Alcohol. Vol. 8 p~. 265-272. e Pergamon Press plc. 1991. Printed in the U.S.
0741-8329/91 $3.00 + .00
The Effects of Chronic Alcohol Consumption Thiamin Deficiency on Radial-Arm Maze Performance in the Rat JUDI HOMEWOOD,
or
N I G E L W. B O N D A N D J A N E C. M c G R E G O R
School o f Behavioural Sciences, Macquarie University, Sydney, Australia N S W 2109
R e c e i v e d 22 A u g u s t 1990; A c c e p t e d 9 J a n u a r y 1991 HOMEWOOD, J., N. W. BOND AND J. C. McGREGOR. The effects of chronic alcohol consumption or thiamin deficiency on radial-arm maze performance in the rat. ALCOHOL 8(41 265-272, 1991.--Experiment I tested the hypothesis that chronic consumption of ethanol, in the presence of good nutrition, is associated with impairments in memory. Rats were fed a vitaminfortified ethanoI-Sustagen diet with 35% ethanol-derived calories for 150 days. Two control groups were used. The first was pair fed a Sustagen fnix in which sucrose had been substituted for ethanol. The second control group received ad lib access to standard laboratory chow and water. All animals were then placed on the chow and water diet for six weeks prior to testing in an eight-arm radial maze. There was a significant, but modest, difference between groups in the number of trials to reach a criterion level of seven correct from the first eight choices and all eight correct in ten choices. However, there were no differences between the groups when delays (0, 5, 20, 60, 120 and 240 minutes) were imposed between the 4th and 5th choices. In Experiment 2. a thiamin-deficient group was compared with pair-fed and ad lib control groups on the same task. The thiamin-deficient group took more trials to reach criterion but the groups did not differ in the delay testing phase. The results are discussed in the context of reference/working memory distinctions, and the literature on remediation of cognitive deficits associated with alcoholism. Alcohol
Thiamine deficiency
Working memory
Reference memory
Korsakoff's syndrome
Rats
tion of the putative etiological factors of alcohol-related memory impairment. However, as Darnton-Hill (9) points out in his recent review, animal studies may not necessarily mimic the human condition in terms of pattern of drinking and poor general health status. Nevertheless, in apparently well-nourished rodents. chronic ethanol consumption has been found to result in loss of hippocampal pyramidal and dentate gyrus granule cells (42) although with abstinence, dendritic spine density may return to control levels (7,24). There are several reports of behavioural impairments in alcohol-treated rats, in paradigms such as shuttlebox avoidance (41), Hebb-Williams maze (5), differential reinforcement of low rates of responding (10) and the radial-arm maze (1), although some studies have failed to find an effect of alcohol exposure (3, 32, 36). Thiamin deficiency has been produced experimentally in a number of species, most commonly the rat (23,25), but also in cats (22) and monkeys (44). There is general agreement that the combination of a thiamin-deficient diet and daily injections of pyrithiamin produces biochemical and neuropathological abnormalities more similar in nature and distribution to those found in human WE than are found with dietary treatment alone (6). In rats, pyrithiamin treatment results in damage to thalamic, hypothalamic, brain stem and cerebellar regions (6). Several authors have reported that thiamin deficiency results in deficits in active ' and passive avoidance learning (23, 25, 39). We have argued elsewhere that the generality of these effects is in doubt (19). Briefly, previous work has been flawed by use of immature animals, lack of internal consistency, and insufficient subjects sur-
STUDIES of human alcoholics have shown that excessive use of ethanol over long periods of time is associated with morphological changes and a variety of cognitive deficits. Recently detoxified alcoholics show a characteristic pattern of impairment on neuropsychological tests with deficits in nonverbal abstracting ability, complex problem solving, perceptual-spatial motor performance, and some tests of learning and memory (29). Neuropathological findings include the presence of cerebral atrophy, as assessed by computed axial tomography examination and autopsy, and reduced brain weight (17, 37, 43). The most extensively studied form of alcohol-related brain damage is Korsakoff's syndrome. Korsakoff's syndrome is characterised by profound anterograde amnesia, in addition to visuo-perceptive and problem solving deficits. Impairments in memory found in alcoholics have usually been considered to result from malnutrition, in particular thiamin deficiency, rather than a direct effect of ethanol per se (38). Thiamin deficiency is manifested clinically by neurological abnormalities termed Wernicke's encephalopathy (WE). Alcoholics are the group most commonly at risk for WE because of inadequate intake, increased need, poor storage, and impaired absorption of thiamin (20). The assessment of th,e role of thiamin deficiency and alcohol in the development of memory impairments in humans i.s complicated by the presence of concurrent liver disease and head injury, together with the possibility that neuropsychologic impairment may precede alcohol abuse (35). An animal model allows eludication of the direct contribu-
265
266
HOMEWOOD, BOND AND McGREGOR
viving to the test phase. Research on animal models of Korsakoff's syndrome has focussed on the acquisition of learned behaviours, rather than on memory or memory processes. Thus, in the present study, we employed the radial-arm maze, as described by Olton and Samuelson (27), as the measure of memory. The maze has two advantages in the present context. First, the "'win-shift" strategy used to efficiently solve the maze is analogous to the "delayed nonmatching to sample" task on which amnesic humans and nonhuman primates are impaired (34). Second, once the task has been acquired, memory mechanisms can be taxed directly by interpolating a delay between the fourth and fifth of the eight choices. It was anticipated that increasing the time over which material had to be retained would magnify possible treatment effects. Olton, Becker and Handelmann (28) suggested there are two types of memorial processes used in the radial-arm maze. Items in reference memory are those that remain useful from trial to trial and may be broadly categorised as the rules of the maze, e.g., food is concealed at the end of the arms and is not replaced if eaten during a daily trial. Working memory items, on the other hand, are useful for only a single daily trial, and must be updated for successful performance on the next trial. The position of each arm visited during a daily trial is believed to be held, in working memory. The technique most commonly used to induce long-term consumption of ethanol with adequate nutrition involves incorporating the ethanol into a liquid diet replete with essential vitamins and minerals (5). However, some doubt exists about the nutritional status of animals treated in this way because recent data suggests intestinal absorption of thiamin may be impaired by long-term ethanol administration (15). In both animals and humans, thiamin status can be assessed by measuring the activity of the thiamin-dependent red blood cell enzyme transketolase (33). The present study comprised two experiments. In the first, we examined the effects of long-term ingestion of alcohol, in the presence of good nutrition. Thiamin levels were assessed in both experimental and control animals. The second experiment was an investigation of the effects of thiamin deficiency in the rat. In both experiments, the radial-arm maze was employed to investigate the effect of treatment on working and reference memory. EXPERIMENT 1 METHOD
Subjects Subjects were 45 experimentally naive male Sprague-Dawley rats obtained from the Central Animal House at Macquarie University. At the start of the study the mean age of the animals was 108 days (range 97-113 days) and the mean weight was 420 grams. The rats were randomly allocated to one of three groups (chow, alcohol or sucrose) and housed individually in a temperature-controlled (23 ~ 2°C) animal house on a normal 12:12 h light:dark cycle (lights on from 0700 to 1900).
Diets The dietary manipulation phase of the experiment lasted for 150 days. Throughout this period the alcohol group was fed, ad lib, a Sustagen-based (Mead-Johnson) liquid diet which contained 5% by volume 95% pure ethanol. Sustagen is replete with thiamin, containing approximately 1 mg thiamin HCI per 100 g. Pentavite (Nicholas), a liquid multivitamin preparation which
contains 1.2 mg thiamin HC1 per ml, was added to the Sustagen mixture at a concentration of 6.9 ml/litre. Animals in the sucrose group were fed an identical liquid diet based on Sustagen and fortified with Pentavite except that sucrose was substituted isocalorically for ethanol. Intake of individual rats in the sucrose group was yoked to the intake of individual rats in the alcohol group on the previous day (pair feeding). The liquid diets provided 35% of daily calories as ethanol or sucrose. Diets were prepared fresh daily and presented in Richter tubes. Consumption was recorded once a day. Over the last ten days of the diet phase the alcohol content of the diet was reduced one percent every two days to avoid adverse effects associated with abrupt withdrawal (30). Throughout this period rats in the chow control group were maintained ad lib on standard Barastoc laboratory chow and water. For a six-week recovery period rats in the alcohol and sucrose groups were fed and watered in a manner identical to the chow group, with unrestricted access to lab chow and water. Following the recovery period all animals were placed on a food deprivation schedule and maintained at 85% of free-feeding body weight. Rats were fed immediately after maze testing. Water was available ad lib in the home cage.
Thiamin Testing During the last month of the dietary manipulation phase, 5 ml of blood was taken from the tail of four animals randomly chosen from each of the three groups. Animals were anaesthetised with 0.33 ml/100 mg body weight Equithesin prior to this procedure. The blood sample had heparin added to prevent clotting and was chilled until analysis. Two indices of thiamin levels were obtained using the method of Smeets, Muller and Wall (33). Thiamin status was estimated through the activity of transkelotase, a red blood cell enzyme whose activity is dependent on levels of the metabolically active form of thiamin, thiamin pyrophosphate (TPP). TPP is an essential coenzyme in transketolase reaction of the pentose pathway. The first measure, erythrocyte transketolase activity (ETKA), assesses the activity of the transkelotase enzyme itself, in terms of the number of micromoles of sedo-heptulose phosphate produced per minute per volume of erythrocytes. Because the activity of the transkelotase enzyme is related directly to the amount of TPP (the active form of thiamin) the higher the activity the better the thiamin nutrition. A low ETKA score may be indicative of thiamin deficiency per se, or reduced binding of the transkelotase enzyme with TPP. Thus a second index of thiamin status (TPP%) is derived, the percentage increase in activity above the original level when TPP is added in vitro. The higher the TPP% effect the greater the level of thiamin deficiency.
Behavioral Testing Rats were approximately 300 days old when behavioural testing began. An eight-arm radial maze, modelled after that described by Olton and Samuelson (27), was employed. The maze was located in a 3 x 3.5 m room, with numerous extramaze visual cues (observer, sink, cupboard, poster, one-way mirror) and was elevated 64 cm above the floor. It was of plywood construction and matt grey in colour. The centre platform was octagonal in shape with a diameter of 85 cm. Guillotine doors operated from a central point and controlled access to the arms. Eight arms radiated at equal angles from the platform. Each arm was 70 cm long and 9 cm wide, with a 3 cm wall around the sides. A barrier 12 cm in height was located near each entrance
ALCOHOL CONSUMPTION AND THIAMIN DEFICIENCY
to prevent animals climbing to adjacent arms without reentering the central platform. A recessed food cup, 1 cm in diameter, was located at the end of each arm. A single trial was run each day and trials were separated by a 48-h period. There were three phases in the behavioural testing: pretraining, trials-to-criterion, and delay testing. Four pretraining trials were given. Currants were placed in each food cup and scattered throughout the maze. The rat was then placed in the maze for a ten-minute period. The guillotine doors were raised to permit the rats to explore. At the end of each trial the maze was wiped clean with a damp cloth. After pretraining, the trials-to-criterion phase of the experiment commenced. A currant was hidden in the food cup at the end of each arm and was not replaced during a trial. The fooddeprived rat was placed in the centre platform with the guillotine doors closed. After a 10-second interval all doors were opened and the rat was allowed to choose an arm. An arm entry was counted when the rat placed all four paws in the arm. After returning to the central platform, the guillotine doors were closed for approximately ten seconds to prevent response chaining, e.g., the rat always choosing an adjacent door. The doors were then opened and the rat permitted to enter another arm. This procedure continued until the rat had entered all eight arms or 10 minutes had elapsed. The rat was then returned to the home cage. Optimal performance consists of recovering all eight currants in eight entries. Entries to arms previously visited were scored as errors. The trials-to-criterion phase of the experiment ended when the rat had made seven correct entries in the first eight choices, and all eight arms correct in the first ten choices, for three consecutive trials. The procedure for the delay testing was as follows. The rat was allowed to make four choices, in a maximum of ten minutes, in the manner outlined above. When the rat entered the centre platform after choice four, it was removed from the maze, returned to its home cage and placed in a holding area. After a specified delay, the rat was again placed in the centre platform with the doors closed for 10 seconds before being allowed to make an additional four choices. Only arms not previously visited during the trial were baited. Delay intervals of 5, 20. 60, 120 and 240 minutes were used. There were three trials at each delay interval. Testing commenced with the 5-minute delay trials. Three blocks of trials with one trial at the four remaining delay intervals were then presented, with order within each block determined by a series of Latin squares. Order of presentation was counterbalanced across the three groups of rats. Each block of delay trials was separated by a no-delay trial, where the rat was allowed to make eight uninterrupted choices. The dependent variable for the delay testing was the number of errors made on choices 5-8, with entries into arms chosen on choices I--4 counted as errors. At the end of testing rats were sacrificed and brains preserved for further study. No macroscopic differences were evident between groups. RESULTS
Mortalio,
267
TABLE
Experiment I
Month I Month 2 Month 3 Month 4 Month 5 Month 6 Month 7
Chow
Alcohol
Sucrose
414 (24) 473 (24) 500
421 (18) 434 (20) 492
422 (23) 422 117) 478
(27)
(18)
(21)
513 130) 532 (30) 543 (33) 551 (28)
521 (22) 543 131) 559 (32) 565 (361
513 121) 521 (26) ' 537 (25) 549 (22)
Standard deviations are in parentheses.
sser (~ = 0.47) correction was performed on monthly body weights during the dietary manipulation phase. As expected, it yielded a highly significant effect for month, F(6,118.4) = 6 2 3 , p
0741-8329/91 $3.00 + .00
The Effects of Chronic Alcohol Consumption Thiamin Deficiency on Radial-Arm Maze Performance in the Rat JUDI HOMEWOOD,
or
N I G E L W. B O N D A N D J A N E C. M c G R E G O R
School o f Behavioural Sciences, Macquarie University, Sydney, Australia N S W 2109
R e c e i v e d 22 A u g u s t 1990; A c c e p t e d 9 J a n u a r y 1991 HOMEWOOD, J., N. W. BOND AND J. C. McGREGOR. The effects of chronic alcohol consumption or thiamin deficiency on radial-arm maze performance in the rat. ALCOHOL 8(41 265-272, 1991.--Experiment I tested the hypothesis that chronic consumption of ethanol, in the presence of good nutrition, is associated with impairments in memory. Rats were fed a vitaminfortified ethanoI-Sustagen diet with 35% ethanol-derived calories for 150 days. Two control groups were used. The first was pair fed a Sustagen fnix in which sucrose had been substituted for ethanol. The second control group received ad lib access to standard laboratory chow and water. All animals were then placed on the chow and water diet for six weeks prior to testing in an eight-arm radial maze. There was a significant, but modest, difference between groups in the number of trials to reach a criterion level of seven correct from the first eight choices and all eight correct in ten choices. However, there were no differences between the groups when delays (0, 5, 20, 60, 120 and 240 minutes) were imposed between the 4th and 5th choices. In Experiment 2. a thiamin-deficient group was compared with pair-fed and ad lib control groups on the same task. The thiamin-deficient group took more trials to reach criterion but the groups did not differ in the delay testing phase. The results are discussed in the context of reference/working memory distinctions, and the literature on remediation of cognitive deficits associated with alcoholism. Alcohol
Thiamine deficiency
Working memory
Reference memory
Korsakoff's syndrome
Rats
tion of the putative etiological factors of alcohol-related memory impairment. However, as Darnton-Hill (9) points out in his recent review, animal studies may not necessarily mimic the human condition in terms of pattern of drinking and poor general health status. Nevertheless, in apparently well-nourished rodents. chronic ethanol consumption has been found to result in loss of hippocampal pyramidal and dentate gyrus granule cells (42) although with abstinence, dendritic spine density may return to control levels (7,24). There are several reports of behavioural impairments in alcohol-treated rats, in paradigms such as shuttlebox avoidance (41), Hebb-Williams maze (5), differential reinforcement of low rates of responding (10) and the radial-arm maze (1), although some studies have failed to find an effect of alcohol exposure (3, 32, 36). Thiamin deficiency has been produced experimentally in a number of species, most commonly the rat (23,25), but also in cats (22) and monkeys (44). There is general agreement that the combination of a thiamin-deficient diet and daily injections of pyrithiamin produces biochemical and neuropathological abnormalities more similar in nature and distribution to those found in human WE than are found with dietary treatment alone (6). In rats, pyrithiamin treatment results in damage to thalamic, hypothalamic, brain stem and cerebellar regions (6). Several authors have reported that thiamin deficiency results in deficits in active ' and passive avoidance learning (23, 25, 39). We have argued elsewhere that the generality of these effects is in doubt (19). Briefly, previous work has been flawed by use of immature animals, lack of internal consistency, and insufficient subjects sur-
STUDIES of human alcoholics have shown that excessive use of ethanol over long periods of time is associated with morphological changes and a variety of cognitive deficits. Recently detoxified alcoholics show a characteristic pattern of impairment on neuropsychological tests with deficits in nonverbal abstracting ability, complex problem solving, perceptual-spatial motor performance, and some tests of learning and memory (29). Neuropathological findings include the presence of cerebral atrophy, as assessed by computed axial tomography examination and autopsy, and reduced brain weight (17, 37, 43). The most extensively studied form of alcohol-related brain damage is Korsakoff's syndrome. Korsakoff's syndrome is characterised by profound anterograde amnesia, in addition to visuo-perceptive and problem solving deficits. Impairments in memory found in alcoholics have usually been considered to result from malnutrition, in particular thiamin deficiency, rather than a direct effect of ethanol per se (38). Thiamin deficiency is manifested clinically by neurological abnormalities termed Wernicke's encephalopathy (WE). Alcoholics are the group most commonly at risk for WE because of inadequate intake, increased need, poor storage, and impaired absorption of thiamin (20). The assessment of th,e role of thiamin deficiency and alcohol in the development of memory impairments in humans i.s complicated by the presence of concurrent liver disease and head injury, together with the possibility that neuropsychologic impairment may precede alcohol abuse (35). An animal model allows eludication of the direct contribu-
265
266
HOMEWOOD, BOND AND McGREGOR
viving to the test phase. Research on animal models of Korsakoff's syndrome has focussed on the acquisition of learned behaviours, rather than on memory or memory processes. Thus, in the present study, we employed the radial-arm maze, as described by Olton and Samuelson (27), as the measure of memory. The maze has two advantages in the present context. First, the "'win-shift" strategy used to efficiently solve the maze is analogous to the "delayed nonmatching to sample" task on which amnesic humans and nonhuman primates are impaired (34). Second, once the task has been acquired, memory mechanisms can be taxed directly by interpolating a delay between the fourth and fifth of the eight choices. It was anticipated that increasing the time over which material had to be retained would magnify possible treatment effects. Olton, Becker and Handelmann (28) suggested there are two types of memorial processes used in the radial-arm maze. Items in reference memory are those that remain useful from trial to trial and may be broadly categorised as the rules of the maze, e.g., food is concealed at the end of the arms and is not replaced if eaten during a daily trial. Working memory items, on the other hand, are useful for only a single daily trial, and must be updated for successful performance on the next trial. The position of each arm visited during a daily trial is believed to be held, in working memory. The technique most commonly used to induce long-term consumption of ethanol with adequate nutrition involves incorporating the ethanol into a liquid diet replete with essential vitamins and minerals (5). However, some doubt exists about the nutritional status of animals treated in this way because recent data suggests intestinal absorption of thiamin may be impaired by long-term ethanol administration (15). In both animals and humans, thiamin status can be assessed by measuring the activity of the thiamin-dependent red blood cell enzyme transketolase (33). The present study comprised two experiments. In the first, we examined the effects of long-term ingestion of alcohol, in the presence of good nutrition. Thiamin levels were assessed in both experimental and control animals. The second experiment was an investigation of the effects of thiamin deficiency in the rat. In both experiments, the radial-arm maze was employed to investigate the effect of treatment on working and reference memory. EXPERIMENT 1 METHOD
Subjects Subjects were 45 experimentally naive male Sprague-Dawley rats obtained from the Central Animal House at Macquarie University. At the start of the study the mean age of the animals was 108 days (range 97-113 days) and the mean weight was 420 grams. The rats were randomly allocated to one of three groups (chow, alcohol or sucrose) and housed individually in a temperature-controlled (23 ~ 2°C) animal house on a normal 12:12 h light:dark cycle (lights on from 0700 to 1900).
Diets The dietary manipulation phase of the experiment lasted for 150 days. Throughout this period the alcohol group was fed, ad lib, a Sustagen-based (Mead-Johnson) liquid diet which contained 5% by volume 95% pure ethanol. Sustagen is replete with thiamin, containing approximately 1 mg thiamin HCI per 100 g. Pentavite (Nicholas), a liquid multivitamin preparation which
contains 1.2 mg thiamin HC1 per ml, was added to the Sustagen mixture at a concentration of 6.9 ml/litre. Animals in the sucrose group were fed an identical liquid diet based on Sustagen and fortified with Pentavite except that sucrose was substituted isocalorically for ethanol. Intake of individual rats in the sucrose group was yoked to the intake of individual rats in the alcohol group on the previous day (pair feeding). The liquid diets provided 35% of daily calories as ethanol or sucrose. Diets were prepared fresh daily and presented in Richter tubes. Consumption was recorded once a day. Over the last ten days of the diet phase the alcohol content of the diet was reduced one percent every two days to avoid adverse effects associated with abrupt withdrawal (30). Throughout this period rats in the chow control group were maintained ad lib on standard Barastoc laboratory chow and water. For a six-week recovery period rats in the alcohol and sucrose groups were fed and watered in a manner identical to the chow group, with unrestricted access to lab chow and water. Following the recovery period all animals were placed on a food deprivation schedule and maintained at 85% of free-feeding body weight. Rats were fed immediately after maze testing. Water was available ad lib in the home cage.
Thiamin Testing During the last month of the dietary manipulation phase, 5 ml of blood was taken from the tail of four animals randomly chosen from each of the three groups. Animals were anaesthetised with 0.33 ml/100 mg body weight Equithesin prior to this procedure. The blood sample had heparin added to prevent clotting and was chilled until analysis. Two indices of thiamin levels were obtained using the method of Smeets, Muller and Wall (33). Thiamin status was estimated through the activity of transkelotase, a red blood cell enzyme whose activity is dependent on levels of the metabolically active form of thiamin, thiamin pyrophosphate (TPP). TPP is an essential coenzyme in transketolase reaction of the pentose pathway. The first measure, erythrocyte transketolase activity (ETKA), assesses the activity of the transkelotase enzyme itself, in terms of the number of micromoles of sedo-heptulose phosphate produced per minute per volume of erythrocytes. Because the activity of the transkelotase enzyme is related directly to the amount of TPP (the active form of thiamin) the higher the activity the better the thiamin nutrition. A low ETKA score may be indicative of thiamin deficiency per se, or reduced binding of the transkelotase enzyme with TPP. Thus a second index of thiamin status (TPP%) is derived, the percentage increase in activity above the original level when TPP is added in vitro. The higher the TPP% effect the greater the level of thiamin deficiency.
Behavioral Testing Rats were approximately 300 days old when behavioural testing began. An eight-arm radial maze, modelled after that described by Olton and Samuelson (27), was employed. The maze was located in a 3 x 3.5 m room, with numerous extramaze visual cues (observer, sink, cupboard, poster, one-way mirror) and was elevated 64 cm above the floor. It was of plywood construction and matt grey in colour. The centre platform was octagonal in shape with a diameter of 85 cm. Guillotine doors operated from a central point and controlled access to the arms. Eight arms radiated at equal angles from the platform. Each arm was 70 cm long and 9 cm wide, with a 3 cm wall around the sides. A barrier 12 cm in height was located near each entrance
ALCOHOL CONSUMPTION AND THIAMIN DEFICIENCY
to prevent animals climbing to adjacent arms without reentering the central platform. A recessed food cup, 1 cm in diameter, was located at the end of each arm. A single trial was run each day and trials were separated by a 48-h period. There were three phases in the behavioural testing: pretraining, trials-to-criterion, and delay testing. Four pretraining trials were given. Currants were placed in each food cup and scattered throughout the maze. The rat was then placed in the maze for a ten-minute period. The guillotine doors were raised to permit the rats to explore. At the end of each trial the maze was wiped clean with a damp cloth. After pretraining, the trials-to-criterion phase of the experiment commenced. A currant was hidden in the food cup at the end of each arm and was not replaced during a trial. The fooddeprived rat was placed in the centre platform with the guillotine doors closed. After a 10-second interval all doors were opened and the rat was allowed to choose an arm. An arm entry was counted when the rat placed all four paws in the arm. After returning to the central platform, the guillotine doors were closed for approximately ten seconds to prevent response chaining, e.g., the rat always choosing an adjacent door. The doors were then opened and the rat permitted to enter another arm. This procedure continued until the rat had entered all eight arms or 10 minutes had elapsed. The rat was then returned to the home cage. Optimal performance consists of recovering all eight currants in eight entries. Entries to arms previously visited were scored as errors. The trials-to-criterion phase of the experiment ended when the rat had made seven correct entries in the first eight choices, and all eight arms correct in the first ten choices, for three consecutive trials. The procedure for the delay testing was as follows. The rat was allowed to make four choices, in a maximum of ten minutes, in the manner outlined above. When the rat entered the centre platform after choice four, it was removed from the maze, returned to its home cage and placed in a holding area. After a specified delay, the rat was again placed in the centre platform with the doors closed for 10 seconds before being allowed to make an additional four choices. Only arms not previously visited during the trial were baited. Delay intervals of 5, 20. 60, 120 and 240 minutes were used. There were three trials at each delay interval. Testing commenced with the 5-minute delay trials. Three blocks of trials with one trial at the four remaining delay intervals were then presented, with order within each block determined by a series of Latin squares. Order of presentation was counterbalanced across the three groups of rats. Each block of delay trials was separated by a no-delay trial, where the rat was allowed to make eight uninterrupted choices. The dependent variable for the delay testing was the number of errors made on choices 5-8, with entries into arms chosen on choices I--4 counted as errors. At the end of testing rats were sacrificed and brains preserved for further study. No macroscopic differences were evident between groups. RESULTS
Mortalio,
267
TABLE
Experiment I
Month I Month 2 Month 3 Month 4 Month 5 Month 6 Month 7
Chow
Alcohol
Sucrose
414 (24) 473 (24) 500
421 (18) 434 (20) 492
422 (23) 422 117) 478
(27)
(18)
(21)
513 130) 532 (30) 543 (33) 551 (28)
521 (22) 543 131) 559 (32) 565 (361
513 121) 521 (26) ' 537 (25) 549 (22)
Standard deviations are in parentheses.
sser (~ = 0.47) correction was performed on monthly body weights during the dietary manipulation phase. As expected, it yielded a highly significant effect for month, F(6,118.4) = 6 2 3 , p
