Critical Periods for the Effects of Alcohol Exposure on Learning in Rats

Transcription

1 Behavioral Neuroscience 2001, Vol. 115, No. 1, Copyright 2001 by the American Psychological Association, Inc /01/$5.00 DOI: // Critical Periods for the Effects of Alcohol Exposure on Learning in Rats Kim Cronise, Melissa D. Marino, Tuan D. Tran, and Sandra J. Kelly University of South Carolina, Columbia Critical periods for alcohol-induced deficits in spatial navigation and passive avoidance learning were investigated with a rat model of fetal alcohol syndrome. Rats were exposed to alcohol prenatally (Gestational Days 1-10 or 11-22) or postnatally (Postnatal Days 2-10) or throughout all 3 periods. Offspring were tested in either a spatial navigation or an avoidance task as juveniles or adults. As juveniles, the combined exposure group took longer to learn the spatial navigation task compared with all other groups. This effect was not seen in adults. Passive avoidance performance was not affected. These results suggest that long-term exposure to alcohol during development has adverse effects on spatial learning. The lack of differences in the short-term exposure groups implies that there may not be 1 critical period of alcohol exposure, but that the adverse effects of alcohol during development may be cumulative on some behaviors. Maternal alcohol consumption during pregnancy has been shown in both the clinical literature and animal studies to produce various physical and behavioral deficits, and severe cases result in a condition called fetal alcohol syndrome (FAS) in humans (Mattson & Riley, 1998). Behavioral dysfunctions are perhaps the most sensitive indicators of the effects of perinatal alcohol exposure on the developing fetus (Vorhees, 1987). In animal models of FAS, some of the most commonly reported behavioral deficits include difficulties in spatial learning ability and response inhibition. The animal models typically entail alcohol exposure during either the prenatal period or early postnatal period, which are roughly equivalent to the first two and third human trimesters, respectively (Bayer, Altman, Russo, & Zhang, 1993). Spatial learning ability has been shown to be adversely affected by both prenatal (Blanchard, Riley, & Hannigan, 1987; Gianoulakis, 1990; Kim et al., 1997; Reyes, Wolfe, & Savage, 1989; Zimmerburg, Sukel, & Stekler, 1991) and postnatal (Goodlett, Kelly, & West, 1987; Goodlett & Johnson, 1997; Kelly, Goodlett, Hulsether, & West, 1988; Tomlinson, Wilce, & Bedi, 1998) alcohol exposure in both juvenile (Blanchard et al., 1987; Goodlett et al., 1987; Goodlett & Johnson, 1997) and adult (Gianoulakis, 1990; Kelly et al., 1988; Kim et al., 1997; Vorhees & Fernandez, 1986) animals. However, many contradictions exist in this body of literature. For example, several studies found that perinatal alcohol exposure induced deficits in both sexes (Gianoulakis, 1990; Goodlett et al., 1987; Reyes Kim Cronise and Tuan D. Tran, Department of Psychology, University of South Carolina, Columbia; Melissa D. Marino, Department of Pharmacology and Physiology, School of Medicine, University of South Carolina, Columbia; Sandra J. Kelly, Department of Pharmacology and Physiology, School of Medicine and Department of Psychology, University of South Carolina, Columbia. This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant A A We acknowledge the technical support of Cindy Gobbel, Lisa Slappey, and Kris Ford. Correspondence concerning this article should be addressed to Sandra J. Kelly, Department of Psychology, University of South Carolina, Columbia, Columbia, South Carolina Electronic mail may be sent to sandrakelly@sc.edu. 138 et al., 1989). In contrast, Blanchard et al. (1987) found the deficits to be more pronounced in females than in males, and Zimmerburg et al. (1991) found spatial learning deficits only in males after prenatal alcohol exposure. Postnatal alcohol exposure has been shown to produce spatial learning deficits in juveniles of both sexes (Goodlett et al., 1987) that are evident only in females in the adult period (Kelly et al., 1988). Another source of contention is the longevity of the deficit. With some studies showing deficits that last into adulthood (Kim et al., 1997; Reyes et al., 1989; Zimmerburg et al., 1991) and others failing to show adulthood deficits {Abel, 1979; Vorhees & Fernandez, 1986; Westergren, Rydenhag, Bassen, Archer, & Conradi, 1996), it is unclear whether alcohol exposure causes long-lasting spatial learning deficits. Similar inconsistencies exist in the literature on alcohol-induced response inhibition deficits. In the passive avoidance task, a putative measure of response inhibition, acquisition deficits have been observed in juveniles in response to prenatal alcohol exposure (Abel, 1982; Lochry & Riley, 1980; Tan, Berman, Abel, & Zajac, 1990; Tan, Abel, & Berman, 1993), but these deficits do not appear to persist into adulthood (Abel, 1982). Retention of this task does not appear to be affected by prenatal alcohol exposure (Lochry & Riley, 1980). A potential problem with this interpretation is that some studies tested only females (Abel, 1982), whereas others tested only males (Tan et al., 1990, 1993). In the only study that investigated the effect of postnatal alcohol exposure on response inhibition, both acquisition and retention of the passive avoidance task were impaired in alcohol-exposed juvenile females but not in males; adults were not tested (Barron & Riley, 1990). It appears from these and other studies that there are certain periods of development during which the fetus is more susceptible to the effects of alcohol. Critical periods of alcohol exposure for passive avoidance performance were suggested by the finding that exposure during the second half of gestation and throughout the entire gestational period produces passive avoidance deficits, whereas exposure during the first half of gestation does not (Driscoll, Chen, & Riley, 1982). The relative importance of the postnatal period for alcohol-induced deficits in passive avoidance is unclear. There has been no systematic examination of critical

2 CRITICAL PERIODS OF ALCOHOL EXPOSURE 139 periods during the prenatal period for deficits in spatial navigation. Goodlett and Johnson (1997) found that Postnatal Days 7 to 9 are critical for deficits in spatial navigation induced by postnatal alcohol exposure. A number of problems exist in attempting to determine critical periods from the results of the previous studies. The methods of alcohol administration vary between studies and may not be equivalent in producing central nervous system dysfunction. Most prenatal alcohol exposure models use a liquid diet to administer the alcohol dose. This method does not produce consistently high blood alcohol concentrations (BACs) and can cause nutritional deficits as a result of decreased intake. Neonatal alcohol exposure has customarily been accomplished with artificial rearing. Although this method allows for strict control over dose and time of alcohol administration, it involves an invasive surgery, and the pup must be reared in isolation, devoid of maternal and sibling contact. These stressors can become confounding factors when using behavioral tests as measures of alcohol's teratogenicity. Also, the BACs induced using artificial rearing are usually much higher than those using a liquid diet procedure. Because the previous studies do not include all of gestation and the period of rapid brain development in the early postnatal period, and the methods of administration and the BACs differ between the different exposure periods, drawing valid conclusions regarding critical periods can be problematic. It has been suggested that peak BAG (Pierce & West, 1986) is the critical factor in producing behavioral deficits. Therefore, an animal model of FAS is needed that closely regulates the dose of alcohol and the time of administration in order to obtain conditions that will be likely to produce behavioral effects. Intragastric intubation can allow for close control of dosage and timing of alcohol administration and eliminate the need for rearing the pups in isolation for the extent of the exposure. Studies from this lab show that this procedure can produce high and consistent BACs that are comparable across both the prenatal and postnatal periods (Tran, Cronise, Marino, Jenkins, & Kelly, 2000). The main purpose of this study was to examine the critical periods of alcohol exposure on spatial learning ability using the Morris water maze task and response inhibition using passive avoidance performance in all three trimester equivalents, alone and in combination, using a rat model of FAS. With this model, we were able to examine not only critical periods but potential sex differences in alcohol's action on behavior as well. In addition, we were able to study the longevity of these behavioral dysfunctions by testing performance in juvenile and adult rats. Method Subjects All subjects were housed in the animal colony (University of South Carolina, Department of Psychology), which is maintained at 22 C with a 12-hr light-dark cycle. One hundred three female Long-Evans rats were housed overnight with proven breeder males. The following morning, vaginal smears were performed to check for the presence of sperm. The morning that sperm was detected was designated as Gestational Day (GD) 1. Once pregnant, dams were housed singly in polypropylene cages with wood shavings. The dams were also assigned to one of six treatment groups (Table 1). Table 1 Treatment Groups Group NC 1C ET1 ET2 ET3 ET123 Gestational Day 1-10 No treatment Gestational Day No treatment Postnatal Day 2-10 No treatment Intubation procedure Intubation procedure Intubation procedure Note. NC = nontreated control; 1C = intubated control; ET = ethanol treated. Procedures The ethanol-treated (ET) 123 dams received intubations of ethanol (4.5 rag/kg in a volume of 20 ml/kg) from GDI through GD22, and the intubated control (1C) dams received intragastric intubations of an isocaloric solution of maltose-dextrin (20 ml/kg) from GDI through GD22. The ET1 and ET2 groups received ethanol intubations on GDI through 10 and GD11 through GD22, respectively, and intubations of maltose-dextrin during the other periods of gestation. During the periods of ethanol treatment, dams of the ET123, ET1, and ET2 groups were allowed access ad libitum to rat chow and water. Food consumption was measured in the ET123 dams to provide data for pair feeding of the other ET and 1C dams. During periods of maltose-dextrin intubations, dams were pair fed to an ET123 dam of similar age, similar weight, and exact gestational day. A nontreated control (NC) group was weighed daily but received no other treatments. On GD20, dams were given paper towels to be used as nesting material. The day of birth (GD23) was designated as Postnatal Day (PD) 1. Dams and pups were not treated in any way on this day. Litters were maintained at 10 pups (5 male and 5 female) whenever possible. On PD2 through PD10, pups were weighed and marked with nontoxic marker for identification. Pups in the ET123 and ET3 groups were intragastrically intubated with 3.0 g/kg ethanol in a volume (.0278 ml/g) of enriched milk (West, Hamre, & Pierce, 1984). Two hours after the first intubation, pups were intubated again with.0278 ml/g of the milk solution alone. Pups in the ET1, ET2, and 1C groups underwent the same intubation procedures but did not receive ethanol or milk. The NC group was weighed and marked daily. On PD10, pups were permanently paw marked with India ink for identification purposes (Geller & Geller, 1966). Pups remained with the dam until PD21, at which time they were weaned and housed in same-sex pairs. Dams BAG We collected 10-/il blood samples from a nick to the tail from the ET123, ET2, and 1C groups on GD20 and from the ET1 group on GD10 3 hr after the alcohol intubation. This time was chosen to assess maximum BACs (Tran et al., 2000). No blood was taken from the NC dams. Samples from the ethanol-treated groups were analyzed for BACs with an enzymatic procedure (Dudek & Abbott, 1984). Pups On PD10, 10-/A1 blood samples were collected by a tail clip from all of the pups, except those in the NC group, 2 hr after the first intubation. This time was chosen to assess maximum BACs (Tran et al., 2000). Blood

3 140 CRONISE, MARINO, TRAM, AND KELLY samples from ET3 and ET123 litters were analyzed for BACs by the same procedure used for the dams. Behavioral Testing Passive Avoidance Procedure Juveniles. Two rats from each litter, one of each sex, were tested on PD23 for acquisition and on PD24 for retention of passive avoidance learning. The testing apparatus consisted of two Plexiglas compartments, one black and one white, separated by a removable door. Both compartments had floors made of stainless steel grids. A Coulbourn Instruments (Allentown, PA) solid-state shock generator delivered a 0.1-mA pulse of distributed shock to the floor of the black compartment. The testing room was illuminated by red light only, except for a light source in the white compartment. The test animal was placed in the white compartment, the light source activated, and the latency for the rat to cross from the white to the black compartment recorded. Once in the black compartment, the door was closed, and a brief shock (1 s) was delivered. Trial length was a maximum of 60 s with an intertrial interval of 60 s. To meet the acquisition criterion, the rat had to remain in the lit compartment for the maximal trial length on two consecutive trials. Both latencies and number of trials to criterion were recorded for each rat. Twenty-four hours after acquisition, the procedure was repeated for the retention phase. Adults. Two additional rats from each litter, one of each sex, were tested on PD120 and PD121. The procedure was the same as that used for the juveniles, except that the maximal trial length was 90 s. Spatial Navigation Procedure Juveniles. Two rats from each litter, 1 of each sex, were tested from PD19 through PD31. The apparatus was a white circular tank (122 cm diameter, 61 cm deep) filled with 24 C water at a depth of cm. The water was made opaque by the addition of nontoxic white tempera paint. In the distal cue condition, a clear Plexiglas platform (15 X 15 cm) was placed in the center of the northeast quadrant 2 cm below the surface of the water. In the proximal cue condition, an opaque black platform was placed in the center of the southwest quadrant with its surface 1 cm above the surface of the water. The rats were tested for 10 sessions in the distal cue condition of the water maze and for 3 sessions in the proximal cue condition. There were eight trials per session, and each trial was separated by 3- to 4-min intervals. On each trial, the start point (N, S, E, or W) occurred in a quasi-random manner, with each point occurring twice in a session. The experimenter, who stood at the same point each trial, traced the path onto a scaled diagram of the tank and recorded the latencies to reach the platform. If the rat located the platform, it was allowed to remain there for 10 s. If the platform was not located, then the rat was placed on the platform for 10s. On s 2, 7, and 10, probe trials were conducted at the end of the sessions. During the probe trials, the platform was removed and the rat was allowed to swim for 45 s. The swim path was traced onto a scale diagram. On the diagram, three concentric annul! were drawn around the platform, which was in the northeast quadrant. The first annulus touched the platform corners. The second and third annuli were one and two platform widths distant from the first annulus, respectively, and all three annuli were contained within the platform quadrant. A second identical set of three annuli was placed in the southwest opposing quadrant as though a platform was located there. Annulus crossings were counted for each quadrant. Proportion of correct annulus crossings was calculated as the ratio of the number of crossings in the platform (northeast) quadrant divided by the total number of crossings of all of the annuli in both the platform and southwest quadrant. The closer the proportion is to 1.0, the more directed the animal's swimming pattern was toward the actual location of the platform. Adults. Two additional rats, 1 of each sex, were tested from each litter from PD90 through PD103. The procedure was the same as that used for the juveniles, except that the ceiling for the adults to find the platform was 90s. Results The data were analyzed using analyses of variance (ANOVAs). For analyses of repeated measures, Greenhouse-Geiser's adjusted degrees of freedom were used to determine significance. When an interaction was significant, analyses of simple main effects were used to describe the interaction. Tukey's post hoc analyses were used to examine main effects and simple main effects, as suggested by May, Masson, and Hunter (1990). The level of significance was p <.05 except when repeated tests of simple main effects were conducted. In these cases, the level of significance was adjusted by dividing.05 by the number of tests conducted. Body Weights for Dams and Pups Body weights were recorded for dams from GDI through GD22. A two-way mixed ANOVA (Group X Gestational Day) revealed no significant differences among groups. Body weights were recorded for pups of all groups from PD2 through PD10. A three-way ANOVA (Group X Sex X Postnatal Day) revealed a significant interaction of group and day, F(40, 5256) = 10.7, using Greenhouse-Geiser adjusted degrees of freedom (p <.001) and a significant interaction of sex and day, F(8, 5256) = 3.9, using Greenhouse-Geiser adjusted degrees of freedom (p <.001). Analyses of simple main effects of group (using p <.005 as the criterion for significance to adjust for nine tests) were significant for each day (PD2-10). Differences in body weight between the ethanol-exposed groups and the control groups, determined by Tukey's post hoc tests, indicated that only the ET123 and ET2 groups differed from the NC and 1C groups. The ET123 group weighed less than the 1C and NC groups on PD2, 3, and 9 (ps <.05). The ET2 group weighed less than the 1C and NC groups on PD2, 3, 4, 5, 6, 7, and 9 (ps <.05). Analyses of simple main effects of sex (using p <.005 as the criterion for significance to adjust for nine tests) were significant for PD2 through PD9. Males weighed more than females on each day. Body Weights for Passive Avoidance Subjects (PD21 and PD90) A two-way between-subjects ANOVA (Group X Sex) on the rats tested in the passive avoidance task as juveniles revealed a significant main effect of group, F(5, 150) = 4.87, p <.0005, on body weight at PD21. Tukey's post hoc analysis demonstrated that the ET123 group weighed less than both the ET3 and the 1C groups and that the NC group weighed less than the ET3 group (Table 2). A two-way between-subjects ANOVA (Group X Sex) on body weights at PD90 of the adults tested in the passive avoidance task revealed a significant main effect of sex, F(l, 165) = ,p<.0001, and group, F(5, 154) = 2.67, p <.03. Tukey's post hoc analysis revealed that the ET2 group weighed less than the 1C group (see Table 2). The control groups did not differ. Females weighed less than males.

4 CRITICAL PERIODS OF ALCOHOL EXPOSURE 141 Table 2 Body Weights (in Grams, M ± SEM) at Time of Testing Group NC 1C ET1 ET2 ET3 ET123 Passive avoidance Morris water maze Juvenile (PD21) Adult (PD90) Juvenile (PD21) Adult (PD90) 43.5 ± ** * ± ± ± ± 20.2* ± ± ± ± ± ± ± ± ± ± ± ± 18.6* ± ± 19.5* Note. PD = postnatal day; NC = nontreated controls; 1C = intubated control; ET = ethanol treated. * and ** indicate significantly different from the 1C and NC groups, respectively (p <.05). Body Weights on Spatial Navigation Subjects (PD21 and PD90) A two-way ANOVA (Group X Sex) on body weights at PD21 of the juvenile animals tested in the spatial navigation task revealed no significant differences among groups (see Table 2). A two-way ANOVA (Group X Sex) on body weights at PD90 of the adults tested in the spatial navigation task revealed a main effect of group, F(5, 153) = 2.33, p <.05. Tukey's post hoc analysis demonstrated that the ET123 and the ET2 groups weighed less than the 1C group (ps <.05) (see Table 2). There was also a significant main effect of sex, F(l, 153) = , p <.0001, indicating that females weighed less than males. BACs A one-way between-subjects ANOVA revealed no significant differences in BACs among groups of dams. The BACs of the dams in the ET1, ET2, and ET123 groups were mg/ dl ± 26.2 SEM, mg/dl ± 28.7 SEM, and mg/dl ± 21.3 SEM, respectively. Separate ANOVAs (Group X Sex) on the BACs at PD10 among groups of rats tested as juveniles or adults in the passive avoidance or spatial navigation task revealed no significant differences among groups or between sexes. The mean BACs were mg/dl ± SEM, mg/dl ± 9.07 SEM, mg/ dl ± SEM, and mg/dl ± SEM for the juvenile rats tested for passive avoidance, adults tested for passive avoidance, juveniles tested for spatial navigation, and adults tested for spatial navigation, respectively. Passive Avoidance: Juveniles Behavioral Findings Number of trials to criteria. A three-way mixed ANOVA (Group X Sex X ) revealed a significant main effect of session, F(l, 136) = 92.10, using Greenhouse-Geiser adjusted degrees of freedom (p <.001), because all groups required more trials to reach criterion during the acquisition phase than during the retention phase. There were no significant differences among groups. Mean trials to criterion were 5.20 ±0.18 SEM for acquisition and 3.28 ± 0.13 SEM for retention. There were no other significant differences. Latencies. Latencies to enter the dark compartment on each trial were analyzed. Separate three-way mixed ANOVAs (Group X Sex X Trial) for each session revealed no significant differences. Mean latencies to enter the dark compartment were as follows: acquisition, s ± 0.57 SEM; retention, s ± 1.19 SEM. Passive Avoidance: Adults Number of trials to criteria. A three-way mixed ANOVA (Group X Sex X ) revealed a significant main effect of session, F(l, 141) = , using Greenhouse-Geiser adjusted degrees of freedom (p <.001), indicating that all groups required more trials to reach criterion during the acquisition phase than during the retention phase. There were no significant differences among groups. Mean number of trials to criteria were as follows: 1, 4.19 ± 0.12 SEM; 2, 2.89 ± 0.09 SEM. Latencies. Latencies to enter the dark compartment on each trial were analyzed. Separate three-way mixed ANOVAs (Group X Sex X Trial) revealed no significant differences among groups. Mean latencies to enter the dark compartments were as follows: 1, s ± 0.77 SEM; 2, s ± 0.96 SEM. Spatial Navigation: Juveniles Distal cue condition. A three-way mixed ANOVA (Group X Sex X ) revealed a significant interaction of group and session, F(45, 1413) = 1.54, using Greenhouse-Geiser adjusted degrees of freedom (p <.03). Analyses of simple main effects of group (using p <.005 as the criterion for significance adjusted for 10 tests) were significant for s 2, 3, 6, and 7. Tukey's post hoc analysis was used to determine differences between the ethanol-exposed groups and the two control groups. Only the ET123 group differed from the control groups. The ET123 had longer latencies than the 1C group in s 2, 3, 6, and 7 and longer latencies than the NC group in s 6 and 7. At no time did the 1C or NC groups differ from each other or from the ET1, ET2, and ET3 groups (Figure 1). Effect size was determined according to Cohen (1988), in which effect sizes of 0.2, 0.5, and 0.8 are considered small, medium, and large, respectively. The effect sizes of the differences between the ET123 and 1C group on s 2, 3, 6, and 7 are 0.83, 0.83, 0.69, and 1.2, respectively.

5 142 CRONISE, MARINO, IRAN, AND KELLY 40 -,»35- - w 25 - Juveniles - ET123 vs. Controls Juveniles - ET1 vs. Controls Juveniles - ET2 vs. Controls Juveniles - ET3 vs. Controls Figure 1. Distal cue condition for juvenile spatial navigation rats. The ethanol-treated (ET) 123 group demonstrated longer latencies than the ET1, ET2, ET3, and nontreated control (NC) groups on s 2 through 8. Stars represent points at which the ET123 group is significantly different from the 1C group. Error bars represent SEM. 1C = intubated control. The effect sizes of the differences between the ET123 and NC group on s 6 and 7 are 0.94 and 0.67, respectively. For the probe trials, a three-way mixed ANOVA (Group X Sex X ) on the proportion of annuli crossings in the platform quadrant revealed a significant main effect of session. Tukey's post hoc analysis demonstrated that all groups crossed fewer annuli in the platform quadrant on 2 than on s 7 and 10. There were no significant differences among groups. The mean proportion of crossings in the correct quadrant were as follows: 2, 0.50 ± 0.02 SEM; 7, 0.70 ± 0.01 SEM; and 10, 0.70 ± 0.01 SEM. Proximal cue condition. A three-way mixed ANOVA (Group X Sex X ) revealed a main effect of session, F(2, 313) = , using Greenhouse-Geiser adjusted degrees of freedom (p <.001). Tukey's post hoc analysis demonstrated that the latencies to reach the black platform decreased across s 11, 12, and 13. There were no significant differences among groups. Spatial Navigation: Adults Distal cue condition. A three-way mixed ANOVA (Group X Sex X ) revealed a significant effect of sex, F(l, 156) = 10.3, p <.001, and session, F(9, 1404) = 264.6, using Greenhouse-Geiser adjusted degrees of freedom (p <.001). In general, females had slower latencies than males, and all animals showed a decline in latencies over time. There were no effects of or interactions with group (Figure 2). Probe trials. For the proportion of annuli crossings in the correct quadrant, a three-way mixed ANOVA (Group X Sex X ) revealed a significant main effect of session, F(2, 310) = 58.77, using Greenhouse-Geiser adjusted degrees of freedom (p <.001). Tukey's post hoc analysis demonstrated that the proportion of crossings in the platform quadrant increased across sessions. There were no significant differences among groups. Proximal cue condition. A three-way mixed ANOVA (Group X Sex X ) revealed a significant interaction of sex and session, F(2, 312) = 4.68, using Greenhouse-Geiser adjusted degrees of freedom (p <.03). Analyses of simple main effects of sex (using p <.01 as the criterion for significance adjusted for three tests) were significant for s 12 and 13. Females took longer to locate the visible platform than males in s 12 and 13. There were no significant differences among groups.

6 CRITICAL PERIODS OF ALCOHOL EXPOSURE 143 Adults--ET123 vs. Controls Adults-ET1 vs. Controls tfi S I 20 S S 15 > " 10 S Adults~ET2 vs. Controls Adults--ET3 vs. Controls in c 15 «25 20 S = ^ Figure 2. Distal cue condition for adult spatial navigation rats. The ethanol-treated (ET) 1 group demonstrated shorter latencies than the intubated control (1C) group on 7. Star represents significance at p <.05. NC = nontreated controls. Error bars represent SEM. Discussion Juvenile rats of both sexes in the ET123 group demonstrated longer latencies during acquisition for the spatial navigation task than all of the other groups, which did not differ. However, this deficit was found to be transient in that no differences were found among the adults. In the proximal cue control condition of the spatial navigation task, adult females took longer to locate the visible platform than males, but there were no differences among groups. In the passive avoidance paradigm, no differences were observed among groups in either the juvenile or adult conditions. The behavioral findings of this study are inconsistent with previous literature on three counts. First, whereas other studies showed juveniles to be impaired at passive avoidance, this study did not demonstrate passive avoidance deficits in juvenile rats (Abel, 1982; Barren & Riley, 1990; Driscoll et al., 1982). Second, whereas persistent spatial deficits in adult animals have been shown (Gianoulakis, 1990; Kelly et al., 1988), this study did not demonstrate spatial deficits in adults. Finally, whereas other studies showed deficits as a result of short-term exposures, this study did not show deficits in either behavioral paradigm as a result of short-term exposure to alcohol (Abel, 1982; Barron & Riley, 1990; Blanchard et al., 1987; Driscoll et al., 1982; Gianoulakis, 1990; Goodlett et al., 1987; Kelly et al., 1988; Tomlinson et al., 1998). These discrepancies may be due to differences between our model and the previous models of alcohol exposure. In the past, studies have used a liquid diet procedure to administer alcohol prenatally and artificial rearing for postnatal administration. There are several side effects of these methods. First, large weight differences have been observed between the pair-fed control dams fed a liquid diet and the ad-lib controls (Hannigan, Abel, & Kruger, 1993; Weinberg, 1985), indicating that the administration procedure induces undernutrition. Although the undernutrition may not have a direct effect on spatial navigation performance, it is quite possible that the undernutrition interacts with the alcohol exposure to potentiate any subthreshold effects. In our model, no weight differences are observed among the dams in the different groups, which indicates that undernutrition is not a potential confounding factor in this study. Second, artificial rearing during the postnatal period causes stress to the pup by implantation of a gastrostomy tube and the deprivation of the neonate of dam-pup and pup-pup interactions. It has been demonstrated that artificial rearing can have large effects on behavior (Kelly, 1996; Kelly, Mahoney, Randich, & West, 1991). Therefore, it is possible that the nutrition and stress effects that result from the use of a liquid diet or artificial rearing may synergize with alcohol to alter subsequent behavior. The current study used intragastric intubation

7 144 CRONISE, MARINO, TRAN, AND KELLY as the method of pre- and postnatal administration of alcohol; this model does not result in weight differences among the dams or social separation in the postnatal period. Although clearly intubations are a stressor to both dams and pups, the stressor is very short lived compared with the chronic stress of being restricted to a liquid diet containing alcohol or artificial rearing. Moreover, this model achieved BACs that were comparable to or surpassed those of previous studies. Because this model avoids the nutritional and stress effects of a liquid diet and the stress and isolation effects associated with artificial rearing, it may assess the direct effects of alcohol on development without synergy with other factors. The failure to replicate alcohol-induced deficits in passive avoidance learning in the present study could also be attributed to the use of a lower level of shock (0.1 ma) than that used in other studies (0.5 ma; Abel, 1982; Barren & Riley, 1990; Lochry & Riley, 1980). The low level of shock was chosen because it results in a slower learning curve, thus increasing the possibility of revealing any group differences in acquisition of the task. It may be that with a higher level of shock alcohol-exposed rats exhibit the enhanced stress response seen in other studies (Kelly et al., 1991; Weinberg, 1989, 1993), which interferes with their acquisition of the passive avoidance response. This suggests that when a deficit in this task is detected in alcohol-exposed animals, it is the result of interference with performance because of an enhanced stress response rather than a deficit in the associative mechanism underlying the task. The current study does show that exposure to alcohol throughout gestation and the postnatal period produces deficits in spatial learning in juveniles. This finding is most robust when the comparison is made with 1C control rats as opposed to the NC rats. Although there were no significant differences between the two control groups, the differential findings concerning the relative performance of the ET123 group compared with the two control groups does suggest that there may be some mild beneficial effects of the intubation procedure on performance. Because the 1C control group is the most relevant control for the ethanol effects, the consistency and size of the differences and between the ET123 and 1C group lend confidence to the findings. According to our findings, the alcohol-induced deficits in spatial navigation do not seem to be enduring. Although most of the literature to date suggests that deficits in spatial navigation do persist into adulthood, a few studies suggest that the deficits may be transient. Westergren et al. (1996) administered alcohol prenatally (GDI 1-20) and did not observe spatial navigation deficits in 90-day-old rats. Also, Kelly et al. (1988) demonstrated spatial deficits in adult females only exposed postnatally to alcohol but did not show any deficits, in either sex, during reversal learning. A strength of the current study was that juveniles and the adults came from the same litters. Thus, the finding that the spatial deficits did not persist into adulthood cannot be attributed to litter or cohort effects. An important finding of this study was the lack of deficits seen as a result of short-term alcohol exposure. Because only the ET123 group demonstrated deficits in spatial navigation, this finding suggests that short-term periods of exposure during development are necessary but not sufficient to alter spatial navigation behavior and that the effects of prolonged exposure (GD1-20 and PD2-10) may be cumulative. Using the same experimental groups as the current study, Tran and Kelly (2000) examined hippocampal cell number. The greatest decreases in CA1 pyramidal cell number were in animals in the ET123 and ET3 group, and there were much smaller decreases in cell number in the ET1 and ET2 groups. The hippocampus is thought to be involved in tasks of spatial learning, although it should be noted that other structures such as the prefrontal and entorhinal cortices also participate in this behavior (for review, see Silva, Giese, & Fedorov, 1998). Because the ET3 rats did not show a deficit in spatial navigation, this suggests that the decrease in CA1 number is necessary but not sufficient to produce the deficit. Other structures may be damaged by alcohol exposure in the ET123 group and contribute to the deficit in spatial navigation in these rats. Because, in this study, both the ET123 and the ET2 groups consistently weighed less than the other groups from PD2 through PD10, the spatial deficit in the ET123 group cannot be attributed to a weight deficit. Furthermore, because the weight differences persisted to PD90 in the ET123 and ET2 groups, spatial deficits in the adult animals would have been expected if the deficit was the result of a weight difference, but this did not occur. From the clinical literature, it can be said that fetal alcohol exposure is often confounded with other factors (Abel, 1995; Abel & Hannigan, 1995). Some of these factors include poor prenatal care, maternal drug abuse, poor nutrition, and smoking. It is possible that the effects of alcohol during development synergize with these factors to produce the robust effects characterized as FAS. This synergy may explain the range of alcohol effects seen in children exposed to alcohol prenatally. It may be that, in mothers who do not abuse other substances or who have adequate prenatal care, lesser effects (called fetal alcohol effects) may be seen. It is also possible that, if all other contributing factors are removed from the experimental design, the effects of alcohol on the developing fetus may be more subtle than previously thought. The subtle effects of alcohol seen in the current study may increase dramatically when combined with such factors as undernutrition, chronic stress, maternal deprivation, and so on. In summary, the current study uses an animal model of FAS that equates BACs across different developmental periods and attempts to minimize disruption by the administration procedure. 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