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Locomotor activity and alcohol preference in alcohol-preferring and -nonpreferring rats following neonatal alcohol exposure
Neurotoxicology and Teratology, Vol. 17, No. 1, pp. 41-48, 1995 Copyright o 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0892-0362/95 $9.50 + .OO
Pergamon 0892-0362(94)00051-4
Locomotor Activity and Alcohol Preference in Alcohol-Preferring and -Nonpreferring Rats Following Neonatal Alcohol Exposure TED
MELCER,’
DAVID
GONZALEZ
AND
EDWARD
P. RILEY
Department of Psychology, San Diego State University, San Diego, CA 92120 Received
9 December
1993; Accepted
14 July 1994
MELCER, T., D. GONZALEZ AND E. P. RILEY. Locomotor activityand alcoholpreference in alcohol-preferringand -nonpreferring ratsfollowing neonatal alcohol exposure. NEUROTOXICOL TERATOL 17(l) 41-48, 1995. -Recently, we reported that the alcohol preferring (P) and nonpreferring (NP) rats, bred for differences in alcohol preference, showed different behavioral effects of neonatal alcohol exposure when tested as juveniles. Following neonatal alcohol exposure, the P line showed a greater increase in activity than the NP line relative to their respective controls. In the present study, P and NP rat pups were separated from their mothers and artificially reared from postnatal day (PND) 4 until PND 12. Pups were implanted with intragastric cannulas on PND 4 and fed a stock milk solution every 2 h via an indwelling cannula. One group had alcohol added to the milk diet during the 4 daytime feeds at a dose of 6 g/kg/day on PND 4-7 and a dose of 3 g/kg/day on PNDs 8 and 9. One control group was artificially reared and fed an isocaloric milk solution and a second control group was reared normally with a surrogate dam. Rats were tested as adults (IO-day-old) for open-field activity and alcohol preference. Neonatal alcohol exposure caused equivalent increases in activity in P and NP rats. In the ethanol preference test, neither the P nor the NP rats showed any effect of neonatal alcohol treatment although there were large line differences in alcohol preference. These data suggest that the increased susceptibility of young P rats to neonatal alcohol exposure, measured by open-field activity, does not extend to adulthood. Furthermore, neonatal alcohol exposure does not appear to alter alcohol preference in either line. Fetal Alcohol Syndrome
P rats
NP rats
Neonatal alcohol/activity
PRENATAL alcohol exposure can produce profound behavioral and physical effects in infants and young children. These effects include growth deficits, physical abnormalities, and altered behavior such as increased body tremors, hand move-
Several investigators have proposed that genetic factors might contribute to this variability in outcomes seen following prenatal alcohol exposure (9,12,26). It is possible that differences in alcohol metabolism or sensitivity to alcohol’s effects might predict the susceptibility of the developing fetus to the effects of fetal alcohol exposure. There are now several reports showing differential outcomes in inbred strains or selectively bred lines that support the role of genetic factors in the etiology of fetal alcohol effects. For instance, long sleep (LS) mice, bred for extreme loss of the righting reflex after ethanol exposure, produced litters that showed greater mortality, growth deficits, and slower passive avoidance learning due to prenatal alcohol exposure than did short sleep (SS) mice, bred for insensitivity to the hypnotic effects of alcohol (13,14, 15,16). Differential effects of neonatal alcohol exposure resulted when comparing Maudsley Reactive (MR) and MS20 rats. The M520 strain is initially more sensitive to acute alco-
ments, and overall body activity as well as impairments in attention and learning (28,29). In cases where these symptoms
occur in a recognized pattern, the child may be diagnosed with Fetal Alcohol Syndrome (FAS). It is important to note that FAS has been estimated to occur in slightly less than 10% of the children of alcohol abusing women (1,4). Similarly, in animal models of fetal alcohol exposure, it is not uncommon to find normally appearing or behaving animals within the same litter as animals that show the effects of in utero alcohol exposure. A critical question is to identify risk factors that make some women or their fetuses more susceptible than others to the effects of equivalent amounts of gestational alcohol exposure. ,-
’ Requests for reprints should be addressed to Ted Melter, Center for Behavioral Teratology, 6363 Alvarado Court, #209, San Diego State University, San Diego, CA 92120. 41
MELCER,
4L
ho1 effects but becomes less sensitive than the MR strain with repeated alcohol exposure (30). The MR rats showed greater reduction in cerebellar weight due to neonatal alcohol exposure than did the M520 rats (16). Recently, we have studied the effects of neonatal alcohol exposure on the behavior of the selectively bred alcohol preferring (P) and alcohol nonpreferring (NP) rat lines (20). These lines were bred for extremes in alcohol preference but also differ in their functional tolerance to alcohol, with the P line showing greater tolerance than the NP line. These two lines were exposed to alcohol neonatally using the “pup-in-thecup” protocol and were then tested in an open field just prior to weaning. Contrary to our expectations, the P rats displayed larger activity increases in the open field as a result of the neonatal alcohol exposure than did the NP rats. Importantly, neonatal alcohol treatment produced equivalent blood alcohol levels in the P and NP lines, eliminating the possibility that differential alcohol effects were caused by differences between lines in the rate of ethanol metabolism (25). More recently, we examined the effects of neonatal alcohol exposure on walking patterns (gait) and balance (walking along elevated parallel bars) in both P and NP rats. Neonatal alcohol exposure altered gait and decreased balance in both lines. There was no evidence of differential susceptibility of the P and NP lines to neonatal alcohol exposure as measured by these motor skills (22). These studies indicate that P rats may be more susceptible to some, but not all, of the consequences of neonatal alcohol exposure than NP rats. The present study followed up on our previous work showing increased activity in both P and NP juvenile rats after neonatal alcohol exposure. Because the P line shows greater functional tolerance to alcohol than the NP line, we were surprised that the P line experienced more adverse effects of neonatal alcohol exposure than the NP line. Increased activity following prenatal alcohol exposure is most commonly reported in weaning rats (18- to 30-day-old) and frequently diminishes in adults (Q although it is again seen in aged rats (2,3). Also, more complex tests can reveal prenatal alcohol effects in adults (24). Similar effects of age have been reported following neonatal alcohol exposure (17,18,19) although increased activity in adult female rats exposed to alcohol neonatally has been reported (19). The current study was conducted to determine if persistent effects on activity could be found in adulthood following neonatal alcohol exposure and if the P and NP rats might be differentially sensitive to these neonatal alcohol effects. The second purpose of this study was to determine if neonatal alcohol exposure might affect subsequent alcohol preference. Previous work on this question has met with mixed results (23). For example, Abel and York (5) did not find any influence of prenatal alcohol exposure on subsequent alcohol intake when rats received free choice between an ethanol solution and water. Similarly, unpublished work from our lab has failed to demonstrate any effect of prenatal alcohol exposure on subsequent alcohol intake or preference. However, Bond and Digiusto (7) found prenatal alcohol exposure resulted in an enhanced intake of alcohol at low concentrations in 45-dayold female rats. A more recent study showed that injection of a 6% alcohol solution into the amniotic fluid of gestation day (CD) 21 rat fetuses increased approach toward alcohol odor and intake of an alcohol solution by S-day-old rat pups (10). Some of the variability in the results of previous studies might be due to genetic differences between rats in alcohol preference. By using lines of rats selectively bred for differential alcohol preference, it might provide an answer to the question
GONZALEZ
AND RILEY
of whether early alcohol exposure influences subsequent alcohol preference. In the present study, P and NP rats pups received alcohol during the brain growth spurt that peaks between postnatal days (PND) 4 to 10 in the rat. In the human fetus, the brain growth spurt occurs during the third trimester of pregnancy and is believed to be a critical period of early brain development (11). The neonatal exposure paradigm in the rat thus models the effects of third trimester alcohol exposure in humans in terms of CNS development. The P and NP received alcohol exposure or control treatment and were tested as adults for open-field activity and then for ethanol preference. METHOD
Subjects
P and NP rat pups were used as subjects. Parent animals were supplied by Drs. T.-K. Li and L. Lumeng of the Indiana University School of Medicine Alcohol Research Center (Indianapolis, IN) and offspring were born at the San Diego State University laboratory of the principal investigator (EPR). All rats were maintained on a 12L : 12D cycle throughout the experiment (lights on at 0700). Mating
Breeder females were group housed and breeder males were individually housed in metal hanging cages. There were no differences between the P and NP groups in terms of the age (70-200 days of age) or body weights (247-340 g) at the time of mating. A few females provided multiple litters for this study and the majority of the animals were multiparous. For mating, one female was placed in the home cage of each male in the evening and left overnight. A check for seminal plugs in the morning was used as evidence of mating and CD 0 was defined as the day a plug was found. Females that did not mate were returned to group housing. Females that mated were housed in plastic breeding cages in a temperature (70°F) and humidity (40% to 60%) controlled nursery with ad lib rat chow (Purina Rat Chow) and water during the remainder of gestation. Breeding cages were checked at 8:30 a.m. and 5:00 p.m. daily for births. Twenty-four hours after a birth, litters were weighed and culled to the 10 heaviest pups (5 males and 5 females whenever possible). The heaviest animals were maintained because this selection procedure improves the outcome of surgery. The surgery to install the stomach cannula for artificial rearing (AR) was conducted on the morning of CD 26 (26 days after mating) which was almost always PND 4. Prior to surgery, pups were removed from the litter, weighed, and the heaviest pups were randomly assigned to one of the following 2 treatment groups: (a) artificial rearing with ethanol exposure (6 g/kg/day) or (b) artificial rearing without ethanol exposure. In this artificially reared control group, maltose calories were substituted isocalorically for ethanol derived calories. The remaining pups received sham surgery (no stomach cannula installed) and normal rearing with a Sprague-Dawley female that had delivered her pups at about the same time the P and NP rats were born. The use of Sprague-Dawley surrogate mothers precluded any effects that might be due to differences in maternal care between the natural P and NP mothers. Litter sizes were maintained at 8 pups using Sprague-Dawley pups where necessary. Neonatal Surgical Procedure
The procedure for surgical implantation of the intragastric cannula has been described in great detail previously for the
NEONATAL
ALCOHOL
43
EXPOSURE
interested reader (6). Each pup was anesthetized with a 50% halothane/50% oxygen mixture and then a stainless steel surgical wire sheathed in polyethylene tubing was maneuvered down the esophagus into the stomach. The lower tip of the wire was pushed out through the stomach and abdominal wall. The feeding cannula, made of Clay Adams polyethylene tubing (PE lo), was attached to the end of the wire protruding from the pup’s mouth. This connection was coated with an antibiotic and corn oil, and the cannula was pulled into the stomach and out through the abdominal wall. A thin plastic disc attached to the end of the cannula formed a seal against the inside of the stomach wall which prevented milk from leaking out. The cannula was then pulled through loose skin about the back of the pup’s neck and washers were attached to the cannula where it entered and exited the back of the neck. The washers absorbed any pull on the cannula at the point of the neck and prevented direct pull on the cannula in the pup’s stomach. The entire surgical procedure required about 2 min per animal. Pups recovered from the anesthetic within 3 min. The sham surgery group received identical surgical procedures except that the cannula was not installed and the pups were returned to a Sprague-Dawley dam after surgery.
received the base milk formula. The AR pups were maintained on PNDs 10 and 11 on the base milk formula without alcohol to allow recovery from acute ethanol effects or withdrawal symptoms that were occasionally observed. The ethanol and maltose groups received isocaloric milk formula throughout the artificial rearing period (PNDs 4-l 1). Pups were weighed daily during the artificial rearing period. On the morning of PN 12, pups from all groups were earmarked for group identification and weighed. Each cannula was cut where it exited the pup’s side and all pups were bathed in a slurry of maternal feces and warm water before being returned to a dam. This slurry procedure virtually eliminated pup rejection by the dam. Pups were returned to SpragueDawley dams. Each of these surrogate dams received a new litter of 8 pups including equal numbers of pups from the AR groups whenever possible. The sham pups were placed with separate surrogate dams to prevent competition with and promote survival of the typically smaller AR pups. Pups were weaned from surrogate dams on PND 21 and housed in metal hanging cages with ad lib access to food and water until behavioral testing. Two animals of the same sex lived in each cage. Activity Testing
Neonatal Rearing and Maintenance From PND 4 until PND 12, the artificially reared pups were individually housed and maintained in Styrofoam cups containing hardwood chips and lined with a piece of synthetic short-haired fur. The fur reportedly minimizes behavioral depression associated with maternal deprivation (31). The cups in which pups lived were placed inside Styrofoam cups partially filled with white washed play sand to keep the housing cup dry and upright. All cups floated in an aquarium tank filled with 49’C aerated water. Every 2 days, the water temperature was decreased by 2OC until the tank temperature was 42’C. This temperature schedule allowed the AR pups to maintain body temperatures similar to those observed in sham control pups in the litter. Each pup’s stomach cannula was connected to a tygon tubing lead attached to a syringe containing a milk-based formula which substituted for mother’s milk (27). The syringes were mounted on an infusion pump (Harvard Instruments) operated on an automated-timed schedule of one 20-min period of milk delivery every 2 h. This schedule resulted in a total of 12 feeds of milk formula daily for each pup. All pups received the same base milk formula during the 8 night-time feeds between the hours of 5:30 p.m. and 7:50 a.m. Ethanol or isocaloric maltose was added to the base diet during the 4 daytime feeds between the hours of 9:30 a.m. and 3:50 p.m. for pups in the alcohol and maltose groups. All AR pups were voided before the first day feed and after each of the 4 daytime feeds on PN days 4 through 7. On PN days 8 through 11, pups were voided before the first feed and after the last feed. The amount of milk administered in milliliters was equivalent to 33% of the weight of the AR pups within each line. Because pups from the P line weighed more than pups from the NP line, two artificial rearing apparatuses were used to allow separate control of the feeding schedules for each line. Ethanol administration was limited to the 4 daytime feeds to represent a “binge drinking” model (19). On PNDs 4 through 7 pups in the ethanol group received a dosage of 6 g/kg/day. To minimize the severity of withdrawal, the ethanol exposed pups received a dosage of 3 g/kg/ day on PNDs 8 and 9. On PNDs IO and 11, all AR pups
All rats were handled for 5 mitt/day for 4 days prior to the commencement of activity testing. Beginning on PND 80, animals were tested for open-field activity for 30 mitt/day for 4 consecutive days. The sample sizes were as follows: P 6 g/kg male = 11; P 6 g/kg female = 10; P maltose male = 13; P maltose female = 7; P sham male = 12; P sham female = 7; NP 6 g/kg male = 12; NP 6 g/kg female = 11; NP maltose male = 10; NP maltose female = 7; NP sham male = 12; NP sham female = 10. Each rat was removed from its home cage and individually placed in one of four automated activity monitors (Omnitech Instruments) for a 30-min test session. Each monitor consisted of a 39 cm x 39 cm chamber with an infrared photobeam grid criss-crossing the apparatus. The monitors were enclosed in sound attenuated boxes with red light illumination (5 W) and white noise. Data on rat movements were detected by photobeam and were automatically recorded by computer. The dependent variables recorded were horizontal activity (number of photobeams crossed), total distance traveled (in inches), and movement time (in seconds). At the conclusion of each test session, rats were returned to their home cage. Testing occurred 5 to 8 h into the animals’ light cycle and individual animals were tested at the same time on each of the 4 test days. Ethanol Preference Test One week after activity testing, the P and NP rats were taken from the hanging cages and housed individually in breeding cages with ad lib access to food and water for 3 days. The sample sizes were as follows: P 6 g/kg male = 11; P 6 g/ kg female = 10; P maltose male = 13; P maltose female = 7; P sham male = 12; P sham female = 7; NP 6 g/kg male = 12; NP 6 g/kg female = 9; NP maltose male = 9; NP maltose female = 6; NP sham male = 12; NP sham female = 9. Five animals from activity testing were inadvertently left out of the ethanol preference test. In the first phase (ethanol exposure), rats received a 10% ethanol solution (v/v) as their sole fluid source for 4 consecutive days. This ethanol exposure phase was used in the initial selective breeding experiments to increase acceptability of eth-
44
MELCER,
anol during later preference testing (21). In the second phase (ethanol preference), rats received a choice between a 10% ethanol solution and water. Fluids were presented in graduated cylinders and the location of the ethanol and water cylinders was alternated daily. The ethanol preference tests were conducted for 9 consecutive days. The number of milliliters intake of water and/or ethanol were recorded daily at 3:00 p.m. The graduated cylinders were then filled with fresh water or 10% ethanol. Food was available ad lib during all phases of testing.
GONZALEZ
ALCOHOL PREFERRING
3000
AND RILEY
RATS
iiiI” ii 3
‘Wg 2500
Maltose
1
Sham
RESULTS
Pup Data
Table 1 shows mean body weights of pups during the neonatal treatment period from PNDs 4 through 10. The means are collapsed across sex because sex did not interact with line or day. An analysis of variance (ANOVA) was conducted on pup weights with sex, line (P or NP rats) and neonatal treatment (ethanol, maltose, sham) as between-subjects factors and postnatal age (PND 4 through 10) as a within-subjects factor. The overall ANOVA indicated main effects of sex, F(1, 110) = 9.65, p < 0.01, because males weighed more than females; line, F(1, 110) = 14.90, p < 0.001, as P pups weighed more than NP pups; and postnatal age, F(6, 660) = 1524.43, p < 0.001, as body weights increased with age. The analysis also revealed significant day x treatment, F(12, 660) = 23.73, p < 0.001, day x line, F(6, 660) = 3.23, p < 0.01 and day x treatment x line interactions, F(12, 660) = 2.96, p < 0.001. Post hoc tests (Newman-Keuls, p < 0.05) were used to analyze the day x treatment x line interaction. As seen in Table 1, sham pups initially weighed less than maltose and ethanol pups, and this effect reversed, particularly in the P line, during the last few days of the neonatal treatment period. Specifically, the sham groups weighed less than the AR groups of their respective lines on PNDs 4 through 7 while the weights of the AR groups were equivalent. On PND 8, the body weights of the P groups were equivalent
ii
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FIG. 1. Mean (k SEM) total distance traveled in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
TABLE 1 MEAN AND SEM (IN PARENTHESES) FOR BODY WEIGHTS IN GRAMS DURING THE ARTIFICIAL REARING PERIOD
Neonatal Treatment 6 g/kg
Maltose
Sham
P rats 4 5 6 7 8 9 10
10.33 (.22) 12.07 (.25) 13.66 (.30) 15.04 (.37) 16.43 (.37) 16.64 (.35) 18.02 (.42)
10.60 (.23) 12.22 (.21) 13.80 (.18) 15.57 (.29) 16.48 (.30) 17.79 (.43) 18.85 (A)
9.07 (.25) 10.64 (.42) 12.21 (.44) 14.35 (.48) 16.16 (.55) 18.55 (.63) 20.93 (.61)
NP rats 4 5 6 7 8 9 10
9.63 (.23) 10.91 (.26) 12.26 (.28) 13.78 (.28) 14.99 (.29) 15.79 (.42) 17.45 (.45)
10.06 (.23) 11.31 (.27) 12.61 (.31) 13.94 (.24) 15.39 (.35) 16.71 (.38) 18.84 (~51)
8.26 (.25) 9.44 (.32) 11.03 (.41) 12.41 (.49) 14.23 (.60) 16.31 (.67) 18.23 (.77)
Postnatal Day
while in the NPs the shams remained lighter than the AR groups. On PNDs 9 and 10 in the P line, the shams weighed more than the maltose or ethanol groups and the maltose group weighed more than the ethanol group. In the NP line, the maltose group weighed more than the ethanol group on PND 9 and more than ethanol or sham groups on PND 10. The NP shams weighed more than ethanol pups on PND 10. Activity Data
Rats were tested for open-field activity beginning at 80 days of age and Figs. 1, 2, and 3 show the results combined across males and females, as sex did not interact with neonatal treatment. As seen in the figures, P and NP rats that received neonatal ethanol exposure (6 g/kg/day) were more active than maltose and sham groups as measured by total distance traveled, horizontal activity, and movement time. We emphasize that alcohol exposure had similar effects on the P and NP lines. The figures also indicate that the NP maltose group had higher activity scores than the NP sham group, while no such artificial rearing effect was evident in the P line. Separate ANOVAs were conducted on activity measures of total distance (inches), horizontal activity (number of photo-
NEONATAL
ALCOHOL
45
EXPOSURE
ALCOHOL PREFERRING
ose groups had higher scores than the sham groups. Figure 2 indicates that the difference between the maltose and sham groups was specific to the NP line but the line x treatment interaction did not reach significance (p c 0.07). The overall ANOVA also showed main effects of sex, F(1, 110) = 98.40, p c 0.001, as females were more active than males; line, F(1, 110) = 19.34, p c 0.001, as P rats were more active than NP rats; and test day, F(3, 330) = 11.09, p < 0.001, as horizontal activity decreased across test days. The ANOVA on movement time (Fig. 3) showed a main effect of neonatal treatment, F(2, 110) = 11.67, p < 0.001. Post hoc tests showed a significant differences among all three treatment groups (6 g/kg vs. maltose; maltose vs. sham; 6 g/ kg vs. sham). Figure 3 indicates differences between the AR groups in the NPs and not the Ps but again the treatment x line interaction only approached significance, F(2, 110) = 2.79 = p < 0.07. The overall ANOVA also showed main effects of line, F(l, 110) = 18.25, p < 0.001, sex, F(1, 110) = 42.56, p < 0.001, and test day, F(3, 330) = 11.95, p < 0.001, and a line x sex x test day interaction, F(3, 330) =
RATS
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ALCOHOL PREFERRING
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FIG. 2. Mean (+ SEM) horizontal activity in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
1200 -
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2
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TEST DAY
beams crossed), and movement time (s) with sex, line and neonatal treatment as between-subjects factors, and test day as the within-subjects factor. Post hoc (Newman-Keuls, p < 0.05) tests were used where necessary to isolate effects from the overall ANOVA. The ANOVA on total distance (Fig. 1) showed a significant neonatal treatment x line interaction, F(2, 110) = 3.69, p < 0.05. Post hoc tests indicated that 6 g/ kg groups of both lines traveled greater distances than their respective maltose or sham controls. However, the distance scores for the P maltose and sham groups were equivalent while the NP maltose group traveled greater distance than the NP sham group. The overall ANOVA showed main effects of sex, F(l, 1IO) = 109.77, p < 0.001, as females traveled farther than males, but there was no treatment x sex interaction; and line, F(l, 110) = 15.36, p < 0.001, as P rats traveled farther than NP rats. There was also a main effect of test day, F(3, 330) = 19.20, p c 0.001 as activity generally decreased across test days. The ANOVA on horizontal activity (Fig. 2) revealed a main effect of neonatal treatment, F(2, 110) = 22.40, p < 0.001. Post hoc tests showed that the 6 g/kg groups had higher horizontal activity scores than maltose or sham groups. The malt-
ALCOHOL NONPREFERRING
RATS
1
_
1400 -
-
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TEST DAY 3. Mean ( f SEM) movement time in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
FIG.
MELCER, TABLE 2 MEAN AND SEM (IN PARENTHESES) FOR BODY WEIGHTS IN GRAMS ON THE LAST DAY OF ACTIVITY TESTING, ETHANOL EXPOSURE, AND ETHANOL PREFERENCE TEST
NeonatalTreatment Maltose
Sham
294 (17.80) n = 21 298 (14.15) n = 23
315 (17.06) n = 20 313 (18.32) n = 17
327 (17.67) n = 19 321 (15.99) n = 22
313 (19.30) n = 21 314 (15.65) n = 21
336 (18.41) n = 20 334 (22.94) n = 15
348 (19.07) n = 19 340 (18.94) n = 21
338 (21.50) n = 21 360 (19.20) n = 21
366 (20.80) n = 20 375 (26.50) n = 15
378 (22.20) n = 19 380 (21.70) n = 21
6 0s Activity P rats NP rats Ethanol Exposure P rats NP rats Ethanol Preference P rats NP rats
Sample sizes shown include both sexes.
4.48, p < 0.01. The P male rats showed a nonsignificant increase in movement time between test days 1 and 4 while P females and NP rats of both sexes decreased movement time across test days. There was no significant interaction between sex and treatment. Activity body weights. Table 2 shows means and SEMs for body weights recorded on the final day of activity testing. An ANOVA with sex, neonatal treatment, and line as betweensubjects factors showed no effects of line and no interaction between sex and treatment and therefore weights are presented averaged across sex. As seen in the table, there was a significant main effect of postnatal treatment, F(2, 110) = 4.07, p < 0.05. Newman-Keuls tests showed the 6 g/kg groups weighed significantly less than the maltose or the sham groups and that the weights of the maltose and sham groups were equivalent. There was also a main effect of sex because males weighed morethanfemales,F(l, 110) = 641.19,p < O.OOl.Noneofthe other main effects or interactions were significant.
GONZALEZ
AND RILEY
males; and test day, F(3, 315) = 93.91, p < 0.001, as intake scores increased across the 4 test days. Finally, the ANOVA also revealed a sex x line interaction, F(1, 105) = 14.62, p < 0.001, as the line differences just described were greater in females than in males, and a day x line interaction, F(3, 315) = 3.62, p < 0.05, as the line difference described above decreased across test days. Ethanol exposure body weights. Table 2 shows means and standard errors for body weights recorded on the last day of ethanol exposure. The weights were averaged across sex as it did not interact with treatment or line. The data were analyzed using ANOVA as just described that showed a significant main effect of neonatal treatment, F(2, 105) = 4.03, p < 0.05. A Newman-Keuls test showed that the 6 g/kg groups weighed less than the maltose or the sham groups while the mean weights of the maltose and sham groups were equivalent. There was also a main effect of sex, F(1, 105) = 580.85, p < 0.001 because males were heavier than females. None of the other effects were significant. Ethanol preference. Figure 4 shows percentage intake of 10% ethanol solution [ml 10% ethanol x lOO/(ml 10% ethanol + ml water)] for the 9 days rats received simultaneous access to the ethanol solution versus water. The means shown are averaged across males and females because there were no effects of sex on ethanol preference. An ANOVA was conducted on percentage ethanol intake with sex, neonatal treatment, and line as between-subjects factors and test day as the within-subjects factor. As seen in Fig. 4, neonatal treatment had no effect on ethanol preference in NP rats. In the P line, the 6 g/kg group showed a nonsignificant enhancement of ethanol preference relative to the control groups on day 1 but not on subsequent test days. The ANOVA showed no significant effects of neonatal treatment. As expected, the P line showed higher percentage ethanol intake than the NP line. The ANOVA revealed main effects of line, F(1,105) =
ETHANOL PREFERENCE TEST
1
Ethanol Exposure Rats received 10% ethanol as their sole source of fluid for 4 days prior to preference testing. An ANOVA was performed on ethanol intake scores corrected for differences in individual body weights [(ml 10% ethanol consumed/gram body weight) x lOOO]with sex, line, and neonatal treatment as betweensubjects factors and test day as the within-subjects factor. There was a main effect of neonatal treatment, fl2, 105) = 7.14, p c 0.001 bdt no interactions between treatment and line, sex or test day. A Newman-Keuls test showed that the 6 g/kg groups (M = 83.3; SE = 2.9) had higher intake scores than the maltose (M = 75.4; SE = 3.4) or the sham groups (M = 67.3; SE = 3.6). The maltose and sham groups were also significantly different. The ANOVA also showed significant main effects of line, fll, 105) = 58.16, p < 0.001, as P rats had higher intake scores than NP rats; sex, F(1, 105) = 57.26, p < 0.001, as females had higher intake scores than
0
12
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5
4
6
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-
fig/kg
--b--
Maltose
--O--
ShamP
P P
&
6glkg
NP
--&-
MaltoseNP
--O-
ShamNP
FIG. 4. Mean (rt SEM) percentage intake 10% ethanol solution (ml etoh x lOO/[ml etoh + ml water]) as a function of neonatal treatment, rat line, and test day. The means shown include both sexes. Rats received simultaneous access to water and 10% ethanol (v/v) beginning at 97 days of age.
NEONATAL
ALCOHOL
EXPOSURE
252.41, p < 0.001, and a test day X line interaction, F(8, 840) = 8.07, p < 0.001, as percentage ethanol intake increased across days in the P rats but not in the NP rats. Finally, there were no effects of sex on ethanol preference. Ethanol preference body weights. Table 2 shows means and SEMs for body weights recorded on the last day of the ethanol preference test. The weights were averaged across males and females because sex did not interact with line or treatment. An ANOVA was conducted on these data including sex, line, and neonatal treatment as between-subjects factors. There was a significant main effect of neonatal treatment on body weight, F(2, 105) = 3.09, p < 0.05. Post hoc tests showed that the 6 g/kg groups weighed less than the maltose or sham groups. There were also main effects of sex, F( 1, 105) = 734.80, p < 0.001, because males weighed more than females, and line, F(1, 105) = 5.10, p -C 0.05, as NP rats weighed more than P rats. DISCUSSION
Neonatal alcohol exposure did not produce differential effects in adult P and NP rats in open field activity or in ethanol preference. P and NP rats of both sexes showed increased activity due to alcohol exposure and the magnitude of the alcohol effect was similar in the two lines. Pups that received alcohol exposure during the peak of the “brain growth spurt” (PNDs 4-9) showed higher open-field activity levels than artificially or normally reared controls when tested as adults (80day-old). Artificial rearing without alcohol treatment also increased activity relative to normal rearing, an effect most apparent in the NP line. In subsequent tests on the same rats, neonatal alcohol exposure increased intake of a 10% ethanol solution when it was the rat’s sole source of fluid and this effect was similar in the P and NP lines. The 6 g/kg groups consumed more 10% ethanol solution (as a function of body weight) than the maltose or the sham groups. The maltose groups had higher intake scores than the sham groups and, as expected, the P rats had higher scores than the NP rats. It is interesting that the line and treatment effects seen in this ethanol intake test parallel those found in the open-field activity tests. However, there were no effects of neonatal treatment on alcohol preference when rats then received free choice between the 10% ethanol solution and water, although the large line difference on which the P and NP rats were bred was very much in evidence. One important effect seen in the previous work with weaning P and NP rats (24) was that neonatal alcohol exposure produced a greater increase in activity in the P line than in NP line. P rats exposed to either 6 g/kg/day or 4 g/kg/day were more active than controls while only NP rats exposed to the 6 g/kg/day dosage were more active than controls. Furthermore, the percent increase in activity was much greater in the P rats than the NP rats following alcohol exposure. In contrast, in the current study the 6 g/kg dose produced a similar increase in activity in the two lines when testing occurred in adulthood. Thus, the differential effects of neonatal alcohol exposure on open-field activity in P and NP rats do not extend into adulthood. It is important to note, however, that neonatal alcohol exposure produced increased activity in adults of both sexes in the present research. In previous research (19), neonatal alcohol exposure increased activity only in adult females while alcohol exposed adult males did not differ from normally reared controls. A recent unpublished study from our lab has also failed to show effects of neonatal alcohol exposure in
adult Sprague-Dawley rats of either sex, although such effects are apparent in weaning age animals. Thus, the present study demonstrates long lasting effects of neonatal alcohol exposure on activity in both sexes. It does need to be pointed out that there was an overall increase in activity in the NP rats following artificial rearing. It appears that some aspect of the artificial rearing protocol, such as maternal separation or nutritional factors, in some way impacted the NP line and caused a general increase in activity levels as measured in the open field. The extent to which the alcohol effect in the NP line may have interacted with the artificial rearing is unknown. No effects of artificial rearing on activity were apparent in the P line and the enhanced activity in this line appears to be more directly related to the alcohol exposure. Neonatal alcohol exposure failed to affect subsequent ethanol preference in adult P or NP rats. Although the 6 g/kg groups of both line consumed more 10% ethanol than controls when it was the only fluid available, there were no reliable differences among treatment groups in either line during the preference test. The NP rats consistently ingested less than 10% of the ethanol solution during the preference test as would be expected, because this line was bred for low ethanol preference. In the P line, the 6 g/kg/day group showed a nonsignificant increase in ethanol preference relative to controls on the first day but not on subsequent days and the neonatal treatment effects were not statistically significant. The absence of neonatal alcohol effects on ethanol preference in these rat lines may not be surprising considering the inconsistent effects of prenatal alcohol exposure on adult rats in previous studies (23). Bond & Dig&to (7) did find increased preference for low alcohol concentrations, up to 6% v/v, following prenatal alcohol exposure but above this concentration preference was unaffected. Also, in that study only young (45-day-old) females were tested. It is possible that using lower concentrations of ethanol solution might have been interesting in the NP line. In a previous study (21), NP rats showed about 25% preference for a 4% ethanol solution. However, the P rats showed almost 100% preference for the 4% solution leaving no room for increased preference as a function of our neonatal alcohol treatment. The baseline preference Ievels for the 10% ethanol solution used in this study did allow both the P and the NP lines room for increased ethanol preference as a function of neonatal alcohol treatment. Neonatal alcohol treatment was also associated with slightly reduced body weights compared to control treatments in adulthood. Again, the two lines appeared to be equally affected in that neonatal treatment did not interact with line. It is interesting to note that, while the artificially reared maltose group of NP rats increased activity relative to sham controls, their body weights were equivalent to the sham group at testing. Furthermore, the activity effect appears to be unrelated to growth deficits since the body weights of NP maltose pups were equal to or greater than NP sham pups during the artificial rearing period. Again, the NP line may be more sensitive than the P line to factors associated with artificial rearing. In comparing the current study with our previous one (25), it is interesting that neonatal alcohol exposure produced differential effects on the P and NP lines at weaning but equal effects on the two lines in adulthood. The present results may be interpreted as a failure to extend this recent report (25) of differential susceptibility of the P and NP lines to the behavioral effects of neonatal alcohol exposure. Alternatively, these previous findings may be age-related as the rats were tested at weaning age in the Riley et al. (25) study and as adults in the present study. The general findings from animal models of
48
MELCER,
FAS indicate that prenatal alcohol effects are most evident at or before weaning age or in aged animals (2,3). This is certainly true with measures of activity (8). However, fetal alcohol effects are seen in adult animals but often more successfully under stressful or challenging circumstances (24). Perhaps the differential sensitivity of the P line to postnatal alcohol exposure reported at weaning age might reappear in adult animals using more challenging behavioral situations
GONZALEZ
AND RILEY
than the open field. In addition, it would be interesting to study the effects of neonatal alcohol exposure on activity in the P and NP rats in old age. ACKNOWLEDGEMENTS
This work was supported in part by grants AA06902 to EPR and AA07611 to T.-K. Li from the National Institute on Alcohol Abuse and Alcoholism.
REFERENCES 1. Abel, E. L. Fetal Alcohol Syndrome. Oradell, NJ: Medical Economics; 1990. 2. Abel, E. L.; Dintcheff, B. A. Effects of prenatal alcohol exposure on behavior of aged rats. Drug Alcohol Depend 16:321-330, 1986a. 3. Abel, E. L.; Dintcheff, B. A. Effects of prenatal alcohol exposure on nose poking in year old rats. Alcohol 3:201-204; 1986b. 4. Abel, E: L.; Sokol, R. J. Incidence of fetal alcohol syndrome and economic impact of FAS-related anomalies. Drug Alcohol Depend. 19:51-70; 1987. 5. Abel, E. L.; York, J. L. Absence of effect of prenatal ethanol on adult emotionality and ethanol consumption in rats. J. Studies Alcohol 40:547-553; 1979. 6. Barron, S.; Kelley, S. J.; Riley, E. P. Neonatal alcohol exposure alters suckling behavior in neonatal rat pups. Pharmacol. Biothem. Behav. 39:423-427; 1991. 7. Bond, N. W.; Di Giusto, E. L. Effects of prenatal alcohol consumption on open-field behaviour and alcohol preference in rats. Psychophamacologia 46:163-165; 1976. 8. Bond, N. W.; Di Giusto, E. L. Prenatal alcohol consumption and open-field behaviour in rats: Effects of age at time of testing. Psvchonharmacology 52:311-312; 1977. 9. Chernoff, G. F. The fetal alcohol syndrome in mice: Maternal variables. Teratol. 22:71-75; 1980. 10. Chotro, M. G.; Molina, J. C. Acute ethanol contamination of the amniotic fluid during gestational day 21: Postnatal changes in alcohol responsiveness in rats. Dev. Psychobiol. 23:535-547; 1990. 11. Dobbing, J.; Sands, J. Quantitative growth and development of the human brain. Arch. Dis. Child. 48757-767: 1973. 12. Gilliam, D.; lrtenkauf, K. Maternal genetic effects on ethanol teratogenesis and dominance of relative embryonic resistance to malformations. Alcohol: Clin. EXD. Res. 14:539-545; 1990. 13. Gilliam, D. M.; Ketch, L.; Dudek, B. C.; Riley, E. P. Ethanol teratogenesis in selectively bred long-sleep and short-sleep mice: A comparison to inbred C57BL/6J mice. Alcohol Clin. Exp. Res. 13:667-672; 1989a. 14. Gilliam, D. M.; Ketch, L.; Dudek, B. C.; Riley, E. P. Ethanol teratogenesis in mice selected for differences in alcohol sensitivity. Alcohol 5:513-519; 1989b. 15. Gilliam, D. M.; Stilman, A.; Dudek, B. C.; Riley, E. P. Fetal alcohol effects in long- and short-sleep mice: activity, passive avoidance and In Utero ethanol levels. Neurotoxicol. Teratol. 9: 349-357, 1987. 16. Goodlett, C. R.; Gilliam, D. M.; Nichols, J.; West, J. Genetic influences on brain growth restriction induced by developmental exposure. Neurotoxicol. 10:321-324; 1989. 17. Grant, K. A.; Choi, E. Y.; Samson, H. H. Neonatal ethanol exposure: Effects on adult behavior and brain growth parameters. Pharmacol. Biochem. Behav. 18: Suppl. 1:331-336; 1983. 18. Kelly, S. J.; Hðer, S. A.; West, J. R. Alterations in sensorimotor development: Relationship to postnatal alcohol exposure. Neurotoxicol. Teratol. 9:243-251; 1987.
19. Kelly, S. J.; Pierce, D. R.; West, J. R. Microencephaly and hyperactivity in adult rats can be induced by neonatal exposure to high blood alcohol concentrations. Exp. Neurol. 96:580-593; 1987. 20. Lumeng, L.; Hawkins, T. D.; Li, T.-K. New strains of rats with alcohol preference and nonpreference. In: Thurman, R. G.; Williamson, J. R.; Drott, H. R.; Chance, B. eds. Alcohol and aldehyde metabolizing systems, vol. 3. New York: Plenum Press; 19771537-544. 21 Li, T.-K.; Lumeng, L.; Mcbride, W. J.; Waller, M. B. Indiana selection studies on alcohol-related behaviors. In: McClearn, G.; Deitrich, R. A.; Erwin, V. G. eds. Development of animal models as pharmacogenetic tools. Research monograph number 6, U.S. Department of Health and Human Services; 1978: 171181. 22. Melter, T.; Gonzalez, D.; Somes, C.; Riley, E. P. Postnatal alcohol exposure and early development of motor skills in alcohol preferring (P) and nonpreferring (NP) rats. Neurotoxicol. Teratol. (in press). 23. Meyer, L.; Riley, E. P. Behavioral teratology of alcohol. In: Riley, E. P.; Vorhees, C. V.; eds. Handbook of behavioral teratology. New York: Plenum Press; 1986:101-140. 24. Riley, E. P. The long-term behavioral effects of prenatal alcohol exposure in rats. Ale. Clin. Exp. Res. 14:670-673; 1990. 25. Riley, E. P.; Barron, S.; Melter, T.; Gonzalez, D. Alterations in activity following alcohol administration during the third trimester equivalent in P and NP rats. Ale. Clin. Exp. Res. 17:12401246; 1993. 26. Riley, E. P.; Lochry, E. A. Genetic influences in the etiology of fetal alcohol syndrome. In: Abel, E. L. ed. Fetal alcohol syndrome, 3, animal studies. Boca Raton, FL: CRC Press; 1982:113130. 27 Samson, H. H.; Diaz, J. Effects of neonatal ethanol exposure on brain development in rodents. In: Abel, E. L. ed. Fetal alcohol syndrome, Ill Animal studies. Boca Raton, FL: CRC Press; 1982: 131-150. 28. Streissguth, A. P. The behavioral teratology of alcohol: Performance, behavioral, and intellectual deficits in prenatally exposed children. In: West, J. R., ed. Alcohol and brain development. New York: Oxford University Press; 1986:3-44. 29. Streissguth, A. P.; Barr, H. M.; Martin, D. C. Alcohol exposure in utero and functional deficits in children during the first four years of life. In: Porter, R.; O’Conner, M.; Whelan, J. eds. Mechanism of alcohol damage in utero. Ciba Foundation Symposium 105; London, Pittman; 1984:176-196. 30. Tabakoff, B.; Culp, S. G. Studies on tolerance development in inbred and heterogenous stock national institutes of health rats. Ale. Clin. Exp. Res. 8:495-499; 1984. 31. Terry, L. M.; Scheinman, J. A.; Hall, W. G. Response deficits in isolate reared rats. Paper presented at the annual meetings of the International Society for Developmental Psychobiology; 1987.
Pergamon 0892-0362(94)00051-4
Locomotor Activity and Alcohol Preference in Alcohol-Preferring and -Nonpreferring Rats Following Neonatal Alcohol Exposure TED
MELCER,’
DAVID
GONZALEZ
AND
EDWARD
P. RILEY
Department of Psychology, San Diego State University, San Diego, CA 92120 Received
9 December
1993; Accepted
14 July 1994
MELCER, T., D. GONZALEZ AND E. P. RILEY. Locomotor activityand alcoholpreference in alcohol-preferringand -nonpreferring ratsfollowing neonatal alcohol exposure. NEUROTOXICOL TERATOL 17(l) 41-48, 1995. -Recently, we reported that the alcohol preferring (P) and nonpreferring (NP) rats, bred for differences in alcohol preference, showed different behavioral effects of neonatal alcohol exposure when tested as juveniles. Following neonatal alcohol exposure, the P line showed a greater increase in activity than the NP line relative to their respective controls. In the present study, P and NP rat pups were separated from their mothers and artificially reared from postnatal day (PND) 4 until PND 12. Pups were implanted with intragastric cannulas on PND 4 and fed a stock milk solution every 2 h via an indwelling cannula. One group had alcohol added to the milk diet during the 4 daytime feeds at a dose of 6 g/kg/day on PND 4-7 and a dose of 3 g/kg/day on PNDs 8 and 9. One control group was artificially reared and fed an isocaloric milk solution and a second control group was reared normally with a surrogate dam. Rats were tested as adults (IO-day-old) for open-field activity and alcohol preference. Neonatal alcohol exposure caused equivalent increases in activity in P and NP rats. In the ethanol preference test, neither the P nor the NP rats showed any effect of neonatal alcohol treatment although there were large line differences in alcohol preference. These data suggest that the increased susceptibility of young P rats to neonatal alcohol exposure, measured by open-field activity, does not extend to adulthood. Furthermore, neonatal alcohol exposure does not appear to alter alcohol preference in either line. Fetal Alcohol Syndrome
P rats
NP rats
Neonatal alcohol/activity
PRENATAL alcohol exposure can produce profound behavioral and physical effects in infants and young children. These effects include growth deficits, physical abnormalities, and altered behavior such as increased body tremors, hand move-
Several investigators have proposed that genetic factors might contribute to this variability in outcomes seen following prenatal alcohol exposure (9,12,26). It is possible that differences in alcohol metabolism or sensitivity to alcohol’s effects might predict the susceptibility of the developing fetus to the effects of fetal alcohol exposure. There are now several reports showing differential outcomes in inbred strains or selectively bred lines that support the role of genetic factors in the etiology of fetal alcohol effects. For instance, long sleep (LS) mice, bred for extreme loss of the righting reflex after ethanol exposure, produced litters that showed greater mortality, growth deficits, and slower passive avoidance learning due to prenatal alcohol exposure than did short sleep (SS) mice, bred for insensitivity to the hypnotic effects of alcohol (13,14, 15,16). Differential effects of neonatal alcohol exposure resulted when comparing Maudsley Reactive (MR) and MS20 rats. The M520 strain is initially more sensitive to acute alco-
ments, and overall body activity as well as impairments in attention and learning (28,29). In cases where these symptoms
occur in a recognized pattern, the child may be diagnosed with Fetal Alcohol Syndrome (FAS). It is important to note that FAS has been estimated to occur in slightly less than 10% of the children of alcohol abusing women (1,4). Similarly, in animal models of fetal alcohol exposure, it is not uncommon to find normally appearing or behaving animals within the same litter as animals that show the effects of in utero alcohol exposure. A critical question is to identify risk factors that make some women or their fetuses more susceptible than others to the effects of equivalent amounts of gestational alcohol exposure. ,-
’ Requests for reprints should be addressed to Ted Melter, Center for Behavioral Teratology, 6363 Alvarado Court, #209, San Diego State University, San Diego, CA 92120. 41
MELCER,
4L
ho1 effects but becomes less sensitive than the MR strain with repeated alcohol exposure (30). The MR rats showed greater reduction in cerebellar weight due to neonatal alcohol exposure than did the M520 rats (16). Recently, we have studied the effects of neonatal alcohol exposure on the behavior of the selectively bred alcohol preferring (P) and alcohol nonpreferring (NP) rat lines (20). These lines were bred for extremes in alcohol preference but also differ in their functional tolerance to alcohol, with the P line showing greater tolerance than the NP line. These two lines were exposed to alcohol neonatally using the “pup-in-thecup” protocol and were then tested in an open field just prior to weaning. Contrary to our expectations, the P rats displayed larger activity increases in the open field as a result of the neonatal alcohol exposure than did the NP rats. Importantly, neonatal alcohol treatment produced equivalent blood alcohol levels in the P and NP lines, eliminating the possibility that differential alcohol effects were caused by differences between lines in the rate of ethanol metabolism (25). More recently, we examined the effects of neonatal alcohol exposure on walking patterns (gait) and balance (walking along elevated parallel bars) in both P and NP rats. Neonatal alcohol exposure altered gait and decreased balance in both lines. There was no evidence of differential susceptibility of the P and NP lines to neonatal alcohol exposure as measured by these motor skills (22). These studies indicate that P rats may be more susceptible to some, but not all, of the consequences of neonatal alcohol exposure than NP rats. The present study followed up on our previous work showing increased activity in both P and NP juvenile rats after neonatal alcohol exposure. Because the P line shows greater functional tolerance to alcohol than the NP line, we were surprised that the P line experienced more adverse effects of neonatal alcohol exposure than the NP line. Increased activity following prenatal alcohol exposure is most commonly reported in weaning rats (18- to 30-day-old) and frequently diminishes in adults (Q although it is again seen in aged rats (2,3). Also, more complex tests can reveal prenatal alcohol effects in adults (24). Similar effects of age have been reported following neonatal alcohol exposure (17,18,19) although increased activity in adult female rats exposed to alcohol neonatally has been reported (19). The current study was conducted to determine if persistent effects on activity could be found in adulthood following neonatal alcohol exposure and if the P and NP rats might be differentially sensitive to these neonatal alcohol effects. The second purpose of this study was to determine if neonatal alcohol exposure might affect subsequent alcohol preference. Previous work on this question has met with mixed results (23). For example, Abel and York (5) did not find any influence of prenatal alcohol exposure on subsequent alcohol intake when rats received free choice between an ethanol solution and water. Similarly, unpublished work from our lab has failed to demonstrate any effect of prenatal alcohol exposure on subsequent alcohol intake or preference. However, Bond and Digiusto (7) found prenatal alcohol exposure resulted in an enhanced intake of alcohol at low concentrations in 45-dayold female rats. A more recent study showed that injection of a 6% alcohol solution into the amniotic fluid of gestation day (CD) 21 rat fetuses increased approach toward alcohol odor and intake of an alcohol solution by S-day-old rat pups (10). Some of the variability in the results of previous studies might be due to genetic differences between rats in alcohol preference. By using lines of rats selectively bred for differential alcohol preference, it might provide an answer to the question
GONZALEZ
AND RILEY
of whether early alcohol exposure influences subsequent alcohol preference. In the present study, P and NP rats pups received alcohol during the brain growth spurt that peaks between postnatal days (PND) 4 to 10 in the rat. In the human fetus, the brain growth spurt occurs during the third trimester of pregnancy and is believed to be a critical period of early brain development (11). The neonatal exposure paradigm in the rat thus models the effects of third trimester alcohol exposure in humans in terms of CNS development. The P and NP received alcohol exposure or control treatment and were tested as adults for open-field activity and then for ethanol preference. METHOD
Subjects
P and NP rat pups were used as subjects. Parent animals were supplied by Drs. T.-K. Li and L. Lumeng of the Indiana University School of Medicine Alcohol Research Center (Indianapolis, IN) and offspring were born at the San Diego State University laboratory of the principal investigator (EPR). All rats were maintained on a 12L : 12D cycle throughout the experiment (lights on at 0700). Mating
Breeder females were group housed and breeder males were individually housed in metal hanging cages. There were no differences between the P and NP groups in terms of the age (70-200 days of age) or body weights (247-340 g) at the time of mating. A few females provided multiple litters for this study and the majority of the animals were multiparous. For mating, one female was placed in the home cage of each male in the evening and left overnight. A check for seminal plugs in the morning was used as evidence of mating and CD 0 was defined as the day a plug was found. Females that did not mate were returned to group housing. Females that mated were housed in plastic breeding cages in a temperature (70°F) and humidity (40% to 60%) controlled nursery with ad lib rat chow (Purina Rat Chow) and water during the remainder of gestation. Breeding cages were checked at 8:30 a.m. and 5:00 p.m. daily for births. Twenty-four hours after a birth, litters were weighed and culled to the 10 heaviest pups (5 males and 5 females whenever possible). The heaviest animals were maintained because this selection procedure improves the outcome of surgery. The surgery to install the stomach cannula for artificial rearing (AR) was conducted on the morning of CD 26 (26 days after mating) which was almost always PND 4. Prior to surgery, pups were removed from the litter, weighed, and the heaviest pups were randomly assigned to one of the following 2 treatment groups: (a) artificial rearing with ethanol exposure (6 g/kg/day) or (b) artificial rearing without ethanol exposure. In this artificially reared control group, maltose calories were substituted isocalorically for ethanol derived calories. The remaining pups received sham surgery (no stomach cannula installed) and normal rearing with a Sprague-Dawley female that had delivered her pups at about the same time the P and NP rats were born. The use of Sprague-Dawley surrogate mothers precluded any effects that might be due to differences in maternal care between the natural P and NP mothers. Litter sizes were maintained at 8 pups using Sprague-Dawley pups where necessary. Neonatal Surgical Procedure
The procedure for surgical implantation of the intragastric cannula has been described in great detail previously for the
NEONATAL
ALCOHOL
43
EXPOSURE
interested reader (6). Each pup was anesthetized with a 50% halothane/50% oxygen mixture and then a stainless steel surgical wire sheathed in polyethylene tubing was maneuvered down the esophagus into the stomach. The lower tip of the wire was pushed out through the stomach and abdominal wall. The feeding cannula, made of Clay Adams polyethylene tubing (PE lo), was attached to the end of the wire protruding from the pup’s mouth. This connection was coated with an antibiotic and corn oil, and the cannula was pulled into the stomach and out through the abdominal wall. A thin plastic disc attached to the end of the cannula formed a seal against the inside of the stomach wall which prevented milk from leaking out. The cannula was then pulled through loose skin about the back of the pup’s neck and washers were attached to the cannula where it entered and exited the back of the neck. The washers absorbed any pull on the cannula at the point of the neck and prevented direct pull on the cannula in the pup’s stomach. The entire surgical procedure required about 2 min per animal. Pups recovered from the anesthetic within 3 min. The sham surgery group received identical surgical procedures except that the cannula was not installed and the pups were returned to a Sprague-Dawley dam after surgery.
received the base milk formula. The AR pups were maintained on PNDs 10 and 11 on the base milk formula without alcohol to allow recovery from acute ethanol effects or withdrawal symptoms that were occasionally observed. The ethanol and maltose groups received isocaloric milk formula throughout the artificial rearing period (PNDs 4-l 1). Pups were weighed daily during the artificial rearing period. On the morning of PN 12, pups from all groups were earmarked for group identification and weighed. Each cannula was cut where it exited the pup’s side and all pups were bathed in a slurry of maternal feces and warm water before being returned to a dam. This slurry procedure virtually eliminated pup rejection by the dam. Pups were returned to SpragueDawley dams. Each of these surrogate dams received a new litter of 8 pups including equal numbers of pups from the AR groups whenever possible. The sham pups were placed with separate surrogate dams to prevent competition with and promote survival of the typically smaller AR pups. Pups were weaned from surrogate dams on PND 21 and housed in metal hanging cages with ad lib access to food and water until behavioral testing. Two animals of the same sex lived in each cage. Activity Testing
Neonatal Rearing and Maintenance From PND 4 until PND 12, the artificially reared pups were individually housed and maintained in Styrofoam cups containing hardwood chips and lined with a piece of synthetic short-haired fur. The fur reportedly minimizes behavioral depression associated with maternal deprivation (31). The cups in which pups lived were placed inside Styrofoam cups partially filled with white washed play sand to keep the housing cup dry and upright. All cups floated in an aquarium tank filled with 49’C aerated water. Every 2 days, the water temperature was decreased by 2OC until the tank temperature was 42’C. This temperature schedule allowed the AR pups to maintain body temperatures similar to those observed in sham control pups in the litter. Each pup’s stomach cannula was connected to a tygon tubing lead attached to a syringe containing a milk-based formula which substituted for mother’s milk (27). The syringes were mounted on an infusion pump (Harvard Instruments) operated on an automated-timed schedule of one 20-min period of milk delivery every 2 h. This schedule resulted in a total of 12 feeds of milk formula daily for each pup. All pups received the same base milk formula during the 8 night-time feeds between the hours of 5:30 p.m. and 7:50 a.m. Ethanol or isocaloric maltose was added to the base diet during the 4 daytime feeds between the hours of 9:30 a.m. and 3:50 p.m. for pups in the alcohol and maltose groups. All AR pups were voided before the first day feed and after each of the 4 daytime feeds on PN days 4 through 7. On PN days 8 through 11, pups were voided before the first feed and after the last feed. The amount of milk administered in milliliters was equivalent to 33% of the weight of the AR pups within each line. Because pups from the P line weighed more than pups from the NP line, two artificial rearing apparatuses were used to allow separate control of the feeding schedules for each line. Ethanol administration was limited to the 4 daytime feeds to represent a “binge drinking” model (19). On PNDs 4 through 7 pups in the ethanol group received a dosage of 6 g/kg/day. To minimize the severity of withdrawal, the ethanol exposed pups received a dosage of 3 g/kg/ day on PNDs 8 and 9. On PNDs IO and 11, all AR pups
All rats were handled for 5 mitt/day for 4 days prior to the commencement of activity testing. Beginning on PND 80, animals were tested for open-field activity for 30 mitt/day for 4 consecutive days. The sample sizes were as follows: P 6 g/kg male = 11; P 6 g/kg female = 10; P maltose male = 13; P maltose female = 7; P sham male = 12; P sham female = 7; NP 6 g/kg male = 12; NP 6 g/kg female = 11; NP maltose male = 10; NP maltose female = 7; NP sham male = 12; NP sham female = 10. Each rat was removed from its home cage and individually placed in one of four automated activity monitors (Omnitech Instruments) for a 30-min test session. Each monitor consisted of a 39 cm x 39 cm chamber with an infrared photobeam grid criss-crossing the apparatus. The monitors were enclosed in sound attenuated boxes with red light illumination (5 W) and white noise. Data on rat movements were detected by photobeam and were automatically recorded by computer. The dependent variables recorded were horizontal activity (number of photobeams crossed), total distance traveled (in inches), and movement time (in seconds). At the conclusion of each test session, rats were returned to their home cage. Testing occurred 5 to 8 h into the animals’ light cycle and individual animals were tested at the same time on each of the 4 test days. Ethanol Preference Test One week after activity testing, the P and NP rats were taken from the hanging cages and housed individually in breeding cages with ad lib access to food and water for 3 days. The sample sizes were as follows: P 6 g/kg male = 11; P 6 g/ kg female = 10; P maltose male = 13; P maltose female = 7; P sham male = 12; P sham female = 7; NP 6 g/kg male = 12; NP 6 g/kg female = 9; NP maltose male = 9; NP maltose female = 6; NP sham male = 12; NP sham female = 9. Five animals from activity testing were inadvertently left out of the ethanol preference test. In the first phase (ethanol exposure), rats received a 10% ethanol solution (v/v) as their sole fluid source for 4 consecutive days. This ethanol exposure phase was used in the initial selective breeding experiments to increase acceptability of eth-
44
MELCER,
anol during later preference testing (21). In the second phase (ethanol preference), rats received a choice between a 10% ethanol solution and water. Fluids were presented in graduated cylinders and the location of the ethanol and water cylinders was alternated daily. The ethanol preference tests were conducted for 9 consecutive days. The number of milliliters intake of water and/or ethanol were recorded daily at 3:00 p.m. The graduated cylinders were then filled with fresh water or 10% ethanol. Food was available ad lib during all phases of testing.
GONZALEZ
ALCOHOL PREFERRING
3000
AND RILEY
RATS
iiiI” ii 3
‘Wg 2500
Maltose
1
Sham
RESULTS
Pup Data
Table 1 shows mean body weights of pups during the neonatal treatment period from PNDs 4 through 10. The means are collapsed across sex because sex did not interact with line or day. An analysis of variance (ANOVA) was conducted on pup weights with sex, line (P or NP rats) and neonatal treatment (ethanol, maltose, sham) as between-subjects factors and postnatal age (PND 4 through 10) as a within-subjects factor. The overall ANOVA indicated main effects of sex, F(1, 110) = 9.65, p < 0.01, because males weighed more than females; line, F(1, 110) = 14.90, p < 0.001, as P pups weighed more than NP pups; and postnatal age, F(6, 660) = 1524.43, p < 0.001, as body weights increased with age. The analysis also revealed significant day x treatment, F(12, 660) = 23.73, p < 0.001, day x line, F(6, 660) = 3.23, p < 0.01 and day x treatment x line interactions, F(12, 660) = 2.96, p < 0.001. Post hoc tests (Newman-Keuls, p < 0.05) were used to analyze the day x treatment x line interaction. As seen in Table 1, sham pups initially weighed less than maltose and ethanol pups, and this effect reversed, particularly in the P line, during the last few days of the neonatal treatment period. Specifically, the sham groups weighed less than the AR groups of their respective lines on PNDs 4 through 7 while the weights of the AR groups were equivalent. On PND 8, the body weights of the P groups were equivalent
ii
1000
I7 0
1
2
3
4
TEST DAY
ALCOHOL NONPREFERRING
*
RATS
3000 5i
-
: ii =
2500
+
!
6glkg Maltose Sham
L
5 $ 6
2000
=
1000
/
6-----------_ 4
TEST DAY
FIG. 1. Mean (k SEM) total distance traveled in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
TABLE 1 MEAN AND SEM (IN PARENTHESES) FOR BODY WEIGHTS IN GRAMS DURING THE ARTIFICIAL REARING PERIOD
Neonatal Treatment 6 g/kg
Maltose
Sham
P rats 4 5 6 7 8 9 10
10.33 (.22) 12.07 (.25) 13.66 (.30) 15.04 (.37) 16.43 (.37) 16.64 (.35) 18.02 (.42)
10.60 (.23) 12.22 (.21) 13.80 (.18) 15.57 (.29) 16.48 (.30) 17.79 (.43) 18.85 (A)
9.07 (.25) 10.64 (.42) 12.21 (.44) 14.35 (.48) 16.16 (.55) 18.55 (.63) 20.93 (.61)
NP rats 4 5 6 7 8 9 10
9.63 (.23) 10.91 (.26) 12.26 (.28) 13.78 (.28) 14.99 (.29) 15.79 (.42) 17.45 (.45)
10.06 (.23) 11.31 (.27) 12.61 (.31) 13.94 (.24) 15.39 (.35) 16.71 (.38) 18.84 (~51)
8.26 (.25) 9.44 (.32) 11.03 (.41) 12.41 (.49) 14.23 (.60) 16.31 (.67) 18.23 (.77)
Postnatal Day
while in the NPs the shams remained lighter than the AR groups. On PNDs 9 and 10 in the P line, the shams weighed more than the maltose or ethanol groups and the maltose group weighed more than the ethanol group. In the NP line, the maltose group weighed more than the ethanol group on PND 9 and more than ethanol or sham groups on PND 10. The NP shams weighed more than ethanol pups on PND 10. Activity Data
Rats were tested for open-field activity beginning at 80 days of age and Figs. 1, 2, and 3 show the results combined across males and females, as sex did not interact with neonatal treatment. As seen in the figures, P and NP rats that received neonatal ethanol exposure (6 g/kg/day) were more active than maltose and sham groups as measured by total distance traveled, horizontal activity, and movement time. We emphasize that alcohol exposure had similar effects on the P and NP lines. The figures also indicate that the NP maltose group had higher activity scores than the NP sham group, while no such artificial rearing effect was evident in the P line. Separate ANOVAs were conducted on activity measures of total distance (inches), horizontal activity (number of photo-
NEONATAL
ALCOHOL
45
EXPOSURE
ALCOHOL PREFERRING
ose groups had higher scores than the sham groups. Figure 2 indicates that the difference between the maltose and sham groups was specific to the NP line but the line x treatment interaction did not reach significance (p c 0.07). The overall ANOVA also showed main effects of sex, F(1, 110) = 98.40, p c 0.001, as females were more active than males; line, F(1, 110) = 19.34, p c 0.001, as P rats were more active than NP rats; and test day, F(3, 330) = 11.09, p < 0.001, as horizontal activity decreased across test days. The ANOVA on movement time (Fig. 3) showed a main effect of neonatal treatment, F(2, 110) = 11.67, p < 0.001. Post hoc tests showed a significant differences among all three treatment groups (6 g/kg vs. maltose; maltose vs. sham; 6 g/ kg vs. sham). Figure 3 indicates differences between the AR groups in the NPs and not the Ps but again the treatment x line interaction only approached significance, F(2, 110) = 2.79 = p < 0.07. The overall ANOVA also showed main effects of line, F(l, 110) = 18.25, p < 0.001, sex, F(1, 110) = 42.56, p < 0.001, and test day, F(3, 330) = 11.95, p < 0.001, and a line x sex x test day interaction, F(3, 330) =
RATS
I
2000
I 0
I 1
b 2
I 3
1 4
1
TEST DAY
ALCOHOL
c 2
T
5000
NONPREFERRING
RATS
T
-
‘VU
*
Maltose
ALCOHOL PREFERRING
o^
1400
ti
s F Z
RATS
*
Maltose
U
Sham
1 1300 -
ki
4
:
5
2000) 0
I 1
I 2
I 3
.
I 4
.
(
TEST DAY
FIG. 2. Mean (+ SEM) horizontal activity in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
1200 -
: P 2
1100 -
!ki 1000
!
0
I
I
1
2
.
1
I
I
3
4
TEST DAY
beams crossed), and movement time (s) with sex, line and neonatal treatment as between-subjects factors, and test day as the within-subjects factor. Post hoc (Newman-Keuls, p < 0.05) tests were used where necessary to isolate effects from the overall ANOVA. The ANOVA on total distance (Fig. 1) showed a significant neonatal treatment x line interaction, F(2, 110) = 3.69, p < 0.05. Post hoc tests indicated that 6 g/ kg groups of both lines traveled greater distances than their respective maltose or sham controls. However, the distance scores for the P maltose and sham groups were equivalent while the NP maltose group traveled greater distance than the NP sham group. The overall ANOVA showed main effects of sex, F(l, 1IO) = 109.77, p < 0.001, as females traveled farther than males, but there was no treatment x sex interaction; and line, F(l, 110) = 15.36, p < 0.001, as P rats traveled farther than NP rats. There was also a main effect of test day, F(3, 330) = 19.20, p c 0.001 as activity generally decreased across test days. The ANOVA on horizontal activity (Fig. 2) revealed a main effect of neonatal treatment, F(2, 110) = 22.40, p < 0.001. Post hoc tests showed that the 6 g/kg groups had higher horizontal activity scores than maltose or sham groups. The malt-
ALCOHOL NONPREFERRING
RATS
1
_
1400 -
-
_r
&g/kg
z: %
g
1300 -
F lE =
1200 -
: % z
1100 -
;: 3 1ooot
I 0
1
I 2
I 3
I
1
4
TEST DAY 3. Mean ( f SEM) movement time in open field as a function of neonatal treatment, rat line, and test day. Rats were tested at 80 days of age and the means shown include both sexes.
FIG.
MELCER, TABLE 2 MEAN AND SEM (IN PARENTHESES) FOR BODY WEIGHTS IN GRAMS ON THE LAST DAY OF ACTIVITY TESTING, ETHANOL EXPOSURE, AND ETHANOL PREFERENCE TEST
NeonatalTreatment Maltose
Sham
294 (17.80) n = 21 298 (14.15) n = 23
315 (17.06) n = 20 313 (18.32) n = 17
327 (17.67) n = 19 321 (15.99) n = 22
313 (19.30) n = 21 314 (15.65) n = 21
336 (18.41) n = 20 334 (22.94) n = 15
348 (19.07) n = 19 340 (18.94) n = 21
338 (21.50) n = 21 360 (19.20) n = 21
366 (20.80) n = 20 375 (26.50) n = 15
378 (22.20) n = 19 380 (21.70) n = 21
6 0s Activity P rats NP rats Ethanol Exposure P rats NP rats Ethanol Preference P rats NP rats
Sample sizes shown include both sexes.
4.48, p < 0.01. The P male rats showed a nonsignificant increase in movement time between test days 1 and 4 while P females and NP rats of both sexes decreased movement time across test days. There was no significant interaction between sex and treatment. Activity body weights. Table 2 shows means and SEMs for body weights recorded on the final day of activity testing. An ANOVA with sex, neonatal treatment, and line as betweensubjects factors showed no effects of line and no interaction between sex and treatment and therefore weights are presented averaged across sex. As seen in the table, there was a significant main effect of postnatal treatment, F(2, 110) = 4.07, p < 0.05. Newman-Keuls tests showed the 6 g/kg groups weighed significantly less than the maltose or the sham groups and that the weights of the maltose and sham groups were equivalent. There was also a main effect of sex because males weighed morethanfemales,F(l, 110) = 641.19,p < O.OOl.Noneofthe other main effects or interactions were significant.
GONZALEZ
AND RILEY
males; and test day, F(3, 315) = 93.91, p < 0.001, as intake scores increased across the 4 test days. Finally, the ANOVA also revealed a sex x line interaction, F(1, 105) = 14.62, p < 0.001, as the line differences just described were greater in females than in males, and a day x line interaction, F(3, 315) = 3.62, p < 0.05, as the line difference described above decreased across test days. Ethanol exposure body weights. Table 2 shows means and standard errors for body weights recorded on the last day of ethanol exposure. The weights were averaged across sex as it did not interact with treatment or line. The data were analyzed using ANOVA as just described that showed a significant main effect of neonatal treatment, F(2, 105) = 4.03, p < 0.05. A Newman-Keuls test showed that the 6 g/kg groups weighed less than the maltose or the sham groups while the mean weights of the maltose and sham groups were equivalent. There was also a main effect of sex, F(1, 105) = 580.85, p < 0.001 because males were heavier than females. None of the other effects were significant. Ethanol preference. Figure 4 shows percentage intake of 10% ethanol solution [ml 10% ethanol x lOO/(ml 10% ethanol + ml water)] for the 9 days rats received simultaneous access to the ethanol solution versus water. The means shown are averaged across males and females because there were no effects of sex on ethanol preference. An ANOVA was conducted on percentage ethanol intake with sex, neonatal treatment, and line as between-subjects factors and test day as the within-subjects factor. As seen in Fig. 4, neonatal treatment had no effect on ethanol preference in NP rats. In the P line, the 6 g/kg group showed a nonsignificant enhancement of ethanol preference relative to the control groups on day 1 but not on subsequent test days. The ANOVA showed no significant effects of neonatal treatment. As expected, the P line showed higher percentage ethanol intake than the NP line. The ANOVA revealed main effects of line, F(1,105) =
ETHANOL PREFERENCE TEST
1
Ethanol Exposure Rats received 10% ethanol as their sole source of fluid for 4 days prior to preference testing. An ANOVA was performed on ethanol intake scores corrected for differences in individual body weights [(ml 10% ethanol consumed/gram body weight) x lOOO]with sex, line, and neonatal treatment as betweensubjects factors and test day as the within-subjects factor. There was a main effect of neonatal treatment, fl2, 105) = 7.14, p c 0.001 bdt no interactions between treatment and line, sex or test day. A Newman-Keuls test showed that the 6 g/kg groups (M = 83.3; SE = 2.9) had higher intake scores than the maltose (M = 75.4; SE = 3.4) or the sham groups (M = 67.3; SE = 3.6). The maltose and sham groups were also significantly different. The ANOVA also showed significant main effects of line, fll, 105) = 58.16, p < 0.001, as P rats had higher intake scores than NP rats; sex, F(1, 105) = 57.26, p < 0.001, as females had higher intake scores than
0
12
3
5
4
6
7
8
9
TEST DAY
-
fig/kg
--b--
Maltose
--O--
ShamP
P P
&
6glkg
NP
--&-
MaltoseNP
--O-
ShamNP
FIG. 4. Mean (rt SEM) percentage intake 10% ethanol solution (ml etoh x lOO/[ml etoh + ml water]) as a function of neonatal treatment, rat line, and test day. The means shown include both sexes. Rats received simultaneous access to water and 10% ethanol (v/v) beginning at 97 days of age.
NEONATAL
ALCOHOL
EXPOSURE
252.41, p < 0.001, and a test day X line interaction, F(8, 840) = 8.07, p < 0.001, as percentage ethanol intake increased across days in the P rats but not in the NP rats. Finally, there were no effects of sex on ethanol preference. Ethanol preference body weights. Table 2 shows means and SEMs for body weights recorded on the last day of the ethanol preference test. The weights were averaged across males and females because sex did not interact with line or treatment. An ANOVA was conducted on these data including sex, line, and neonatal treatment as between-subjects factors. There was a significant main effect of neonatal treatment on body weight, F(2, 105) = 3.09, p < 0.05. Post hoc tests showed that the 6 g/kg groups weighed less than the maltose or sham groups. There were also main effects of sex, F( 1, 105) = 734.80, p < 0.001, because males weighed more than females, and line, F(1, 105) = 5.10, p -C 0.05, as NP rats weighed more than P rats. DISCUSSION
Neonatal alcohol exposure did not produce differential effects in adult P and NP rats in open field activity or in ethanol preference. P and NP rats of both sexes showed increased activity due to alcohol exposure and the magnitude of the alcohol effect was similar in the two lines. Pups that received alcohol exposure during the peak of the “brain growth spurt” (PNDs 4-9) showed higher open-field activity levels than artificially or normally reared controls when tested as adults (80day-old). Artificial rearing without alcohol treatment also increased activity relative to normal rearing, an effect most apparent in the NP line. In subsequent tests on the same rats, neonatal alcohol exposure increased intake of a 10% ethanol solution when it was the rat’s sole source of fluid and this effect was similar in the P and NP lines. The 6 g/kg groups consumed more 10% ethanol solution (as a function of body weight) than the maltose or the sham groups. The maltose groups had higher intake scores than the sham groups and, as expected, the P rats had higher scores than the NP rats. It is interesting that the line and treatment effects seen in this ethanol intake test parallel those found in the open-field activity tests. However, there were no effects of neonatal treatment on alcohol preference when rats then received free choice between the 10% ethanol solution and water, although the large line difference on which the P and NP rats were bred was very much in evidence. One important effect seen in the previous work with weaning P and NP rats (24) was that neonatal alcohol exposure produced a greater increase in activity in the P line than in NP line. P rats exposed to either 6 g/kg/day or 4 g/kg/day were more active than controls while only NP rats exposed to the 6 g/kg/day dosage were more active than controls. Furthermore, the percent increase in activity was much greater in the P rats than the NP rats following alcohol exposure. In contrast, in the current study the 6 g/kg dose produced a similar increase in activity in the two lines when testing occurred in adulthood. Thus, the differential effects of neonatal alcohol exposure on open-field activity in P and NP rats do not extend into adulthood. It is important to note, however, that neonatal alcohol exposure produced increased activity in adults of both sexes in the present research. In previous research (19), neonatal alcohol exposure increased activity only in adult females while alcohol exposed adult males did not differ from normally reared controls. A recent unpublished study from our lab has also failed to show effects of neonatal alcohol exposure in
adult Sprague-Dawley rats of either sex, although such effects are apparent in weaning age animals. Thus, the present study demonstrates long lasting effects of neonatal alcohol exposure on activity in both sexes. It does need to be pointed out that there was an overall increase in activity in the NP rats following artificial rearing. It appears that some aspect of the artificial rearing protocol, such as maternal separation or nutritional factors, in some way impacted the NP line and caused a general increase in activity levels as measured in the open field. The extent to which the alcohol effect in the NP line may have interacted with the artificial rearing is unknown. No effects of artificial rearing on activity were apparent in the P line and the enhanced activity in this line appears to be more directly related to the alcohol exposure. Neonatal alcohol exposure failed to affect subsequent ethanol preference in adult P or NP rats. Although the 6 g/kg groups of both line consumed more 10% ethanol than controls when it was the only fluid available, there were no reliable differences among treatment groups in either line during the preference test. The NP rats consistently ingested less than 10% of the ethanol solution during the preference test as would be expected, because this line was bred for low ethanol preference. In the P line, the 6 g/kg/day group showed a nonsignificant increase in ethanol preference relative to controls on the first day but not on subsequent days and the neonatal treatment effects were not statistically significant. The absence of neonatal alcohol effects on ethanol preference in these rat lines may not be surprising considering the inconsistent effects of prenatal alcohol exposure on adult rats in previous studies (23). Bond & Dig&to (7) did find increased preference for low alcohol concentrations, up to 6% v/v, following prenatal alcohol exposure but above this concentration preference was unaffected. Also, in that study only young (45-day-old) females were tested. It is possible that using lower concentrations of ethanol solution might have been interesting in the NP line. In a previous study (21), NP rats showed about 25% preference for a 4% ethanol solution. However, the P rats showed almost 100% preference for the 4% solution leaving no room for increased preference as a function of our neonatal alcohol treatment. The baseline preference Ievels for the 10% ethanol solution used in this study did allow both the P and the NP lines room for increased ethanol preference as a function of neonatal alcohol treatment. Neonatal alcohol treatment was also associated with slightly reduced body weights compared to control treatments in adulthood. Again, the two lines appeared to be equally affected in that neonatal treatment did not interact with line. It is interesting to note that, while the artificially reared maltose group of NP rats increased activity relative to sham controls, their body weights were equivalent to the sham group at testing. Furthermore, the activity effect appears to be unrelated to growth deficits since the body weights of NP maltose pups were equal to or greater than NP sham pups during the artificial rearing period. Again, the NP line may be more sensitive than the P line to factors associated with artificial rearing. In comparing the current study with our previous one (25), it is interesting that neonatal alcohol exposure produced differential effects on the P and NP lines at weaning but equal effects on the two lines in adulthood. The present results may be interpreted as a failure to extend this recent report (25) of differential susceptibility of the P and NP lines to the behavioral effects of neonatal alcohol exposure. Alternatively, these previous findings may be age-related as the rats were tested at weaning age in the Riley et al. (25) study and as adults in the present study. The general findings from animal models of
48
MELCER,
FAS indicate that prenatal alcohol effects are most evident at or before weaning age or in aged animals (2,3). This is certainly true with measures of activity (8). However, fetal alcohol effects are seen in adult animals but often more successfully under stressful or challenging circumstances (24). Perhaps the differential sensitivity of the P line to postnatal alcohol exposure reported at weaning age might reappear in adult animals using more challenging behavioral situations
GONZALEZ
AND RILEY
than the open field. In addition, it would be interesting to study the effects of neonatal alcohol exposure on activity in the P and NP rats in old age. ACKNOWLEDGEMENTS
This work was supported in part by grants AA06902 to EPR and AA07611 to T.-K. Li from the National Institute on Alcohol Abuse and Alcoholism.
REFERENCES 1. Abel, E. L. Fetal Alcohol Syndrome. Oradell, NJ: Medical Economics; 1990. 2. Abel, E. L.; Dintcheff, B. A. Effects of prenatal alcohol exposure on behavior of aged rats. Drug Alcohol Depend 16:321-330, 1986a. 3. Abel, E. L.; Dintcheff, B. A. Effects of prenatal alcohol exposure on nose poking in year old rats. Alcohol 3:201-204; 1986b. 4. Abel, E: L.; Sokol, R. J. Incidence of fetal alcohol syndrome and economic impact of FAS-related anomalies. Drug Alcohol Depend. 19:51-70; 1987. 5. Abel, E. L.; York, J. L. Absence of effect of prenatal ethanol on adult emotionality and ethanol consumption in rats. J. Studies Alcohol 40:547-553; 1979. 6. Barron, S.; Kelley, S. J.; Riley, E. P. Neonatal alcohol exposure alters suckling behavior in neonatal rat pups. Pharmacol. Biothem. Behav. 39:423-427; 1991. 7. Bond, N. W.; Di Giusto, E. L. Effects of prenatal alcohol consumption on open-field behaviour and alcohol preference in rats. Psychophamacologia 46:163-165; 1976. 8. Bond, N. W.; Di Giusto, E. L. Prenatal alcohol consumption and open-field behaviour in rats: Effects of age at time of testing. Psvchonharmacology 52:311-312; 1977. 9. Chernoff, G. F. The fetal alcohol syndrome in mice: Maternal variables. Teratol. 22:71-75; 1980. 10. Chotro, M. G.; Molina, J. C. Acute ethanol contamination of the amniotic fluid during gestational day 21: Postnatal changes in alcohol responsiveness in rats. Dev. Psychobiol. 23:535-547; 1990. 11. Dobbing, J.; Sands, J. Quantitative growth and development of the human brain. Arch. Dis. Child. 48757-767: 1973. 12. Gilliam, D.; lrtenkauf, K. Maternal genetic effects on ethanol teratogenesis and dominance of relative embryonic resistance to malformations. Alcohol: Clin. EXD. Res. 14:539-545; 1990. 13. Gilliam, D. M.; Ketch, L.; Dudek, B. C.; Riley, E. P. Ethanol teratogenesis in selectively bred long-sleep and short-sleep mice: A comparison to inbred C57BL/6J mice. Alcohol Clin. Exp. Res. 13:667-672; 1989a. 14. Gilliam, D. M.; Ketch, L.; Dudek, B. C.; Riley, E. P. Ethanol teratogenesis in mice selected for differences in alcohol sensitivity. Alcohol 5:513-519; 1989b. 15. Gilliam, D. M.; Stilman, A.; Dudek, B. C.; Riley, E. P. Fetal alcohol effects in long- and short-sleep mice: activity, passive avoidance and In Utero ethanol levels. Neurotoxicol. Teratol. 9: 349-357, 1987. 16. Goodlett, C. R.; Gilliam, D. M.; Nichols, J.; West, J. Genetic influences on brain growth restriction induced by developmental exposure. Neurotoxicol. 10:321-324; 1989. 17. Grant, K. A.; Choi, E. Y.; Samson, H. H. Neonatal ethanol exposure: Effects on adult behavior and brain growth parameters. Pharmacol. Biochem. Behav. 18: Suppl. 1:331-336; 1983. 18. Kelly, S. J.; Hðer, S. A.; West, J. R. Alterations in sensorimotor development: Relationship to postnatal alcohol exposure. Neurotoxicol. Teratol. 9:243-251; 1987.
19. Kelly, S. J.; Pierce, D. R.; West, J. R. Microencephaly and hyperactivity in adult rats can be induced by neonatal exposure to high blood alcohol concentrations. Exp. Neurol. 96:580-593; 1987. 20. Lumeng, L.; Hawkins, T. D.; Li, T.-K. New strains of rats with alcohol preference and nonpreference. In: Thurman, R. G.; Williamson, J. R.; Drott, H. R.; Chance, B. eds. Alcohol and aldehyde metabolizing systems, vol. 3. New York: Plenum Press; 19771537-544. 21 Li, T.-K.; Lumeng, L.; Mcbride, W. J.; Waller, M. B. Indiana selection studies on alcohol-related behaviors. In: McClearn, G.; Deitrich, R. A.; Erwin, V. G. eds. Development of animal models as pharmacogenetic tools. Research monograph number 6, U.S. Department of Health and Human Services; 1978: 171181. 22. Melter, T.; Gonzalez, D.; Somes, C.; Riley, E. P. Postnatal alcohol exposure and early development of motor skills in alcohol preferring (P) and nonpreferring (NP) rats. Neurotoxicol. Teratol. (in press). 23. Meyer, L.; Riley, E. P. Behavioral teratology of alcohol. In: Riley, E. P.; Vorhees, C. V.; eds. Handbook of behavioral teratology. New York: Plenum Press; 1986:101-140. 24. Riley, E. P. The long-term behavioral effects of prenatal alcohol exposure in rats. Ale. Clin. Exp. Res. 14:670-673; 1990. 25. Riley, E. P.; Barron, S.; Melter, T.; Gonzalez, D. Alterations in activity following alcohol administration during the third trimester equivalent in P and NP rats. Ale. Clin. Exp. Res. 17:12401246; 1993. 26. Riley, E. P.; Lochry, E. A. Genetic influences in the etiology of fetal alcohol syndrome. In: Abel, E. L. ed. Fetal alcohol syndrome, 3, animal studies. Boca Raton, FL: CRC Press; 1982:113130. 27 Samson, H. H.; Diaz, J. Effects of neonatal ethanol exposure on brain development in rodents. In: Abel, E. L. ed. Fetal alcohol syndrome, Ill Animal studies. Boca Raton, FL: CRC Press; 1982: 131-150. 28. Streissguth, A. P. The behavioral teratology of alcohol: Performance, behavioral, and intellectual deficits in prenatally exposed children. In: West, J. R., ed. Alcohol and brain development. New York: Oxford University Press; 1986:3-44. 29. Streissguth, A. P.; Barr, H. M.; Martin, D. C. Alcohol exposure in utero and functional deficits in children during the first four years of life. In: Porter, R.; O’Conner, M.; Whelan, J. eds. Mechanism of alcohol damage in utero. Ciba Foundation Symposium 105; London, Pittman; 1984:176-196. 30. Tabakoff, B.; Culp, S. G. Studies on tolerance development in inbred and heterogenous stock national institutes of health rats. Ale. Clin. Exp. Res. 8:495-499; 1984. 31. Terry, L. M.; Scheinman, J. A.; Hall, W. G. Response deficits in isolate reared rats. Paper presented at the annual meetings of the International Society for Developmental Psychobiology; 1987.