Deficits on a spatial navigation task following prenatal exposure to ethanol

Neurotox&ologyand Teratology, Vol. 9, pp. 253--258.©PergamonJournals Ltd., 1987. Printedin the U.S.A.

0892-0362/87$3.00 + .00

Deficits on a Spatial Navigation Task Following Prenatal Exposure to Ethanol B. A. BLANCHARD, E. P. R I L E Y A N D J. H. H A N N I G A N 1 Center for Behavioral Teratology, and Department o f Psychology The University at Albany, State University o f New York 1400 Washington Avenue, Albany, N Y 12222 R e c e i v e d 6 O c t o b e r 1986 BLANCHARD, B. A., E. P. RILEY AND J. H. HANN1GAN. Deficits on a spatial navigation task following prenatal exposure to ethanol. NEUROTOXICOL TERATOL 9(3) 253-258, 1987.--Performance on a Morris water task was examined in young rats whose mothers consumed a liquid diet consisting of 35% ethanol-derived calories (EDC) during pregnancy. Offspring of pair-fed (0% EDC) and ad lib lab chow (LC) dams served as controls. Rats were required to find a platform submerged below the surface in a pool of opaque water. A trial ended when the rat remained on the platform for 15 sec, or had been in the tank for 180 sec without reaching the platform. Subjects received 5 trials daily for 3 consecutive days, followed by reversal training on Day 4. Groups did not differ in swimming ability. On Day 1 there were no group differences among females in latency to reach the platform or in distance traveled, but male 35% EDC and 0% EDC animals had shorter latencies than LC controls. On Day 2, latencies and distance traveled of LC and 0% EDC controls decreased while 35% EDC animals showed no change from Day 1, so that alcohol-exposed rats took longer to reach the platform and traveled a greater distance than controls. On Day 3, 35% EDC females took longer than controls to reach the platform, and 35% EDC animals of both sexes traveled a greater distance than controls. Search patterns on the first reversal trial on Day 4 suggest the differences are in spatial processing and not learning per se, but more so in alcohol-exposed males than females. The impaired performance on this task suggests that prenatal alcohol exposure alters the ability to process spatial information. Fetal ethanol exposure Morris maze Hippocampal dysfunction Rats

Morris water-tank task

CHRONIC maternal alcohol consumption during pregnancy produces a pattern of anomalies in the offspring, including pre- and postnatal growth deficits, craniofacial defects and central nervous system dysfunctions, which has been termed Fetal Alcohol Syndrome (FAS; [2, 3, 19, 21]). Offspring exposed to lower levels of alcohol during gestation often do not exhibit the morphological defects associated with FAS, but are nonetheless at risk for functional deficits such as hyperactivity and mental retardation [34]. Morphological and functional abnormalities similar to those observed in humans have been reported in laboratory animals following prenatal alcohol exposure [3, 34]. Among the behavioral alterations observed in rats exposed prenatally to alcohol are overactivity [10,33], deficits in passive [22,30] and active [ 1,11] avoidance, and in taste aversion and appetitive odor conditioning [7,28]. The neural changes underlying the behavioral deficits following fetal alcohol exposure are unclear. Neuroanatomical alterations in hippocampal pyramidal cell number and arborization [4,5] and mossy fiber branching [37] have been reported. A functional importance of these changes in hippocampal structure is suggested by the fact that animals exposed prenatally to alcohol display a number of behavioral similarities to animals with hippocampal lesions. For exam-

Spatial navigation

pie, both ethanol-exposed and hippocampal-lesioned animals show increased activity, exploration and reactivity in an open field, deficits in spontaneous alternation and poorer performance than controls on passive avoidance tasks [29]. On other tasks, however, alcohol-exposed and hippocampal-lesioned animals perform quite differently. For example, adult rats exposed prenatally to alcohol display an increased grooming response to stress under some conditions [17], while animals with hippocampal lesions show decreased grooming relative to control animals [27]. Alcoholexposed animals perform more poorly than non-exposed animals on a two-way avoidance task, while hippocampal lesioned animals perform better than controls [29]. While there are several factors which may explain the differences between the behavioral effects of fetal alcohol exposure and of hippocampal lesions (e.g., different manifestations of CNS damage or processes of recovery of function), the use of tasks sensitive to hippocampal dysfunction could clarify hippocampal involvement in the effects of prenatal exposure to alcohol. The hippocampus plays an important role in spatial learning and memory [25,26]. Tasks requiring the use of spatial information are sensitive to hippocampal dysfunction. One task that has been used successfully to examine the role of

1Requests for reprints should be addressed to John H. Hannigan, Ph.D.

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the hippocampus in spatial performance is the Morris water task [23]. Normal rats placed into a pool of opaque water learn quickly to escape by locating and climbing onto a platform hidden just below the surface of the water. Rats with hippocampal damage take longer to learn to reach the platform and travel more circuitous routes. This deficit is not due to motor impairments [24] nor is it due entirely to differences in sensory abilities [25], suggesting disruption of central spatial processing per se [25]. If some of the behavioral deficits observed in fetal alcohol-exposed animals are due to hippocampal dysfunction, then those animals should show deficits on tasks which are sensitive to hippocampal dysfunction. The present study examined the ability of young (22-26-day-old) rats to learn and recall the position of a platform in a modified Morris task. We hypothesized that prenatal alcohol-exposed offspring would perform more poorly than controls on this task. Specifically, we predicted that alcohol-exposed rats would require more trials to learn the response, exhibit longer latencies to escape to the platform, and travel a more circuitous path to reach the platform than non-exposed control animals. In addition, since both hippocampal-lesioned and fetal alcohol-exposed animals have been characterized as displaying a basic deficit in response inhibition [29], we examined specific aspects of performance in the Morris task as indices of impaired response inhibition. We hypothesized that the impaired ability of alcohol-exposed rats to inhibit responding would interfere with learning to escape the water tank. Specifically, we predicted that the alcohol-exposed rats would be more likely to j u m p off the escape platform within the time period the animals are required to wait on the platform before removal from the water tank. METHOD

The procedures used to generate alcohol-exposed offspring have been detailed elsewhere [31]. Briefly, male and female Long Evans hooded rats (Blue Spruce Farms, Altamont, NY) were mated overnight and pregnant dams were housed individually in an separate nursery and assigned to one of three prenatal treatment groups. One group received free access to a liquid diet consisting of 35% ethanol-derived calories (EDC) on Days 6 through 20 of pregnancy. A second group was pair-fed a liquid diet with sucrose substituted isocalorically for ethanol (0% EDC) and served as a yoked nutritional control for any effects of the liquid diet. A third group (LC) received ad lib lab chow and water throughout pregnancy. The liquid diets consisted of water, chocolate Sustacal (Mead Johnson, Inc.), Vitamin Diet Fortification Mixture and Salt Mixture XIV (ICN Nutritional Biochemicals) with either 95% ethanol or sucrose added. The liquid diets provided approximately 1.3 kcal/ml and were the sole source of nutrition during the period they were administered. Just prior to expected parturition cages were inspected twice daily for births. Pups were weighed on the day following birth, inspected for physical anomalies and the litters culled randomly to 10 pups, maintaining an equal number of males and females whenever possible. Pups remained with their dams until weaning at 21 days of age when they were housed individually and maintained on a 12-hr light-dark cycle with free access to food and water throughout testing.

Behavioral Testing Testing began at 22 days of age for male and female rats from each prenatal treatment group ( n ' s = 5 - 9 animals per

cell). No more than one animal from any litter was assigned to any given cell of the design to randomize possible litter effects. An experimenter remained in the testing room (2225°C) during testing to handle the animal. Another experimenter in an adjacent room observed the animal via a remote video system and recorded its behavior with an event recorder (S&K Electronics, Toronto, Canada) and by tracing the animal's path on a sheet of acetate placed over the image of the tank on a video monitor. The experimenters were blind to the prenatal history and sex of the animals. On Day 1 each animal was placed into a circular (55 cm dia.) galvanized tub (30 cm in height) filled with 10-cm-deep water (25-26°C) made opaque with 1 I milk. The animal was allowed to swim for 2 min then removed from the water and returned to a holding cage for 5 min at which time training trials began. The tank was divided into four quadrants (unmarked to rat's view) and a small platform (6.5 cm dia.) was placed into one quadrant (the " g o a l " quadrant) 1.5 cm below the surface of the water. The animal was placed, facing outward, into one of the other three quadrants (the " s t a r t " quadrant) and allowed to swim until it had found the platform or until a 180 sec ceiling was reached. An animal successfully escaped when it remained on the platform for 15 sec. The rat was removed from the tank then, or when 180 sec had elapsed, and was placed into a holding cage for a 1-min intertrial interval at which time the next trial was begun from a different " s t a r t " quadrant. Each animal received 5 trials daily for three days. The platform was placed into a different " g o a l " quadrant for different animals, and remained the same for each subject for its first 15 trials (Days 1-3). The sequence of varying the " s t a r t " quadrant over successive trials was such that each day began with a different " s t a r t " quadrant, no animal was placed into the same quadrant on two consecutive trials, and over 15 trials each animal started from each of three " s t a r t " quadrants 5 times. The use of particular sequences was balanced across groups. On the last trial of the third test day (Trial 15) the animal was not removed from the platform after 15 sec. The latency (up to 180 sec) to step off the platform was recorded to test the hypothesis that alcohol-exposed rats suffer from deficits in response inhibition, also a characteristic of animals with hippocampal damage [29]. On the fourth day, the platform was moved to the quadrant opposite the original " g o a l " quadrant for each animal, and the rat was given 5 trials (Trials 16 through 20) starting in the quadrant that had been used for the " g o a l " on the first 15 trials. The proportion of distance traveled in the original goal quandrant was noted [35]. On Day 5, the animal started in the center of the tank and was allowed to swim freely for a single 2-min trial (Trial 21) with the platform removed. The manipulations on Days 4 and 5 were designed to further challenge the animal's ability to navigate in space. Following the last trial each day, the rat was damped dry with a paper towel and placed into a warm (40°C) cage for 10-15 rain before being returned to its home cage in the colony. The dependent measures were latency to begin swimming, latency to reach the platform, number of trials on which an animal stepped off the platform before 15 sec elapsed, distance traveled before reaching the platform, latency to step off the platform on trial 15, the occurrence of any component of the grooming sequence (e.g., paw licking, face washing, flank licking or genital grooming) while remaining on the platform on Trial 15, and proportion of distance traveled in each quadrant on the final trial in which the platform was removed.

SPATIAL NAVIGATION DEFICITS AND ETHANOL

255

22 Days Old 2.0

Males

F~mPles 1.6

1.2

"~ 0.8 ÷l

LC

== 0.4

0% EDC

o 0

LC

35% EDC •

Days

FIG. 1. Mean latency to find platform on each day of training (5 trials/day) for females and males from all three prenatal treatment groups. LC controls are indicated by circles (n's=8 and 7 for females and males, respectively), 0% EDC animals by squares (n's=6 and 5 for females and males), and 35% EDC animals by closed triangles (n's=9 and 8 for females and males).

RESULTS

Dam and Litter Characteristics The ~ubjects in this experiment were chosen randomly from a larger pool of animals for which dam and litter characteristics are reported. Mothers in the 35% EDC group consumed 12.76_+0.24 g/kg ethanol daily during the period of ethanol administration. Analysis of variance followed by Fisher's least significant difference (LSD) comparisons (0 <0.05) indicated that dams in the 35% and 0% EDC groups gained significantly less weight (29.5% and 30.1%, respectively) than LC dams (38.2%) over the course of pregnancy, F(2,96)= 10.76, p <0.0005 for overall Treatment effect. There were no significant effects of Treatment on gestation length or litter size. Pup data were analyzed similarly with prenatal Treatment and Sex as factors (3x2). There was a significant effect of Treatment on pup body weight, F(2,192)=35.77, p<0.0001 due to 35% EDC pups weighing less than 0% EDC pups, who were in turn lighter than LC pups. There were no group differences in sex ratio. Body weights at testing were analyzed by a 3 × 2 x 5 mixed design analysis of variance, with Treatment and Sex as crossed factors and Day as a repeated factor. Analysis of variance followed by comparisons using Fisher's LSD test (0<0.05) indicated that 35% EDC animals weighed significantly less than LC animals, F(2,37)=7.49, p<0.005 for overall Treatment effect. LC and 0% EDC animals did not differ from each other.

Behavioral Testing Day 1-3. Prior to examining latency and distance traveled in finding the platform, it was necessary to determine if the integrity o f the swimming response was altered by fetal alcohol exposure. There were no significant group differences in mean swim velocity (cm/sec) over all trials, suggesting that swimming ability was not disrupted in alcohol-exposed animals. Initial latency to begin swimming was analyzed also by mixed analysis of variance ( 3 × 2 x 3 x 5 ) with Treatment and Sex as crossed factors and Day and Trial as repeated

35% EDC

FIG. 2. Examples of swim patterns of LC and 35% EDC animals. Represented are the trials on Day 2 on which the distance traveled by the animal depicted was closest to the Day 2 mean for each group.

factors. There was a significant effect of Treatment on latency to begin swimming, F(2,37)=6.16, p<0.005, which LSD comparisons (0<0.05) indicated was due to alcoholexposed animals floating for a longer period of time than LC and ~ e EDC controls on any trial. However, the magnitude of this mean difference was relatively small (approximately 0.4 sec on any trial) and did not account for differences in latency to find the platform (see below). The mean latency scores are presented in Fig. 1. Latency data were normalized by a log transformation and analyzed as described for initial float duration. Analysis revealed a significant Treatment x Sex × Day interaction, F(4,74)=2.66, p<0.05. LSD tests (0<0.05) indicated that among females there were no group differences in mean latency (collapsed across Trials) on Day 1, but on Days 2 and 3 alcohol-exposed females exhibited significantly longer mean latencies to reach the platform than LC and 0% EDC females. Thirty-five percent and 0% EDC males had significantly shorter latencies than LC males on Day 1, but on Day 2, 35% EDC males had longer latencies than LC controls. By Day 3, 35% EDC males performed at control levels. LC and 0% EDC males did not differ from each other on Days 2 and 3. There was also a significant Treatment x Day × Trial interaction, F(16,296)=1.73, p<0.05, which LSD comparisons (0 <0.05) indicated was due to 35% EDC animals having shorter latencies than LC controls on Trial 2 (Day 1) only, but higher latencies than LC and 0% EDC controls on several different trials on Days 2 and 3. Distance scores were normalized by a log transformation and analyzed as described for initial float duration. Overall analysis revealed a significant Treatment x Day interaction, F(4,296)=2.82, p<0.05. Further LSD comparisons (0<0.05) indicated that while LC and 0% EDC animals traveled a significantly shorter distance on Day 2 than on Day 1, 35% EDC animals showed no decrease on Day 2. In addition, 35% EDC animals traveled a greater mean distance (collapsed across Trials) than controls on Day 3. There was also a significant Treatment x Day x Trial interaction, F(16,296)=1.84, p<0.05). LSD comparisons (0<0.05) indicated that 35% EDC animals traveled further than controls on several trials on Day 2. Figure 2 depicts the search patterns for two animals representative of the LC and 35% EDC animals on Day 2. Number of failures to reach the platform, step down latency on Trial 15, number of " j u m p offs" and proportion of total time spent floating were each analyzed by 3 x 2

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analysis o f variance (Treatment x Sex). There were no group differences on any of these measures. However, a significantly greater percentage of 35% EDC animals (64.7%) than of controls (20.0 and 27.3% for L C ' s and 0% E D C ' s , respectively) groomed while remaining on the platform for the step down test on Trial 15, X2(2)=7.92, p<0.025. Day 4 (reversal and probe). Latency (log sec) to find the platform, distance traveled (log cm) and initial float duration were each analyzed by mixed analysis of variance (3 x 2 x 5), with Treatment and Sex as crossed factors and Trial as a repeated factor. The only significant group difference was for initial float duration, F(2,37)=4.06, p<0.05, which LSD comparisons (p<0.05) indicated was due to 35% EDC animals floating for a longer period of time upon being placed into the water for each trial. This was a replication of the findings on Days 1-3. The use of spatial strategies was probed by measuring the distribution of searching on Trial 16. Since the platform was present in the quadrant opposite the original goal, the analysis of the distribution of distance traveled among the three remaining quadrants is presented, although the same pattern o f results was obtained when all quadrants were analysed. The proportion of distance traveled in the original goal quandrant was compared to chance performance (0.33) for each group using independent one-sample t-tests (p<0.05). A significantly greater proportion of distance (ranging from 0.46 to 0.59) was traveled in the original goal quadrant by LC and 0% EDC animals (males and females), as well as 35% EDC females, than would be expected if there were no preference for that quadrant, t's(4-8)=2.83 to 4.17. However, 35% EDC males showed no preference for the original goal quadrant, not differing significantly from chance. Day 5 (no platform). On Day 5 the dependent variables measured were distance traveled, proportion of distance traveled in the original goal quadrant and the proportion traveled in the reversed goal quadrant. Data were analyzed with Treatment and Sex as factors and there were no group differences for any of the dependent measures. DISCUSSION

The results show rats exposed prenatally to alcohol do not learn to locate a submerged platform in a Morris-type water-tank task as quickly as control rats. Day I performance was similar for all groups of females, but among males 0% and 35% EDC animals had shorter latencies than LC animals. Controls showed improved performance on each successive day. Alcohol-exposed males and females did not improve their performance between Days 1 and 2 and took longer than control animals to reach the platform on Day 2. By Day 3, 35% EDC males had slightly higher latencies than control animals, but the difference did not reach statistical signficance. However, 35% EDC females continued to take longer than controls to reach the platform. Alcoholexposed animals traveled a greater distance than controls on Days 2 and 3. The slight differences between the information on group effects provided by the latency measures and the distance socres may be due to the manner in which distance traveled was scored. On the few trials where an animal traveled a relatively great distance before reaching the platform (e.g., over 15 m) there were a number of overlapping lines on the tracing of the animal's path which made scoring difficult. While every effort was made to verify the accuracy o f these

scores, we believed it prudent in these rare circumstances to consider latency a more reliable reflection of performance than distance. The increased latency of 35% EDC animals to reach the platform did not appear to be due to differences in the quality of swimming itself since there were no group differences in swim speed. Although 35% EDC animals floated significantly longer at the beginning of the trials, the difference was not large enough to explain the differences in latency to find the platform. We do not believe the effect observed in alcohol-exposed animals was due to prenatal malnutrition, since pair-fed controls (0% EDC rats) did not show a similar deficit. Also, it is important to note that the deficits seen in the fetal alcohol-exposed rats are not likely to be general deficits in learning. This is indicated by: (1) the presence of no group differences in the number of failures to reach the platform; and (2) the ability of all prenatal groups to learn to wait on the platform 15 sec to be removed from the water tank. The results suggest an impairment in processing spatial information in rats exposed prenatally to alcohol, implicating involvement of the hippocampus in this aspect of the effects of fetal alcohol exposure [24--26]. These findings are consistent with reports that fetal alcohol exposure disrupts hippocampal structure and function [4, 5, 12, 29, 37] since a number of studies have demonstrated the importance of the hippocampus in spatial navigation tasks (e.g., [24,25]). Similar deficits in a Morris task were reported in young (24 days) rats given hippocampal lesions within 48 hours after birth [13]. However, we caution that there may be additional CNS involvement in the effects of fetal alcohol exposure on this task (e.g., [20]). The current latency results show also that spatial information processing may be slightly more affected by prenatal alcohol exposure in female than in male offspring. Yet in examining the first trial on Day 4 (Trial 16, Reversal), LC and 0% EDC rats of both sexes showed a greater preference for the quadrant in which the platform had been located than would be expected by chance alone. Fetal alcohol-exposed males, on the other hand, despite showing latencies near those of controls, were no more likely to search the goal quadrant than would be expected by chance alone. Alcoholexposed females behaved like controls in this " p r o b e " of spatial preference. Sex differences in susceptibility to fetal alcohol effects have been reported previously (e.g., [6, 8, 9]), although, specific to the dependent variables examined, either males or females have been reported to be more susceptible. Fetal alcohol exposure may delay maturation of systems involved in spatial learning and memory. Dyck et al. [13] reported that the performance of normal animals on a Morris task was indistinguishable from that of neonatally hippocampal-lesioned animals until around postnatal day 24, when control animals began to perform quite well. In the present study, differences in performance became apparent on Day 2, when subjects were 23 days of age. At that time, the ability of controls to locate the platform began to improve, while 35% EDC animals continued to perform at Day 1 levels. The delayed improvement in performance of 35% EDC animals may reflect an ethanol-induced delay in hippocampal development rather than a long-lasting deficit in spatial abilities per se. Delays have been reported in the development of mouse hippocampal pyramidal cells following perinatal ethanol expsoure [12]. We should note in passing that we had examined the performance of rats beginning at 23 days of age and found no

SPATIAL N A V I G A T I O N DEFICITS AND E T H A N O L significant differences in their performance from that of the 22-day-old animals reported here suggesting that our ability to resolve maturational differences following fetal alcohol exposure may not be great for this task. This would be more consistent with the results of Schenk [32] who reports a much later development of spatial navigational abilities. In fact, the performance of controls animals in the present study did not reach the levels of performance typically seen in adult animals on this task (e.g., [23]). While this may have been due to restricted access to visual cues, since the water level was well below the rim of the tank, it may be that such young animals simply do not display adult levels of performance. One alternative explanation for the observed effect is that the poorer performance of alcohol-exposed rats may be due to differential responsiveness to the stress of the test situation. An apparent deficit on a Morris task in malnourished rats was eliminated when extensive experience with swimming preceded training, suggesting that a stress-induced change in performance was an important factor [16]. Since alcohol-exposed rats show increased responsiveness on a number of measures to a variety of stressors, including swimming (e.g., [17,36]), deficits in the present experiment may reflect altered reactions to stress rather than impaired spatial information processing. Our data on grooming in the present study support this since grooming in rats increases in response to stress [14,18]. The greater percentage of 35% EDC animals grooming while on the platform on Trail 15 suggests-that more alcohol-exposed animals than controls found the situation to be stressful.

257 Finally, the present results do not support the hypothesis that fetal alcohol-exposed rats suffer from a response inhibition deficit since very few animals at all left the platform during the 15-sec wait to be removed from the tank. In hindsight it may be that leaving a safe platform to reenter water which rats find aversive might not be indicative of a response inhibition deficit. In summary, rats exposed prenatally to alcohol may have impaired spatial abilities, such that a somewhat longer training period is required for alcohol-exposed animals to learn the task. We believe these data are consistent with the hypothesis that some of the behavioral deficits associated with prenatal alcohol exposure are related to hippocampal dysfunction. Interestingly, similar deficits on this task were found recently using a postnatal alcohol exposure regime which has also been shown to result in hippocampal abnormalities ([ 15]; personal communication from C. R. Goodlett). Prenatal alcohol exposure may delay maturation of structures involved in spatial learning. Alternatively, alcohol exposure in utero may alter learning ability in stressful situations. Further examination of fetal alcohol-exposed animals at different stages of development and under different conditions of stress and cue-availability in a Morris task will help to clarify the nature of the deficit, and the extent of hippocampal involvement in the dysfunction. ACKNOWLEDGEMENTS This work was supported in part by NIAAA grants No. 00077 to E.P.R. and No. 06721 to J.H.H.

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