Journal of Neuroscience Research 3: 34 1-35 1 ( 1 978)
Biochemical Markers of Ethanol Effects on Brain W. R. Klemm and R. L. Engen Department of Biology, Texas A&M University, College Station (W. R.K.); and Department of Veterinary Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Iowa State University, Ames (R.L.E.)
Because sialic acid is a potential biochemical marker of membrane development or alteration, we compared acute and chronic ethanol effects on sialic acid. Experiments were conducted with 50 adult male Wistar rats (- 400 gm), housed in groups of five. Rats drank ad libitum a vitamin-fortified diet (Nutrament) that was adulterated with ethanol; ethanol intake averaged for each rat 10-18 gm/kg/day. Controls were fed an equal total volume, made isocaloric with sucrose. Rats were sacrificed weekly for four weeks, and an acute challenge dose of ethanol (2 gm/kg, intraperitoneally) was given 45 minutes before sacrifice of both control and ethanol-consuming rats. Some controls were challenged only with saline. We replicated our earlier findings of regional differences in sialic acid and in cerebellar deoxyribose (measured as a necessary adjunct in the autoanalyzer modification of the Warren-Delmotte methods) In the saline-challenged controls, levels of both compounds were higher at four weeks than after one week. Similar increases occurred also in the chronic ethanol-consuming group, but not in the ethanol-challenged controls, which had significantly lower values. Results in saline-challenged controls suggest that the chronic treatment either 1) created a tolerance which protected cells from damage by the challenge dose of ethanol, or 2) killed neurons, thus promoting proliferation of glial cells. Key words: sialic acid, 2deoxyribose, brain, ethanol, tolerance, maturation, development
This study is the first investigation of ethanol effect on levels of sialic acid (SA) in whole-brain tissue. Sialic acid is a generic term for a family of nitrogen-containing acidic sugars that are key components of structural polysaccharides. These polysaccharides are covalently bound to both lipids (gangliosides) and to proteins (glycoproteins); and the A preliminary report of this research was made at the Seventh Annual Meeting of the Society for Neuroscience, November 7, 1977, Anaheim, California. Address reprint requests to Dr W.R.Klemm, Department of Biology, Texas A&M University, College Station, TX 11843.
0360-4012/78/0305/6-0341$02.30
0 1978 Alan R. Liss, Inc
342
Klemm and Engen
oligosaccharide side chains of these macromolecules commonly contain negatively charged SA residues [Lehninger, 1975; Rosenberg and Schengrund, 19761 . There are several valid reasons for investigating the effect of ethanol on SA. First, the gangliosidic and glycoprotein parent structures are integral components of cell membranes, and SA residues are exposed terminal groups projecting into extracellular space [Wherret and McIlwain, 1962; Brunngraber et al, 1967; Warren, 19761. These stereochemical features enable SA to interact with the cationic groups of many metals, drugs, hormones, and neurotransmitters [Rosenberg and Schengrund, 19761 . The terminal OH group of ethanol makes it ideal for forming hydrogen bonds with the nitrogen source of electronegativity of SA; moreover, ethanol has many actions on diverse neurotransmitter systems [Kalant, 1975; Rahwan, 19741. Secondly, SA is a component of Na+,K+-ATPase, the enzyme which mediates active transport in nerve cells and a compound which is markedly inhibited by ethanol [Israel et al, 1966; Grenell, 19721 and which in turn underlies much of the profound influence of ethanol on nerve impulse activity and associated functions [Klemm, in press]. Therefore, we chose to evaluate the effects of both acute and chronic ethanol consymption of SA levels in various sections of the brain of rats. Several clear differential effects were observed. The SA analysis method also included a measure of 2-deoxyribose (DR) levels, which also proved to be a sensitive index of ethanol effect. METHODS Voluntary Ethanol Intake
A principal prerequisite was to develop a method for causing voluntary intake of large amounts of ethanol. Several prior reports indicated that restricting intake to a liquid diet adulterated with ethanol caused intake levels as large as 12 gm ethanol/kg/day after several weeks [Lieber et al, 1963; Pilstrom et al, 19731. We wished to produce high levels of intake immediately, and so chose to use a liquid diet which had a robust Dutch chocolate flavor that rats like; consumption of food in rats is greatly influenced by taste [Ernits and Corbit, 19731 . Another feature we incorporated was group housing, in the expectation that social interactions would facilitate ethanol intake (Randall and Lester, 1975). Experiments were conducted with 50 adult male Wistar rats, housed in groups of five in plastic cages (18 X 10 X 8 cm), bedding. At the beginning, rats averaged 407 gm in weight. To promote excessive consumption of ethanol, rats had restricted access to commercial rat pellets for one month, with a 13% average weight loss. Then rats were placed on experimental diets and were weighed every other day. The basic medium for supplying ethanol was Nutrament, Dutch Chocolate flavor. This is a human diet product, manufactured by Mead Johnson and Co; it contains 23 gm of protein and 360 calories per 12 oz serving. The diet solutions were fortified with a commercial vitamin diet fortification mix (3 gm/liter), manufactured by ICN Pharmaceuticals Co. Two groups of rats received Nutrament only, with enough added sucrose to make the diet isocaloric with the diet of rats that received Nutrament with ethanol. The amount of ethanol in the diet was gradually increased, from a beginning level of 5% (876 total calories/liter, 30% from ethanol), to 5.8% after three days (873 calories/liter, 35% from ethanol), to 7.5% at seven days (875 calories/liter, 45% from ethanol), to 15% at 18 days (1,75 1 calories/liter, 45% from ethanol and 28% from sucrose).
Biochemical Indices of Ethanol Effects
34 3
The concentration of ethanol in the diet was gradually increased in the hope that this would lead to greater intake of ethanol. As it turned out, rats were consuming almost maximum levels with the lowest concentration, 5%. As the ethanol concentration was changed, the diet of control rats was adjusted accordingly with added sucrose so that calories consumed each day were equivalent for all groups. The ethanol group’s supply of diet was replenished as often as necessary to keep rats satiated. Control groups were limited to the average fluid volume consumed by the ethanol groups on the preceding day. Calculations for assuring that all groups had equal caloric intake were based on the following caloric densities for each substance: Nutrament, 1.03 calories/ml; ethanol (95%.w/v), 5.25 calories/ml; and sucrose (87% w/v solution), 3.5 calories/ml. Experimental Design
Selected groups of rats were killed every seven days for the four weeks of the experiment. Forty-five minutes prior to killing all rats were given a challenge acute dose of ethanol (2 gm/kg, intraperitoneally) or a comparable volume of saline as a control. Salinechallenged controls were killed at the first and fouth weeks. Ethanol-challenged rats, both controls and those consuming ethanol continuously, were killed each week (one hatch of sanples was Lost due to lahnratory accident). Sample Preparation and Analysis
Rats were killed by decapitation, and the brains were removed and cut into four sections within two minutes; the sections were then frozen in liquid nitrogen. The sections were identified as: 1) forebrain (cut was immediately behind optic chiasma and extended through the hippocampus); 2) midbrain (caudal border of inferior colliculi); 3) medulla; and 4) cerebellum. Tissue content of total sialic acid was determined by a continuous-flow colorimetric method [Engen et al, 19741, which basically combined the the two established procedures, by Warren [I9591 and by Delmotte [1968], with a two-channel autoanalyzer. In this analysis, 2-deoxyribose interferes with the color reaction, but calculations were corrected by exploiting the differences in the characteristic absorption maxima for sialic acid and 2-deoxyribose (DR) (530 and 550 nm, respectively). Samples of mixed standards and of brain were prepared as acid hydrolysates, as described by both Warren and Delmotte. Sialic acid standards of 2 0 , 4 0 , 8 0 , and 100 mg/liter and DR standards of 1 , 2 , 4 , and 8 mg/liter were prepared in trichloroacetic acid (50 gm/liter of physiological saline). Mixed standards were prepared by mixing equal volumes of SA and DR standards. Brain samples were prepared by mixing 0.2 gm of sonified or homogenized brain in 9.8 ml of the previously mentioned trichloroacetic acid solution. All standards and tissue samples were hydrolyzed in glass-stoppered centrifuge tubes for one hour at 80°C. Data were analyzed by regression analysis of variance and subsequept Duncan’s test (Kramer modification for unbalanced data). RESULTS Ethanol Intake and Body Weight Maintenance
The diet was very palatable, and rats consumed massive quantities of ethanol, even at the outset (Fig 1). Increasing concentrations consistently decreased total fluid intake; however, the absolute amount of ethanol consumed was relatively consistent, with a
344
Klemm and Engen IS 0
1
rron
11
6
I1
16
DIET
21
26
DAYS
Fig 1. Excessive consumption of ethanol in rats. Mean daily voluntary fluid intake for each rat that was given ethanol-adulterated Nutrament and for control rats given Nutrament with sufficient sucrose added to provide equal number of calories. The one-day phase shift in the control diet curve is due to limiting fluid intake in control rats to the mean intake of ethanol-drinking rats on the preceding day. Graph shows that total intake was depressed as ethanol concentration was increased from 5 to 15%, but the absolute amount of ethanol ingested remained relatively constant. Note the especially high levels of ethanol consumption, ranging after the first day from 10 up to 18 gm/kg/day, average per rat. Note also that high consumption levels were achieved at the very beginning of the test and maintained throughout.
maximum range of 10 to 18 gm/kg/day for each rat. This is a much greater intake than usually achieved in voluntary intake studies, and the rats might have consumed even more if they had not spent so much time asleep. We have achieved even greater average daily intake levels (-19 gm/kg) in another unpublished study in which a 7.5% ethanol Nutrament diet, with added sucrose, was fed to juvenile rats. The very high consumption is attributed to the palatibility of the diet. Rats were quite aggressive in drinking from a newly filled drinking tube, and would actually fight over access to the fluid. Body weight remained about the same in all groups, indicating the adequacy of our attempt to maintain equivalent nutrition (Fig 2). Over the entire test period, all groups had a slight weight loss. About 85 calories/rat/day were consumed during the early stages of the experiment when a 5% diet was used, with later intake at about 60 calories/rat/day with a 15% ethanol diet. Behavioral Signs of Tolerance
The rats which were drinking ethanol chronically had developed some tolerance even after one week, as indicated by responses of acute challenge dose of alcohol. All control rats were collapsed and hypokinetic within 15 minutes; but all ethanol-consuming rats were standing and moving their heads about, albeit with signs of intoxication. The typical decapitation convulsions were greatly suppressed or totally absent in rats that had chronically consumed ethanol.
Biochemical Indices of Ethanol Effects
345
L
c
r
9
:
300
9
c . CONTROL DIET CONTROL DIET * - - a ETOH DIET
z
q u,
T
200
.
.
.
1
.
1
1
1
1
1
1
1
DAYS
Fig 2. Body weight graph, illustrating that all groups had a similar degree of mild weight loss over the experimental period. Rats in the two control groups received the Same diet, but animals were sacrificed at different times.
The rats that had been consuming ethanol for four weeks were withdrawn from
ethanol during the night preceding the morning’s sacrifice. These rats were highly irritable when taken from the cage, and despite the fact that they had been handled every other day for weighing, they attacked the handler who had been doing the weighing. During attempts to inject them, they scratched vigorously and two of the five rats inflicted bite wounds. The injection of ethanol had a marked calming effect within ten minutes. Control Data
The general regional differences which we had previously reported [Engen and Klemm, 19781 were replicated; SA levels were highest in the forebrain and lowest in the medulla, whereas DR levels were about the same everywhere except for an approximate fourfold increase in the cerebellum. Quite unexpectedly, there were clearly significant increases in both SA and DR in most regions in the rats that were sacrificed three weeks after the first group (Fig 3). The only known differences between the two groups were a three-week age difference and the time of exposure to the diet regimen and the communal living environment. To test for the age factor, data from the rats sacrificed at the first week were compared to data from the same strain of much younger rats in a previous study [Engen and Klemm, 19781; levels of SA were significantly higher in the older rats, but DR levels were lower in the older rats (Table I). Effects of Alcohol
Whereas levels of SA tended to increase in controls over the experimental period, control rats that were given a challenge dose of ethanol 45 minutes before sacrifice had lower levels, especially in the forebrain and medulla (Fig 4). On the other hand, rats which drank ethanol in their daily diet responded differently to the challenge dose of ethanol. The SA levels were not suppressed by the acute ethanol dose, and in fact levels were more comparable to those of saline-challenged controls (Fig 4). The differences among treatments were highly significant [F(2,104); P < 0.0051 ,which was attributable
346
K l e m m and E n g e n
SlALlC ACID 6 0
0 1st
WEEK
5 0
4 0
30 FOREBRAIN
MIDBRAIN
CEREBELLUM
MEDULLA
Fig 3. Brain area differences in total sialic acid and 2-deoxyribose (mean pmoles/gm f SE) in salinechallenged control rats. Also illustrated is the increase in both chemicals which occurred in the group that received three more weeks of exposure to the communal housing and the liquid control diet.
TABLE 1. Comparison With Younger Controls in Previous Study
Brain region Forebrain Midbrain Cerebellum Medulla
Sialic acid xpm/gm i- S.E. Young adultsa Mature adultsb 4.55 4.33 3.43 2.69
f
t k
t
0.08 0.15 0.07 0.08
5.44 i 0.15 5.13 f 0.21 4.01 f 0.14 3.13 i 0.15
P
Biochemical Markers of Ethanol Effects on Brain W. R. Klemm and R. L. Engen Department of Biology, Texas A&M University, College Station (W. R.K.); and Department of Veterinary Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Iowa State University, Ames (R.L.E.)
Because sialic acid is a potential biochemical marker of membrane development or alteration, we compared acute and chronic ethanol effects on sialic acid. Experiments were conducted with 50 adult male Wistar rats (- 400 gm), housed in groups of five. Rats drank ad libitum a vitamin-fortified diet (Nutrament) that was adulterated with ethanol; ethanol intake averaged for each rat 10-18 gm/kg/day. Controls were fed an equal total volume, made isocaloric with sucrose. Rats were sacrificed weekly for four weeks, and an acute challenge dose of ethanol (2 gm/kg, intraperitoneally) was given 45 minutes before sacrifice of both control and ethanol-consuming rats. Some controls were challenged only with saline. We replicated our earlier findings of regional differences in sialic acid and in cerebellar deoxyribose (measured as a necessary adjunct in the autoanalyzer modification of the Warren-Delmotte methods) In the saline-challenged controls, levels of both compounds were higher at four weeks than after one week. Similar increases occurred also in the chronic ethanol-consuming group, but not in the ethanol-challenged controls, which had significantly lower values. Results in saline-challenged controls suggest that the chronic treatment either 1) created a tolerance which protected cells from damage by the challenge dose of ethanol, or 2) killed neurons, thus promoting proliferation of glial cells. Key words: sialic acid, 2deoxyribose, brain, ethanol, tolerance, maturation, development
This study is the first investigation of ethanol effect on levels of sialic acid (SA) in whole-brain tissue. Sialic acid is a generic term for a family of nitrogen-containing acidic sugars that are key components of structural polysaccharides. These polysaccharides are covalently bound to both lipids (gangliosides) and to proteins (glycoproteins); and the A preliminary report of this research was made at the Seventh Annual Meeting of the Society for Neuroscience, November 7, 1977, Anaheim, California. Address reprint requests to Dr W.R.Klemm, Department of Biology, Texas A&M University, College Station, TX 11843.
0360-4012/78/0305/6-0341$02.30
0 1978 Alan R. Liss, Inc
342
Klemm and Engen
oligosaccharide side chains of these macromolecules commonly contain negatively charged SA residues [Lehninger, 1975; Rosenberg and Schengrund, 19761 . There are several valid reasons for investigating the effect of ethanol on SA. First, the gangliosidic and glycoprotein parent structures are integral components of cell membranes, and SA residues are exposed terminal groups projecting into extracellular space [Wherret and McIlwain, 1962; Brunngraber et al, 1967; Warren, 19761. These stereochemical features enable SA to interact with the cationic groups of many metals, drugs, hormones, and neurotransmitters [Rosenberg and Schengrund, 19761 . The terminal OH group of ethanol makes it ideal for forming hydrogen bonds with the nitrogen source of electronegativity of SA; moreover, ethanol has many actions on diverse neurotransmitter systems [Kalant, 1975; Rahwan, 19741. Secondly, SA is a component of Na+,K+-ATPase, the enzyme which mediates active transport in nerve cells and a compound which is markedly inhibited by ethanol [Israel et al, 1966; Grenell, 19721 and which in turn underlies much of the profound influence of ethanol on nerve impulse activity and associated functions [Klemm, in press]. Therefore, we chose to evaluate the effects of both acute and chronic ethanol consymption of SA levels in various sections of the brain of rats. Several clear differential effects were observed. The SA analysis method also included a measure of 2-deoxyribose (DR) levels, which also proved to be a sensitive index of ethanol effect. METHODS Voluntary Ethanol Intake
A principal prerequisite was to develop a method for causing voluntary intake of large amounts of ethanol. Several prior reports indicated that restricting intake to a liquid diet adulterated with ethanol caused intake levels as large as 12 gm ethanol/kg/day after several weeks [Lieber et al, 1963; Pilstrom et al, 19731. We wished to produce high levels of intake immediately, and so chose to use a liquid diet which had a robust Dutch chocolate flavor that rats like; consumption of food in rats is greatly influenced by taste [Ernits and Corbit, 19731 . Another feature we incorporated was group housing, in the expectation that social interactions would facilitate ethanol intake (Randall and Lester, 1975). Experiments were conducted with 50 adult male Wistar rats, housed in groups of five in plastic cages (18 X 10 X 8 cm), bedding. At the beginning, rats averaged 407 gm in weight. To promote excessive consumption of ethanol, rats had restricted access to commercial rat pellets for one month, with a 13% average weight loss. Then rats were placed on experimental diets and were weighed every other day. The basic medium for supplying ethanol was Nutrament, Dutch Chocolate flavor. This is a human diet product, manufactured by Mead Johnson and Co; it contains 23 gm of protein and 360 calories per 12 oz serving. The diet solutions were fortified with a commercial vitamin diet fortification mix (3 gm/liter), manufactured by ICN Pharmaceuticals Co. Two groups of rats received Nutrament only, with enough added sucrose to make the diet isocaloric with the diet of rats that received Nutrament with ethanol. The amount of ethanol in the diet was gradually increased, from a beginning level of 5% (876 total calories/liter, 30% from ethanol), to 5.8% after three days (873 calories/liter, 35% from ethanol), to 7.5% at seven days (875 calories/liter, 45% from ethanol), to 15% at 18 days (1,75 1 calories/liter, 45% from ethanol and 28% from sucrose).
Biochemical Indices of Ethanol Effects
34 3
The concentration of ethanol in the diet was gradually increased in the hope that this would lead to greater intake of ethanol. As it turned out, rats were consuming almost maximum levels with the lowest concentration, 5%. As the ethanol concentration was changed, the diet of control rats was adjusted accordingly with added sucrose so that calories consumed each day were equivalent for all groups. The ethanol group’s supply of diet was replenished as often as necessary to keep rats satiated. Control groups were limited to the average fluid volume consumed by the ethanol groups on the preceding day. Calculations for assuring that all groups had equal caloric intake were based on the following caloric densities for each substance: Nutrament, 1.03 calories/ml; ethanol (95%.w/v), 5.25 calories/ml; and sucrose (87% w/v solution), 3.5 calories/ml. Experimental Design
Selected groups of rats were killed every seven days for the four weeks of the experiment. Forty-five minutes prior to killing all rats were given a challenge acute dose of ethanol (2 gm/kg, intraperitoneally) or a comparable volume of saline as a control. Salinechallenged controls were killed at the first and fouth weeks. Ethanol-challenged rats, both controls and those consuming ethanol continuously, were killed each week (one hatch of sanples was Lost due to lahnratory accident). Sample Preparation and Analysis
Rats were killed by decapitation, and the brains were removed and cut into four sections within two minutes; the sections were then frozen in liquid nitrogen. The sections were identified as: 1) forebrain (cut was immediately behind optic chiasma and extended through the hippocampus); 2) midbrain (caudal border of inferior colliculi); 3) medulla; and 4) cerebellum. Tissue content of total sialic acid was determined by a continuous-flow colorimetric method [Engen et al, 19741, which basically combined the the two established procedures, by Warren [I9591 and by Delmotte [1968], with a two-channel autoanalyzer. In this analysis, 2-deoxyribose interferes with the color reaction, but calculations were corrected by exploiting the differences in the characteristic absorption maxima for sialic acid and 2-deoxyribose (DR) (530 and 550 nm, respectively). Samples of mixed standards and of brain were prepared as acid hydrolysates, as described by both Warren and Delmotte. Sialic acid standards of 2 0 , 4 0 , 8 0 , and 100 mg/liter and DR standards of 1 , 2 , 4 , and 8 mg/liter were prepared in trichloroacetic acid (50 gm/liter of physiological saline). Mixed standards were prepared by mixing equal volumes of SA and DR standards. Brain samples were prepared by mixing 0.2 gm of sonified or homogenized brain in 9.8 ml of the previously mentioned trichloroacetic acid solution. All standards and tissue samples were hydrolyzed in glass-stoppered centrifuge tubes for one hour at 80°C. Data were analyzed by regression analysis of variance and subsequept Duncan’s test (Kramer modification for unbalanced data). RESULTS Ethanol Intake and Body Weight Maintenance
The diet was very palatable, and rats consumed massive quantities of ethanol, even at the outset (Fig 1). Increasing concentrations consistently decreased total fluid intake; however, the absolute amount of ethanol consumed was relatively consistent, with a
344
Klemm and Engen IS 0
1
rron
11
6
I1
16
DIET
21
26
DAYS
Fig 1. Excessive consumption of ethanol in rats. Mean daily voluntary fluid intake for each rat that was given ethanol-adulterated Nutrament and for control rats given Nutrament with sufficient sucrose added to provide equal number of calories. The one-day phase shift in the control diet curve is due to limiting fluid intake in control rats to the mean intake of ethanol-drinking rats on the preceding day. Graph shows that total intake was depressed as ethanol concentration was increased from 5 to 15%, but the absolute amount of ethanol ingested remained relatively constant. Note the especially high levels of ethanol consumption, ranging after the first day from 10 up to 18 gm/kg/day, average per rat. Note also that high consumption levels were achieved at the very beginning of the test and maintained throughout.
maximum range of 10 to 18 gm/kg/day for each rat. This is a much greater intake than usually achieved in voluntary intake studies, and the rats might have consumed even more if they had not spent so much time asleep. We have achieved even greater average daily intake levels (-19 gm/kg) in another unpublished study in which a 7.5% ethanol Nutrament diet, with added sucrose, was fed to juvenile rats. The very high consumption is attributed to the palatibility of the diet. Rats were quite aggressive in drinking from a newly filled drinking tube, and would actually fight over access to the fluid. Body weight remained about the same in all groups, indicating the adequacy of our attempt to maintain equivalent nutrition (Fig 2). Over the entire test period, all groups had a slight weight loss. About 85 calories/rat/day were consumed during the early stages of the experiment when a 5% diet was used, with later intake at about 60 calories/rat/day with a 15% ethanol diet. Behavioral Signs of Tolerance
The rats which were drinking ethanol chronically had developed some tolerance even after one week, as indicated by responses of acute challenge dose of alcohol. All control rats were collapsed and hypokinetic within 15 minutes; but all ethanol-consuming rats were standing and moving their heads about, albeit with signs of intoxication. The typical decapitation convulsions were greatly suppressed or totally absent in rats that had chronically consumed ethanol.
Biochemical Indices of Ethanol Effects
345
L
c
r
9
:
300
9
c . CONTROL DIET CONTROL DIET * - - a ETOH DIET
z
q u,
T
200
.
.
.
1
.
1
1
1
1
1
1
1
DAYS
Fig 2. Body weight graph, illustrating that all groups had a similar degree of mild weight loss over the experimental period. Rats in the two control groups received the Same diet, but animals were sacrificed at different times.
The rats that had been consuming ethanol for four weeks were withdrawn from
ethanol during the night preceding the morning’s sacrifice. These rats were highly irritable when taken from the cage, and despite the fact that they had been handled every other day for weighing, they attacked the handler who had been doing the weighing. During attempts to inject them, they scratched vigorously and two of the five rats inflicted bite wounds. The injection of ethanol had a marked calming effect within ten minutes. Control Data
The general regional differences which we had previously reported [Engen and Klemm, 19781 were replicated; SA levels were highest in the forebrain and lowest in the medulla, whereas DR levels were about the same everywhere except for an approximate fourfold increase in the cerebellum. Quite unexpectedly, there were clearly significant increases in both SA and DR in most regions in the rats that were sacrificed three weeks after the first group (Fig 3). The only known differences between the two groups were a three-week age difference and the time of exposure to the diet regimen and the communal living environment. To test for the age factor, data from the rats sacrificed at the first week were compared to data from the same strain of much younger rats in a previous study [Engen and Klemm, 19781; levels of SA were significantly higher in the older rats, but DR levels were lower in the older rats (Table I). Effects of Alcohol
Whereas levels of SA tended to increase in controls over the experimental period, control rats that were given a challenge dose of ethanol 45 minutes before sacrifice had lower levels, especially in the forebrain and medulla (Fig 4). On the other hand, rats which drank ethanol in their daily diet responded differently to the challenge dose of ethanol. The SA levels were not suppressed by the acute ethanol dose, and in fact levels were more comparable to those of saline-challenged controls (Fig 4). The differences among treatments were highly significant [F(2,104); P < 0.0051 ,which was attributable
346
K l e m m and E n g e n
SlALlC ACID 6 0
0 1st
WEEK
5 0
4 0
30 FOREBRAIN
MIDBRAIN
CEREBELLUM
MEDULLA
Fig 3. Brain area differences in total sialic acid and 2-deoxyribose (mean pmoles/gm f SE) in salinechallenged control rats. Also illustrated is the increase in both chemicals which occurred in the group that received three more weeks of exposure to the communal housing and the liquid control diet.
TABLE 1. Comparison With Younger Controls in Previous Study
Brain region Forebrain Midbrain Cerebellum Medulla
Sialic acid xpm/gm i- S.E. Young adultsa Mature adultsb 4.55 4.33 3.43 2.69
f
t k
t
0.08 0.15 0.07 0.08
5.44 i 0.15 5.13 f 0.21 4.01 f 0.14 3.13 i 0.15
P
