Effects of prenatal alcohol exposure on nose poking in year-old rats

Alcohol, Vol. 3, pp. 201-204, 1986. ©AnkhoInternationalInc. Printedin the U.S.A.

0741-8329/86$3.00 + .00

Effects of Prenatal Alcohol Exposure on Nose Poking in Year-Old Rats E R N E S T L. A B E L

D e p a r t m e n t s o f Obstetrics and Gynecology, and Psychology Wayne State University, Detroit, M I 48201 AND B A R B A R A A. D I N T C H E F F

R e s e a r c h Institute on Alcoholism, Buffalo, N Y 14203 R e c e i v e d 16 S e p t e m b e r 1985 ABEL, E. L. AND B. A. DINTCHEFF. Effects of prenatal alcohol exposure on nose poking in year-old rats. ALCOHOL 3(3) 201-204, 1986.--Rats were prenatally exposed to alcohol (3.5 g/kg/twice daily) on gestation days 11-21. Control animals were vehicle-treated and pair-fed or untreated and fed ad lib. When animals were more than 1 year of age, they were tested for nose-poking behavior with female mice or empty cages and then male mice or empty cages as test stimuli. Alcohol exposed animals made significantly more nose pokes than pair fed or ad lib fed controls. Alcohol exposed females made significantly more nose pokes with female mice as test stimuli whereas males made fewer nose pokes when tested with female mice as test stimuli. Testing in the presence of male mice did not affect nose poking significantly. Nose poking of female alcohol-exposed animals was not affected by female mouse bedding relative to testing with empty cages but was increased in male alcohol-exposed males relative to empty cages. Prenatal

Alcohol

Nose poking

Rat

P R E N A T A L alcohol exposure is associated with a number of behavioral anomalies in animals including increased activity [2-4], impaired shuttle box performance [1,5], impaired passive avoidance [2,7], impaired maze learning [5] and perseverative behavior [2,7]. Riley and coworkers [7,8] have also reported that rats prenatally exposed to alcohol engage in more nose-poking and head dipping behavior than pair-fed controls and this effect does not appear to be dependent on the age of animals when tested [7] as are some of the other behaviors previously mentioned [2]. Riley et al. [8] also reported that head dipping was not affected by testing under the high or low stimulus complexity provided by inanimate objects (a coin and soap) which could be observed but not touched. In a previous study [3] we found that prenatal alcohol exposure increased activity in animals tested later than 1 year of age. The present study was conducted to determine if the previously reported effect on nose-poking also occurs when animals are tested at a much later age than that studied by Riley and coworkers [7,8] and to determine if stimulus complexity consisting of animate objects (caged mice) would differentially affect nose poking behavior of rats prenatally exposed to alcohol.

tamont, 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 alcohol-treated group. A third group was nontreated and was allowed ad lib access to food and water. At birth, pups were weighed and culled to 8 pups per litter and the entire litter was placed with a nontreated surrogate dam that had given birth within 24 hours of experimental mothers. Animals were weaned at 21 days of age, remained with a similar sex littermate for 5--6 months, and then were housed individually. Animals were tested at 57-62 weeks of age. Although there were initially 20 litters of 1 male and female tested per group (littermates were used in other tests), there were a few deaths in various litters so that some groups contained 17-20 males or females per group at time of testing. (Only 1 male and female per litter were tested to avoid litter effects.) Animals were chosen at random for each test condition. The nose poking apparatus was a 38x38x25.5 cm wooden box which contained two 4.5 cm (diameter) holes on one of the walls. The holes were located 3 cm from the floor, 7 cm from each side wall, and were 15 cm apart. The box was covered by a Plexiglas lid. If the animal poked its head

EXPERIMENT 1 METHOD Nulliparous Long Evans rats (Blue Spruce Farms, AI-

JRequests for reprints should be addressed to E. L. Abel. Wayne State University, Mott Center, 275 E. Hancock, Detroit, MI 48201.

201

2O2

A B E L AND D I N T C H E F F

35

Males -if" Fema e Mice

30

Males Male Mice

Males No Mice

30,

25-

25-

20~

o

\~1c4 ~J

(D ul o 7"

.

.

.

\\

10-

~5

.

$

Females

.Q

~ Female Mice

E

•

20

'

15~2,~

5

251

/ ~

6

10~

35 Z I x 30

\\

20-

\

15-

"~ o O_

Males w No Mice

Females No Mice

m o :7

Females

Male Mice

~ No Mice

.Q

E

•

ix

J~

Females

• •

:30-

25201510-

5

Time (rain)

i

i

i

i

5

10

15

20

i

2L5 30

I

i

i

=

i

5

10

15

20

i

2~5 30

Time (min)

FIG. 1. Nose poking behavior in the presence or absence of female mice by male and female rats prenatally exposed to alcohol (Q) compared to pair fed ((D) and ad lib (E3) treated animals.

FIG. 2. Nose poking behavior in the presence or absence of male mice by male and female rats prenatally exposed to alcohol (O) compared to pair fed (©) and ad lib ([]) treated animals.

through the hole, a photobeam was interrupted and a counter was activated. The counter was programmed so that total number of pokes in 5 min were recorded. Testing lasted for 30 min. Half the animals were tested in the presence of 2 cages each containing 4 adult female Swiss Webster mice. The cages were located at a distance of 3 cm from each opening. The mice were con[ined in Plexiglas cages 18.8x29.5 cm. The other half was tested with empty cages. Following testing with female mice or empty cages as test stimuli, the experiment was repeated with cages containing male mice (2 per cage) or an empty cage. The same animals were tested in both conditions but animals were chosen randomly for testing in the presence of female or male mice or empty cages. The data were analyzed for each mouse test condition. Internal analyses of group means were performed by Duncan Multiple Range Tests [5].

made 110-+8 nose pokes compared to 86-+6 nose pokes for animals tested with empty cages. The main effect of sex was not significant. The treatment × sex x test stimulus interaction was significant, F(2,103)=5.96, p<0.004. Decomposition of this interaction indicated that treatment differences were significant only for male animals tested with empty cages (i.e., no female mice), F(2,25)=4.29, p<0.03, and for females tested in the presence of female mice, F(2,26)=6.00, p<0.007. For males tested with empty cages, alcohol exposed males made more nose pokes than pair fed (/9<0.03) and ad lib mice (0<0.004) but did not differ from controls when tested in front of female mice. Pair fed animals did not differ significantly from controls in any test condition. F o r female rats tested with female mice, alcohol-exposed animals made significantly more nose pokes than pair fed (0<0.05) and ad lib fed animals 09<0.05). Pair fed animals did not differ from ad lib controls. Nose poking decreased over time for all groups, F(5,515)= 19.4, p<0.001. The only variable to interact significantly with time was sex, F(5,515)=3.56, p<0.004.

RESULTS Testing in the Presence o f Female Mice vs. No Mice

The data are presented in Fig. 1. Treatment differences were significant, F(2,103)=7.36, p<0.001. Alcohol exposed animals made an average (-+S.E.) of 120-+16 nose pokes compared to 96-+ 10 nose pokes for pair fed controls (0<0.05) and 79-+8 for ad lib controls (0<0.001). Pair fed animals did not differ significantly from ad lib controls. The main effect of female mice test stimuli was significant, F(1,103)=7.63, p<0.007. Animals tested in the presence of female mice

Testing in the Presence o f Male Mice vs. No Mice

The data are presented in Fig. 2. Treatment differences were significant, F(2,103)=7.03, p<0.002. Alcohol exposed animals made an average (+-S.E.) of 135-+!1 nose pokes compared to 114+10 nose pokes for pair fed controls (0<0.03) and 100-+9 for ad lib controls (0<0.001). Pair fed controls did not differ from ad lib controls. The main effect of male mice test stimuli was not signifi-

PRENATAL ALCOHOL AND NOSE POKING

203

Males

other half was tested with an identical cage except that it had not been previously occupied by female mice.

Males

No Bedding

Bedding

W Female 35

Testing in the Presence o f Female Mouse Bedding vs. No Bedding

30 25 20. 15

o O.

5 i

o Z

i

~ 4s

~

~

i

Females w Female

i

i

i

i

Females --

Bedding

i

, g

,0

IX3530 25 2015. 10.

The main treatment effect was significant, F(2,106)=6.6, p<0.002. Alcohol exposed animals made an average (---S.E.) of 132±12 nose pokes compared to 106---10 for pair fed animals and 93_+9 for ad lib fed animals. Females made more nose pokes than males, F(1,106)= 14.7, p<0.001. The main effect of bedding was not significant nor was the group x bedding interaction. The sex x bedding interaction was significant, F(1,106)=9.41, p<0.003. There was a significant group x sex interaction, F(2,106)=4.04, p<0.02. Nosepoking decreased over time, F(5,530)=25.3, p<0.001. The group x bedding × period interaction was significant, F(10,530)=2.14, p<0.02. The group × sex x bedding × period interaction was also significant, F(10,830)=1.9, p<0.04. Decomposition of this effect indicated that group differences among females were only significant when testing with female bedding compared to no bedding at the 2nd 5 min periods. Group differences among males were significant (p<0.001) when testing with female bedding versus no bedding at all 5 rain time periods except the 4th and 5th.

5

5

10

25

30

5

L

i

i

i

i

10

15

20

25

30

Time (rain)

FIG. 3. Nose poking behavior in the presence or absence of female mouse bedding by male and female rats prenatally exposed to alcohol (0) compared to pair fed (O) and ad lib (I-q)treated animals.

cant. The main effect of sex was significant, F(I, 103)= 11.4, p<0.001, with females making more nose pokes than males. The treatment x sex interaction was also significant, F(2,103)=3.41, p<0.04. This interaction arose because male alcohol-exposed animals made more nose pokes than pair fed males (p <0.05) and ad lib males (p<0.05) whereas female alcohol exposed animals did not differ significantly from pair fed or ad lib fed females. Nose poking decreased over time for all groups, F(5,515)=15.7, p<0.001. None of the other interactions were significant.

EXPERIMENT 2 The previous study showed that alcohol-exposed rats made more nose pokes than pair-fed or ad lib controls and that the presence of female mice as test stimuli reduced nose poking for male alcohol rats and increased it for female alcohol-exposed rats. To test the role of odors in this effect, the same experiment was repeated except that instead of using female mice as stimuli, the bedding from cages previously occupied by female mice was used. METHOD

All animals previously studied were tested one week after the last animal was tested in the previous study. Conditions were identical to those described above except that for half the animals a cage previously occupied for 4 days by 4 adult female mice was placed in front of each opening whereas the

GENERAL DISCUSSION The results of this study corroborate the main finding of Riley et al.'s [8] report showing that prenatal alcohol exposure increases nose poking behavior and also suggests that this effect is longlasting. Plonsky and Riley [7] studied head dipping and found a significant increase in head dipping associated with prenatal alcohol exposure when animals were tested as late as 95 days of age. In the present study, animals were more than a year old at time of testing. The results of the present study however, differed from Riley et al. [8] in that test stimuli did affect nose poking behavior in alcohol exposed animals. This effect depended on the sex of the animals being tested. Females prenatally exposed to alcohol engaged in more nose poking if allowed the opportunity to view female mice but this increase did not occur if the bedding from a cage previously occupied by female mice was the stimulus. This implies that female alcohol-exposed animals in the first study were responding to the movement of female animals rater than the odors of female mice. Male alcohol-exposed animals, on the other hand, made more nose pokes than controls when tested in the absence of female mice. Testing males with female bedding increased nose poking compared to testing in front of an empty cage. This suggests that for male alcohol-exposed animals, males' movement decreased nose poking but odor increased it. One possibility to explain this apparent inconsistency is that movement caused males to engage in fewer nose pokes because they spent more time per poke looking at moving animals. Since we did not measure duration of nose pokes, we are unable to state whether the relative effects of testing with female mice may have been due to shorter or longer durations of individual nose pokes nor are we able to comment on the transitions between holes. These results imply that nose poking in older animals prenatally exposed to alcohol is affected by test stimuli, but differently for males and females.

204

ABEL AND DINTCHEFF ACKNOWLEDGEMENTS We thank D. Dintcheff, C. Matyjasik, M. McGowan, M. J. Noble and C. Poveromo for technical assistance. This study was supported by grant No. AA05631 from the National Institue on Alcoholism and Alcohol Abuse to E. L. Abel.

REFERENCES 1. Abel, E. L. Prenatal effects of alcohol on adult learning in rats. Pharmacol Biochem Behav 10: 239-243, 1979. 2. Abel, E. L. In utero alcohol exposure and developmental delay of response inhibition. Alcohol Clin Exp Res 6: 369-376, 1982. 3. Abel, E. L. and B. A. Dintcheff. Effects of prenatal alcohol exposure on behavior of aged rats. Drug Alcohol Depend, in press. 4. Bond, N. W. and E. L. DiGiusto. Effects of prenatal alcohol consumption on open-field behavior and alcohol preference in rats. Psychopharmacologia 46: 163-168, 1976. 5. Bond, N. W. and E. L. DiGiusto. Avoidance conditioning and Hebb-Williams maze performance in rats treated prenatally with alcohol. Psychopharmacology (Berlin) 58: 69-71, 1978.

6. Edwards, A. L. Experimental Design in Psychological Research. New York: Rinehart and Winston, 1965. 7. Plonsky, M. and E. P. Riley. Head-dipping behaviors in rats exposed to alcohol prenatally as a function of age at testing. Neurobehav Toxicol Teratol 5: 309-314, 1983. 8. Riley, E. P., N. R. Shapiro and E. A. Lochry. Nose-poking and head-dipping behaviors in rats prenatally exposed to alcohol. Pharmacol Biochem Behav 11: 513-519, 1979. 9. Riley, E. P., E. A. Lochry and N. R. Shapiro. Response perseveration in rats exposed to alcohol prenatally. Pharmacol Bioehem Behav 10: 255-259, 1979. 10. Riley, E. P., E. A. Lochry and N. R. Shapiro, Lack of response inhibition in rats prenatally exposed to alcohol. Psyehopharmacology (Berlin) 62: 47-52, 1979,