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Idebenone improves learning and memory impairment induced by cholinergic or serotonergic dysfunction in rats
Arch. Gerontol. Geriatr., 8 (1989) 225-239 Elsevier
225
A G G 00253
Idebenone improves learning and memory impairment induced by cholinergic or serotonergic dysfunction in rats Naoki Yamazaki a Masahiko Nomura b Akinobu Nagaoka a
and Yuji Nagawa
a
a Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532, Japan and b Department of Physiology, School of Medicine, Fujita Gakuen Health University, Toyoake-City, Aichi 470-11, Japan (Received 2 August 1988; revised version received 31 October 1988; accepted 1 November 1988)
Summary The effects of idebenone, a cerebral metabolic enhancer, on learning and memory impairment in two rat models with central cholinergic or serotonergic dysfunction were investigated using positively reinforced learning tasks. A delayed alternation task using a T maze was employed to test the effect of idebenone on short-term memory impairment induced by a cholinergic antagonist, scopolamine. A correct response, defined as a turn toward the arm opposite to that in the forced run, was rewarded with food pellets. Scopolamine (0.2 and 0.5 mg/kg, i.p.) significantly decreased the correct responses to the chance level in the 60-s-delayed alternation task. The scopolamine (0.2 mg/kg, i.p.)-induced impairment of short-term memory was improved by idebenone (3-30 mg/kg, i.p.) or an acetylcholinesterase inhibitor, physostigmine (0.1 and 0.2 mg/kg, i.p.), administered simultaneously. The central serotonergic dysfunction model was produced by giving rats a diet deficient in tryptophan, a precursor of serotonin. The rats fed on a tryptophan-deficient diet (TDD) showed a slower learning process in the operant brightness discrimination task (mult VI15 EXT) than did rats fed on a normal diet. Idebenone (60 mg/kg/day) admixed with the TDD decreased the number of lever-pressing responses emitted during the extinction periods. The percentage of correct responses was significantly higher in the idebenonetreated group than in the control TDD group. These results suggest that idebenone may improve both the impairment of short-term memory induced by a decreased cholinergic activity and the retardation of discrimination learning induced by central serotonergic dysfunction.
Idebenone; Short-term memory; Operant discrimination learning; Cholinergic dysfunction; Serotonergic dysfunction; Rats
Correspondence should be addressed to Naoki Yamazaki, Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 2-17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan. 0167-4943/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
226 Introduction
A decrease of brain metabolism can easily lead to a deterioration in neurologic and mental function. Dysfunctions in the brain induced by a deficiency in neurotransmitters involve impairment of cognitive function in animals. For example, an experimentally-induced decrease in cholinergic activity impairs learning and memory in animals (Kubanis and Zornetzer, 1981; Bartus et al., 1982), and patients with presenile and senile dementia of the Alzheimer type show a decline in cholinergic markers in the cortex and hippocampus that correlate with the degree of cognitive and memory impairment (Perry et al.. 1978; Coyle et al., 1983). These findings and many others imply that the impairment in learning and memory is attributable to a decrease in central cholinergic function. A decline in central serotonergic neurotransmission also involves changes in sleep and wakefulness (Jouvet, 1973), aggression (Hodge and Butcher, 1974), ingestion behavior (Coscina et al., 1972; Blundell, 1977), nociception (Tenen, 1967), and learned behavior (Srebro and Lorens, 1975; Altman et al., 1984). Nomura et al. (1982) reported that rats fed on a tryptophan (a precursor of serotonin)-deficient diet showed impaired operant learning behavior. In the present study, we used two rat models with a decline in central cholinergic or serotonergic activity to examine the effects of idebenone, a cerebral metabolic enhancer, occasionally called a nootropic drug, on impaired learning and memory. Idebenone reverses decline in the contents of acetylcholine and serotonin in rat brains under a transient cerebral ischemia, and improves memory impairment of avoidance learning in animal models of vascular and Alzheimer types of senile dementia (Nagaoka, 1987). However, the effect of idebenone on learning and memory of the appetitive type remains nuclear. Therefore, we investigated possible effects of idebenone on impaired learning and memory motivated with food. E X P E R I M E N T I: I N V E S T I G A T I O N OF A C H O L I N E R G I C D E F I C I T M O D E L A decline in the cerebral cholinergic system was produced by administering a cholinergic blocker, scopolamine. The effect of idebenone on the scopolamine-induced memory impairment was examined in a delayed alternation task, regarded as one of the tests for short-term memory (Feldman and Gordon, 1979). (Results described here have been reported elsewhere; Yamazaki et al., 1985.)
Materials and Methods
Subjects Male rats of the Wistar strain (Charles River, Japan) were used; they were 6 weeks old at the start of the training of the delayed alternation task. They were housed individually, maintained on a food-deprivation schedule throughout the experiment, and fed on a diet sufficient to maintain approximately 85% of initial body weight at the end of the daily session.
227
Apparatus The apparatus was a T-maze with a grid floor and a clear plastic top, the sides were 12 cm high and alleys were 12 cm wide. The start alley was 44 cm long and each arm was 50 cm long. Guillotine doors were mounted at the entrance to each arm and were used to control the direction of the turning response on forced runs. A food cup, containing pellets (90 mg) as reinforcers, was located 3 cm from the floor at each arm end.
Procedure Habituation to the T-maze was permitted for 5 rain with pellets in both food cups and the guillotine doors opened. On the next 2 days, 3 - 4 trials of running training from the start point to both food cups, filled with pellets, was performed. Then each rat was trained in pairs of 10 trials of alternation training in a session. On the first (forced) run in the pair trial, the guillotine door was closed and blocked one of the arms of the maze; the unblocked arm had pellets in the food cup. After they ate the pellets, the rats were returned to the starting box and immediately given a free-choice run with no delay. On this run, both guillotine doors were opened, and pellets were present only in the arm opposite to the arm i'ewarded on the forced run. If the rat entered the rewarded side, a correct response was recorded, and the rat was returned to a plastic waiting cage after it had consumed the pellets. If the rat entered the same arm as on the forced run, an incorrect response was recorded, and the rat was returned to the starting box and allowed an other opportunity to enter the rewarded side. The correction runs were continued until the correct arm was chosen. The intertrial interval was 1 min, during which time the rats were put in the waiting cage. An equal number of left-correct and right-correct trials were presented during each session. This alternation training was continued until all rats reached a learning criterion of 90% or more correct responses over the last two training sessions. The delayed alternation training was carried out after the alternation training was completed. In this training, a delay between the forced run and the free-choice run was introduced for 0, 30, 60, and 120 s in a random order. Two trials for each delay (eight trials per session) were made for four consecutive sessions. During the delay, the rats were placed in the waiting cage. Based on the results of this preliminary experiment, a 60-s delay and eight trials in a session were used in the following drug testing. Another 16 rats, which had been trained in the 60-s-delayed alternation task, were divided into four groups (four rats in each group) and the effect of scopolamine (SCP) on the task was tested. The experiment was arranged in a Latin square design (4 x 4) in which all groups received four drug conditions in a counter-balanced order. The drug conditions included saline, or 0.1, 0.2, or 0.5 m g / k g i.p., of SCP. The drug or saline was given 20 min before the test, which was run four times, once every 3rd day for 10 days. After testing SCP, the same rats were divided into two groups (eight rats each) and the effect of SCP (0.2 m g / k g i.p.) was further
228 tested in the non-delayed (0 s delay) alternation task using a Latin square design (2 x 2). Another 15 rats, which had been trained in the 60-s-delayed alternation task, were used to test the effect of physostigmine (PHY) on the SCP (0.2 m g / k g i.p.)-induced memory impairment. The rats were divided into five groups (three in each group) and each group was exposed to five drug conditions in a Latin square design (5 x 5). The drug conditions included saline, SCP, or 0.05, 0.1, or 0.2 m g / k g i.p., of P H Y combined with SCP. The drug or saline was given 20 rain before the test which was run five times, once every 3rd day for 13 days. An additional rat. which had been tested only with saline in the same way, was prepared for the next experiment to test the effect of PHY alone on the same task. Rats were re-divided into four groups (four in each group) to match the percentage of correct response of groups in an additional session of the same task made 3 - 6 days after the task; each group was exposed to four drug conditions in a Latin square design (4 × 4). The drug conditions were saline, or 0.05, 0.1, or 0.2 m g / k g i.p., of PHY. The effect of idebenone (3, 10 and 30 m g / k g i.p.) on the task with and without SCP was examined in the same way as in the test of P H Y using other rats.
Drugs The drugs used in these studies were scopolamine hydrobromide (Wako, Japan), physostigmine (Eserine Sulfate, Wako, Japan) and 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-l,4-benzoquinone (idebenone, Takeda, Japan). SCP and P H Y were dissolved in saline and idebenone was suspended in a 5% arabic gum solution. These drugs were administered in a volume of 0.2 m l / 1 0 0 g body weight.
Statistics The percentage of correct responses in each drug exposure was analyzed by the A N O V A for Latin square design. Newman-Keuls tests were performed to compare the correct responses in each of drug exposures, and the differences in the percentages of correct responses from the chance level (50%) were examined by the critical ratio test.
Results In the first experiment to test the effect of varying the delay, the percentage of correct responses decreased as the delay increased. The A N O V A indicated a significant decline in the percentage of correct responses over the delay, F~3,4s)--36.93, p < 0.001. The percentage of correct responses after a 0-s delay was 90%, whereas it was significantly reduced to 78%, 63%, and 61% when the delay was 30, 60, and 120 s ( p < 0.05, p < 0.01 and p < 0.01), respectively. In only the 120-s delay did the percentage of correct responses not differ significantly from the
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Fig. 1. The effect of scopolamine (SCP) on the 60-s-delayed alternation task in rats (n =16). Mean ( d: SE) percentage of correct responses is shown. SCP was given 20 min before the test, which was run four times, once every 3rd day for 10 days. Each rat received four doses including saline (SAL) in a counter-balanced order under a Latin square design. * p < 0.05, * * p < 0.01 (Newman-Keuls test) vs. SAL control. chance level (50%). Therefore, we chose a delay of 60 s for the following experiments. SCP reduced the percentage of correct responses in a dose-dependent manner, when compared with the control level (64%) obtained with saline (Fig. 1). The decreases of the percentage of correct responses after 0.2 and 0.5 m g / k g of SCP were significant, but they were not statistically different from the chance level. Slight decreases in running speed and difficulty in swallowing pellets as a consequence of the blocking action on salivary secretions occurred dose-dependently. However, the incidence of animals with neurologic signs in the group receiving 0.2 m g / k g of SCP (1/16) was less than that in the group receiving 0.5 m g / k g (4/16). The percentage of correct responses in the group receiving SCP (0.2 m g / k g ) in the non-delay task was 88%, which did not significantly differ from the saline level (93%); this observation indicated that this dose of SCP did not influence the performance of the rats. P H Y dose-dependently improved the SCP-induced decline of the percentage of correct responses (Fig. 2,A). The A N O V A indicated that the main effect of drug treatment was significant, F4,4o = 5.95, p < 0.05. In this experiment, the percentage of correct responses with SCP alone was 44%, whereas P H Y (0.1 and 0.2 m g / k g ) significantly enhanced the correct responses to the level of the non-SCP condition. Significant ( p < 0.05) differences in the percentage of correct responses from the chance level were obtained in the non-SCP condition and at the higher two doses of PHY. The decline in running speed observed after SCP was administered, almost recovered when P H Y was administered. Furthermore, in the experiment performed without SCP, P H Y increased the correct responses dose-dependently (Fig. 2,B). The A N O V A indicated that the main effect of the drug was significant, F3,36 = 6.64, p < 0.005. Significant increases of correct responses were obtained at 0.1 and 0.2 m g / k g of PHY.
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Fig. 2. The effect of physostigmine (PHY) on the 60-s-delayed alternation task in rats administered scopolamine (SCP) and in those without the drug. PHY was given simultaneously with SCP (panel A: n = 15). The effect of PHY alone was further tested in the same rats without SCP (panel B: n = 16). The number of rats used in the second experiment was 16 as one rat that had been treated with saline (SAL) in the first experiment was added to the second. Both experiments were carried out according to a Latin square design. * p < 0.05, ** p < 0.01 (Newman-Keuls test) vs. SCP-administered condition without PHY (panel A), or vs. (SAL) control (panel B).
Idebenone reversed the SCP-induced decline of the correct responses (Fig. 3,A). The A N O V A indicated that the main effect of the drug was significant, F4.4o = 8.58, p < 0.001. Idebenone at doses of 3 and 10 m g / k g significantly increased the percentage of correct responses to 60% and 71%, respectively, compared with the level of saline (48%). The highest dose of idebenone also increased the correct responses significantly but the extent was less than that at 10 mg/kg. Significant ( p < 0.05) differences in the percentage of correct responses from the chance level were obtained in the non-SCP condition at the higher two doses of idebenone.
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Fig. 3. The effect of idebenone (IDB) on the 60-s-delayed alternation task in rats administered SCP and in those without the drug. IDB was given simultaneously with SCP (panel A: n = 15). The effect of IDB alone was further tested in the same rats without SCP (panel B). The number of rats used in the second experiment was 16. Both experiments were carried out according to a Latin square design, * p < 0.05, • * p < 0.01 (Newman-Keuls test) vs. SCP administered condition without P H ¥ .
231 Furthermore, in the test performed without SCP, idebenone alone did not increase the correct responses (Fig. 3,B).
Discussion
The present experiment demonstrated that the extension of the delay in the alternation task reduced the correct responses, suggesting that the consolidation of short-term memory is time dependent. The negative correlation agrees with previous findings (Roberts, 1974; Feldman and Gordon, 1979). The low level of the percentage of correct responses, near 60%, in the 60-s-delayed alternation task seems close to the chance level, but in fact the two levels were significantly different. Therefore, it is considered that the 60-s delay in the task reduced, but did not deplete, short-term memory. Cholinergic blockers like SCP impair many types of learning and memory. In the present study, SCP markedly reduced the correct responses to the chance level in the 60-s-delayed condition but not in the non-delayed condition. This result is consistent with the finding that an interaction exists between the effect of anticholinergics and the length of the delay in delayed matching to sample task in monkeys (Bartus and Johnson, 1976; Penetar and McDonough, 1983). Thus, SCP reduces short-term memory. PHY reversed the SCP-induced impairment of short-term memory by increasing the correct responses. The reversal effect of PHY may be explained easily by its inhibition of acetylcholinesterase and the resultant increase of acetylcholine at the receptor sites. Furthermore, PHY administered alone enhanced this task. This result suggests that PHY may enhance a putative process of short-term memory (Wagner et al., 1973) by means of activating the central cholinergic system. Similarly, PHY improved the performance in a delayed matching to sample task in aged monkeys in which the brain cholinergic function might be reduced (Bartus et al., 1980). These findings support the notion that memory function is closely correlated with the cholinergic system in the brain. Idebenone reversed the SCP-induced impairment of short-term memory; the peak effect was observed at a dose of 10 mg/kg. Consequently, the overall dose-response relation was an inverted U. Though the mechanism underlying the inverted U curve is unclear, the same relation was found when idebenone was administered to rats with amnesia induced by a transient cerebral ischemia (Yamazaki et al., 1984). Idebenone did not improve short-term memory in untreated rats. This suggests that idebenone improves memory impairment in rats with cholinergic dysfunction, but not in rats whose cholinergic function is normal. EXPERIMENT II: INVESTIGATION OF A SEROTONIN DEFICIT MODEL A decline in the cerebral serotonergic system was produced by feeding a diet deficient of tryptophan to rats after they were weaned. The effect of idebenone on impaired learning in these rats was examined in brightness discrimination learning
232
of the operant type. (Results described here have been reported elsewhere; Nomura, 1985.)
Materials and Methods
Subjects Thirty male Wistar rats (Clea, Japan) were used, aged 3 weeks when the tryptophan-deficient diet ( T D D ) was begun. The diet was composed of corn, corn oil, Harper's salt, vitamin mixture, L-lysine, and choline (Fernstorm and Wurtman, 1971). Twenty rats fed on the T D D were divided into two groups (10 each); one group was administered idebenone (60 m g / k g / d a y ) admixed with the diet. Ten rats fed on a normal diet (MF, Oriental Yeast Co., Japan) were used as controls. After being on the diet for 8 weeks, the rats were individually housed and kept on a food-deprivation schedule throughout the experiment to maintain body weight at 85% of the initial free feeding level.
Apparatus Two operant chambers containing a single lever (Ralph Gerbrand's) were used. An indicator lamp with a diameter of 5 cm was mounted on the center of the front wall 10 cm above a food tray. The intensity of the light at the surface of the lamp was 5 X 10 4 foot Lambert; it was possible to decrease the intensity by 1/1000 by inserting a filter (Kodak, ND4.0). A lever was mounted 2 cm from the right edge of the front wall and 2 cm above the floor. Pressing the lever was reinforced by a food pellet (40 mg). The entire assembly was placed in a dark room in which the white noise (60 dB) was diffused to mask external noises; the behavior of the rats was monitored in an adjacent room by a supersensitive videocamera. Presentations of light and reinforcement, and the recording of responses were automatically controlled by a microcomputer.
Procedure One week after the deprivation schedule was begun, the rats were given one session of shaping in the operant chamber in which every lever-pressing response was reinforced. The rats were further trained to respond to the lever in a continuous reinforcement schedule until they got 40 reinforcements within 4 rain in a session. All rats reached this criterion within a week. Then, a variable interval (VI) schedule was introduced, under which a reinforcement was delivered by the response at a mean interval of 5 s (VI5). After this schedule was completed, the VI value was increased to 10 s with 40 reinforcements a session. When the rate of response became constant, the discrimination training was begun. A multiple schedule was used in the discrimination training (mult VI15 EXT). This consisted of VI15 reinforcement for lever presses during periods when the indicator light was bright
233 (S ÷ periods) and of no reinforcement for presses during periods when the light was dark (S- periods). The duration of an S ÷ or S - period was 20 s, and each period was repeated 20 times in a session. A blackout, arranged by turning out all the lights in the chamber for 5 s, was inserted whenever the period was exchanged. The order of appearance of an S ÷ or S - period in a session was determined by Gellerman sequence (Gellerman, 1933). The rats were trained in this schedule for 30 consecutive sessions. The percentage of correct responses, R + / ( R ÷ + R - ) X 100, was calculated from the number of correct responses (R ÷) during the S ÷ period and that of incorrect responses ( R - ) during the S - period in a session. Statistics
Paired t tests were performed to compare the mean ( + SE) number of responses in each session with that in the first session. Non-paired t tests were used to compare the percentage of correct responses in the idebenone-treated and the control groups.
Results In rats fed on the normal diet, the number of correct responses (R +), which was 120 in the first session, increased as the training sessions progressed, and reached approximately 220-250 (Fig. 4). In contrast to R ÷, R - , which was approximately 120 in the first session, decreased as the training progressed, and finally reached 30. The total responses (R ÷ + R - ) increased gradually from approximately 230 to 280. The fact that differentiation of R ÷ and R - was observed after the third session indicates that the brightness discrimination learning was easily established. The percentage of correct responses in this group increased from the chance level to 85% after the 20th session. In rats fed on a tryptophan-deficient diet (TDD), the number of R + and R - in the first session was approximately 100. The increase of R + was much greater than that of the group fed on the normal diet; the final number of R + in the T D D group reached 350 (Fig. 5). Significant ( p < 0.05) increases of R ÷ in the T D D group were obtained after the 5th session. R - in the T D D group, however, did not decrease to the extent observed in the normal rats. The final level of R - was approximately 100, and a significant difference from the first session was not obtained in any session after the second. The total responses increased significantly ( p < 0.05) after the 6th session. Though the percentage of correct responses increased with the training, it did not reach the 80% level. In rats administered idebenone continuously, the number of R ÷ was 100 at the first session and increased with training; a significant ( p < 0.05) increase was obtained after the second session (Fig. 6). The final level of R + in this group was 340, which was almost the same as that in the T D D group. On the other hand, though the number of R - varied from 100 to 120 during the first 10 sessions, it
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Fig. 4. Discrimination learning to press a lever for food in the mult VI15 EXT schedule in rats fed on a normal diet. Rats were trained to discriminate a light period (S +, 20 s) from a dark period (S-, 20 s). Responses of pressing the lever in the S + periods were reinforced (V|15 schedule). R + represents the mean (_+ SE) responses during S + periods and R - represents the mean number of responses during S periods. R + was reinforced under the VI15 schedule. R - was not reinforced (EXT schedule). S + was 1000 times brighter than S . Each period was repeated 20 times in a session. The percentage of correct responses is derived from the ratio of R + divided by total responses (R + + R ). Ten rats were used.
d e c r e a s e d r e m a r k a b l y t h e r e a f t e r , a n d r e a c h e d less t h a n 50 o n the final session, S i g n i f i c a n t ( p < 0.05) d e c r e a s e s w e r e f o u n d at s e s s i o n s a f t e r t h e 24th. A d i f f e r e n t i a t i o n was d e f i n i t e l y o b s e r v e d a f t e r t h e 7 t h session. T h e p e r c e n t a g e o f c o r r e c t r e s p o n s e s i n c r e a s e d r a p i d l y in the l a t e r sessions w h i l e its r a t e o f i n c r e a s e w a s g r a d u a l d u r i n g t h e first 20 sessions. T h e p e r c e n t a g e of c o r r e c t r e s p o n s e s in t h e i d e b e n o n e - t r e a t e d g r o u p was s i g n i f i c a n t l y h i g h e r ( p < 0.05) t h a n t h a t in t h e T D D g r o u p a f t e r the 2 3 r d session.
Discussion I n the p r e s e n t study, the rats w e r e f e d o n a T D D a f t e r t h e y h a d b e e n w e a n e d . I n t h e s e rats, t h e b r a i n c o n t e n t s of t r y p t o p h a n , s e r o t o n i n , a n d its m e t a b o l i t e 5 - h y d r o x y i n d o l e a c e t i c a c i d ( 5 - H I A A ) d e c r e a s e d b y a p p r o x i m a t e l y 70% of t h o s e of t h e
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Fig. 5. Discrimination learning to press a lever for food in the mult VI15 EXT schedule in rats fed on a tryptophan-deficient diet. Ten rats were used. For further explanation, cf. Fig. 4.
rats fed on a normal diet (Nomura, 1981). The progress of the discrimination learning in the T D D rats was almost the same as that in the normal rats in the early phase of the training, but was obviously retarded in the later phase. The impairment of the discrimination learning in the T D D group was mainly due to the failure to decrease R - even after repeated training; this failure resulted in a lowered level of the percentage of correct responses. In addition, the fact that R + in the T D D rats were more than that in the normal rats may indicate that the rats with serotonergic dysfunction tended to respond at a high rate. This is supported by the finding that the disturbance in serotonergic activities by lesioning the midbrain raphe and administering p-chlorophenylalanine increased motility in the rats (Jacobs et al., 1975). Therefore, the impairment of the discrimination learning observed in the rats with serotonergic dysfunction might be, in part, related to the inability to inhibit responses. Idebenone improved the impairment of the discrimination learning in the T D D rats. The rate of increase and the level finally reached in the percentage of correct
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responses in the idebenone-treated group were c o m p a r a b l e to those observed in the rats fed on a normal diet. I d e b e n o n e might i m p r o v e insufficient serotonergic activity by increasing the lowered content of serotonin in the ischemic brain. Thus, it is speculated that i d e b e n o n e enhanced the serotonergic s y s t e m of the T D D rats by the repeated discrimination training during which serotonergic activity increased as learning progressed.
General Discussion The present experiments demonstrated the beneficial effects of i d e b e n o n e on s c o p o l a m i n e - i n d u c e d impairment of short-term m e m o r y and T D D - i n d u c e d impairm e n t of operant discrimination learning in rats. Since i d e b e n o n e ameliorates the decrease of cholinergic turnover induced by transient cerebral i s c h e m i a in rats
237 (Kakihana et al., 1984), it was expected that the drug might improve the impairment of short-term memory induced by the cholinergic blocker, scopolamine. This hypothesis was clearly proved correct by the present results. The effect was observed in rats with cholinergic dysfunction but not in normal rats. From the point of view of its clinical application, it is important that idebenone may exert its effect on a pathogenic condition related to hypofunction of the cerebral cholinergic system, which is observed in presenile dementia and senile dementia of Alzheimer type. The second experiment indicated that the rats fed on the TDD were less able to learn brightness discrimination. Idebenone, administered continuously from weaning, improved the decreased progress of discrimination learning. Idebenone activates brain serotonergic function in normal and cerebral ischemic rats. In normal rats, idebenone at a high dose of 100 mg/kg i.p. increased the content of the 5-HIAA, a metabolite of serotonin, and facilitated the decrease of the serotonin content induced by p-chlorophenylalanine, a serotonin synthesis inhibitor (Narumi et al., 1985). In rats with cerebral ischemia, idebenone at a low dose of 10 mg/kg normalized the decrease of the 5-HIAA content. These findings suggest that idebenone may facilitate the turnover of serotonin in the brain. It is reasonable to speculate that the effect of idebenone to reinstate the learning ability is partly due to amelioration of the serotonergic dysfunction induced by the TDD. The fact that idebenone ameliorates the impairment of memory and learning found in a passive avoidance task using shocks (Yamazaki et al., 1984; Kiyota et al., 1985), and the present finding that idebenone ameliorates memory and learning impairment in tasks using positive reinforcements, clearly confirm that the ameliorating effects of idebenone on the impairment of learning and memory do not depend upon the type of reinforcers. A close interaction between the cholinergic and serotonergic systems in the brain has been proposed (Butcher and Woolf, 1982). Some physiologic and behavioral processes in which acetylcholine is implicated are the same as those in which serotonin appears to play a role, and possible neural mechanisms for this communication may exist. In fact, a transient cerebral ischemia in rats produces a decline in cholinergic (Kakihana et al., 1984) and serotonergic (Narumi et al., 1985) activities. Idebenone improves the amnesia in rats induced by cerebral ischemia (Yamazaki et al., 1984). Therefore, it is clear that idebenone improves learning and memory impairment in which the cholinergic and/or serotonergic transmission is decreased by certain experimental manipulations. The neurochemical mechanisms underlying the effect of idebenone are considered to be based upon improvement in cerebral energy metabolism; idebenone accelerates net ATP formation in brain mitochondria by activating the electron-transfer system and by inhibiting the production of lipid peroxide in the mitochondrial membrane (Suno and Nagaoka, 1985). On the basis of these findings it is expected that idebenone may partly improve mental or cognitive dysfunction by enhancing the brain energy metabolism in patients whose cholinergic and/or serotonergic activities are decreased.
238
Acknowledgement T h e a u t h o r s w i s h to t h a n k D r . J . R . M i l l e r f o r c o m m e n t s o n t h e m a n u s c r i p t .
References Altman, H.J., Nordy, D.A. and Ogren, S.O. (1984): Role of serotonin in memory: faciliation by alaproclate and zimeldine. Psychopharmacology, 84, 496-502. Bartus, R.T. and Johnson, H.R. (1976): Short-term memory in the rhesus monkey: disruption from the anti-cholinergic scopolamine. Pharmacol. Biochem. Behav., 5, 39-46. Bartus, R.T., Dean, R.L. and Beer, B. (1980): Memory deficits in aged cubus monkeys and facilitation with central cholinomimetics. Neurobiol. Aging, 1, 145-152. Bartus, R.T., Dean R.L., Beer, B. and Lippa, A.S. (1982): The cholinergic hypothesis of geriatric memory dysfunction. Science, 217, 408-417. Blundell, J.E. (1977): Is there a role for serotonin in feeding? Int. J. Obesity, l, 15-42. Butcher, E L and Woolf, N.J. (1982): Cholinergic and serotonergic systems in the brain and spinal cord: anatomic organization, role in intercellular communication processes, and interactive mechanisms. In: Chemical Transmission in the Brain. The Role of Amines, Amino Acids and Peptides. pp. 3 40, Progress in Brain Research, Vot. 55. Editors: R.M. Buijs, P. P~vet and D.F. Swaab. Elsevier Biomedical Press, Amsterdam. Coscina, D.V., Grant, L.D., Balagura, S. and Grossman, S.P. (1972): Hyperdipsia after serotonin depleting midbrain lesions. Nature New Biol., 235, 63-64. Coyle, J.T., Price, D.L. and DeLong, M.R. (1983): Alzheimer's disease: a disorder of cortical cholinergic i nnervation. Science, 19, 1184-1190. Feldman, D. and Gordon, W.C. (1979): The alleviation of short-term retention decrements with reactivation. Learn. Motiv., 10, 198-210. Fernstorm, J.D. and Wurtman, R.J. (1971): Effect of chronic corn consumption on serotonin content of rat brain. Nature New Biol., 234, 62-64. Gellerman, L.W. (1933): Chance orders of alternating stimulus in visual discrimination experiments. J. Genet. Psychol., 42, 206 208. Hodge, G.K. and Butcher, L i . (1974): 5-Hydroxytryptamine correlates of isolation-induced aggression in mice. Eur, J. Pharmacol., 28. 326 337. Jacobs, B.L., Trimbach, C., Eubanks, E.E. and Trulson, M. (1975): Hippocampal mediation of raphe lesion and PCPA-induced hyperactivity in the rat. Brain Res., 94~ 253-261. Jouvet, M. (1973): Serotonin and sleep in the cat. In: Serotonin and Behavior, pp. 385-400. Editor: J. Barchas and E. Usdin. Academic Press, New York. Kakihana, M., Yamazaki, N. and Nagaoka, A. (1984): Effect of idebenone (CV-2619) on the concentrations of acetylcholine and choline in various brain regions of rats with cerebral ischemia. Jpn. J. Pharmacol., 36, 357-363. Kiyota, Y., Hamajo, K., Miyamoto. M. and Nagaoka, A. (1985): Effect of idebenone (CV-2619) on memory impairment observed in passive avoidance task in rats with cerebral embolization. Jpn. J. Pharmacol., 37, 300-302. Kubanis, P. and Zornetzer, S.F. (1981): Age-related behavioral and neurobiological changes: a review with an emphasis on memory. Behav. Neural Biol., 31, 115 172. Nagaoka, A. (1987): Idebenone. In: New Cardiovascular Drugs, pp. 217-235. Editor: A. Scriabine. Raven Press, New York. Narumi, S., Nagai, Y., Kakihana, M., Yamazaki, N.~ Nagaoka~ A. and Nagawa, Y. (1985): Effects of idebenone (CV-2619) on metabolism of monoamines, especially serotonin, in the brain of normal rats and rats with cerebral ischemia. Jpn. J. Pharmacol., 37, 235-244. Nomura, M. (1981): The effects of tryptophan on learning ability and indoleamine metabolites in the rat brain. Neurochem. Res., 6, 115-116.
239 Nomura, M. (1985): Effect of idebenone (CV-2619) on brightness discrimination learning in rats with central serotonergic dysfunction. Jpn. J. Psychopharmacol., 5, 243-249, abstract in English. Nomura, M., Okamura, K. and Otsuji, M. (1982): Influence of serotonin on the operant learning in rats. Annu. Anim. Psychol. 32, 36. Penetar, D.M. and McDonough, Jr., J.H. (1983): Effects of cholinergic drugs on delayed-matching-tosample performance on rhesus monkeys. Pharmacol. Biochem. Behav., 19, 963-967. Perry, E.K., Tomlinson, B.E., Blessed, G., Bergmann, K., Gibson, P.H. and Perry, R.H. (1978): Correlation of cbolinergic abnormalities with senile plaques and mental test scores in senile dementia. Br. Med. J., 2, 1457-1459. Roberts, W.A. (1974): Spaced repetition facilitates short-term retention in the rat. J. Comp. Physiol. Psychol., 86, 164-171. Srebro, B. and Lorens, S.A. (1975): Behavioral effects of selective midbrain raphe lesions in the rat. Brain Res., 89, 303-325. Suno, M. and Nagaoka, A. (1985): Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition. Biochem. Biophys. Res. Commun., 125, 1046-1052. Tenen, S.S. (1967): The effects of p-chlorophenylalanine, a serotonin depletor, on avoidance acquisition, pain sensitivity and related behavior in the rat. Psychopharmacologia, 10, 204-219. Wagner, A.R., Rudy, J.W. and Whitlow, J.W. (1973): Rehearsal in animal conditioning. J. Exp. Psychol., 97, 407-426. Yamazaki, N., Take, Y., Nagaoka, A and Nagawa, Y. (1984): Beneficial effect of idebenone (CV-2619) on cerebral ischemia-induced amnesia in rats. Jpn. J. Pharmacol., 36, 349-356. Yamazaki, N., Nagaoka, A. and Nagawa, Y. (1985): Effect of idebenone on scopolamine-induced impairment of short-term memory in rats. Jpn. J. Psychopharmacol., 5, 321-328, abstract in English.
225
A G G 00253
Idebenone improves learning and memory impairment induced by cholinergic or serotonergic dysfunction in rats Naoki Yamazaki a Masahiko Nomura b Akinobu Nagaoka a
and Yuji Nagawa
a
a Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532, Japan and b Department of Physiology, School of Medicine, Fujita Gakuen Health University, Toyoake-City, Aichi 470-11, Japan (Received 2 August 1988; revised version received 31 October 1988; accepted 1 November 1988)
Summary The effects of idebenone, a cerebral metabolic enhancer, on learning and memory impairment in two rat models with central cholinergic or serotonergic dysfunction were investigated using positively reinforced learning tasks. A delayed alternation task using a T maze was employed to test the effect of idebenone on short-term memory impairment induced by a cholinergic antagonist, scopolamine. A correct response, defined as a turn toward the arm opposite to that in the forced run, was rewarded with food pellets. Scopolamine (0.2 and 0.5 mg/kg, i.p.) significantly decreased the correct responses to the chance level in the 60-s-delayed alternation task. The scopolamine (0.2 mg/kg, i.p.)-induced impairment of short-term memory was improved by idebenone (3-30 mg/kg, i.p.) or an acetylcholinesterase inhibitor, physostigmine (0.1 and 0.2 mg/kg, i.p.), administered simultaneously. The central serotonergic dysfunction model was produced by giving rats a diet deficient in tryptophan, a precursor of serotonin. The rats fed on a tryptophan-deficient diet (TDD) showed a slower learning process in the operant brightness discrimination task (mult VI15 EXT) than did rats fed on a normal diet. Idebenone (60 mg/kg/day) admixed with the TDD decreased the number of lever-pressing responses emitted during the extinction periods. The percentage of correct responses was significantly higher in the idebenonetreated group than in the control TDD group. These results suggest that idebenone may improve both the impairment of short-term memory induced by a decreased cholinergic activity and the retardation of discrimination learning induced by central serotonergic dysfunction.
Idebenone; Short-term memory; Operant discrimination learning; Cholinergic dysfunction; Serotonergic dysfunction; Rats
Correspondence should be addressed to Naoki Yamazaki, Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 2-17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan. 0167-4943/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
226 Introduction
A decrease of brain metabolism can easily lead to a deterioration in neurologic and mental function. Dysfunctions in the brain induced by a deficiency in neurotransmitters involve impairment of cognitive function in animals. For example, an experimentally-induced decrease in cholinergic activity impairs learning and memory in animals (Kubanis and Zornetzer, 1981; Bartus et al., 1982), and patients with presenile and senile dementia of the Alzheimer type show a decline in cholinergic markers in the cortex and hippocampus that correlate with the degree of cognitive and memory impairment (Perry et al.. 1978; Coyle et al., 1983). These findings and many others imply that the impairment in learning and memory is attributable to a decrease in central cholinergic function. A decline in central serotonergic neurotransmission also involves changes in sleep and wakefulness (Jouvet, 1973), aggression (Hodge and Butcher, 1974), ingestion behavior (Coscina et al., 1972; Blundell, 1977), nociception (Tenen, 1967), and learned behavior (Srebro and Lorens, 1975; Altman et al., 1984). Nomura et al. (1982) reported that rats fed on a tryptophan (a precursor of serotonin)-deficient diet showed impaired operant learning behavior. In the present study, we used two rat models with a decline in central cholinergic or serotonergic activity to examine the effects of idebenone, a cerebral metabolic enhancer, occasionally called a nootropic drug, on impaired learning and memory. Idebenone reverses decline in the contents of acetylcholine and serotonin in rat brains under a transient cerebral ischemia, and improves memory impairment of avoidance learning in animal models of vascular and Alzheimer types of senile dementia (Nagaoka, 1987). However, the effect of idebenone on learning and memory of the appetitive type remains nuclear. Therefore, we investigated possible effects of idebenone on impaired learning and memory motivated with food. E X P E R I M E N T I: I N V E S T I G A T I O N OF A C H O L I N E R G I C D E F I C I T M O D E L A decline in the cerebral cholinergic system was produced by administering a cholinergic blocker, scopolamine. The effect of idebenone on the scopolamine-induced memory impairment was examined in a delayed alternation task, regarded as one of the tests for short-term memory (Feldman and Gordon, 1979). (Results described here have been reported elsewhere; Yamazaki et al., 1985.)
Materials and Methods
Subjects Male rats of the Wistar strain (Charles River, Japan) were used; they were 6 weeks old at the start of the training of the delayed alternation task. They were housed individually, maintained on a food-deprivation schedule throughout the experiment, and fed on a diet sufficient to maintain approximately 85% of initial body weight at the end of the daily session.
227
Apparatus The apparatus was a T-maze with a grid floor and a clear plastic top, the sides were 12 cm high and alleys were 12 cm wide. The start alley was 44 cm long and each arm was 50 cm long. Guillotine doors were mounted at the entrance to each arm and were used to control the direction of the turning response on forced runs. A food cup, containing pellets (90 mg) as reinforcers, was located 3 cm from the floor at each arm end.
Procedure Habituation to the T-maze was permitted for 5 rain with pellets in both food cups and the guillotine doors opened. On the next 2 days, 3 - 4 trials of running training from the start point to both food cups, filled with pellets, was performed. Then each rat was trained in pairs of 10 trials of alternation training in a session. On the first (forced) run in the pair trial, the guillotine door was closed and blocked one of the arms of the maze; the unblocked arm had pellets in the food cup. After they ate the pellets, the rats were returned to the starting box and immediately given a free-choice run with no delay. On this run, both guillotine doors were opened, and pellets were present only in the arm opposite to the arm i'ewarded on the forced run. If the rat entered the rewarded side, a correct response was recorded, and the rat was returned to a plastic waiting cage after it had consumed the pellets. If the rat entered the same arm as on the forced run, an incorrect response was recorded, and the rat was returned to the starting box and allowed an other opportunity to enter the rewarded side. The correction runs were continued until the correct arm was chosen. The intertrial interval was 1 min, during which time the rats were put in the waiting cage. An equal number of left-correct and right-correct trials were presented during each session. This alternation training was continued until all rats reached a learning criterion of 90% or more correct responses over the last two training sessions. The delayed alternation training was carried out after the alternation training was completed. In this training, a delay between the forced run and the free-choice run was introduced for 0, 30, 60, and 120 s in a random order. Two trials for each delay (eight trials per session) were made for four consecutive sessions. During the delay, the rats were placed in the waiting cage. Based on the results of this preliminary experiment, a 60-s delay and eight trials in a session were used in the following drug testing. Another 16 rats, which had been trained in the 60-s-delayed alternation task, were divided into four groups (four rats in each group) and the effect of scopolamine (SCP) on the task was tested. The experiment was arranged in a Latin square design (4 x 4) in which all groups received four drug conditions in a counter-balanced order. The drug conditions included saline, or 0.1, 0.2, or 0.5 m g / k g i.p., of SCP. The drug or saline was given 20 min before the test, which was run four times, once every 3rd day for 10 days. After testing SCP, the same rats were divided into two groups (eight rats each) and the effect of SCP (0.2 m g / k g i.p.) was further
228 tested in the non-delayed (0 s delay) alternation task using a Latin square design (2 x 2). Another 15 rats, which had been trained in the 60-s-delayed alternation task, were used to test the effect of physostigmine (PHY) on the SCP (0.2 m g / k g i.p.)-induced memory impairment. The rats were divided into five groups (three in each group) and each group was exposed to five drug conditions in a Latin square design (5 x 5). The drug conditions included saline, SCP, or 0.05, 0.1, or 0.2 m g / k g i.p., of P H Y combined with SCP. The drug or saline was given 20 rain before the test which was run five times, once every 3rd day for 13 days. An additional rat. which had been tested only with saline in the same way, was prepared for the next experiment to test the effect of PHY alone on the same task. Rats were re-divided into four groups (four in each group) to match the percentage of correct response of groups in an additional session of the same task made 3 - 6 days after the task; each group was exposed to four drug conditions in a Latin square design (4 × 4). The drug conditions were saline, or 0.05, 0.1, or 0.2 m g / k g i.p., of PHY. The effect of idebenone (3, 10 and 30 m g / k g i.p.) on the task with and without SCP was examined in the same way as in the test of P H Y using other rats.
Drugs The drugs used in these studies were scopolamine hydrobromide (Wako, Japan), physostigmine (Eserine Sulfate, Wako, Japan) and 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-l,4-benzoquinone (idebenone, Takeda, Japan). SCP and P H Y were dissolved in saline and idebenone was suspended in a 5% arabic gum solution. These drugs were administered in a volume of 0.2 m l / 1 0 0 g body weight.
Statistics The percentage of correct responses in each drug exposure was analyzed by the A N O V A for Latin square design. Newman-Keuls tests were performed to compare the correct responses in each of drug exposures, and the differences in the percentages of correct responses from the chance level (50%) were examined by the critical ratio test.
Results In the first experiment to test the effect of varying the delay, the percentage of correct responses decreased as the delay increased. The A N O V A indicated a significant decline in the percentage of correct responses over the delay, F~3,4s)--36.93, p < 0.001. The percentage of correct responses after a 0-s delay was 90%, whereas it was significantly reduced to 78%, 63%, and 61% when the delay was 30, 60, and 120 s ( p < 0.05, p < 0.01 and p < 0.01), respectively. In only the 120-s delay did the percentage of correct responses not differ significantly from the
229
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Fig. 1. The effect of scopolamine (SCP) on the 60-s-delayed alternation task in rats (n =16). Mean ( d: SE) percentage of correct responses is shown. SCP was given 20 min before the test, which was run four times, once every 3rd day for 10 days. Each rat received four doses including saline (SAL) in a counter-balanced order under a Latin square design. * p < 0.05, * * p < 0.01 (Newman-Keuls test) vs. SAL control. chance level (50%). Therefore, we chose a delay of 60 s for the following experiments. SCP reduced the percentage of correct responses in a dose-dependent manner, when compared with the control level (64%) obtained with saline (Fig. 1). The decreases of the percentage of correct responses after 0.2 and 0.5 m g / k g of SCP were significant, but they were not statistically different from the chance level. Slight decreases in running speed and difficulty in swallowing pellets as a consequence of the blocking action on salivary secretions occurred dose-dependently. However, the incidence of animals with neurologic signs in the group receiving 0.2 m g / k g of SCP (1/16) was less than that in the group receiving 0.5 m g / k g (4/16). The percentage of correct responses in the group receiving SCP (0.2 m g / k g ) in the non-delay task was 88%, which did not significantly differ from the saline level (93%); this observation indicated that this dose of SCP did not influence the performance of the rats. P H Y dose-dependently improved the SCP-induced decline of the percentage of correct responses (Fig. 2,A). The A N O V A indicated that the main effect of drug treatment was significant, F4,4o = 5.95, p < 0.05. In this experiment, the percentage of correct responses with SCP alone was 44%, whereas P H Y (0.1 and 0.2 m g / k g ) significantly enhanced the correct responses to the level of the non-SCP condition. Significant ( p < 0.05) differences in the percentage of correct responses from the chance level were obtained in the non-SCP condition and at the higher two doses of PHY. The decline in running speed observed after SCP was administered, almost recovered when P H Y was administered. Furthermore, in the experiment performed without SCP, P H Y increased the correct responses dose-dependently (Fig. 2,B). The A N O V A indicated that the main effect of the drug was significant, F3,36 = 6.64, p < 0.005. Significant increases of correct responses were obtained at 0.1 and 0.2 m g / k g of PHY.
230
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Fig. 2. The effect of physostigmine (PHY) on the 60-s-delayed alternation task in rats administered scopolamine (SCP) and in those without the drug. PHY was given simultaneously with SCP (panel A: n = 15). The effect of PHY alone was further tested in the same rats without SCP (panel B: n = 16). The number of rats used in the second experiment was 16 as one rat that had been treated with saline (SAL) in the first experiment was added to the second. Both experiments were carried out according to a Latin square design. * p < 0.05, ** p < 0.01 (Newman-Keuls test) vs. SCP-administered condition without PHY (panel A), or vs. (SAL) control (panel B).
Idebenone reversed the SCP-induced decline of the correct responses (Fig. 3,A). The A N O V A indicated that the main effect of the drug was significant, F4.4o = 8.58, p < 0.001. Idebenone at doses of 3 and 10 m g / k g significantly increased the percentage of correct responses to 60% and 71%, respectively, compared with the level of saline (48%). The highest dose of idebenone also increased the correct responses significantly but the extent was less than that at 10 mg/kg. Significant ( p < 0.05) differences in the percentage of correct responses from the chance level were obtained in the non-SCP condition at the higher two doses of idebenone.
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Fig. 3. The effect of idebenone (IDB) on the 60-s-delayed alternation task in rats administered SCP and in those without the drug. IDB was given simultaneously with SCP (panel A: n = 15). The effect of IDB alone was further tested in the same rats without SCP (panel B). The number of rats used in the second experiment was 16. Both experiments were carried out according to a Latin square design, * p < 0.05, • * p < 0.01 (Newman-Keuls test) vs. SCP administered condition without P H ¥ .
231 Furthermore, in the test performed without SCP, idebenone alone did not increase the correct responses (Fig. 3,B).
Discussion
The present experiment demonstrated that the extension of the delay in the alternation task reduced the correct responses, suggesting that the consolidation of short-term memory is time dependent. The negative correlation agrees with previous findings (Roberts, 1974; Feldman and Gordon, 1979). The low level of the percentage of correct responses, near 60%, in the 60-s-delayed alternation task seems close to the chance level, but in fact the two levels were significantly different. Therefore, it is considered that the 60-s delay in the task reduced, but did not deplete, short-term memory. Cholinergic blockers like SCP impair many types of learning and memory. In the present study, SCP markedly reduced the correct responses to the chance level in the 60-s-delayed condition but not in the non-delayed condition. This result is consistent with the finding that an interaction exists between the effect of anticholinergics and the length of the delay in delayed matching to sample task in monkeys (Bartus and Johnson, 1976; Penetar and McDonough, 1983). Thus, SCP reduces short-term memory. PHY reversed the SCP-induced impairment of short-term memory by increasing the correct responses. The reversal effect of PHY may be explained easily by its inhibition of acetylcholinesterase and the resultant increase of acetylcholine at the receptor sites. Furthermore, PHY administered alone enhanced this task. This result suggests that PHY may enhance a putative process of short-term memory (Wagner et al., 1973) by means of activating the central cholinergic system. Similarly, PHY improved the performance in a delayed matching to sample task in aged monkeys in which the brain cholinergic function might be reduced (Bartus et al., 1980). These findings support the notion that memory function is closely correlated with the cholinergic system in the brain. Idebenone reversed the SCP-induced impairment of short-term memory; the peak effect was observed at a dose of 10 mg/kg. Consequently, the overall dose-response relation was an inverted U. Though the mechanism underlying the inverted U curve is unclear, the same relation was found when idebenone was administered to rats with amnesia induced by a transient cerebral ischemia (Yamazaki et al., 1984). Idebenone did not improve short-term memory in untreated rats. This suggests that idebenone improves memory impairment in rats with cholinergic dysfunction, but not in rats whose cholinergic function is normal. EXPERIMENT II: INVESTIGATION OF A SEROTONIN DEFICIT MODEL A decline in the cerebral serotonergic system was produced by feeding a diet deficient of tryptophan to rats after they were weaned. The effect of idebenone on impaired learning in these rats was examined in brightness discrimination learning
232
of the operant type. (Results described here have been reported elsewhere; Nomura, 1985.)
Materials and Methods
Subjects Thirty male Wistar rats (Clea, Japan) were used, aged 3 weeks when the tryptophan-deficient diet ( T D D ) was begun. The diet was composed of corn, corn oil, Harper's salt, vitamin mixture, L-lysine, and choline (Fernstorm and Wurtman, 1971). Twenty rats fed on the T D D were divided into two groups (10 each); one group was administered idebenone (60 m g / k g / d a y ) admixed with the diet. Ten rats fed on a normal diet (MF, Oriental Yeast Co., Japan) were used as controls. After being on the diet for 8 weeks, the rats were individually housed and kept on a food-deprivation schedule throughout the experiment to maintain body weight at 85% of the initial free feeding level.
Apparatus Two operant chambers containing a single lever (Ralph Gerbrand's) were used. An indicator lamp with a diameter of 5 cm was mounted on the center of the front wall 10 cm above a food tray. The intensity of the light at the surface of the lamp was 5 X 10 4 foot Lambert; it was possible to decrease the intensity by 1/1000 by inserting a filter (Kodak, ND4.0). A lever was mounted 2 cm from the right edge of the front wall and 2 cm above the floor. Pressing the lever was reinforced by a food pellet (40 mg). The entire assembly was placed in a dark room in which the white noise (60 dB) was diffused to mask external noises; the behavior of the rats was monitored in an adjacent room by a supersensitive videocamera. Presentations of light and reinforcement, and the recording of responses were automatically controlled by a microcomputer.
Procedure One week after the deprivation schedule was begun, the rats were given one session of shaping in the operant chamber in which every lever-pressing response was reinforced. The rats were further trained to respond to the lever in a continuous reinforcement schedule until they got 40 reinforcements within 4 rain in a session. All rats reached this criterion within a week. Then, a variable interval (VI) schedule was introduced, under which a reinforcement was delivered by the response at a mean interval of 5 s (VI5). After this schedule was completed, the VI value was increased to 10 s with 40 reinforcements a session. When the rate of response became constant, the discrimination training was begun. A multiple schedule was used in the discrimination training (mult VI15 EXT). This consisted of VI15 reinforcement for lever presses during periods when the indicator light was bright
233 (S ÷ periods) and of no reinforcement for presses during periods when the light was dark (S- periods). The duration of an S ÷ or S - period was 20 s, and each period was repeated 20 times in a session. A blackout, arranged by turning out all the lights in the chamber for 5 s, was inserted whenever the period was exchanged. The order of appearance of an S ÷ or S - period in a session was determined by Gellerman sequence (Gellerman, 1933). The rats were trained in this schedule for 30 consecutive sessions. The percentage of correct responses, R + / ( R ÷ + R - ) X 100, was calculated from the number of correct responses (R ÷) during the S ÷ period and that of incorrect responses ( R - ) during the S - period in a session. Statistics
Paired t tests were performed to compare the mean ( + SE) number of responses in each session with that in the first session. Non-paired t tests were used to compare the percentage of correct responses in the idebenone-treated and the control groups.
Results In rats fed on the normal diet, the number of correct responses (R +), which was 120 in the first session, increased as the training sessions progressed, and reached approximately 220-250 (Fig. 4). In contrast to R ÷, R - , which was approximately 120 in the first session, decreased as the training progressed, and finally reached 30. The total responses (R ÷ + R - ) increased gradually from approximately 230 to 280. The fact that differentiation of R ÷ and R - was observed after the third session indicates that the brightness discrimination learning was easily established. The percentage of correct responses in this group increased from the chance level to 85% after the 20th session. In rats fed on a tryptophan-deficient diet (TDD), the number of R + and R - in the first session was approximately 100. The increase of R + was much greater than that of the group fed on the normal diet; the final number of R + in the T D D group reached 350 (Fig. 5). Significant ( p < 0.05) increases of R ÷ in the T D D group were obtained after the 5th session. R - in the T D D group, however, did not decrease to the extent observed in the normal rats. The final level of R - was approximately 100, and a significant difference from the first session was not obtained in any session after the second. The total responses increased significantly ( p < 0.05) after the 6th session. Though the percentage of correct responses increased with the training, it did not reach the 80% level. In rats administered idebenone continuously, the number of R ÷ was 100 at the first session and increased with training; a significant ( p < 0.05) increase was obtained after the second session (Fig. 6). The final level of R + in this group was 340, which was almost the same as that in the T D D group. On the other hand, though the number of R - varied from 100 to 120 during the first 10 sessions, it
234
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Fig. 4. Discrimination learning to press a lever for food in the mult VI15 EXT schedule in rats fed on a normal diet. Rats were trained to discriminate a light period (S +, 20 s) from a dark period (S-, 20 s). Responses of pressing the lever in the S + periods were reinforced (V|15 schedule). R + represents the mean (_+ SE) responses during S + periods and R - represents the mean number of responses during S periods. R + was reinforced under the VI15 schedule. R - was not reinforced (EXT schedule). S + was 1000 times brighter than S . Each period was repeated 20 times in a session. The percentage of correct responses is derived from the ratio of R + divided by total responses (R + + R ). Ten rats were used.
d e c r e a s e d r e m a r k a b l y t h e r e a f t e r , a n d r e a c h e d less t h a n 50 o n the final session, S i g n i f i c a n t ( p < 0.05) d e c r e a s e s w e r e f o u n d at s e s s i o n s a f t e r t h e 24th. A d i f f e r e n t i a t i o n was d e f i n i t e l y o b s e r v e d a f t e r t h e 7 t h session. T h e p e r c e n t a g e o f c o r r e c t r e s p o n s e s i n c r e a s e d r a p i d l y in the l a t e r sessions w h i l e its r a t e o f i n c r e a s e w a s g r a d u a l d u r i n g t h e first 20 sessions. T h e p e r c e n t a g e of c o r r e c t r e s p o n s e s in t h e i d e b e n o n e - t r e a t e d g r o u p was s i g n i f i c a n t l y h i g h e r ( p < 0.05) t h a n t h a t in t h e T D D g r o u p a f t e r the 2 3 r d session.
Discussion I n the p r e s e n t study, the rats w e r e f e d o n a T D D a f t e r t h e y h a d b e e n w e a n e d . I n t h e s e rats, t h e b r a i n c o n t e n t s of t r y p t o p h a n , s e r o t o n i n , a n d its m e t a b o l i t e 5 - h y d r o x y i n d o l e a c e t i c a c i d ( 5 - H I A A ) d e c r e a s e d b y a p p r o x i m a t e l y 70% of t h o s e of t h e
235
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Fig. 5. Discrimination learning to press a lever for food in the mult VI15 EXT schedule in rats fed on a tryptophan-deficient diet. Ten rats were used. For further explanation, cf. Fig. 4.
rats fed on a normal diet (Nomura, 1981). The progress of the discrimination learning in the T D D rats was almost the same as that in the normal rats in the early phase of the training, but was obviously retarded in the later phase. The impairment of the discrimination learning in the T D D group was mainly due to the failure to decrease R - even after repeated training; this failure resulted in a lowered level of the percentage of correct responses. In addition, the fact that R + in the T D D rats were more than that in the normal rats may indicate that the rats with serotonergic dysfunction tended to respond at a high rate. This is supported by the finding that the disturbance in serotonergic activities by lesioning the midbrain raphe and administering p-chlorophenylalanine increased motility in the rats (Jacobs et al., 1975). Therefore, the impairment of the discrimination learning observed in the rats with serotonergic dysfunction might be, in part, related to the inability to inhibit responses. Idebenone improved the impairment of the discrimination learning in the T D D rats. The rate of increase and the level finally reached in the percentage of correct
236
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Fig. 6. The effect of idebenone (60 mg/kg/day) on the discrimination learning to press a lever for food in the mult VI15 EXT schedule in rats fed on a tryptophan-deficient diet. Ten rats were used. For further explanation, cf. Fig. 4. * p < 0.05 (t test) vs. responses in the first session (upper panel), and vs. rats fed on tryptophan-deficient diet (lower panel).
responses in the idebenone-treated group were c o m p a r a b l e to those observed in the rats fed on a normal diet. I d e b e n o n e might i m p r o v e insufficient serotonergic activity by increasing the lowered content of serotonin in the ischemic brain. Thus, it is speculated that i d e b e n o n e enhanced the serotonergic s y s t e m of the T D D rats by the repeated discrimination training during which serotonergic activity increased as learning progressed.
General Discussion The present experiments demonstrated the beneficial effects of i d e b e n o n e on s c o p o l a m i n e - i n d u c e d impairment of short-term m e m o r y and T D D - i n d u c e d impairm e n t of operant discrimination learning in rats. Since i d e b e n o n e ameliorates the decrease of cholinergic turnover induced by transient cerebral i s c h e m i a in rats
237 (Kakihana et al., 1984), it was expected that the drug might improve the impairment of short-term memory induced by the cholinergic blocker, scopolamine. This hypothesis was clearly proved correct by the present results. The effect was observed in rats with cholinergic dysfunction but not in normal rats. From the point of view of its clinical application, it is important that idebenone may exert its effect on a pathogenic condition related to hypofunction of the cerebral cholinergic system, which is observed in presenile dementia and senile dementia of Alzheimer type. The second experiment indicated that the rats fed on the TDD were less able to learn brightness discrimination. Idebenone, administered continuously from weaning, improved the decreased progress of discrimination learning. Idebenone activates brain serotonergic function in normal and cerebral ischemic rats. In normal rats, idebenone at a high dose of 100 mg/kg i.p. increased the content of the 5-HIAA, a metabolite of serotonin, and facilitated the decrease of the serotonin content induced by p-chlorophenylalanine, a serotonin synthesis inhibitor (Narumi et al., 1985). In rats with cerebral ischemia, idebenone at a low dose of 10 mg/kg normalized the decrease of the 5-HIAA content. These findings suggest that idebenone may facilitate the turnover of serotonin in the brain. It is reasonable to speculate that the effect of idebenone to reinstate the learning ability is partly due to amelioration of the serotonergic dysfunction induced by the TDD. The fact that idebenone ameliorates the impairment of memory and learning found in a passive avoidance task using shocks (Yamazaki et al., 1984; Kiyota et al., 1985), and the present finding that idebenone ameliorates memory and learning impairment in tasks using positive reinforcements, clearly confirm that the ameliorating effects of idebenone on the impairment of learning and memory do not depend upon the type of reinforcers. A close interaction between the cholinergic and serotonergic systems in the brain has been proposed (Butcher and Woolf, 1982). Some physiologic and behavioral processes in which acetylcholine is implicated are the same as those in which serotonin appears to play a role, and possible neural mechanisms for this communication may exist. In fact, a transient cerebral ischemia in rats produces a decline in cholinergic (Kakihana et al., 1984) and serotonergic (Narumi et al., 1985) activities. Idebenone improves the amnesia in rats induced by cerebral ischemia (Yamazaki et al., 1984). Therefore, it is clear that idebenone improves learning and memory impairment in which the cholinergic and/or serotonergic transmission is decreased by certain experimental manipulations. The neurochemical mechanisms underlying the effect of idebenone are considered to be based upon improvement in cerebral energy metabolism; idebenone accelerates net ATP formation in brain mitochondria by activating the electron-transfer system and by inhibiting the production of lipid peroxide in the mitochondrial membrane (Suno and Nagaoka, 1985). On the basis of these findings it is expected that idebenone may partly improve mental or cognitive dysfunction by enhancing the brain energy metabolism in patients whose cholinergic and/or serotonergic activities are decreased.
238
Acknowledgement T h e a u t h o r s w i s h to t h a n k D r . J . R . M i l l e r f o r c o m m e n t s o n t h e m a n u s c r i p t .
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