- Email: [email protected]
Effects of prenatal alcohol exposure on behavior of aged rats
Drug and Alcohol
Dependence,
Elsevier
Publishers
Scientific
16 (1986) Ireland
EFFECTS OF PRENATAL AGED RATS
ERNEST
L. ABEL*
Research Institute (Received
July
and B.A.
ALCOHOL EXPOSURE
ON BEHAVIOR
OF
DINTCHEFF
on Alcoholism,
26th,
321
321-330
Ltd.
1021 Main Street,
Buffalo,
NY 14203 (U.S.A.)
1985)
SUMMARY
Pregnant rats were intubated with alcohol (3.5 g/kg, twice daily) on gestation days 11-21. Control animals were intubated with an isocaloric sucrose solution and were pair-fed and pair-watered to alcohol-treated dams. At birth, offspring were placed with non-treated surrogate dams. When animals were slightly more than 1 year of age, they were tested for passive avoidance learning, spontaneous alternation and activity. Groups did not differ in passive avoidance learning or spontaneous alteration but animals prenatally exposed to alcohol were more active than controls. Additional studies showed that this increased activity was not affected by testing animals in the presence of environmental stimuli such as objects which could be manipulated, or by odors from mouse shavings from male and female mice. Key words: Prenatal alcohol - Activity - Passive avoidance - Spontaneous alternation INTRODUCTION
Prenatal exposure to alcohol results in a number of behaviorally teratogenie effects in humans and animals including hyperactivity [l-4] perseverative behavior [1,5], motor dysfunction [6,7], mental retardation and varying degrees of learning impairment [4,8-lo]. For some behaviors, these effects do not appear to be permanent, suggesting that prenatal alcohol exposure produces a developmental delay in CNS maturation rather than permanent impairment [lo]. For example, Abel [l] found that animals prenatally exposed to alcohol perseverated more and perfomed worse in a passive avoidance learning task at 16, and 42 days of age but not at 114 days of age. Bond and DiGiusto [ 111 reported that rats pre*Present address: 48021, U.S.A.
Mott
Center,
Wayne
State
0376-8716/86/$03.50 0 Elsevier Scientific Publishers Ireland Printed and Published in Ireland
Ltd.
University,
275 E. Hancock,
Detroit,
MI
322
natally exposed to alcohol were more active than controls at 28 and 56 days of age but not at 112 days of age. Lee et al. [12] noted T-maze learning deficits at 17 but not at 80 days of age in similarly exposed rats. On the other hand, Abel [8] reported that rats prenatally exposed to alcohol performed worse than controls in a shock avoidance learning task at 5 months of age [ 81. Rats prenatally exposed to alcohol also engaged in more headdipping behavior than controls at each age they were tested up to 95 days of age [ 131. These latter results in animals are in agreement with studies in humans showing relatively stable lower IQ scores in patients with fetal alcohol syndrome [ 4,141. One reason some studies report long-lasting effects whereas others do not is that some of the methods used to test animals are less sensitive than others [ 131. Alternatively, the age periods studied have been relatively short and have not allowed for permanent effects to be identified. In the present series of studies, we examined three behaviors (spontaneous alternation, Rotorod and activity) previously regarded as being among those not permanently affected by prenatal alcohol exposure [ 1,111. Animals in this study were more than 1 year old at time of testing. To my knowledge, there are no previous reports of animals prenatally exposed to alcohol that have been tested at these ages. METHODS
Nulliparous Long Evans rats (Blue Spruce Farms, Altamont, NY) were bred in our laboratory. Upon detection of a sperm plug (conception day 0) dams were placed in individual Plexiglas cages with wooden shavings and assigned to one of three groups. The Alcohol group was intubated with 3.5 g/kg alcohol (15%, w/v) twice daily on gestation days 11-21. Pair-fed controls were intubated twice daily with an isocaloric sucrose solution and were pair-fed (Teklad 10% pregnancy lab chow) and pair-watered to the alcoholtreated group. A third group was non-treated and was allowed ad lib access to food and water. At birth, pups were weighed and culled to 8 pups/litter and the entire litter was placed with a non-treated surrogate dam that had given birth within 24 h of experimental mothers. Animals were weaned at 21 days of age, remained with similar sex litter mates for 5-6 months, and then were housed individually. Although there were initially 20 litters of 1 male and female/group, there were a few deaths in various litters so that some groups contained 17-20 males or females at time of testing. All animals were tested in each condition. Experiment
1
Spontaneous alternation. Male and female animals were tested for spontaneous alternation in a T-maze beginning at 56 weeks of age. Animals were
323
removed from their home cage and placed in the start alley and were permitted to explore the maze. After entry into one of the arms of the maze, a gate was lowered to prevent exit from the alley and the animal was confined for 30 s. The animal was then placed back into its cage for 30 s, the T-maze was cleaned with a damp sponge, and the animal was tested again. There were a total of five trials. Rotarod. Females were individually tested within 1 week of spontaneous alternation testing, for their ability to remain on a Rotarod (a l&cm rotating drum which accelerated linearly from an initial speed of 4 rev./min) until they fell onto a platform (30 cm below). (Males could not be tested because their higher body weights caused the motors to malfunction). Depression of the platform activated a switch, halting the drum and a digital timer. Animals were given an initial test to familiarize them with the apparatus, followed by three test trials. Activity. Activity was monitored by placing animals in an Omnitech activity monitor for 30 min. The 30-min session was divided into 6 5-min periods. Both horizontal locomotor activity and rearing were automatically recorded. RESULTS
Detailed information concerning maternal and pup data has been presented elsewhere [ 151 and will only be summarized here. Offspring born to dams prenatally exposed to alcohol weighed less at birth than pair-fed and ad lib controls. A number of deaths occurred in each group, but group differences in postnatal mortality were not significant. Group sizes and body weights at 11 months, 12 months and 15 months of age are presented in Table I. As indicated by the table, both males and females prenatally exposed to alcohol weighed less at each age than pair-fed or ad-lib controls. The latter did not differ significantly. Males weighed significantly more than females at each age (P < 0.05) and the treatment by sex interaction was not significant at any age. Spontaneous alternation. Group differences in the number of trials until the first alternation or in number of alternations were not statistically significant. The main effect of sex or the group X sex interaction were also not statistically significant. Rotarod. Group differences in time on the Rotarod were not statistically significant. Activity. Effects on activity are shown in Fig. 1. The main effect of treatment was statistically significant (F = 4.79, d.f. = 2,110, P < 0.01). Females were more active thanmales (P= 121, d.f. = l,llO,P< 0.001). The group X sex interaction was not significant. Activity decreased during the 30-min test session (F = 270, d.f. = 5, P < 0.001). The group X period interaction was not statistically significant, but the sex X period interaction was (F = 28.6, d.f. = 5,550, P < 0.001). The latter occurred because the decrease in activity
606 -+ 23 (2V
334 It 11 (19)
in brackets
Females
%umbers
age
indicate
343 t 12 (191
645 k 18 (19)
55 wks
litters
347 + 14 (16)
634 + 26 (17) 348 + 15 (16)
631 -+ 25 (18)
66 wks
EXPOSED
per group.
64 wks
OF RATS PRENATALLY
Males
48 wks
Alcohol
BODY WEIGHTS
TABLE I
377 * 10 (20)
687 + 14 (19)
48 wks
Pair-fed
398 2 10 (19)
716 + 16 (19) 406 * 13 (18)
704 + 30 (17)
64 wks
(2 _+S.E. 1
55 wks
age
TO ALCOHOL
398 * 14 (18)
717 _+19 (19)
66 wks
372 ? 8.5 (20)
103 + 21 (20)
48 wks
397 + 10 (20)
731 * 24 (19)
55 wks
Ad lib control age
395 + 10 (19)
133 f 31 (19)
64 wks
394
750 + 25 (17)
66 wks
325
1000
800
600
400
600
400
200
C
I
5
10
I
15
20
25
30
Minutes Fig, 1. Mean activity counts for 15-month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. iV = 17--2O/sex per group. n-m, alcohol; o-o, pair-fed; OV, ad lib control.
was greater for females. The group X sex X period interaction was not significant. Rearing behavior did not resemble activity in terms of statistically significant differences. Group differences were not statistically significant; females reared more than males (F = 62.9, d.f. = 1,110, P < 0.001); the group X sex interaction was not significant. Rearing decreased as testing continued (F = 145, d.f. = 5,550, P< 0.001) and only the sex X period interaction was significant (F = 6.83, d.f. = 5,550, P< 0.001).
326 DISCUSSION
These data indicate that prenatal alcohol exposure has long-lasting effects on some measures and not others. The permanent effects on body weight have been previously noted [8,16]. The present study indicates that at the dosage used in this study this is undoubtedly an irreversible effect. Previous studies have shown that this effect on body weight is not due to metabolic differences in utilization of nutrients [ 171 and attempts to provide optimal postnatal nutritional conditions [18] have not been able to overcome alcohol’s effects on intrauterine growth retardation. Henderson and co-workers [ 191 have reported that animals prenatally exposed to alcohol had less DNA content in brain, heart and kidney, indicative of fewer cells in such organs. As a result they are unable to ‘catch up’ in body weight after alcohol exposure. The absence of any significant effects on spontaneous alteration and Rotorod corroborate our previous reports [1,20]. The effect on activity, however, is in contrast with Bond and Digiusto’s [ 111 report that the initial increases in activity associated with prenatal alcohol exposure do not persist into adulthood. There are numerous methodological differences between the latter and the present study which preclude direct comparisons such as difference in age of testing, method of alcohol exposure and testing apparatus. Experiment
2
Since a significant difference in activity was noted in the previous study, we examined this difference further by retesting animals in the same apparatus except that for this second exposure, ‘toys’ were placed in the apparatus. The rationale for this procedure was that control animals might have been less active because they were less stimulated by the environment. If this were so, the presence of objects might act to increase their activity more than that of alcohol exposed animals. Conversely, a more stimulating test environment could act to increase activity to a greater degree in alcohol-exposed animals. METHOD
The animals and test method were identical to those used in the previous study. Animals were tested 1 week later. The ‘toys’ were a l/4 inch aluminum weighing pan (Fisher Scientific), a glass stopper (Standard taper No. 16), and a small paper binding clip (IDL Mfg Co, Carlstadt, NJ, No. 20). RESULTS
The effect of testing animals in the presence of ‘toys’ is shown in Fig. 2. Treatment differences were statistically significant (F = 3.98, d.f. = 2,102, significant (F = 10.0, d.f. = 5,510, P < 0.001). The period X sex interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other
327
300
200 m
2
0’
100
v h
3
;;
1000-
5 N
‘g
800 -
Lc 600
-
400
-
200
-
0:
1
2
3
4
5
6
Periods Fig. 2. Mean activity counts for l&month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. Animals were tested in the presence of ‘toys’. N = 17-20/sex per group. n -m, Alcohol, o0, pair-fed; o -0, ad lib control.
d.f =
166,P<
0.004).
Pair-fed
animals
did not differ significantly
from ad lib
controls. Females were more active than males (F = 64.3, d.f. = 1,102, P < 0.001). The groups X sex interaction was not significant. The sex X period interaction was not significant. The sex X period interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other interactions was significant.
328
Treatment differences in rearing were not significant. Females reared more than males (F = 22.1, d.f. = 1102, P < 0.001). The effect of periods was significant (F = 10.0, d.f. = 5,510, P < 0.001). The period X sex interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other interactions was significant. DISCUSSION
Animals prenatally exposed to alcohol were more active than controls as noted in the previous study. The presence of ‘toys’ in the test apparatus did not affect differences between groups. Activity levels were lower than those noted when animals were previously tested but this was probably due to prior exposure to the apparatus. Experiment
3
In a related series of studies on the effects of odors on aged animals prenatally exposed to alcohol (Abel and Dintcheff, unpublished), we noted that the presence of shavings taken from mouse cages significantly increase ‘nose-poking’ activity. Buelke-Same and co-workers [ 211 reported that activity levels of animals prenatally exposed to sodium salicylate were differently affected by odors. To examine the effects of odor on activity, we retested animals in the presence of male or female mouse shavings to see if activity of alcohol-exposed animals would be affected differentially than activity of control animals by the presence of mouse shavings from male and female mice. METHOD
Animals were tested in the same apparatus for the same duration. Bedding material from cages occupied for 3 days by mature male or female mice (C57BL) (4/cage) was placed into the Omnitech activity cages prior to testing. Half the animals were tested in the presence of male shavings and the other half were tested with female shavings. RESULTS
The effects of male and female mouse shavings on activity are shown in Fig. 3. The treatment factor was significant (F = 51.6, d.f. = 2,102, P< 0.008) with animals prenatally exposed to alcohol being more active than animals in the other two groups, Females were more active than males (F = 108, d.f. = 1,102, P < 0.001). The main effect of shavings was not statistically significant, nor did any factor interact significantly with shavings. None of the other main interactions was significant. The main effect of period was significant (F = 98, d.f. = 5,510, P < 0.001). The only factor interacting
329
Females
with
800
600
400
Females Female
200
I
I
I
I
I
with Shavings
-
I
I
I
,
25
30
Males with Female Shavings
Males with Male Shavings
,-
4oc
2oc
0
5
10
15
20
25
30
5
10
15
20
Minutes Fig. 3. Mean activity counts for 15-month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. Animals were tested in the presence of soiled bedding taken from male and female mouse cages. N = 9-lo/sex per group. n -m , Alcohol; o0, pair-fed; 09, ad lib control.
significantly with periods was sex (F = 5.79, d.f. = 5,510, P < 0.001). (Inspection of Fig. 3 suggests that this was due to a more rapid decrease in activity on the part of females as testing continued). DISCUSSION
The results of this study indicate that odor did not affect activity levels significantly. Bedding odors have been found to differentially affect activity
330
levels in animals subjected to other prenatal treatments [21] but the present study indicates that this does not occur in conjunction with alcohol as well. This observation underscores Plonsky and Riley’s [ 131 comment that many of the test conditions used to examine the effects of prenatal exposure to drugs may be affected not only by the age of the animals tested, but also by the condition under which animals are tested. GENERAL DISCUSSION
Prenatal alcohol exposure can have ephemeral or long-lasting effects depending on, among other things, amount of exposure, type of test and testing conditions [ 1,9,11--131. The present study corroborates our previous report that initial changes in spontaneous alternation and passive avoidance learning produced by prenatal alcohol exposure are not permanent. On the other hand, effects on activity do not dissipate with age. The present study also indicates that odors do not influence behavior in relatively old animals. ACKNOWLEDGMENTS
I thank B. Dintcheff, D. Dintcheff, C. Matyjasik, M. McGowan and M. Nobel for technical assistance. This project was supported by a grant from the National Institute on Alcoholism and Alcohol Abuse (AA 05631). REFERENCES 1 E.L. Abel, Alcoholism: Clin. Exp. Res., 6 (1982) 369. 2 G.L. Osborne, W.F. Caul and K. Fernandez, Pharmacol. Biochem. Behav., 12 (1980) 393. 3 B.A. Shaywitz, G.G. Griffith and J.B. Warshaw, Neurobehav. Toxicol., 1 (1979) 113. 4 A.P. Streissguth, C.S. Herman and D.W. Smith, J. Pediatr., 92 (1978) 363. 5 E.P. Riley, E.A. Lochry and N.R. Shapiro, Pharmacol. Biochem. Behav., 10 (1979a) 255. E.L. Abel and B.A. Dintcheff, J. Pharmacol. Exp. Ther., 916 (1978) 207. N.L. Golden et al., Pediatrics, 70 (1982) 931. E.L. Abel, Pharmacol. Biochem. Behav., 10 (1979) 239. E.L. Abel, Fetal Alcohol Syndrome and Fetal Alcohol Effects, Plenum Press, New York, 1984. 10 E.P. Riley et al., Pharmacol. Biochem. Behav., 11 (1979b) 513. 11 N.W. Bond and E.L. DiGiusto, Psychopharmacology, 52 (1977) 311. 12 M.H. Lee, R. Haddad and A. Rabe, Neurobehav. Toxicol., 2 (1980) 189. 13 M. Plonsky and E.P. Riley, Neurobehav. Toxicol. Teratol., 5 (1983) 309. 14 S. Iosub et al., Pediatrics, 68 (1981) 475. 15 E.L. Abel and B.A. Dintcheff, Neurobehav. Toxicol. Teratol., in press. 16 A.A. Monjan and W. Mandell, Neurobehav. Toxicol., 2 (1980) 213. 17 E.L. Abel, Neurobehav. Toxicol. Teratol., 3 (1981) 49. 18 M. Lee and J. Leichter, Growth, 44 (1980) 327. 19 G.I. Henderson et al., Alcoholism: Clin. Exp. Res., 3 (1979) 99. 20 E.L. Abel, R. Bush and B.A. Dintcheff, Science, 212 (1981) 1531. 21 J. Buelke-Sam et al., Neurobehav. Toxicol. Teratol., 6 (1984) 171.
Dependence,
Elsevier
Publishers
Scientific
16 (1986) Ireland
EFFECTS OF PRENATAL AGED RATS
ERNEST
L. ABEL*
Research Institute (Received
July
and B.A.
ALCOHOL EXPOSURE
ON BEHAVIOR
OF
DINTCHEFF
on Alcoholism,
26th,
321
321-330
Ltd.
1021 Main Street,
Buffalo,
NY 14203 (U.S.A.)
1985)
SUMMARY
Pregnant rats were intubated with alcohol (3.5 g/kg, twice daily) on gestation days 11-21. Control animals were intubated with an isocaloric sucrose solution and were pair-fed and pair-watered to alcohol-treated dams. At birth, offspring were placed with non-treated surrogate dams. When animals were slightly more than 1 year of age, they were tested for passive avoidance learning, spontaneous alternation and activity. Groups did not differ in passive avoidance learning or spontaneous alteration but animals prenatally exposed to alcohol were more active than controls. Additional studies showed that this increased activity was not affected by testing animals in the presence of environmental stimuli such as objects which could be manipulated, or by odors from mouse shavings from male and female mice. Key words: Prenatal alcohol - Activity - Passive avoidance - Spontaneous alternation INTRODUCTION
Prenatal exposure to alcohol results in a number of behaviorally teratogenie effects in humans and animals including hyperactivity [l-4] perseverative behavior [1,5], motor dysfunction [6,7], mental retardation and varying degrees of learning impairment [4,8-lo]. For some behaviors, these effects do not appear to be permanent, suggesting that prenatal alcohol exposure produces a developmental delay in CNS maturation rather than permanent impairment [lo]. For example, Abel [l] found that animals prenatally exposed to alcohol perseverated more and perfomed worse in a passive avoidance learning task at 16, and 42 days of age but not at 114 days of age. Bond and DiGiusto [ 111 reported that rats pre*Present address: 48021, U.S.A.
Mott
Center,
Wayne
State
0376-8716/86/$03.50 0 Elsevier Scientific Publishers Ireland Printed and Published in Ireland
Ltd.
University,
275 E. Hancock,
Detroit,
MI
322
natally exposed to alcohol were more active than controls at 28 and 56 days of age but not at 112 days of age. Lee et al. [12] noted T-maze learning deficits at 17 but not at 80 days of age in similarly exposed rats. On the other hand, Abel [8] reported that rats prenatally exposed to alcohol performed worse than controls in a shock avoidance learning task at 5 months of age [ 81. Rats prenatally exposed to alcohol also engaged in more headdipping behavior than controls at each age they were tested up to 95 days of age [ 131. These latter results in animals are in agreement with studies in humans showing relatively stable lower IQ scores in patients with fetal alcohol syndrome [ 4,141. One reason some studies report long-lasting effects whereas others do not is that some of the methods used to test animals are less sensitive than others [ 131. Alternatively, the age periods studied have been relatively short and have not allowed for permanent effects to be identified. In the present series of studies, we examined three behaviors (spontaneous alternation, Rotorod and activity) previously regarded as being among those not permanently affected by prenatal alcohol exposure [ 1,111. Animals in this study were more than 1 year old at time of testing. To my knowledge, there are no previous reports of animals prenatally exposed to alcohol that have been tested at these ages. METHODS
Nulliparous Long Evans rats (Blue Spruce Farms, Altamont, NY) were bred in our laboratory. Upon detection of a sperm plug (conception day 0) dams were placed in individual Plexiglas cages with wooden shavings and assigned to one of three groups. The Alcohol group was intubated with 3.5 g/kg alcohol (15%, w/v) twice daily on gestation days 11-21. Pair-fed controls were intubated twice daily with an isocaloric sucrose solution and were pair-fed (Teklad 10% pregnancy lab chow) and pair-watered to the alcoholtreated group. A third group was non-treated and was allowed ad lib access to food and water. At birth, pups were weighed and culled to 8 pups/litter and the entire litter was placed with a non-treated surrogate dam that had given birth within 24 h of experimental mothers. Animals were weaned at 21 days of age, remained with similar sex litter mates for 5-6 months, and then were housed individually. Although there were initially 20 litters of 1 male and female/group, there were a few deaths in various litters so that some groups contained 17-20 males or females at time of testing. All animals were tested in each condition. Experiment
1
Spontaneous alternation. Male and female animals were tested for spontaneous alternation in a T-maze beginning at 56 weeks of age. Animals were
323
removed from their home cage and placed in the start alley and were permitted to explore the maze. After entry into one of the arms of the maze, a gate was lowered to prevent exit from the alley and the animal was confined for 30 s. The animal was then placed back into its cage for 30 s, the T-maze was cleaned with a damp sponge, and the animal was tested again. There were a total of five trials. Rotarod. Females were individually tested within 1 week of spontaneous alternation testing, for their ability to remain on a Rotarod (a l&cm rotating drum which accelerated linearly from an initial speed of 4 rev./min) until they fell onto a platform (30 cm below). (Males could not be tested because their higher body weights caused the motors to malfunction). Depression of the platform activated a switch, halting the drum and a digital timer. Animals were given an initial test to familiarize them with the apparatus, followed by three test trials. Activity. Activity was monitored by placing animals in an Omnitech activity monitor for 30 min. The 30-min session was divided into 6 5-min periods. Both horizontal locomotor activity and rearing were automatically recorded. RESULTS
Detailed information concerning maternal and pup data has been presented elsewhere [ 151 and will only be summarized here. Offspring born to dams prenatally exposed to alcohol weighed less at birth than pair-fed and ad lib controls. A number of deaths occurred in each group, but group differences in postnatal mortality were not significant. Group sizes and body weights at 11 months, 12 months and 15 months of age are presented in Table I. As indicated by the table, both males and females prenatally exposed to alcohol weighed less at each age than pair-fed or ad-lib controls. The latter did not differ significantly. Males weighed significantly more than females at each age (P < 0.05) and the treatment by sex interaction was not significant at any age. Spontaneous alternation. Group differences in the number of trials until the first alternation or in number of alternations were not statistically significant. The main effect of sex or the group X sex interaction were also not statistically significant. Rotarod. Group differences in time on the Rotarod were not statistically significant. Activity. Effects on activity are shown in Fig. 1. The main effect of treatment was statistically significant (F = 4.79, d.f. = 2,110, P < 0.01). Females were more active thanmales (P= 121, d.f. = l,llO,P< 0.001). The group X sex interaction was not significant. Activity decreased during the 30-min test session (F = 270, d.f. = 5, P < 0.001). The group X period interaction was not statistically significant, but the sex X period interaction was (F = 28.6, d.f. = 5,550, P < 0.001). The latter occurred because the decrease in activity
606 -+ 23 (2V
334 It 11 (19)
in brackets
Females
%umbers
age
indicate
343 t 12 (191
645 k 18 (19)
55 wks
litters
347 + 14 (16)
634 + 26 (17) 348 + 15 (16)
631 -+ 25 (18)
66 wks
EXPOSED
per group.
64 wks
OF RATS PRENATALLY
Males
48 wks
Alcohol
BODY WEIGHTS
TABLE I
377 * 10 (20)
687 + 14 (19)
48 wks
Pair-fed
398 2 10 (19)
716 + 16 (19) 406 * 13 (18)
704 + 30 (17)
64 wks
(2 _+S.E. 1
55 wks
age
TO ALCOHOL
398 * 14 (18)
717 _+19 (19)
66 wks
372 ? 8.5 (20)
103 + 21 (20)
48 wks
397 + 10 (20)
731 * 24 (19)
55 wks
Ad lib control age
395 + 10 (19)
133 f 31 (19)
64 wks
394
750 + 25 (17)
66 wks
325
1000
800
600
400
600
400
200
C
I
5
10
I
15
20
25
30
Minutes Fig, 1. Mean activity counts for 15-month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. iV = 17--2O/sex per group. n-m, alcohol; o-o, pair-fed; OV, ad lib control.
was greater for females. The group X sex X period interaction was not significant. Rearing behavior did not resemble activity in terms of statistically significant differences. Group differences were not statistically significant; females reared more than males (F = 62.9, d.f. = 1,110, P < 0.001); the group X sex interaction was not significant. Rearing decreased as testing continued (F = 145, d.f. = 5,550, P< 0.001) and only the sex X period interaction was significant (F = 6.83, d.f. = 5,550, P< 0.001).
326 DISCUSSION
These data indicate that prenatal alcohol exposure has long-lasting effects on some measures and not others. The permanent effects on body weight have been previously noted [8,16]. The present study indicates that at the dosage used in this study this is undoubtedly an irreversible effect. Previous studies have shown that this effect on body weight is not due to metabolic differences in utilization of nutrients [ 171 and attempts to provide optimal postnatal nutritional conditions [18] have not been able to overcome alcohol’s effects on intrauterine growth retardation. Henderson and co-workers [ 191 have reported that animals prenatally exposed to alcohol had less DNA content in brain, heart and kidney, indicative of fewer cells in such organs. As a result they are unable to ‘catch up’ in body weight after alcohol exposure. The absence of any significant effects on spontaneous alteration and Rotorod corroborate our previous reports [1,20]. The effect on activity, however, is in contrast with Bond and Digiusto’s [ 111 report that the initial increases in activity associated with prenatal alcohol exposure do not persist into adulthood. There are numerous methodological differences between the latter and the present study which preclude direct comparisons such as difference in age of testing, method of alcohol exposure and testing apparatus. Experiment
2
Since a significant difference in activity was noted in the previous study, we examined this difference further by retesting animals in the same apparatus except that for this second exposure, ‘toys’ were placed in the apparatus. The rationale for this procedure was that control animals might have been less active because they were less stimulated by the environment. If this were so, the presence of objects might act to increase their activity more than that of alcohol exposed animals. Conversely, a more stimulating test environment could act to increase activity to a greater degree in alcohol-exposed animals. METHOD
The animals and test method were identical to those used in the previous study. Animals were tested 1 week later. The ‘toys’ were a l/4 inch aluminum weighing pan (Fisher Scientific), a glass stopper (Standard taper No. 16), and a small paper binding clip (IDL Mfg Co, Carlstadt, NJ, No. 20). RESULTS
The effect of testing animals in the presence of ‘toys’ is shown in Fig. 2. Treatment differences were statistically significant (F = 3.98, d.f. = 2,102, significant (F = 10.0, d.f. = 5,510, P < 0.001). The period X sex interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other
327
300
200 m
2
0’
100
v h
3
;;
1000-
5 N
‘g
800 -
Lc 600
-
400
-
200
-
0:
1
2
3
4
5
6
Periods Fig. 2. Mean activity counts for l&month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. Animals were tested in the presence of ‘toys’. N = 17-20/sex per group. n -m, Alcohol, o0, pair-fed; o -0, ad lib control.
d.f =
166,P<
0.004).
Pair-fed
animals
did not differ significantly
from ad lib
controls. Females were more active than males (F = 64.3, d.f. = 1,102, P < 0.001). The groups X sex interaction was not significant. The sex X period interaction was not significant. The sex X period interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other interactions was significant.
328
Treatment differences in rearing were not significant. Females reared more than males (F = 22.1, d.f. = 1102, P < 0.001). The effect of periods was significant (F = 10.0, d.f. = 5,510, P < 0.001). The period X sex interaction was also significant (F = 2.54, d.f. = 5,510, P < 0.03). None of the other interactions was significant. DISCUSSION
Animals prenatally exposed to alcohol were more active than controls as noted in the previous study. The presence of ‘toys’ in the test apparatus did not affect differences between groups. Activity levels were lower than those noted when animals were previously tested but this was probably due to prior exposure to the apparatus. Experiment
3
In a related series of studies on the effects of odors on aged animals prenatally exposed to alcohol (Abel and Dintcheff, unpublished), we noted that the presence of shavings taken from mouse cages significantly increase ‘nose-poking’ activity. Buelke-Same and co-workers [ 211 reported that activity levels of animals prenatally exposed to sodium salicylate were differently affected by odors. To examine the effects of odor on activity, we retested animals in the presence of male or female mouse shavings to see if activity of alcohol-exposed animals would be affected differentially than activity of control animals by the presence of mouse shavings from male and female mice. METHOD
Animals were tested in the same apparatus for the same duration. Bedding material from cages occupied for 3 days by mature male or female mice (C57BL) (4/cage) was placed into the Omnitech activity cages prior to testing. Half the animals were tested in the presence of male shavings and the other half were tested with female shavings. RESULTS
The effects of male and female mouse shavings on activity are shown in Fig. 3. The treatment factor was significant (F = 51.6, d.f. = 2,102, P< 0.008) with animals prenatally exposed to alcohol being more active than animals in the other two groups, Females were more active than males (F = 108, d.f. = 1,102, P < 0.001). The main effect of shavings was not statistically significant, nor did any factor interact significantly with shavings. None of the other main interactions was significant. The main effect of period was significant (F = 98, d.f. = 5,510, P < 0.001). The only factor interacting
329
Females
with
800
600
400
Females Female
200
I
I
I
I
I
with Shavings
-
I
I
I
,
25
30
Males with Female Shavings
Males with Male Shavings
,-
4oc
2oc
0
5
10
15
20
25
30
5
10
15
20
Minutes Fig. 3. Mean activity counts for 15-month-old rats prenatally exposed to alcohol compared to pair-fed and ad lib fed controls. Animals were tested in the presence of soiled bedding taken from male and female mouse cages. N = 9-lo/sex per group. n -m , Alcohol; o0, pair-fed; 09, ad lib control.
significantly with periods was sex (F = 5.79, d.f. = 5,510, P < 0.001). (Inspection of Fig. 3 suggests that this was due to a more rapid decrease in activity on the part of females as testing continued). DISCUSSION
The results of this study indicate that odor did not affect activity levels significantly. Bedding odors have been found to differentially affect activity
330
levels in animals subjected to other prenatal treatments [21] but the present study indicates that this does not occur in conjunction with alcohol as well. This observation underscores Plonsky and Riley’s [ 131 comment that many of the test conditions used to examine the effects of prenatal exposure to drugs may be affected not only by the age of the animals tested, but also by the condition under which animals are tested. GENERAL DISCUSSION
Prenatal alcohol exposure can have ephemeral or long-lasting effects depending on, among other things, amount of exposure, type of test and testing conditions [ 1,9,11--131. The present study corroborates our previous report that initial changes in spontaneous alternation and passive avoidance learning produced by prenatal alcohol exposure are not permanent. On the other hand, effects on activity do not dissipate with age. The present study also indicates that odors do not influence behavior in relatively old animals. ACKNOWLEDGMENTS
I thank B. Dintcheff, D. Dintcheff, C. Matyjasik, M. McGowan and M. Nobel for technical assistance. This project was supported by a grant from the National Institute on Alcoholism and Alcohol Abuse (AA 05631). REFERENCES 1 E.L. Abel, Alcoholism: Clin. Exp. Res., 6 (1982) 369. 2 G.L. Osborne, W.F. Caul and K. Fernandez, Pharmacol. Biochem. Behav., 12 (1980) 393. 3 B.A. Shaywitz, G.G. Griffith and J.B. Warshaw, Neurobehav. Toxicol., 1 (1979) 113. 4 A.P. Streissguth, C.S. Herman and D.W. Smith, J. Pediatr., 92 (1978) 363. 5 E.P. Riley, E.A. Lochry and N.R. Shapiro, Pharmacol. Biochem. Behav., 10 (1979a) 255. E.L. Abel and B.A. Dintcheff, J. Pharmacol. Exp. Ther., 916 (1978) 207. N.L. Golden et al., Pediatrics, 70 (1982) 931. E.L. Abel, Pharmacol. Biochem. Behav., 10 (1979) 239. E.L. Abel, Fetal Alcohol Syndrome and Fetal Alcohol Effects, Plenum Press, New York, 1984. 10 E.P. Riley et al., Pharmacol. Biochem. Behav., 11 (1979b) 513. 11 N.W. Bond and E.L. DiGiusto, Psychopharmacology, 52 (1977) 311. 12 M.H. Lee, R. Haddad and A. Rabe, Neurobehav. Toxicol., 2 (1980) 189. 13 M. Plonsky and E.P. Riley, Neurobehav. Toxicol. Teratol., 5 (1983) 309. 14 S. Iosub et al., Pediatrics, 68 (1981) 475. 15 E.L. Abel and B.A. Dintcheff, Neurobehav. Toxicol. Teratol., in press. 16 A.A. Monjan and W. Mandell, Neurobehav. Toxicol., 2 (1980) 213. 17 E.L. Abel, Neurobehav. Toxicol. Teratol., 3 (1981) 49. 18 M. Lee and J. Leichter, Growth, 44 (1980) 327. 19 G.I. Henderson et al., Alcoholism: Clin. Exp. Res., 3 (1979) 99. 20 E.L. Abel, R. Bush and B.A. Dintcheff, Science, 212 (1981) 1531. 21 J. Buelke-Sam et al., Neurobehav. Toxicol. Teratol., 6 (1984) 171.