 3 ] , w h i c h i n h i b i t s c h o l i n e s t e r a s e activity. In a previous study, N a k a j i m a [12] f o u n d t h a t corticosteroids a t t e n u a t e the amnesic e f f e c t o f c y c l o h e x i m i d e . Mice were t r a i n e d in a passive a v o i d a n c e a p p a r a t u s a f t e r i n j e c t i o n of c y c l o h e x i m i d e , a n d t h e n given an a d d i t i o n a l i n j e c t i o n o f e i t h e r h y d r o c o r t i s o n e or c o r t i c o s t e r o n e . In a later test, these animals d e m o n s t r a t e d good a v o i d a n c e behavior, t h a t is, the usual amnesic e f f e c t o f c y c l o h e x i m i d e was absent. In a s u b s e q u e n t s t u d y , C o t t r e l l a n d N a k a j i m a [2] s h o w e d t h a t c y c l o h e x i m i d e , at a dosage t h a t p r o d u c e d amnesia, also suppressed the f o o t s h o c k - i n d u c e d s e c r e t i o n o f corticosteroids. U n d e r these c o n d i t i o n s , i n j e c t i o n of h y d r o c o r t i s o n e i n t o the h i p p o c a m p u s a t t e n u a t e d the a m n e s i c effect of c y c l o h e x i m i d e . The p r e s e n t e x p e r i m e n t s were c o n d u c t e d t o d e t e r m i n e w h e t h e r h y d r o c o r t i s o n e w o u l d have an a n t i - a m n e s i c e f f e c t on E C S - i n d u c e d a m n e s i a as it does o n the c y c l o h e x i m i d e i n d u c e d amnesia. EXPERIMENT 1
Method Male Swiss a l b i n o mice (Charles River, C D - 1 s t r a i n ) were o b t a i n e d f r o m C a n a d i a n Breeding F a r m s ( M o n t r e a l ) . They weighed 3 0 - 3 8 g at the b e g i n n i n g of the e x p e r i m e n t .
Supported by the National Research Council of Canada grant A0233. The author is grateful to P. A. Cummings and N. P. Phelps for their technical assistance, and to G. A. Cottrell for her critical comments on the manuscript. 607
608
NAKAJIMA
behavior was tested 7 days later by placing each mouse in the small compartment again. The step-through latency was recorded up to a maximum of 600 sec. The latency scores were subjected to analysis of variance, and post hoc comparisons were made according to the method of Rodger [13]. The expected probability of the Type I error was 0.05. In order to confirm the validity of the statistical treatments, the same sets of data were subjected to non-parametric tests. In both Experiments 1 and 2, the overall differences were significant at p<0.01 by the Kruskal-Wallis one-way analysis of variance. Those groupdifferences which were indicated by the method of Rodger [ 13] were all significant at p< 0.01 by the Mann-Whitney U tests.
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Results The mean step-through latency in the training trial was 7.0 sec, and there was no significant difference between the animals injected with hydrocortisone before training and those injected after training. A typical reaction to ECS was an immediate tonic extension of the 4 legs and forward flexion of the head. Of the 60 mice initially assigned to the 6 ECS groups, 16 died and 3 escaped ECS by a brisk movement of the head. These animals were replaced with additional animals so that each group consisted of 10 mice. There was no observable difference in the pattern of convulsions or in the mortality rate among the animals injected with the hormone before ECS, those injected after ECS, and those without injection. The mean latency of each group in the test trial is shown in Fig. 1. The differences among the groups were statistically significant, F(11,108) = 6.46. The No-ECS groups all showed relatively long latencies, indicating one-trial acquisition of the passive avoidance task. There was no significant difference among the No-ECS groups. Administration of ECS produced retrograde amnesia of passive avoidance as indicated by a short latency of the No-Injection ECS group. Three other groups that were injected with hydrocortisone succinate either 15 rain before, 1 min after, or 24 hr after the training trial also demonstrated relatively short latencies. The remaining two ECS groups that were injected either 1 or 5 min before training (marked with asterisks in Fig. 1) demonstrated latencies significantly longer than the latencies of the other 4 ECS groups (p<0.05). The fact that these 2 groups of animals demonstrated relatively long latencies suggest that the amnesic effect of ECS was attenuated by the hormone injected. The attenuation, however, was not complete: the latencies of the two groups were still significantly shorter than those of the No-ECS groups (p<0.05). EXPERIMENT 2 It is possible that the incomplete anti-amnesic effect observed in Experiment 1 was due to the use of an inappropriate form of steroid. Hydrocortisone-21-sodium succinate was used in Experiment I because it readily dissolves in physiological saline. However, the amnesic effect of cycloheximide was more effectively attenuated by a suspension of hydrocortisone than a solution of its sodium succinate ester [12]. Therefore, hydrocortisone suspension was used in Experiment 2 in the hope that it would be more effective in attenuating the amnesic effect of ECS.
-15'
-5' -1' *1' 24h Nolnj. Training- Injection Interval
FIG. 1. Mean step-through latency (and standard error) of each group in the test trial of Experiment 1. ECS was given within 1 rain of footshock in the training trial. In the two ECS groups marked with asterisks, the latencies were significantly (p<0.05) longer than in the other four ECS groups, but shorter than in the No-ECS groups. Method Ten groups of mice (10 per group) were trained in the passive avoidance apparatus and tested 7 days later as in Experiment 1. Hydrocortisone (Sigma Chemical Co.) was suspended in physiological saline containing 5% Tween-80 (Fisher Scientific), and subcutaneously injected 30 mg/kg. This dosage is equimolar to 42 mg/kg hydrocortisone succinate. ECS was given either immediately (within 1 min), 2 min or 5 min after the training trial (Fig. 2). Two groups were assigned to each of the 3 training-ECS intervals and to the No-ECS control: one group without injection and the other group with hydrocortisone injection 5 min prior to training. One of the remaining 2 groups was injected with saline, containing 5% Tween-80, 5 rain before training and given ECS immediately after training. The other group was injected with hydrocortisone immediately after training (marked as AT in Fig. 2) and given ECS 2 min after training. Results The mean step-through latency in the training trial was 8.3 sec, and there was no statistically significant difference between the hydrocortisone-injected groups and uninjected groups. Out of 80 animals that received ECS, 21 died and were replaced with additional animals. Figure 2 shows the step-through latencies of the 10 groups in the test trial. Analysis of variance revealed significant differences among the groups, F(9,90) = 3.37. Both of the No-ECS groups demonstrated good avoidance response, and the three ECS groups without injection (white columns) showed a gradient of amnesic effect depending on the time interval between training and ECS. Three groups which received ECS after 2 min or less (marked with asterisks in Fig. 2) showed significantly shorter latencies than the No-ECS control groups (p<0.05). It should be noted that none of these 3 groups received
AMNESIA AND HYDROCORTISONE
609 EXPERIMENT 3
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In the preceding experiments, the amnesic effect of ECS was absent if a sufficient amount of corticosteroid was injected prior to the administration of ECS. There is a possibility that corticosteroids reduce the excitability of neurons so that ECS fails to elicit seizure activity in the brain. However there are some studies which indicate the contrary, that is, corticosteroids enhance the susceptibility of the brain to convulsive stimulation [7,8]. To examine the effect of hydrocortisone on seizure activity, electrographic recording was conducted in a situation similar to the training of a passive avoidance task. Convulsive stimulation was applied through implanted electrodes to ensure that the locus of stimulation remained the same on different days with and without hormone injection.
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hydrocortisone injection. All of the hydrocortisone-injected groups demonstrated long latencies, which were not significantly different from the latencies of the No-ECS control groups. It is clear that the amnesic effect of ECS was completely abolished by prior injection of hydrocortisone regardless of the training-ECS interval. The hormone was more effective as a suspension than as a solution of an ester. It may be argued that the hydrocortisone-injected animals showed long step-through latencies, not because the effect of ECS was attenuated, but because the hormone enhanced the animal's sensitivity to footshock. An answer to this argument is provided by the AT group, which received the hormone injection after footshock. The mean latency of the AT group was similar to the latencies of the groups injected before footshock, and of the No-ECS groups. Thus, the long latencies in the hydrocortisone injected groups are not attributable to an enhanced shock sensitivity. Another possible argument is that hydrocortisone may have suppressed the animal's activity in the avoidance apparatus 7 days after injection, regardless of footshock and regardless of ECS. In order to test such a possibility, an additional group of l 0 mice was injected with hydrocortisone and 5 min later placed in the avoidance apparatus. Upon entering the dark compartment, however, the mice were not given footshock but simply removed from the apparatus and returned to the home cage without ECS. Seven days later, they were tested in the same way as the animals in the other groups. The mean step-through latency of this group was 7.3 sec, and the possibility that the injected animals may have been slow to move simply because of the injection was ruled out.
Ten mice were implanted with electrodes under sodium pentobarbital anesthesia (75 mg/kg, IP). For the delivery of convulsive stimulation, screw electrodes were bilaterally tapped into the skull, 2 mm anterior to the bregma, and 2.5 mm lateral to the midline. The screws extended approximately 0.5 mm into the anterior cortex. A bipolar recording electrode, made of twisted strands of stainlesssteel wire (0.2 mm in dia. and insulated except at the tip cross-section), was implanted into the dorsal hippocampus, 1.5 mm posterior to the bregma, 1.5 mm lateral to the midline. One pole of the bipolar electrode was implanted 2 mm deep into the brain, and the other pole was 0.5 mm shorter. Recording of electrical activity was started after a recovery period of 1 0 - 1 4 days using a polygraph (Grass Instruments, Model 79). The mouse was placed in a box (10 x 15 cm) with a grid floor, and electrical activity was recorded from the anterior cortex and the hippocampus for approximately half an hour. On the following day, 5 of the animals were injected with hydrocortisone suspension (30 mg/kg, SC), given 0.35 mA footshock for 1 sec, and then stimulated through the cortical electrodes. The time interval between the injection and footshock was 5 min, and the interval between the footshock and brain stimulation was 30 sec, so that the timing of the experimental treatments was similar to the timing in the ECS < I min group in Experiment 2. The remaining 5 animals were given footshock and brain stimulation without hydrocortisone injection. Two days later, the animals which did not receive the injection were now injected with the hormone suspension, and those which had the injection before were recorded without injection. The rest of the procedure was exactly the same as before. In order to avoid any inadvertent stimulation, the recording cable was completely disconnected from the animal prior to the administration of footshock, and reconnected after the end of the shock. During the brair/ stimulation, the cortical electrodes were connected to a constant current unit (Grass Instruments, Model C C U - 1 ) driven by a stimulator (Grass Instruments, Model S D - 9 ) , while the hippocampal electrode remained disconnected. The stimulation was a 0.2 sec train of 1 msec rectangular pulses, 100 pulses per sec, and 3 mA peak to peak. The intensity of the stimulation had been determined in a preliminary experiment using 8 mice, and was sufficient to
610
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FIG. 3. Electrographic records of a representative animal in Experiment 3. Artifacts on the records indicate the time when the recording cable was disconnected from the polygraph. Actual administration of footshock or ECS was some time after the onset of the artifact. The records 10 min and 30 min after footshock indicate the course of recovery from ECS. A: No injection, B: Hydrocortisone injection 5 min before footshock. produce a type of convulsion and amnesia similar to those observed in Experiments 1 and 2. At the conclusion of the recording experiment, the animals were anesthetized with sodium pentobarbital (75 mg/kg, IP), and the brains were removed. After fixation in 10% Formalin, the brains were sectioned and stained to identify the sites of the electrodes. Results Two animals died of convulsion, and the results are based on 8 animals. Examination of the brain sections indicated that the electrodes were located in the anterior cortex and the h i p p o c a m p u s as originally intended. The electrographic records of a representative animal are shown in Fig. 3. The brain stimulation p r o d u c e d after-discharges in the anterior c o r t e x and the hippocampus. The hippocampal after-discharge was followed by post-ictal depression and sporadic spike discharges. Each animal showed a characteristic pattern o f seizure activity regardless o f whether h y d r o c o r t i s o n e had been injected or not. In some animals the duration of the primary after-discharge was slightly shorter on the injection day, but in other animals it was shorter on the no-injection day. Whether the injection was given on the first or second day of brain stimulation was not related to the pattern of discharges in any discernible way. Definitely, h y d r o c o r t i s o n e injection did not prevent seizure activity either in the c o r t e x or in the hippocampus. DISCUSSION Subcutaneous injection of h y d r o c o r t i s o n e abolished the amnesic effect of ECS in a passive avoidance situation. The h o r m o n e injection alone, w i t h o u t ECS, had no effect on the acquisition of the avoidance task; the convulsive shock was strong enough to produce a gradient of amnesia in uninjected control animals. There was no indication that hydrocortisone suppressed seizure activity in the brain in any appreciably degree. Nevertheless, the amnesic effect of ECS was absent in the animals that were injected with the h o r m o n e before the administration of ECS. The results suggest that hydrocortisone p r o t e c t e d neurons in the brain from the disruptive action of ECS.
The mechanism underlying the protective action of hydrocortisone is not clear. One possible explanation would be that the h o r m o n e facilitated accumulation of some substance into the neuron - possibly a transmitter substance that would otherwise be exhausted by the high rate of firing during convulsion. In this regard, it is interesting to note that h y d r o c o r t i s o n e facilitates the rate of norepinephrine uptake by brain slices incubated in vitro [ 11 ]. The finding that exogenous h y d r o c o r t i s o n e attenuates the amnesic effect of ECS suggests that endogenous corticosteroids (adrenocortical h o r m o n e s ) would also attenuate the ECS-induced amnesia. It follows that if the secretion of adrenocortical h o r m o n e s is suppressed, an administration of ECS more than several minutes after training would still produce retrograde amnesia. The results of experients c o n d u c t e d by Andry and Luttges [1], and more recently by Flood and his associates [ 9 ] , are congruent with this inference. They found that ECS given 30 min or more after training p r o d u c e d amnesia in the mice injected with c y c l o h e x i m i d e [ 1 ] or anisomycin [ 9 ] , but not in saline-injected control animals. In these experiments, c y c l o h e x i m i d e and anisomycin were administered in order to suppress protein synthesis in the brain, but these drugs would have suppressed steroidogenesis in the adrenal cortex as well. Therefore, the behavioral results may have come from the adrenal rather than cerebral effect of the drugs. Recently, Nakajima (unpublished preliminary study) observed that a delayed administration of ECS produces amnesia in the mice injected with aminoglutethimide, which suppresses adrenocortical function without affecting cerebral protein synthesis. Implications of this observation have to be considered very carefully, in view of the fact that a m i n o g l u t e t h i m i d e alone does not produce amnesia [5,141. It is still indecisive whether hydrocortisone itself is the effective anti-amnesic substance. The a m o u n t of exogenous hydrocortisone required to abolish the amnesic effects of ECS and c y c l o h e x i m i d e is far larger than the c o n c e n t r a t i o n of endogenous corticosteroids normally observed after a stress of f o o t s h o c k [ 12,14]. It is possible that the effective anti-amnesic substance may be a minute a m o u n t of aldosterone or d e o x y c o r t i c o s t e r o n e that happened to be contained in the suspension of hydrocortisone. The im-
AMNESIA AND HYDROCORTISONE
611
p o r t a n c e o f these m i n e r a l o c o r t i c o i d s in the p e r f o r m a n c e o f a v o i d a n c e tasks has b e e n d e m o n s t r a t e d r e c e n t l y [ 1 0 ] . A n o t h e r possibility is t h a t a h i g h dose o f h y d r o c o r t i s o n e m a y facilitate or suppress the s e c r e t i o n o f various
h y p o p h y s e a l p e p t i d e h o r m o n e s , w h i c h in t u r n i n f l u e n c e avoidance behavior [4]. At present, however, information is n o t sufficient to favor a n y o f these a l t e r n a t i v e possibilities.
REFERENCES 1. Andry, D. K. and M. W. Luttges. Memory traces: Experimental separation by cycloheximide and electroconvulsive shock. Science 178: 5 1 8 - 5 2 0 , 1972. 2. Cottrell, G. A. and S. Nakajima. Effect of corticosteroids in the hippocampus on passive avoidance behavior in the rat. Pharmac. Biochern. Behav. 7: 277-280, 1977. 3. Davis, J. W., R. K. Thomas and H. E. Adams. Interactions of scopolamine and physostigmine with ECS and one-trial learning. Physiol. Behav. 6: 219-222, 1971. 4. De Wied, D. Peptides and behavior. Life ScL 20: 195-204, 1977. 5. Dunn, A. J. and S. Leibmann. The amnestic effect of protein synthesis inhibitors is not due to the inhibition of adrenal corticosteroidogenesis. Behav. Biol. 1 9 : 4 1 1 - 4 1 6 , 1977. 6. Essman, W. B. Effect of tricyanoaminopropene on the amnesic effect of electroconvulsive shock. Psychopharmacologia 9: 4 2 6 - 4 3 3 , 1966. 7. Feldman, S. Convulsive phenomena produced by intraventricular administration of hydrocortisone in cats. Epilepsia 7: 271-282, 1966. 8. Feldman, S. and J. M. Davidson. Effect of hydrocortisone on electrical activity, arousal thresholds and evoked potentials in the brains of chronically implanted rabbits. J. Neurol. Sci. 3: 4 6 2 - 4 7 2 , 1966.
9. Flood, J. F., E. L. Bennett, A. E. Orme and M. E. Jarvik. Protein synthesis dependent gradient of ECS retrograde amnesia. Behav. Biol. 21: 307-328, 1977. 10. Gray, P. Effect of prestimulation on avoidance responding in rats, and hormonal dependence of the effect. J. cornp, physiol. Psychol. 90: 1-17, 1976. 11. Maas, J. W. and M. Mednieks. Hydrocortisone-mediated increase of norepinephrine uptake by brain slices. Science 171: 178-179, 1971. 12. Nakajima, S. Amnesic effect of cycloheximide in the mouse mediated by adrenocortical hormones. J. comp. physiol. Psychol. 88: 378-385, 1975. 13. Rodger, R. S. The number of non-zero, post hoc contrasts from ANOVA and error-rate. I. Br. J. Math. Stat. Psychol. 28: 71-78, 1975. 14. Squire, L. R., S. St. John and H. P. Davis. Inhibitors of protein synthesis and memory: Dissociation of amnesic effects and effects on adrenal steroidogenesis. Brain Res. 112: 200-206, 1976. 15. Weissman, A. Drugs and retrograde amnesia. Int. Rev. Neurobiol. 10: 167-198, 1967.
Attenuation of Amnesia by Hydrocortisone in the M o u s e I SHINSHU NAKAJIMA
Department o f Psychology, Dalhousie Universi~, Halifax, Nova Scotia, Canada ( R e c e i v e d 10 O c t o b e r 1 9 7 7 ) NAKAJIMA, S. Attenuation of amnesia by hydrocortisone in the mouse. PHYSIOL. BEHAV. 20(5) 6 0 7 - 6 1 1 , 1978. Effects of hydrocortisone on the retrograde amnesia produced by electroconvulsive shock (ECS) was studied in 228 mice. The animals were given ECS after one-trial training in a step-through apparatus, and tested for passive avoidance behavior 7 days later. The amnesic effect of ECS was absent in the animals that were injected with hydrocortisone SC prior to the administration of ECS. Hydrocortisone alone, without ECS, had no significant effect on passive avoidance. There was no indication that the hormone suppressed the ECS-induced seizure activity in the brain. The results suggest that hydrocortisone protected the animals from amnesia by acting on the brain before the onset of the disruptive action of ECS. Amnesia
Corticosteroids
ECS
Hydrocortisone
Memory
Passive avoidance
They were h o u s e d in plastic cages, 5 - 8 t o a cage, a n d given free access to f o o d a n d water. The a n i m a l c o l o n y was i l l u m i n a t e d from 0 8 : 0 0 - 2 0 : 0 0 hr, a n d the b e h a v i o r a l tests were c o n d u c t e d b e t w e e n 0 9 : 3 0 - 1 2 : 3 0 h r to m i n i m i z e t h e effects of circadian f l u c t u a t i o n of h y p o p h y s e a l - a d r e n a l functions. Details of the s t e p - t h r o u g h passive a v o i d a n c e task h a v e been given in a previous p a p e r [ 12] ; a b r i e f o u t l i n e o f t h e p r o c e d u r e is as follows. Each m o u s e was p l a c e d in a small, well-illuminated c o m p a r t m e n t , facing away f r o m a d o o r way. When the animal t u r n e d a r o u n d t o w a r d t h e d o o r , it was o p e n e d a n d a t i m e r started. When the a n i m a l s t e p p e d t h r o u g h the d o o r w a y i n t o a relatively large, d a r k c o m p a r t m e n t , the t i m e r was s t o p p e d , a n d f o o t s h o c k (0.35 m A , AC) was delivered t h r o u g h m e t a l plates c o v e r i n g t h e f l o o r a n d the walls. The animal was allowed t o r e t r e a t to t h e small c o m p a r t m e n t , where it was p i c k e d u p a n d given ECS i m m e d i a t e l y , or r e t u r n e d to the h o m e cage w i t h o u t ECS. The ECS was 60 Hz AC, 10 m A in i n t e n s i t y , lasting for 0.2 sec, a n d was applied to the f o r e h e a d j u s t a b o v e the eyes, using r o u n d e d tips of stainless-steel r o d s (1.5 m m dia., 10 m m a p a r t ) as electrodes. Twelve groups of mice (10 per g r o u p ) were given one trial of f o o t s h o c k training: 6 groups receiving ECS a n d the o t h e r 6 w i t h o u t ECS. H y d r o c o r t i s o n e - 2 1 - s o d i u m s u c c i n a t e (Sigma Chemical Co.) was dissolved in p h y s i o l o g i c a l saline and i n j e c t e d s u b c u t a n e o u s l y 42 m g / k g e i t h e r 15, 5 or 1 m i n before, or 1 rain or 24 h r a f t e r the t r a i n i n g trial. One ECS group a n d one No-ECS group were assigned to e a c h of the 5 training-injection intervals. The r e m a i n i n g 2 groups, one ECS and one No-ECS, did n o t receive a n y i n j e c t i o n a n d served as No-Injection c o n t r o l groups. Passive avoidance
T H E R E are several drugs t h a t a t t e n u a t e the amnesic e f f e c t of e l e c t r o c o n v u l s i v e s h o c k (ECS). Neural s t i m u l a n t s such as s t r y c h n i n e and p e n t y l e n e t e t r a z o l , at relatively low dosage levels, reduce the a m n e s i c e f f e c t if t h e y are i n j e c t e d before the a d m i n i s t r a t i o n o f ECS [ 1 5 ] . Similarly, t h e a m n e s i c effect o f ECS is a t t e n u a t e d b y t r i c y a n o a m i n o p r o p e n e [ 6 ] , w h i c h facilitates b i o s y n t h e s i s of r i b o n u c l e i c acid, a n d by p h y s o s t i g m i n e [ 3 ] , w h i c h i n h i b i t s c h o l i n e s t e r a s e activity. In a previous study, N a k a j i m a [12] f o u n d t h a t corticosteroids a t t e n u a t e the amnesic e f f e c t o f c y c l o h e x i m i d e . Mice were t r a i n e d in a passive a v o i d a n c e a p p a r a t u s a f t e r i n j e c t i o n of c y c l o h e x i m i d e , a n d t h e n given an a d d i t i o n a l i n j e c t i o n o f e i t h e r h y d r o c o r t i s o n e or c o r t i c o s t e r o n e . In a later test, these animals d e m o n s t r a t e d good a v o i d a n c e behavior, t h a t is, the usual amnesic e f f e c t o f c y c l o h e x i m i d e was absent. In a s u b s e q u e n t s t u d y , C o t t r e l l a n d N a k a j i m a [2] s h o w e d t h a t c y c l o h e x i m i d e , at a dosage t h a t p r o d u c e d amnesia, also suppressed the f o o t s h o c k - i n d u c e d s e c r e t i o n o f corticosteroids. U n d e r these c o n d i t i o n s , i n j e c t i o n of h y d r o c o r t i s o n e i n t o the h i p p o c a m p u s a t t e n u a t e d the a m n e s i c effect of c y c l o h e x i m i d e . The p r e s e n t e x p e r i m e n t s were c o n d u c t e d t o d e t e r m i n e w h e t h e r h y d r o c o r t i s o n e w o u l d have an a n t i - a m n e s i c e f f e c t on E C S - i n d u c e d a m n e s i a as it does o n the c y c l o h e x i m i d e i n d u c e d amnesia. EXPERIMENT 1
Method Male Swiss a l b i n o mice (Charles River, C D - 1 s t r a i n ) were o b t a i n e d f r o m C a n a d i a n Breeding F a r m s ( M o n t r e a l ) . They weighed 3 0 - 3 8 g at the b e g i n n i n g of the e x p e r i m e n t .
Supported by the National Research Council of Canada grant A0233. The author is grateful to P. A. Cummings and N. P. Phelps for their technical assistance, and to G. A. Cottrell for her critical comments on the manuscript. 607
608
NAKAJIMA
behavior was tested 7 days later by placing each mouse in the small compartment again. The step-through latency was recorded up to a maximum of 600 sec. The latency scores were subjected to analysis of variance, and post hoc comparisons were made according to the method of Rodger [13]. The expected probability of the Type I error was 0.05. In order to confirm the validity of the statistical treatments, the same sets of data were subjected to non-parametric tests. In both Experiments 1 and 2, the overall differences were significant at p<0.01 by the Kruskal-Wallis one-way analysis of variance. Those groupdifferences which were indicated by the method of Rodger [ 13] were all significant at p< 0.01 by the Mann-Whitney U tests.
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Results The mean step-through latency in the training trial was 7.0 sec, and there was no significant difference between the animals injected with hydrocortisone before training and those injected after training. A typical reaction to ECS was an immediate tonic extension of the 4 legs and forward flexion of the head. Of the 60 mice initially assigned to the 6 ECS groups, 16 died and 3 escaped ECS by a brisk movement of the head. These animals were replaced with additional animals so that each group consisted of 10 mice. There was no observable difference in the pattern of convulsions or in the mortality rate among the animals injected with the hormone before ECS, those injected after ECS, and those without injection. The mean latency of each group in the test trial is shown in Fig. 1. The differences among the groups were statistically significant, F(11,108) = 6.46. The No-ECS groups all showed relatively long latencies, indicating one-trial acquisition of the passive avoidance task. There was no significant difference among the No-ECS groups. Administration of ECS produced retrograde amnesia of passive avoidance as indicated by a short latency of the No-Injection ECS group. Three other groups that were injected with hydrocortisone succinate either 15 rain before, 1 min after, or 24 hr after the training trial also demonstrated relatively short latencies. The remaining two ECS groups that were injected either 1 or 5 min before training (marked with asterisks in Fig. 1) demonstrated latencies significantly longer than the latencies of the other 4 ECS groups (p<0.05). The fact that these 2 groups of animals demonstrated relatively long latencies suggest that the amnesic effect of ECS was attenuated by the hormone injected. The attenuation, however, was not complete: the latencies of the two groups were still significantly shorter than those of the No-ECS groups (p<0.05). EXPERIMENT 2 It is possible that the incomplete anti-amnesic effect observed in Experiment 1 was due to the use of an inappropriate form of steroid. Hydrocortisone-21-sodium succinate was used in Experiment I because it readily dissolves in physiological saline. However, the amnesic effect of cycloheximide was more effectively attenuated by a suspension of hydrocortisone than a solution of its sodium succinate ester [12]. Therefore, hydrocortisone suspension was used in Experiment 2 in the hope that it would be more effective in attenuating the amnesic effect of ECS.
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FIG. 1. Mean step-through latency (and standard error) of each group in the test trial of Experiment 1. ECS was given within 1 rain of footshock in the training trial. In the two ECS groups marked with asterisks, the latencies were significantly (p<0.05) longer than in the other four ECS groups, but shorter than in the No-ECS groups. Method Ten groups of mice (10 per group) were trained in the passive avoidance apparatus and tested 7 days later as in Experiment 1. Hydrocortisone (Sigma Chemical Co.) was suspended in physiological saline containing 5% Tween-80 (Fisher Scientific), and subcutaneously injected 30 mg/kg. This dosage is equimolar to 42 mg/kg hydrocortisone succinate. ECS was given either immediately (within 1 min), 2 min or 5 min after the training trial (Fig. 2). Two groups were assigned to each of the 3 training-ECS intervals and to the No-ECS control: one group without injection and the other group with hydrocortisone injection 5 min prior to training. One of the remaining 2 groups was injected with saline, containing 5% Tween-80, 5 rain before training and given ECS immediately after training. The other group was injected with hydrocortisone immediately after training (marked as AT in Fig. 2) and given ECS 2 min after training. Results The mean step-through latency in the training trial was 8.3 sec, and there was no statistically significant difference between the hydrocortisone-injected groups and uninjected groups. Out of 80 animals that received ECS, 21 died and were replaced with additional animals. Figure 2 shows the step-through latencies of the 10 groups in the test trial. Analysis of variance revealed significant differences among the groups, F(9,90) = 3.37. Both of the No-ECS groups demonstrated good avoidance response, and the three ECS groups without injection (white columns) showed a gradient of amnesic effect depending on the time interval between training and ECS. Three groups which received ECS after 2 min or less (marked with asterisks in Fig. 2) showed significantly shorter latencies than the No-ECS control groups (p<0.05). It should be noted that none of these 3 groups received
AMNESIA AND HYDROCORTISONE
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In the preceding experiments, the amnesic effect of ECS was absent if a sufficient amount of corticosteroid was injected prior to the administration of ECS. There is a possibility that corticosteroids reduce the excitability of neurons so that ECS fails to elicit seizure activity in the brain. However there are some studies which indicate the contrary, that is, corticosteroids enhance the susceptibility of the brain to convulsive stimulation [7,8]. To examine the effect of hydrocortisone on seizure activity, electrographic recording was conducted in a situation similar to the training of a passive avoidance task. Convulsive stimulation was applied through implanted electrodes to ensure that the locus of stimulation remained the same on different days with and without hormone injection.
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hydrocortisone injection. All of the hydrocortisone-injected groups demonstrated long latencies, which were not significantly different from the latencies of the No-ECS control groups. It is clear that the amnesic effect of ECS was completely abolished by prior injection of hydrocortisone regardless of the training-ECS interval. The hormone was more effective as a suspension than as a solution of an ester. It may be argued that the hydrocortisone-injected animals showed long step-through latencies, not because the effect of ECS was attenuated, but because the hormone enhanced the animal's sensitivity to footshock. An answer to this argument is provided by the AT group, which received the hormone injection after footshock. The mean latency of the AT group was similar to the latencies of the groups injected before footshock, and of the No-ECS groups. Thus, the long latencies in the hydrocortisone injected groups are not attributable to an enhanced shock sensitivity. Another possible argument is that hydrocortisone may have suppressed the animal's activity in the avoidance apparatus 7 days after injection, regardless of footshock and regardless of ECS. In order to test such a possibility, an additional group of l 0 mice was injected with hydrocortisone and 5 min later placed in the avoidance apparatus. Upon entering the dark compartment, however, the mice were not given footshock but simply removed from the apparatus and returned to the home cage without ECS. Seven days later, they were tested in the same way as the animals in the other groups. The mean step-through latency of this group was 7.3 sec, and the possibility that the injected animals may have been slow to move simply because of the injection was ruled out.
Ten mice were implanted with electrodes under sodium pentobarbital anesthesia (75 mg/kg, IP). For the delivery of convulsive stimulation, screw electrodes were bilaterally tapped into the skull, 2 mm anterior to the bregma, and 2.5 mm lateral to the midline. The screws extended approximately 0.5 mm into the anterior cortex. A bipolar recording electrode, made of twisted strands of stainlesssteel wire (0.2 mm in dia. and insulated except at the tip cross-section), was implanted into the dorsal hippocampus, 1.5 mm posterior to the bregma, 1.5 mm lateral to the midline. One pole of the bipolar electrode was implanted 2 mm deep into the brain, and the other pole was 0.5 mm shorter. Recording of electrical activity was started after a recovery period of 1 0 - 1 4 days using a polygraph (Grass Instruments, Model 79). The mouse was placed in a box (10 x 15 cm) with a grid floor, and electrical activity was recorded from the anterior cortex and the hippocampus for approximately half an hour. On the following day, 5 of the animals were injected with hydrocortisone suspension (30 mg/kg, SC), given 0.35 mA footshock for 1 sec, and then stimulated through the cortical electrodes. The time interval between the injection and footshock was 5 min, and the interval between the footshock and brain stimulation was 30 sec, so that the timing of the experimental treatments was similar to the timing in the ECS < I min group in Experiment 2. The remaining 5 animals were given footshock and brain stimulation without hydrocortisone injection. Two days later, the animals which did not receive the injection were now injected with the hormone suspension, and those which had the injection before were recorded without injection. The rest of the procedure was exactly the same as before. In order to avoid any inadvertent stimulation, the recording cable was completely disconnected from the animal prior to the administration of footshock, and reconnected after the end of the shock. During the brair/ stimulation, the cortical electrodes were connected to a constant current unit (Grass Instruments, Model C C U - 1 ) driven by a stimulator (Grass Instruments, Model S D - 9 ) , while the hippocampal electrode remained disconnected. The stimulation was a 0.2 sec train of 1 msec rectangular pulses, 100 pulses per sec, and 3 mA peak to peak. The intensity of the stimulation had been determined in a preliminary experiment using 8 mice, and was sufficient to
610
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FIG. 3. Electrographic records of a representative animal in Experiment 3. Artifacts on the records indicate the time when the recording cable was disconnected from the polygraph. Actual administration of footshock or ECS was some time after the onset of the artifact. The records 10 min and 30 min after footshock indicate the course of recovery from ECS. A: No injection, B: Hydrocortisone injection 5 min before footshock. produce a type of convulsion and amnesia similar to those observed in Experiments 1 and 2. At the conclusion of the recording experiment, the animals were anesthetized with sodium pentobarbital (75 mg/kg, IP), and the brains were removed. After fixation in 10% Formalin, the brains were sectioned and stained to identify the sites of the electrodes. Results Two animals died of convulsion, and the results are based on 8 animals. Examination of the brain sections indicated that the electrodes were located in the anterior cortex and the h i p p o c a m p u s as originally intended. The electrographic records of a representative animal are shown in Fig. 3. The brain stimulation p r o d u c e d after-discharges in the anterior c o r t e x and the hippocampus. The hippocampal after-discharge was followed by post-ictal depression and sporadic spike discharges. Each animal showed a characteristic pattern o f seizure activity regardless o f whether h y d r o c o r t i s o n e had been injected or not. In some animals the duration of the primary after-discharge was slightly shorter on the injection day, but in other animals it was shorter on the no-injection day. Whether the injection was given on the first or second day of brain stimulation was not related to the pattern of discharges in any discernible way. Definitely, h y d r o c o r t i s o n e injection did not prevent seizure activity either in the c o r t e x or in the hippocampus. DISCUSSION Subcutaneous injection of h y d r o c o r t i s o n e abolished the amnesic effect of ECS in a passive avoidance situation. The h o r m o n e injection alone, w i t h o u t ECS, had no effect on the acquisition of the avoidance task; the convulsive shock was strong enough to produce a gradient of amnesia in uninjected control animals. There was no indication that hydrocortisone suppressed seizure activity in the brain in any appreciably degree. Nevertheless, the amnesic effect of ECS was absent in the animals that were injected with the h o r m o n e before the administration of ECS. The results suggest that hydrocortisone p r o t e c t e d neurons in the brain from the disruptive action of ECS.
The mechanism underlying the protective action of hydrocortisone is not clear. One possible explanation would be that the h o r m o n e facilitated accumulation of some substance into the neuron - possibly a transmitter substance that would otherwise be exhausted by the high rate of firing during convulsion. In this regard, it is interesting to note that h y d r o c o r t i s o n e facilitates the rate of norepinephrine uptake by brain slices incubated in vitro [ 11 ]. The finding that exogenous h y d r o c o r t i s o n e attenuates the amnesic effect of ECS suggests that endogenous corticosteroids (adrenocortical h o r m o n e s ) would also attenuate the ECS-induced amnesia. It follows that if the secretion of adrenocortical h o r m o n e s is suppressed, an administration of ECS more than several minutes after training would still produce retrograde amnesia. The results of experients c o n d u c t e d by Andry and Luttges [1], and more recently by Flood and his associates [ 9 ] , are congruent with this inference. They found that ECS given 30 min or more after training p r o d u c e d amnesia in the mice injected with c y c l o h e x i m i d e [ 1 ] or anisomycin [ 9 ] , but not in saline-injected control animals. In these experiments, c y c l o h e x i m i d e and anisomycin were administered in order to suppress protein synthesis in the brain, but these drugs would have suppressed steroidogenesis in the adrenal cortex as well. Therefore, the behavioral results may have come from the adrenal rather than cerebral effect of the drugs. Recently, Nakajima (unpublished preliminary study) observed that a delayed administration of ECS produces amnesia in the mice injected with aminoglutethimide, which suppresses adrenocortical function without affecting cerebral protein synthesis. Implications of this observation have to be considered very carefully, in view of the fact that a m i n o g l u t e t h i m i d e alone does not produce amnesia [5,141. It is still indecisive whether hydrocortisone itself is the effective anti-amnesic substance. The a m o u n t of exogenous hydrocortisone required to abolish the amnesic effects of ECS and c y c l o h e x i m i d e is far larger than the c o n c e n t r a t i o n of endogenous corticosteroids normally observed after a stress of f o o t s h o c k [ 12,14]. It is possible that the effective anti-amnesic substance may be a minute a m o u n t of aldosterone or d e o x y c o r t i c o s t e r o n e that happened to be contained in the suspension of hydrocortisone. The im-
AMNESIA AND HYDROCORTISONE
611
p o r t a n c e o f these m i n e r a l o c o r t i c o i d s in the p e r f o r m a n c e o f a v o i d a n c e tasks has b e e n d e m o n s t r a t e d r e c e n t l y [ 1 0 ] . A n o t h e r possibility is t h a t a h i g h dose o f h y d r o c o r t i s o n e m a y facilitate or suppress the s e c r e t i o n o f various
h y p o p h y s e a l p e p t i d e h o r m o n e s , w h i c h in t u r n i n f l u e n c e avoidance behavior [4]. At present, however, information is n o t sufficient to favor a n y o f these a l t e r n a t i v e possibilities.
REFERENCES 1. Andry, D. K. and M. W. Luttges. Memory traces: Experimental separation by cycloheximide and electroconvulsive shock. Science 178: 5 1 8 - 5 2 0 , 1972. 2. Cottrell, G. A. and S. Nakajima. Effect of corticosteroids in the hippocampus on passive avoidance behavior in the rat. Pharmac. Biochern. Behav. 7: 277-280, 1977. 3. Davis, J. W., R. K. Thomas and H. E. Adams. Interactions of scopolamine and physostigmine with ECS and one-trial learning. Physiol. Behav. 6: 219-222, 1971. 4. De Wied, D. Peptides and behavior. Life ScL 20: 195-204, 1977. 5. Dunn, A. J. and S. Leibmann. The amnestic effect of protein synthesis inhibitors is not due to the inhibition of adrenal corticosteroidogenesis. Behav. Biol. 1 9 : 4 1 1 - 4 1 6 , 1977. 6. Essman, W. B. Effect of tricyanoaminopropene on the amnesic effect of electroconvulsive shock. Psychopharmacologia 9: 4 2 6 - 4 3 3 , 1966. 7. Feldman, S. Convulsive phenomena produced by intraventricular administration of hydrocortisone in cats. Epilepsia 7: 271-282, 1966. 8. Feldman, S. and J. M. Davidson. Effect of hydrocortisone on electrical activity, arousal thresholds and evoked potentials in the brains of chronically implanted rabbits. J. Neurol. Sci. 3: 4 6 2 - 4 7 2 , 1966.
9. Flood, J. F., E. L. Bennett, A. E. Orme and M. E. Jarvik. Protein synthesis dependent gradient of ECS retrograde amnesia. Behav. Biol. 21: 307-328, 1977. 10. Gray, P. Effect of prestimulation on avoidance responding in rats, and hormonal dependence of the effect. J. cornp, physiol. Psychol. 90: 1-17, 1976. 11. Maas, J. W. and M. Mednieks. Hydrocortisone-mediated increase of norepinephrine uptake by brain slices. Science 171: 178-179, 1971. 12. Nakajima, S. Amnesic effect of cycloheximide in the mouse mediated by adrenocortical hormones. J. comp. physiol. Psychol. 88: 378-385, 1975. 13. Rodger, R. S. The number of non-zero, post hoc contrasts from ANOVA and error-rate. I. Br. J. Math. Stat. Psychol. 28: 71-78, 1975. 14. Squire, L. R., S. St. John and H. P. Davis. Inhibitors of protein synthesis and memory: Dissociation of amnesic effects and effects on adrenal steroidogenesis. Brain Res. 112: 200-206, 1976. 15. Weissman, A. Drugs and retrograde amnesia. Int. Rev. Neurobiol. 10: 167-198, 1967.