Behavioural Brabz Research, 51 (1992) 157-164 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92/$05.00
157
BBR 01364
Learning behaviour in chronic vitamin E-deficient and -supplemented rats: radial arm maze learning and passive avoidance response Yukio Ichitani n, Hiroshige Okaichi b, Toshikazu Yoshikawa c and Yasuhiko Ibata a Departments of aA natonO' and ch~ternal Medicine, Kyoto Prefectural UnirersiO' of Medicine, Kyoto (JapapO and bDepartment of Psychology, Faculty of Letters, Doshisha Unlversio,, Kyoto (Japat0 (Received 1 },larch 1992) (Revised version received 15 June 1992) (Accepted 31 July 1992)
Key words: Vitamin E; Radial maze learning; Passive avoidance response; Spatial memory; Scopolamine; Aging; Rat
The effects of long-term vitamin E deficiency and supplementation on learning behaviour were investigated. Rats were fed vitamin E-deficient [ V E ( - )], -supplemented [VE( + )], or control standard food beginning after the age of 4 weeks. They were trained in an eight-arm radial maze learning task at the age of 17 months, and in a step-through passive avoidance response (PAR) task at the age of 25 months. In the radial maze task, both V E ( - ) and VE( + ) animals required as many trials to reach the learning criterion as control animals. Scopolamine injection (0.250.5 mg/kg) after acquisition of the task decreased the number of correct choices dose-dependently; however, the degree of the drug effect on VE( - ) and VE(+ ) rats did not differ from that on control rats. On the other hand, V E ( - ) animals showed significantly lower rate of avoidance response and VE(+ ) animals tended to show higher rate of avoidance response in the PAR task than did control animals. These results suggest that long-term vitamin E deficiency or supplementation does not influence general ability to acquire and maintain memory tasks in rats, but that it may affect learning behaviour, depending on the kind of task in which animals were trained.
INTRODUCTION
Although vitamin E (~-tocopherol) deficiency in humans and animals has been reported to produce a variety of dysfunctions such as reduced growth rate, testicular degeneration, fetal resorption, myopathy, muscle degeneration, anemia, and axonal dystrophy 8']6'22'26'28"32, little is known about its effects on the central nervous system (CNS). One of the most consistent relationships that has been shown between vitamin E deficiency and changes in the CNS is that neuronal lipofuscin accumulation with advancing age can be markedly accelerated by vitamin E deprivation 21'34'35. The fact that aging in animals causes learning impairment in many kinds of memory tasks 2~ indicates the possibility that lipofuscin accumulation in the CNS is related to the deterioration or dysfunction of highly.cognitive processes like memory or learning. However, there has been little investigation of the ef-
Correspondence: Yukio Ichitani, Present address: Institute of Psychology, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
fects of chronic vitamin E deficiency on animal behaviour, especially on memory and learning. It has been demonstrated in vitro that vitamin E is a lipid-soluble antioxidant which inhibits the peroxidation of unsaturated fatty acids 13. It serves as a free radical scavenger and is able to protect cell membranes against oxidative damage by terminating free radicalgenerated chain reactions. In addition, evidence supporting this type of biological action of vitamin E has been reported recently, even in in vivo experiments6'14"37-39. If memory and learning deficits in aged animals are also attributable to the deleterious side attacks of free radicals on cell constituents, as is speculated from Harman's theory of aging 15, it may be possible that vitamin E supplementation slows down the aging process and improves learning performance by protecting from the deleterious oxidative effects of free radicals, while vitamin E deficiency exacerbates learning performance in aged animals. In the present study, therefore, we investigated the effects of both chronic vitamin E deficiency and supplementation on performance in two kinds of memory tasks, employing aged rats which had been reared with vitamin E-controlled diets.
158 Radial maze learning behaviour 29 and passive avoidance response (PAR) were tcstcd since many previous studies have shown behavioural deficits in aged animals in these tasks 2"3'7'23"25'27"36, and it has been suggested that performance of these tasks requires normal functioning of the hippocampus 4"~8"t9"3~ where most or markedly increased lipofuscin accumulation has bccn found 1~ In addition, the effect of scopolamine, a muscarinic receptor antagonist, on the performance of radial maze task was also tested, since this drug has been shown to severely impair radial maze performa n c c 5.9,17.
MATERIALS AND METIIODS
Anhnals and diet Male Sprague-Dawley rats were purchased from Charles-River Japan Co. Ltd., at the age of 4 weeks; they were then housed in groups (4-5 rats per cage) under constant temperature and humidity conditions, with a 12-h light-dark cycle. All the behavioural testings and sacrifice for biochemical analysis were conductcd in the latter half of the light phase. The rats were fed a vitamin E-deficient [ V E ( - ) ] diet, a vitamin E-supplemented [VE( + )] diet, or a control standard diet until all the experiments ended. Table I shows the components of the V E ( - ) diet, which contained less than 0.1 mg D-~-tocopherol per 100 g food. The control diet was prepared by adding 2 mg of De-~-tocopherol acetate (vitamin E 2 1.U.) to 100 g of the basal VE( - ) diet, and the V E ( + ) diet was prepared by adding 58.5 mg DU-7-tocopherol nicotinate (vitamin E 50 I.U.)
TABLE I
Compos#ion of vitambt E-deficient diet used in this study
Corn starch Vitamin-freecasein ~-Mah starch Powdered cellulose Mineral mixture Granulated sugar Vitamin mixture* Stripped corn oil
36% 25% 10% 8% 6% 5% 2% 8~ 1oo%
* The vitamin mixture in 100g diet contains !,000 I.U. vitamin A, 200 I.U. vitamin D3, 2.4 mg vitamin BI, 8.0 mg vitamin B:, 1.6 mg vitamin B6, 1.0 ttg vitamin B~:, 60.0 nag vitamin C, 10.4mg vitamin K3, 0.04 mg biotin, 0.4 mg folic acid, 10.0rag Ca-pantothenate, 10.0 mg p-aminobenzoic acid, 12.0mg niacin, 12.0mg inositol, and 400.0 mg cholin-CI.The vitamin E content of this diet is less than 0.1 mg of D-:~-tocopherol]100g diet fi)od.
to 100 g of the basal VE( - ) diet. Water was given ad libitum. At the age of 17 months, 10 animals from each diet group were randomly selected for radial maze learning training. Body weights of VE( - ), control, and VE( + ) animals before starting the food deprivation schedule were 4 6 4 + 1 3 g (mean +S.E.M.), 4 9 0 + 1 2 g , and 452+ 17 g, respectively, and there was no significant difference between them. Two control rats were discarded during the acquisition training because they did not run down the maze at all, and one VE( + ) rat was discarded due to sudden death during drug testing. At the age of 25 months, animals were trained in a step-through passive avoidance response (PAR) task. All the rats used in the radial maze task and which were alive at this time were used, as well as 15 additional rats which had been fed vitamin E-controlled diets for the same period, but had not experienced radial maze learning. Total numbers of animals trained for the PAR test were I0, 11, and 14 for VE( - ), control, and VE( + ) group, respectively. Mean body weights ( + S.E.M.) of V E ( - ), control, and VE( + ) animals at PAR training were 630 + 28 g, 643 +_29 g, 574 + 18 g, respectively, and there was no significant difference between them. To test the effects of a shorter period of feeding with V E ( - ) and VE(+ ) diets, an additional 18 rats were used (1l = 6 for each diet). They were housed and fed for 4 months according to the procedures outlined above for the other animals, and they were tested for PAR task at the age of 5 months. After PAR testing they were used to determine brain ~-tocopherol level. Apparatus For radial maze learning, the apparatus was a wooden, elevated (55 cm above the floor), eight-arm radial maze. Each arm (70 cm long and 9 cm wide) extended from an octagonally shaped central platform (35 cm across). A hole (1.3 cm in diameter and 0.4 cm deep) at the distal end of each arm served as a reward well. The maze was placed in a soundproof room with some extramaze visual cues: fluorescent ceiling lights, a desk and a chair, a rack of rat cages, shelves, and doors with a curtain. The experimenter kept a constant position beside the maze and observed and checked animal behaviour. Illumination on the central platform was 800 lux. For PAR learning, a two-compartment PAR apparatus made of polyvinyl chloride resin was used. It consisted of a small, bright compartment (25 • 10 (W) • 25 (H) cm, 1,000 lux) and a large, dark compartment (30x 30 • 30 (H) cm, less than 10 lux) covercd with an opaque ceiling; the two compartments were connected by a guillotine door (8 • 8 cm). The
159 inside of the apparatus was painted gray. The floor of the dark compartment consisted of stainless-steel grids which could be electrified.
Procedure Radial maze learnhzg. During the training for radial maze learning and the following drug tests, rats were housed individually. The amount of food each rat received was adjusted daily (12-18 g per day) so that their body weights were maintained at about 60-75~o ofthose before starting food deprivation schedule. Each rat was handled for 5 min a day for 3 consecutive days. Then, as adaptation to the apparatus, rats were allowed to explore the maze in groups for 30-min periods for 3 days, with some additional days for some rats. During this period, a 23~o sucrose solution was placcd in a small glass dish at the centre of the maze. From the next day, the animals received acquisition training of one trial per day, in which they were allowed to run down all 8 arms to obtain sugared water (0.2 ml of 23 ~o sucrose solution for each arm). The animal was left in the maze until all 8 portions had been consumed, until 16 choices had been made, or until 5 rain had elapsed, whichever occurred first. A correct choice was defined as catering an arm not chosen previously on that trial and licking the sugared water. Reentering an arm after the reward had already been consumed was recorded as an error. The acquisition training was completed when the animals exhibitcd 5 consecutive criterional trials: 7 or more correct choices out ofthe first 8 choices. After completion of the acquisition training, the effects of the following 4 drug pretreatment conditions were tested; scopolamine (scopolamine hydrobromide, 0.25 mg/kg and 0.5 mg/kg), methylscopolamine (scopolamine methylbromide, 0.5 mg/kg), and physiological saline. Methylscopolamine, which does not pass through the blood-brain barrier, was used to test the peripheral effect of scopolamine. Drugs were dissolved in physiological saline and administered i.p. in a volume of I ml/kg body weight 30 rain prior to testing. In order to exclude the possibility that a drug treatment retained its effect until the next drug treatment, drug tests were carried out after confirmation that each rat had achieved a criterional trial without drug treatment on the preceding day. Each rat was tested once under a particular treatment condition; the order of treatment was random. Passi're avoidance response (PAR). On the first day (acquisition day), the animal was placed in the bright compartment so that it was facing away from the guillotine door, and it was allowed to explore the compartment. When the rat faced the guillotine door, it was opened by the experimenter. As soon as the rat entered
the dark compartment from the bright one, i.e. all 4 legs were in the compartment, the animal was confined in it by closing the door. Ten s later, a 3.0-s electric shock of 0.5 mA (1,400 V, A.C.) was delivered twice, at a 10-s interval, to the grid floor through a shock generator-scrambler (Neuroscience Co. Inc., Tokyo). Fifteen s later, the animal was returned to the home cage. Time from opening the guillotine door to entering the dark compartment was measured as response latency. The retention test began 48 h later. Each rat was placed again in the bright compartment and all the procedures were the same as the first day's, except that foot shock was not delivered. The animals which did not enter the dark compartment within 3 rain were taken from the apparatus and retestcd for retention 4, 7, 18, and 26 days after the acquisition day until they showed the response of entering the dark compartment. For testing young animals (4-month diet group), an electric shock of 0.2 mA was employed, since a preliminary experiment showed that the same intensity as was used in the aged rats (0.5 mA) was so strong for the young rats that they maintained more than 80~o avoidance even 26 days after the shock experience.
o~-'Ibcopherol determhlation Brain ~-tocopherol content was determined by the following procedure, using rats fed vitamin E-controlled diets for 4 months. Immediately after decapitation, brains were removed and the regions of the cerebral cortex (cortex), striatum, and medulla oblongata + pons (medulla oblongata) were dissected out xz on an ice-cooled plate, c~-Tocopherol level was determined by using a modification of the method of Abe ct al. t. The tissue samples were homogenized in 10 mM sodium phosphate buffer containing 0.01~ butylated hydroxytoluene and 30 mM KCI. The tocopherols were extracted from 1.0 ml of brain homogenate (brain 100 #g) with 5.0 ml of n-hexane after the addition of 2.0 ml of ethanol containing 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMC) 2 #g as an internal standard. Four ml of n-hexane extracts was evaporated under N2 gas at 60 ~ for 20 min. The residue was dissolved in 200 ill ofn-hexane. Twenty tll of the solution was injected into a high-performance liquid chromatograph (LC-6A, Shimazu, Kyoto) colunm (Shim-pack FLC-SIL, 5 cm • 4.6 mm i.d., particle size 3 ILm, Shimazu) using n-hexane/dioxane/ethanol (97.7/2.0/0.3, v/v) as eluant, at a flow rate of 1.5 ml/min. The eluted tocopherols were determined separately by using a spectrofluorometer (Ex. 298 nm, Era. 325 nm, RF-530, Shimazu). Protein was measured according to the method of Lowry et al. 24.
160 8-
Statistical anab'sis Results were analyzed using analysis of variance (ANOVA) and t-test in principle. For the analysis of data in PAR, non-parametric methods were used: response latency was analyzed by H- and U-tests, and percent avoidance was analyzed by Z2-test.
7"
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RESULTS
Radial ann maze leamhzg Mean trials to criterion in each diet group, not including the 5 criterional trials, are shown in Fig. la, and the harmonic mean running times per choice in the criterional 5 trials are shown in Fig. lb. The running time scores were obtained from the total running time for a trial divided by the number of total choices in that trial. There was no statistically significant difference between the 3 diet groups. The same statistical result was obtained from the analysis of the total number of errors to reach criterion. The learning processes of each group are shown in Fig. 2 as the number of corrcct choices per first 8 responses. ANOVA showed significant effects of blocks of trials (F= 58.02, dr= 11/275, P
157
BBR 01364
Learning behaviour in chronic vitamin E-deficient and -supplemented rats: radial arm maze learning and passive avoidance response Yukio Ichitani n, Hiroshige Okaichi b, Toshikazu Yoshikawa c and Yasuhiko Ibata a Departments of aA natonO' and ch~ternal Medicine, Kyoto Prefectural UnirersiO' of Medicine, Kyoto (JapapO and bDepartment of Psychology, Faculty of Letters, Doshisha Unlversio,, Kyoto (Japat0 (Received 1 },larch 1992) (Revised version received 15 June 1992) (Accepted 31 July 1992)
Key words: Vitamin E; Radial maze learning; Passive avoidance response; Spatial memory; Scopolamine; Aging; Rat
The effects of long-term vitamin E deficiency and supplementation on learning behaviour were investigated. Rats were fed vitamin E-deficient [ V E ( - )], -supplemented [VE( + )], or control standard food beginning after the age of 4 weeks. They were trained in an eight-arm radial maze learning task at the age of 17 months, and in a step-through passive avoidance response (PAR) task at the age of 25 months. In the radial maze task, both V E ( - ) and VE( + ) animals required as many trials to reach the learning criterion as control animals. Scopolamine injection (0.250.5 mg/kg) after acquisition of the task decreased the number of correct choices dose-dependently; however, the degree of the drug effect on VE( - ) and VE(+ ) rats did not differ from that on control rats. On the other hand, V E ( - ) animals showed significantly lower rate of avoidance response and VE(+ ) animals tended to show higher rate of avoidance response in the PAR task than did control animals. These results suggest that long-term vitamin E deficiency or supplementation does not influence general ability to acquire and maintain memory tasks in rats, but that it may affect learning behaviour, depending on the kind of task in which animals were trained.
INTRODUCTION
Although vitamin E (~-tocopherol) deficiency in humans and animals has been reported to produce a variety of dysfunctions such as reduced growth rate, testicular degeneration, fetal resorption, myopathy, muscle degeneration, anemia, and axonal dystrophy 8']6'22'26'28"32, little is known about its effects on the central nervous system (CNS). One of the most consistent relationships that has been shown between vitamin E deficiency and changes in the CNS is that neuronal lipofuscin accumulation with advancing age can be markedly accelerated by vitamin E deprivation 21'34'35. The fact that aging in animals causes learning impairment in many kinds of memory tasks 2~ indicates the possibility that lipofuscin accumulation in the CNS is related to the deterioration or dysfunction of highly.cognitive processes like memory or learning. However, there has been little investigation of the ef-
Correspondence: Yukio Ichitani, Present address: Institute of Psychology, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
fects of chronic vitamin E deficiency on animal behaviour, especially on memory and learning. It has been demonstrated in vitro that vitamin E is a lipid-soluble antioxidant which inhibits the peroxidation of unsaturated fatty acids 13. It serves as a free radical scavenger and is able to protect cell membranes against oxidative damage by terminating free radicalgenerated chain reactions. In addition, evidence supporting this type of biological action of vitamin E has been reported recently, even in in vivo experiments6'14"37-39. If memory and learning deficits in aged animals are also attributable to the deleterious side attacks of free radicals on cell constituents, as is speculated from Harman's theory of aging 15, it may be possible that vitamin E supplementation slows down the aging process and improves learning performance by protecting from the deleterious oxidative effects of free radicals, while vitamin E deficiency exacerbates learning performance in aged animals. In the present study, therefore, we investigated the effects of both chronic vitamin E deficiency and supplementation on performance in two kinds of memory tasks, employing aged rats which had been reared with vitamin E-controlled diets.
158 Radial maze learning behaviour 29 and passive avoidance response (PAR) were tcstcd since many previous studies have shown behavioural deficits in aged animals in these tasks 2"3'7'23"25'27"36, and it has been suggested that performance of these tasks requires normal functioning of the hippocampus 4"~8"t9"3~ where most or markedly increased lipofuscin accumulation has bccn found 1~ In addition, the effect of scopolamine, a muscarinic receptor antagonist, on the performance of radial maze task was also tested, since this drug has been shown to severely impair radial maze performa n c c 5.9,17.
MATERIALS AND METIIODS
Anhnals and diet Male Sprague-Dawley rats were purchased from Charles-River Japan Co. Ltd., at the age of 4 weeks; they were then housed in groups (4-5 rats per cage) under constant temperature and humidity conditions, with a 12-h light-dark cycle. All the behavioural testings and sacrifice for biochemical analysis were conductcd in the latter half of the light phase. The rats were fed a vitamin E-deficient [ V E ( - ) ] diet, a vitamin E-supplemented [VE( + )] diet, or a control standard diet until all the experiments ended. Table I shows the components of the V E ( - ) diet, which contained less than 0.1 mg D-~-tocopherol per 100 g food. The control diet was prepared by adding 2 mg of De-~-tocopherol acetate (vitamin E 2 1.U.) to 100 g of the basal VE( - ) diet, and the V E ( + ) diet was prepared by adding 58.5 mg DU-7-tocopherol nicotinate (vitamin E 50 I.U.)
TABLE I
Compos#ion of vitambt E-deficient diet used in this study
Corn starch Vitamin-freecasein ~-Mah starch Powdered cellulose Mineral mixture Granulated sugar Vitamin mixture* Stripped corn oil
36% 25% 10% 8% 6% 5% 2% 8~ 1oo%
* The vitamin mixture in 100g diet contains !,000 I.U. vitamin A, 200 I.U. vitamin D3, 2.4 mg vitamin BI, 8.0 mg vitamin B:, 1.6 mg vitamin B6, 1.0 ttg vitamin B~:, 60.0 nag vitamin C, 10.4mg vitamin K3, 0.04 mg biotin, 0.4 mg folic acid, 10.0rag Ca-pantothenate, 10.0 mg p-aminobenzoic acid, 12.0mg niacin, 12.0mg inositol, and 400.0 mg cholin-CI.The vitamin E content of this diet is less than 0.1 mg of D-:~-tocopherol]100g diet fi)od.
to 100 g of the basal VE( - ) diet. Water was given ad libitum. At the age of 17 months, 10 animals from each diet group were randomly selected for radial maze learning training. Body weights of VE( - ), control, and VE( + ) animals before starting the food deprivation schedule were 4 6 4 + 1 3 g (mean +S.E.M.), 4 9 0 + 1 2 g , and 452+ 17 g, respectively, and there was no significant difference between them. Two control rats were discarded during the acquisition training because they did not run down the maze at all, and one VE( + ) rat was discarded due to sudden death during drug testing. At the age of 25 months, animals were trained in a step-through passive avoidance response (PAR) task. All the rats used in the radial maze task and which were alive at this time were used, as well as 15 additional rats which had been fed vitamin E-controlled diets for the same period, but had not experienced radial maze learning. Total numbers of animals trained for the PAR test were I0, 11, and 14 for VE( - ), control, and VE( + ) group, respectively. Mean body weights ( + S.E.M.) of V E ( - ), control, and VE( + ) animals at PAR training were 630 + 28 g, 643 +_29 g, 574 + 18 g, respectively, and there was no significant difference between them. To test the effects of a shorter period of feeding with V E ( - ) and VE(+ ) diets, an additional 18 rats were used (1l = 6 for each diet). They were housed and fed for 4 months according to the procedures outlined above for the other animals, and they were tested for PAR task at the age of 5 months. After PAR testing they were used to determine brain ~-tocopherol level. Apparatus For radial maze learning, the apparatus was a wooden, elevated (55 cm above the floor), eight-arm radial maze. Each arm (70 cm long and 9 cm wide) extended from an octagonally shaped central platform (35 cm across). A hole (1.3 cm in diameter and 0.4 cm deep) at the distal end of each arm served as a reward well. The maze was placed in a soundproof room with some extramaze visual cues: fluorescent ceiling lights, a desk and a chair, a rack of rat cages, shelves, and doors with a curtain. The experimenter kept a constant position beside the maze and observed and checked animal behaviour. Illumination on the central platform was 800 lux. For PAR learning, a two-compartment PAR apparatus made of polyvinyl chloride resin was used. It consisted of a small, bright compartment (25 • 10 (W) • 25 (H) cm, 1,000 lux) and a large, dark compartment (30x 30 • 30 (H) cm, less than 10 lux) covercd with an opaque ceiling; the two compartments were connected by a guillotine door (8 • 8 cm). The
159 inside of the apparatus was painted gray. The floor of the dark compartment consisted of stainless-steel grids which could be electrified.
Procedure Radial maze learnhzg. During the training for radial maze learning and the following drug tests, rats were housed individually. The amount of food each rat received was adjusted daily (12-18 g per day) so that their body weights were maintained at about 60-75~o ofthose before starting food deprivation schedule. Each rat was handled for 5 min a day for 3 consecutive days. Then, as adaptation to the apparatus, rats were allowed to explore the maze in groups for 30-min periods for 3 days, with some additional days for some rats. During this period, a 23~o sucrose solution was placcd in a small glass dish at the centre of the maze. From the next day, the animals received acquisition training of one trial per day, in which they were allowed to run down all 8 arms to obtain sugared water (0.2 ml of 23 ~o sucrose solution for each arm). The animal was left in the maze until all 8 portions had been consumed, until 16 choices had been made, or until 5 rain had elapsed, whichever occurred first. A correct choice was defined as catering an arm not chosen previously on that trial and licking the sugared water. Reentering an arm after the reward had already been consumed was recorded as an error. The acquisition training was completed when the animals exhibitcd 5 consecutive criterional trials: 7 or more correct choices out ofthe first 8 choices. After completion of the acquisition training, the effects of the following 4 drug pretreatment conditions were tested; scopolamine (scopolamine hydrobromide, 0.25 mg/kg and 0.5 mg/kg), methylscopolamine (scopolamine methylbromide, 0.5 mg/kg), and physiological saline. Methylscopolamine, which does not pass through the blood-brain barrier, was used to test the peripheral effect of scopolamine. Drugs were dissolved in physiological saline and administered i.p. in a volume of I ml/kg body weight 30 rain prior to testing. In order to exclude the possibility that a drug treatment retained its effect until the next drug treatment, drug tests were carried out after confirmation that each rat had achieved a criterional trial without drug treatment on the preceding day. Each rat was tested once under a particular treatment condition; the order of treatment was random. Passi're avoidance response (PAR). On the first day (acquisition day), the animal was placed in the bright compartment so that it was facing away from the guillotine door, and it was allowed to explore the compartment. When the rat faced the guillotine door, it was opened by the experimenter. As soon as the rat entered
the dark compartment from the bright one, i.e. all 4 legs were in the compartment, the animal was confined in it by closing the door. Ten s later, a 3.0-s electric shock of 0.5 mA (1,400 V, A.C.) was delivered twice, at a 10-s interval, to the grid floor through a shock generator-scrambler (Neuroscience Co. Inc., Tokyo). Fifteen s later, the animal was returned to the home cage. Time from opening the guillotine door to entering the dark compartment was measured as response latency. The retention test began 48 h later. Each rat was placed again in the bright compartment and all the procedures were the same as the first day's, except that foot shock was not delivered. The animals which did not enter the dark compartment within 3 rain were taken from the apparatus and retestcd for retention 4, 7, 18, and 26 days after the acquisition day until they showed the response of entering the dark compartment. For testing young animals (4-month diet group), an electric shock of 0.2 mA was employed, since a preliminary experiment showed that the same intensity as was used in the aged rats (0.5 mA) was so strong for the young rats that they maintained more than 80~o avoidance even 26 days after the shock experience.
o~-'Ibcopherol determhlation Brain ~-tocopherol content was determined by the following procedure, using rats fed vitamin E-controlled diets for 4 months. Immediately after decapitation, brains were removed and the regions of the cerebral cortex (cortex), striatum, and medulla oblongata + pons (medulla oblongata) were dissected out xz on an ice-cooled plate, c~-Tocopherol level was determined by using a modification of the method of Abe ct al. t. The tissue samples were homogenized in 10 mM sodium phosphate buffer containing 0.01~ butylated hydroxytoluene and 30 mM KCI. The tocopherols were extracted from 1.0 ml of brain homogenate (brain 100 #g) with 5.0 ml of n-hexane after the addition of 2.0 ml of ethanol containing 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMC) 2 #g as an internal standard. Four ml of n-hexane extracts was evaporated under N2 gas at 60 ~ for 20 min. The residue was dissolved in 200 ill ofn-hexane. Twenty tll of the solution was injected into a high-performance liquid chromatograph (LC-6A, Shimazu, Kyoto) colunm (Shim-pack FLC-SIL, 5 cm • 4.6 mm i.d., particle size 3 ILm, Shimazu) using n-hexane/dioxane/ethanol (97.7/2.0/0.3, v/v) as eluant, at a flow rate of 1.5 ml/min. The eluted tocopherols were determined separately by using a spectrofluorometer (Ex. 298 nm, Era. 325 nm, RF-530, Shimazu). Protein was measured according to the method of Lowry et al. 24.
160 8-
Statistical anab'sis Results were analyzed using analysis of variance (ANOVA) and t-test in principle. For the analysis of data in PAR, non-parametric methods were used: response latency was analyzed by H- and U-tests, and percent avoidance was analyzed by Z2-test.
7"
.O I
6-
o
"~ 5"6 ~0 4 0
o""
.... o---
Y
....7
Con!
;il,
3-
RESULTS
Radial ann maze leamhzg Mean trials to criterion in each diet group, not including the 5 criterional trials, are shown in Fig. la, and the harmonic mean running times per choice in the criterional 5 trials are shown in Fig. lb. The running time scores were obtained from the total running time for a trial divided by the number of total choices in that trial. There was no statistically significant difference between the 3 diet groups. The same statistical result was obtained from the analysis of the total number of errors to reach criterion. The learning processes of each group are shown in Fig. 2 as the number of corrcct choices per first 8 responses. ANOVA showed significant effects of blocks of trials (F= 58.02, dr= 11/275, P
