Psychopharmacology
Psychopharmacology 62, 247-251 (1979)
9
by Springer-Verlag 1979
Effects of Ethyl Alcohol on Development and Social Behaviour in the Offspring of Laboratory Mice Franzeska G. Ewart and Margaret G. Cutler* Department of Biological Sciences, Glasgow College of Technology, Glasgow G40BA, Scotland
Abstract. Behavioural and developmental effects o f exposure to a low level of ethyl alcohol f r o m conception to a d u l t h o o d have been examined in mice. A 5 % solution of alcohol in the drinking fluid o f male and female adults did not impair fertility. The offspring of treated mice were significantly lighter at birth than their controls and remained so t h r o u g h o u t their life span. N o congenital malformations were observed. Behaviour o f the offspring at 3 - 4 weeks o f age and in a d u l t h o o d was examined by ethological analysis o f encounters between alcohol-treated and control mice o f the same sex. In male and female juvenile mice receiving 5 % alcohol, the frequency and duration o f social investigation were significantly lower than those o f control partners and the duration of non-social behaviour was raised. Individual elements of 'investigate' and 'sniff' were significantly reduced in frequency. In alcohol-treated adults, the only behavioural difference f r o m controls was an increased frequency o f flight elements occurring in males. It is suggested that longterm exposure to alcohol m a y lead to behavioural adaptation.
Key words: ,Ethyl alcohol Growth -
Mouse offspring -
Behaviour
It has been suggested that chronic exposure to alcohol by pregnant w o m e n m a y lead to retarded physical and mental development in their children, sometimes in association with specific congenital malformations, and these developmental effects have been called the 'foetal alcohol s y n d r o m e ' (Bianchine and Taylor, 1974; Ferrier et al., 1973; Jones and Smith, 1973; Kaminski et al., 1975). However, the causal role played by ethyl alcohol has been questioned since the n u m b e r o f reported eases is small relative to the total n u m b e r of * To whom offprint requests should be sent
women alcoholics, and factors other than alcohol ingestion m a y well play a part. O n this account, an animal model needs to be employed to examine effects o f ethyl alcohol on the outcome o f pregnancy and on the growth and behaviour of the offspring. In these experiments effects o f a non-ataxic dose o f ethyl alcohol have been examined in the laboratory mouse. Effects on growth and development have been examined by standard procedures and behavioural effects have been assessed by the ethological techniques described by Silverman (1965) and G r a n t and Mackintosh (1963). These techniques provide a m e t h o d o f quantifying changes in unrestrained social behaviour produced by drugs or other psychoactive compounds, and have been f o u n d in other experiments to be highly sensitive and to give reproducible results (Cutler et al., 1975a).
Materials and Methods Animals. Male and female CFW mice supplied by Tucks Ltd. were used in these experiments. Mice were maintained under a reversed lighting regime with a strong white light from 3 P.M. to 3 A.M. and darkness for the remainder of the period. There was an ad libitum supply of drink and Diet 41B. Experimental Procedure. For the first 3 weeks of the experiment, mice were housed in groups of ten animals of the same sex in stock boxes measuring 38 • 15 • 13 cm. The mice had been randomly distributed into two groups, one of which received 5 % ethyl alcohol as the sole drinking fluid whereas the other was given tap water. After equilibration to the new regime for 3 weeks, breeding was commenced. The mice were housed in pairs of one male with one female from the same treatment group in transparent observation cages measuring 33 x 15 x 13 cm and treatment was continued as before. When females had reached an advanced stage of pregnancy, nest material was added to the cages. On the day of birth all offspring were examined and counted. Pups were weighed daily from 1 day of age until weaning and the mean weights of the litters recorded. A note was made of the age of the pups when fur growth was first observed, when their eyes opened and when they began to explore the cage. At 6 days after birth the size of each litter was culled to six animals.
0033-3158/79/0062/0247/$01.00
248
Psychopharmacology 62 (1979)
Table 1. Categories adopted for the description of behaviour and
their respective postural elements (adapted from Grant and Mackintosh, 1963) Category
Elements
Non-Social
Explore, scan, wash, self-groom, scratch, eat, dig, push-dig, on-bars, off-bars, immobility Attend, nose, investigate, sniff, push-under, groom, stretchattend, follow, attempted mount, mount, genital groom, crawl over
Social investigation and sexual behaviour
Aggression
Flight
Threat, thrust, attack, bite, aggressive groom, chase, offensive upright posture, offensive sideways posture Flag, evade, retreat, flee, oblique, freeze, defensive upright posture, defensive sideways posture
At 21 - 30 days of age, depending on their size, pups were weaned, sexed and housed in groups of 10-15 animals in stock boxes measuring 38 x 25 x 18 cm. The offspring of alcohol-treated mice were given 5 % ethyl alcohol as their drinking fluid and the offspring of the control mice received tap water. An ad libitum supply of Diet 41B was given to all animals. The behaviour of the offspring was examined shortly after weaning when they were 3 - 4 weeks old and when they were adults (at 1 8 - 2 4 weeks of age). The behaviour of each of the mice was examined for a 5-rain period when encountering an unfamiliar mouse in a clean transparent observation cage with sawdust flooring and a metal grid roof. On each occasion encounters were between an alcohol-treated mouse and an untreated control partner of the same sex. For indentification purposes, alcohol-treated mice were marked on their backs with water-based felt pen and controls with strokes of water. Encounters between control mice marked with a felt pen and untreated controls were also examined to find out if the marking had any effect on behaviour. Elements of behaviour shown by the mice during each observation were recorded on a tape recorder using the check list of elements described by Grant and Mackintosh (1966). The major elements of behaviour are listed in Table 1 within four behavioural categories: non-social behaviour, social investigation and sexual behaviour, aggression and flight, these being grouped as described by Dixon and Mackintosh (1971). The behavioural categories are derived from cluster analysis and factor analysis of the sequence of elements seen in encounters between normal mice. For each category of behaviour, the total frequency and total duration of elements in each observation were recorded on a machine especially constructed for that purpose. A record was also made of the frequency with which each individual element occurred within an observation. The data were summarised by the means for each group.
Results
Effects of Alcohol on Fertility in Mice and on the Rate of Development in Their Offspring Table 2 shows that treatment with 5 % ethyl alcohol was without significant effect on the period of gestation in
Table 2. Effects of chronic alcohol administration to breeding mice on the outcome of pregnancy and development of the litters No. in group
Treated group (N= 15)
Control group (N= 15)
Mean duration of pregnancy, days Females bearing litters, % Mean no. born/litter
25 81 11.6
24 93 11.6
Mean weight of pups (g) at: 1 day 5 days 10 days 15 days 20 days
1.5"* 2.3** 4.3** 5.9** 7.3**
1.8 2.9 5.5 7.2 8.8
Mean age of pups at first appearance of fur, days
7.0*
5.8
Mean age of pups at eye opening, days
15.8
15.1
** P < 0.01, *P< 0.05 between treated and control mice by Student's t-test
breeding females or on the size of their litters. Development of the pups was affected. The first appearance of fur growth occurred at a significantly later age in alcohol-treated pups than in controls, although the age at which eye opening occurred was not significantly affected by the treatment. The pups in the alcohol-treated group were significantly lighter than their controls. The difference in weight between the groups was significant at 99 % confidence limits when the pups were weighed at 1 day of age, and the subsequent rate of weight gain up to an age of 21 days was lower among alcohol-treated pups than among controls, the coefficient of linear regression of body weight against age being 0.33 in the treated group and 0.39 in the control group. From the regression curves the mean weights at birth appeared as 0.89 g in the treated group and 1.19 g in the controls. Figure 1 shows that mouse pups from the group treated with alcohol were both shorter and smaller than controls of the same age. In adulthood, mice from the group receiving alcohol continued to be lighter than their controls. At the age of 1 8 - 24 weeks, both males and females of the treated group weighed significantly less than controls at 99 % confidence limits, the mean weight for females being 27.7 g in the treated and 31.3 g in the control group, and the mean weights for males being 33.0 g in the treated and 36.3 g in the control group. The fluid intake of mice receiving alcohol in their drinking water was similar to that of the controls, averaging approximately 7 - 9 ml/24 h. The mean daily intake of alcohol by treated mice amounted to 17 mg/g body weight on average.
F. G. Ewart and M. G. Cutler: Alcohol on Mouse Development and Behaviour
249
Table 3. Effects of chronic alcohol administration on social behaviour in juvenile mice aged 3 - 4 weeks Sex group No. examined
Female treated 20
Partner 20
Male treated 12
Mean frequency of behavioural elements: Non-social behaviour I06.2 111.2 103.2 Social investigation 23.5** 37.2 22.3* Sniff 2.5* 4.6 2.3 Investigate 6.0** 11.4 5.8* Flight 0.1 0.1 0 Aggression 0 0 0 Mean duration of behavioural elements (s): Non-social behaviour 268.0** 240.0 270.2* Social investigation 32.0** 59.7 29.8* Flight 0.02 0.03 0 Aggression 0 0 0
Partner 12
105.8 32.5 2.6 10.2 0 0
245.8 54.2 0 0
** P
Psychopharmacology 62, 247-251 (1979)
9
by Springer-Verlag 1979
Effects of Ethyl Alcohol on Development and Social Behaviour in the Offspring of Laboratory Mice Franzeska G. Ewart and Margaret G. Cutler* Department of Biological Sciences, Glasgow College of Technology, Glasgow G40BA, Scotland
Abstract. Behavioural and developmental effects o f exposure to a low level of ethyl alcohol f r o m conception to a d u l t h o o d have been examined in mice. A 5 % solution of alcohol in the drinking fluid o f male and female adults did not impair fertility. The offspring of treated mice were significantly lighter at birth than their controls and remained so t h r o u g h o u t their life span. N o congenital malformations were observed. Behaviour o f the offspring at 3 - 4 weeks o f age and in a d u l t h o o d was examined by ethological analysis o f encounters between alcohol-treated and control mice o f the same sex. In male and female juvenile mice receiving 5 % alcohol, the frequency and duration o f social investigation were significantly lower than those o f control partners and the duration of non-social behaviour was raised. Individual elements of 'investigate' and 'sniff' were significantly reduced in frequency. In alcohol-treated adults, the only behavioural difference f r o m controls was an increased frequency o f flight elements occurring in males. It is suggested that longterm exposure to alcohol m a y lead to behavioural adaptation.
Key words: ,Ethyl alcohol Growth -
Mouse offspring -
Behaviour
It has been suggested that chronic exposure to alcohol by pregnant w o m e n m a y lead to retarded physical and mental development in their children, sometimes in association with specific congenital malformations, and these developmental effects have been called the 'foetal alcohol s y n d r o m e ' (Bianchine and Taylor, 1974; Ferrier et al., 1973; Jones and Smith, 1973; Kaminski et al., 1975). However, the causal role played by ethyl alcohol has been questioned since the n u m b e r o f reported eases is small relative to the total n u m b e r of * To whom offprint requests should be sent
women alcoholics, and factors other than alcohol ingestion m a y well play a part. O n this account, an animal model needs to be employed to examine effects o f ethyl alcohol on the outcome o f pregnancy and on the growth and behaviour of the offspring. In these experiments effects o f a non-ataxic dose o f ethyl alcohol have been examined in the laboratory mouse. Effects on growth and development have been examined by standard procedures and behavioural effects have been assessed by the ethological techniques described by Silverman (1965) and G r a n t and Mackintosh (1963). These techniques provide a m e t h o d o f quantifying changes in unrestrained social behaviour produced by drugs or other psychoactive compounds, and have been f o u n d in other experiments to be highly sensitive and to give reproducible results (Cutler et al., 1975a).
Materials and Methods Animals. Male and female CFW mice supplied by Tucks Ltd. were used in these experiments. Mice were maintained under a reversed lighting regime with a strong white light from 3 P.M. to 3 A.M. and darkness for the remainder of the period. There was an ad libitum supply of drink and Diet 41B. Experimental Procedure. For the first 3 weeks of the experiment, mice were housed in groups of ten animals of the same sex in stock boxes measuring 38 • 15 • 13 cm. The mice had been randomly distributed into two groups, one of which received 5 % ethyl alcohol as the sole drinking fluid whereas the other was given tap water. After equilibration to the new regime for 3 weeks, breeding was commenced. The mice were housed in pairs of one male with one female from the same treatment group in transparent observation cages measuring 33 x 15 x 13 cm and treatment was continued as before. When females had reached an advanced stage of pregnancy, nest material was added to the cages. On the day of birth all offspring were examined and counted. Pups were weighed daily from 1 day of age until weaning and the mean weights of the litters recorded. A note was made of the age of the pups when fur growth was first observed, when their eyes opened and when they began to explore the cage. At 6 days after birth the size of each litter was culled to six animals.
0033-3158/79/0062/0247/$01.00
248
Psychopharmacology 62 (1979)
Table 1. Categories adopted for the description of behaviour and
their respective postural elements (adapted from Grant and Mackintosh, 1963) Category
Elements
Non-Social
Explore, scan, wash, self-groom, scratch, eat, dig, push-dig, on-bars, off-bars, immobility Attend, nose, investigate, sniff, push-under, groom, stretchattend, follow, attempted mount, mount, genital groom, crawl over
Social investigation and sexual behaviour
Aggression
Flight
Threat, thrust, attack, bite, aggressive groom, chase, offensive upright posture, offensive sideways posture Flag, evade, retreat, flee, oblique, freeze, defensive upright posture, defensive sideways posture
At 21 - 30 days of age, depending on their size, pups were weaned, sexed and housed in groups of 10-15 animals in stock boxes measuring 38 x 25 x 18 cm. The offspring of alcohol-treated mice were given 5 % ethyl alcohol as their drinking fluid and the offspring of the control mice received tap water. An ad libitum supply of Diet 41B was given to all animals. The behaviour of the offspring was examined shortly after weaning when they were 3 - 4 weeks old and when they were adults (at 1 8 - 2 4 weeks of age). The behaviour of each of the mice was examined for a 5-rain period when encountering an unfamiliar mouse in a clean transparent observation cage with sawdust flooring and a metal grid roof. On each occasion encounters were between an alcohol-treated mouse and an untreated control partner of the same sex. For indentification purposes, alcohol-treated mice were marked on their backs with water-based felt pen and controls with strokes of water. Encounters between control mice marked with a felt pen and untreated controls were also examined to find out if the marking had any effect on behaviour. Elements of behaviour shown by the mice during each observation were recorded on a tape recorder using the check list of elements described by Grant and Mackintosh (1966). The major elements of behaviour are listed in Table 1 within four behavioural categories: non-social behaviour, social investigation and sexual behaviour, aggression and flight, these being grouped as described by Dixon and Mackintosh (1971). The behavioural categories are derived from cluster analysis and factor analysis of the sequence of elements seen in encounters between normal mice. For each category of behaviour, the total frequency and total duration of elements in each observation were recorded on a machine especially constructed for that purpose. A record was also made of the frequency with which each individual element occurred within an observation. The data were summarised by the means for each group.
Results
Effects of Alcohol on Fertility in Mice and on the Rate of Development in Their Offspring Table 2 shows that treatment with 5 % ethyl alcohol was without significant effect on the period of gestation in
Table 2. Effects of chronic alcohol administration to breeding mice on the outcome of pregnancy and development of the litters No. in group
Treated group (N= 15)
Control group (N= 15)
Mean duration of pregnancy, days Females bearing litters, % Mean no. born/litter
25 81 11.6
24 93 11.6
Mean weight of pups (g) at: 1 day 5 days 10 days 15 days 20 days
1.5"* 2.3** 4.3** 5.9** 7.3**
1.8 2.9 5.5 7.2 8.8
Mean age of pups at first appearance of fur, days
7.0*
5.8
Mean age of pups at eye opening, days
15.8
15.1
** P < 0.01, *P< 0.05 between treated and control mice by Student's t-test
breeding females or on the size of their litters. Development of the pups was affected. The first appearance of fur growth occurred at a significantly later age in alcohol-treated pups than in controls, although the age at which eye opening occurred was not significantly affected by the treatment. The pups in the alcohol-treated group were significantly lighter than their controls. The difference in weight between the groups was significant at 99 % confidence limits when the pups were weighed at 1 day of age, and the subsequent rate of weight gain up to an age of 21 days was lower among alcohol-treated pups than among controls, the coefficient of linear regression of body weight against age being 0.33 in the treated group and 0.39 in the control group. From the regression curves the mean weights at birth appeared as 0.89 g in the treated group and 1.19 g in the controls. Figure 1 shows that mouse pups from the group treated with alcohol were both shorter and smaller than controls of the same age. In adulthood, mice from the group receiving alcohol continued to be lighter than their controls. At the age of 1 8 - 24 weeks, both males and females of the treated group weighed significantly less than controls at 99 % confidence limits, the mean weight for females being 27.7 g in the treated and 31.3 g in the control group, and the mean weights for males being 33.0 g in the treated and 36.3 g in the control group. The fluid intake of mice receiving alcohol in their drinking water was similar to that of the controls, averaging approximately 7 - 9 ml/24 h. The mean daily intake of alcohol by treated mice amounted to 17 mg/g body weight on average.
F. G. Ewart and M. G. Cutler: Alcohol on Mouse Development and Behaviour
249
Table 3. Effects of chronic alcohol administration on social behaviour in juvenile mice aged 3 - 4 weeks Sex group No. examined
Female treated 20
Partner 20
Male treated 12
Mean frequency of behavioural elements: Non-social behaviour I06.2 111.2 103.2 Social investigation 23.5** 37.2 22.3* Sniff 2.5* 4.6 2.3 Investigate 6.0** 11.4 5.8* Flight 0.1 0.1 0 Aggression 0 0 0 Mean duration of behavioural elements (s): Non-social behaviour 268.0** 240.0 270.2* Social investigation 32.0** 59.7 29.8* Flight 0.02 0.03 0 Aggression 0 0 0
Partner 12
105.8 32.5 2.6 10.2 0 0
245.8 54.2 0 0
** P
