Effects of prenatal ethanol exposure on hippocampal theta activity in the rat

Alcohol, Vol. 14, No. 3, pp. 231-235, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/97 $17.00 + .00

PII S0741-8329(96)00147-4

ELSEVIER

Effects of Prenatal Ethanol Exposure on Hippocampal Theta Activity in the Rat BERNADEq-TE

M. C O R T E S E , S C O T T E. K R A H L , R O B E R T A N D J O H N H. H A N N I G A N

F. B E R M A N

C. S. Mott Center for Human Growth & Development, Departments of Obstetrics and Gynecology, and Psychology, Wayne State University, Detroit, M I 48201 R e c e i v e d 6 July 1996; A c c e p t e d 26 July 1996 CORTESE, B. M., S. E. KRAHL, R. F. BERMAN AND J. H. HANNIGAN. Effects of prenatal ethanol exposure on hippocampal theta activityin the rat. ALCOHOL 14(3) 231-235, 1997. This study examined the effects of prenatal ethanol exposure on hippocampal theta activity in adult rats. Subjects were randomly selected from four prenatal treatment conditions: untreated, 0, 3, or 5 g/kg/day ethanol. At approximately 90 days of age, all subjects were surgically implanted with a bipolar electrode in the CA1 region of the hippocampus. Four epochs of hippocampal theta rhythm activity were recorded while the subjects were moving and four more while still, and a normalized theta score was computed and compared among groups. The 5 g/kg male group demonstrated a significantly higher theta score than controls, indicating either an increase in type I (movement-associated) theta and/or a decrease in type II (information-processing) theta activity. These results are consistent with prior reports that prenatal ethanol exposure alters hippocampal function and support clinical indications that monitoring the EEG of children may prove to be useful in the diagnosis of fetal alcohol syndrome and/or the detection of alcohol-related birth defects. ©1997 Elsevier Science Inc. Alcohol

EEG

FAS

Fetal alcohol syndrome

Hippocampus

FOR an ethanol-affected newborn to be diagnosed with full fetal alcohol syndrome (FAS), the following must be met: 1) growth retardation below the 10th percentile in height, weight, and/or length after correction for gestational age; 2) characteristic facial dysmorphology, typically reported as short palpebral fissures (i.e., small eye slits), epicanthic folds, hypoplastic midface, indistinct philtrum, and/or thin vermilion; and 3) central nervous system (CNS) involvement, such as neurological and/or structural abnormalities, or behavioral dysfunction that may include hyperactivity, attention deficits, and mental retardation (1,19,36). Because of intrinsic individual differences of each mother and infant, differences in the amount or pattern of ethanol intake, and the presence or absence of various risk factors such as poverty or use of other drugs of abuse (2), newborns born to frankly alcoholic mothers do not always meet the criteria for an FAS diagnosis. The terms fetal alcohol effects and alcohol-related birth defects (ARBDs) are used to describe cases where a newborn exhibits only some of the characteristics of FAS (36). There may also be difficulties in the diagnosis of ARBDs because subtle CNS effects may not be readily evident through physical or neurological examinations shortly after

Theta rhythm

birth (19,21). Because the effects of prenatal ethanol exposure can include a range of subtle emotional or cognitive disabilities, it would be useful to develop techniques for assessing CNS dysfunction associated with both FAS and ARBDs that are similar in objectivity to the criteria for growth retardation and facial dysmorphology. For example, Rintelman et al. (34) reported that auditory evoked potentials in day-old human infants are sensitive to delays in CNS development, although this test may not be specific to the effects of ethanol. The electroencephalogram (EEG), long used to study brain and behavioral dysfunction, may be a useful diagnostic tool to detect FAS and ARBDs (4). Ioffe et al. (22) and Chernick et al. (10), for example, reported hypersynchrony in the cortical EEGs of newborns born to alcoholic mothers as a specific effect of prenatal ethanol exposure. Researchers have also demonstrated differences in the EEGs of children with emotional and behavioral problems, including children diagnosed with attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD) (3,26,27). Typically, overall cortical E E G records of A D D children are described as abnormal or immature (27). Other reports have shown spe-

Requests for reprints should be addressed to John H. Hannigan, Ph.D., C. S. Mott Center for Human Growth & Development, Wayne State University, 275 East Hancock Avenue, Detroit, MI 48201. Tel: (313) 577-8671;Fax: (313) 577-8554. 231

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cific increases in theta or rhythmic slow wave (RSA) activity in cortical E E G s of A D D children (26). The connection between attention and theta rhythms stems from clinical and animal research, suggesting that all mammalian species exhibit high levels of hippocampal theta during information-processing behaviors, such as exploration of a novel environment and the orientative reflex elicited by a novel stimulus (31). According to Bland (7), hippocampal theta activity in rats has been linked to both movement during voluntary activities and to processing of sensory information independent of movement. Because the behavioral effects of fetal ethanol exposure include both hyperactivity and perhaps attentional deficits in both people (1,30) and rats (39), it is possible that locomotor activity and information processing deficits produced by prenatal ethanol exposure may be detectible as differences in E E G pattern, and specifically in hippocampal theta activity in rats. E E G recordings taken from children diagnosed with F A S have been reported to be moderately abnormal, with a dominant theta pattern (28). This same group reported that prenatal ethanol exposure altered E E G in humans and hippocampal E E G in laboratory animals, and suggested that EEG, along with more traditional measures, may serve as a biological marker for prenatal ethanol exposure and aid in the diagnosis of F A S / A R B D s (28). Few labs have examined the effects of prenatal ethanol exposure on hippocampal electrophysiological activity in awake animals [cf. (4)]. In one such study, rats exposed to ethanol in utero demonstrated longer P1 and N1 latencies when auditory event-related potentials were measured in the hippocampus, whereas latencies were unaffected in the amygdala and cortex (23). To date, we are aware of no study examining the effects of prenatal ethanol exposure on hippocampal theta activity in an experimental m9del of FAS/ARBDs. The present study examines these effects in adult rats. Given that hyperactivity and attentional deficits are commonly associated with FAS/ ARBDs, we hypothesized that relative hippocampal theta activity would be greater in rats prenatally exposed to ethanol as compared to controls. METHOD

Breeding

Male and female Long-Evans hooded rats (Charles River Labs, Portage, MI) were housed singly and maintained on a 12 h/12 h light/dark schedule with ad lib access to rat chow and water, except as noted below. During breeding, one female rat was placed overnight into a male's cage. The presence of a vaginal plug the next morning was taken as evidence of fertilization, indicating gestational day 0. On gestational day 8, the dams were assigned randomly to one of four treatment groups. The first three groups received either 0, 3, or 5 g/ kg ethanol intubations once daily on gestational days 8-20. The 0 and 3 g/kg groups were pair-fed to the 5 g/kg group to control for nonspecific nutritional effects. Another untreated control group was given ad lib access to food and water throughout the gestational period. After birth, the pups were counted, weighed, and culled to a litter size of 10, keeping equal numbers of males and females when possible. On postnatal day (PD) 21, the pups were weaned, weighed, and housed in same-sex groups of two to three rats per plastic cage. Surgery

At about PD 90, one male and one female rat from each litter were anesthetized (60 mg/kg pentobarbital; IP), the head

shaved, and the animal placed into a Kopf stereotaxic device. Using aseptic techniques, a midline scalp incision was made, the periosteum deflected, and holes drilled through the skull, including one overlying the CA1 region of hippocampus (4.3 mm posterior to bregma and 2.5 mm to the right of the midline). Small screws were threaded into three holes. The dura was reflected and the tips of two twisted wires from a Tefloncoated stainless steel tripolar electrode (Plastics One, Roanoke, VA) were lowered into hippocampus (3.5 mm below dura). The third wire of the electrode was soldered to a skull screw over the cerebellum to serve as a ground. The entire assembly was then anchored to the skull with dental acrylic. The animals were singly housed and allowed to recover for at least 1 week. All procedures were approved by the University's IACUC. Electrophysiological Procedures

Between 1000 and 1300 h, rats were individually placed into a 25 x 25 cm acrylic chamber. The lead from a Grass E E G amplifier (Quincy, MA; model 7E) was attached to the tripolar electrode assembly. Output from the amplifier, halfamplitude filtered below 1 Hz and above 35 Hz, was saved by computer using an RC Electronics A/D converter and software (Santa Barbara, CA). Immediately after being placed in the novel test chamber, four 10-s epochs of hippocampal E E G activity were collected while the animal was engaged in locomotion, and four 10-s epochs were collected while the animal was still. The experimenter was blind to the animals prenatal histories. Estrous cycle was not measured in the females. After the data collection was complete, animals were sacrificed with an overdose of pentobarbital (200 mg/kg), and the brains removed and placed in a 10% sucrose/formalin solution. The brains were later sectioned, stained with cresyl violet, and the location of the recording electrode determined. Only those animals whose electrode tips were within CA1 were used in the subsequent data analyses. Data Analysis

The eight epochs collected from each rat were subjected to spectral analysis. Because spectral power can be influenced by a number of factors, including differences in the position of the electrode and signal amplification, raw spectral power was not compared directly among animals (15). A normalized theta score was computed for each animal according to a method derived from Vanderwolf and Baker (40). The spectral power for the 5-10 Hz (theta) range was averaged for the four moving and four still epochs for each animal, and a ratio of the spectral power in theta range to the spectral power of the full range for those epochs was computed. The percent increase in the relative theta power during movement, as compared to the relative theta power while the animal was still, was then used as the theta (0) score. Specifically, OM = Os =

mean 5-10 Hz power during movement mean full power during movement mean 5-10 Hz power while still mean full power while still OM - Os x 100

O scor e --

OS

The mean theta scores for the 0, 3, and 5 g/kg ethanol groups and the untreated control group were compared using a 4 (prenatal treatment) x 2 (gender) A N O V A (a < 0.05).

233

PRENATAL ETHANOL AND HIPPOCAMPAL THETA TABLE 1 EFFECTS OF PRENATALTREATMENT ON MATERNAL WEIGHT GAIN, LITTER SIZE, AND BIRTH WEIGHT Subject Gender

Prenatal Treatment

Female

Untreated 0 g/kg 3 g/kg 5 g/kg Untreated 0 g/kg 3 g/kg 5 g/kg

Male

N

Maternal WeightGain (% Increase-+SE)

Overall Litter Size (mean _+SE)

Birth Weight(g) (mean -+ SE)

8 7 9 7 7 5 8 6

37.2 -4-3.4 40.7 -+ 5.4 29.8 -+ 2.4 32.6 _+2.1 44.8 --_4.2 42.5 --- 4.4 36.3 _+3.0 34.2 _+3.9

12.5 _+ 1.7 12.6 + 1.3 11.8 _+1.2 11.7 4- 1.2 13.3 _+1.6 13.6 _+0.7 13.4 _ 1.4 13.0 ___2.1

6.0 - 0.2 5.6 + 0.4 6.1 -+ 0.2 6.0 _+0.2 6.3 +_0.2 6.1 - 0.2 6.4 -4-0.2 6.7 -+ 0.2

RESULTS Maternal and Litter Data Maternal weight gain was not significantly altered during gestation as a result of the different ethanol doses and diets. Similarly, no significant birth weight or litter size differences were detected among the prenatal treatment groups for either male or female offspring (see Table 1). Theta Scores Significant main effects on the theta scores were found for both gender, F(1, 49) = 4.94, p < 0.05, and prenatal ethanol exposure, F(3, 49) = 3.41, p < 0.05. Post hoc comparisons indicated that the 5 g/kg ethanol male group had a significantly higher theta score than both the untreated and 0 g/kg male control groups. The 3 g/kg ethanol male group did not differ significantly from the 5 g/kg male group, nor were there any significant differences among the untreated, 0 g/kg, or 3 g/kg male groups. No significant differences were found among any of the female groups (see Fig. 1). DISCUSSION

Prenatal ethanol exposure altered hippocampal theta rhythm in male rat offspriag. A significant main effect of gender on the hippocampal theta score was demonstrated when the data were collapsed across ethanol groups. Specifically, the male 5 g/kg ethanol group alone showed a significantly higher theta score than either control group. Because there were no significant differences between males and females in the untreated and 0 g/kg control groups, the gender effect was attributed to the differential effects of ethanol on male offspring. Gender differences such as these among animals exposed prenatally to ethanol have been reported in behavioral (41,44), neuroimmunological (42), psychopharmacological (18), and neurochemical (6) studies. In the hippocampus, two types of theta activity have been described [for a review, see (7)]. Type I theta has been linked to movement because of its expression during voluntary motor activities, including walking, running, and rearing. Type II theta is associated with information processing of sensory stimulation, independent of movement. Because the theta score is a ratio of theta activity collected while the animal is moving to that collected while the animal is still, the results of the present study suggest that there may be either more type I theta, and/or less type II theta activity in the 5 g/kg male group compared to controls. As a result, the higher theta

score demonstrated by the 5 g/kg male group may be interpreted as reflecting an increase in locomotor activity and/or reflecting a decrease in information processing or cognitive activity, relative to controls. A review of the literature supports the presence of motor activity, cognitive and attentional problems in rats and children that have been exposed prenatally to ethanol. For example, among the most commonly reported effects of ethanol exposure in utero is an increase in offspring activity levels (29). These findings are demonstrated in rats and other laboratory animals in the open field maze, running wheels, and exploratory chambers (12,33,37). Along with hyperactivity, human studies have found distractibility, impulsiveness, and short attention spans among the cognitive and behavioral problems of children with FAS (8,9,11,16,30,35,38). The present results, consistent with a report by Kaneko et al. (24) assessing hippocampal evoked responses, suggest that altered hippocampal electrophysiologicat function in rats may underlie some of these behavioral changes. There is a preliminary report that similar associations between abnormal cortical E E G and hyperactivity may also exist clinically in FAS children (23). If this holds, E E G may provide a useful diagnostic tool.

THETA SCORE

I

40

l l Untreated [ ] 0g/kg II

30 Mean (+SEa) 2o

10

n=

8

7

9

7

FEMALES

7

5

8

6

MALES

FIG. 1. Effects of prenatal treatment on normalized theta scores. The bars represent SE. *Significantlydifferent from untreated and 0 g/kg ethanol control groups, p < 0.05.

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O n e limitation of the present study is that the methods did not allow the unambiguous separation of type I and type II theta activity because movement-related theta activity includes both types. Separate analysis of type I and type II theta did not reveal consistent systematic changes in either due to prenatal alcohol exposure, although as we noted above, use of the normalized theta score is preferred to minimize the impact of large individual differences in raw spectral power (15,40). A separation of theta subtypes would allow us to determine the degree to which the higher theta score demonstrated by the 5 g/kg male group was caused by increased type I theta, decreased type II theta, or both. Type I and type II theta can be distinguished by their sensitivity to cholinergic agonists and antagonists, due to the septohippocampal cholinergic input that influences the generation of type II theta in the hippocampus (15). Type II, or atropinesensitive, theta can be induced or blocked by cholinergic agonists and antagonists, respectively, whereas type I, or atropine-resistant, theta is not affected by these. Because there are studies showing alterations in cholinergic function in rats as a result of prenatal ethanol exposure (13,14,32), the increased theta score in the 5 g/kg male ethanol group might be

attributed to a compromised cholinergic system in these animals and thus a decrease in type II theta. H o w e v e r , other studies have not found an effect of prenatal alcohol exposure on the cholinergic system of adult offspring (5,17,25,43). Because adult subjects were used in the present study, we hypothesize that the increased theta score in the 5g/kg male ethanol group is most likely due to an increase in type I theta. This issue remains to be resolved and we are currently assessing hippocampal theta activity in prenatal ethanol-exposed rats challenged with cholinergic agents. In conclusion, the present results indicate that fetal ethanol exposure alters hippocampal theta activity in adult male, but not female rats. A l t h o u g h the mechanism(s) of this effect have yet to be elucidated, the present results warrant further study of E E G in children with F A S / A R B D s . ACKNOWLEDGEMENTS The authors wish to thank Loraine Treas, John Hotra, Jennifer Hackett, and John DiCerbo for technical assistance, and Michael Kruger for statistical analysis. This work was supported by Fetal Alcohol Research Center (P50 AA07607), training (T32 AA07531), and research (R01 AA06721) grants from NIAAA.

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