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Effects of physostigmine on shuttle avoidance in rats exposed prenatally to ethanol
Alcohol, Vol. 5, pp. 27-31. ©PergamonJournals Ltd., 1988.Printed in the U.S.A.
0741-8329/88$3.00 + .00
Effects of Physostigmine on Shuttle Avoidance in Rats Exposed Prenatally to Ethanol B. A. B L A N C H A R D
A N D E. P. R I L E Y 1
Center f o r Behavioral Teratology and Department o f Psychology State University o f N e w York at Albany, Albany, N Y 12222 R e c e i v e d 6 M a y 1987; A c c e p t e d 3 A u g u s t 1987 BLANCHARD, B. A. AND E. P. RILEY. Effects of physostigmine on shuttle avoidance in rats exposed prenatally to ethanol. ALCOHOL $(1) 27-31, 1988.--Shuttle avoidance performance following pretreatment with physostigmine was assessed in 85- to 100-day-old rats whose mothers consumed a liquid diet consisting of 35% ethanol-derived calories (EDC) during pregnancy. Offspring of pair-fed (0% El)C) and ad lib lab chow (LC) dams served as controls. Animals received either 0, 0.1 or 0.2 mg/kg physostigmine sulfate prior to acquisition training in a shuttle avoidance apparatus. Training consisted of 50 trials/day for 4 days. Thirty-five percent EDC rats made fewer avoidances than controls during acquisition training. Treatment with physostigmine reduced the number of avoidances made, and did so similarly for all prenatal treatment groups. Escape latencies were not affected by prenatal treatment, although they were increased by physostigmine administration prior to training. Neither prenatal treatment nor physostigmine treatment affected activity as measured by the number of intertrial crossings while in the apparatus. These data indicate that alcohol-exposed animals did not respond differentially to physostigmine relative to controls, suggesting that cholinergic dysfunction may not underlie the prenatal alcohol-induced deficit in active avoidance. Prenatal alcohol exposure
Shuttle avoidance
Cholinergic
F E T A L Alcohol Sydrome is comprised of a number of characteristic morphological, physiological and neurobehavioral abnormalities resulting from chronic maternal alcohol con. sumption during pregnancy [4, 17, 19, 34]. While the full range of symptoms is primarily seen following high levels of prenatal alcohol exposure, behavioral deficits in the absence of morphological anomalies may be associated with lower levels of exposure [2, 4, 33, 34]. The effects reported in humans have been demonstrated in laboratory animals as well (for reviews, see [2, 4, 21, 34]), providing models with which to explore the mechanisms of alcohol damage to the developing organism. Behavioral deficits produced in rats by fetal ethanol exposure include hyperactivity in an open field [3, 8, 9, 32] and poorer performance on a number of learning tasks, including passive [20,29] and active [1, 10, 30, 32] avoidance, taste aversion [26] and appetitive odor conditioning [5]. A number of the behavioral deficits seen following fetal ethanol exposure are consistent with a response inhibition deficit hypothesis [3, 21, 28, 29]. Central cholinergic systems appear to be involved in response inhibition [11,12], and alcohol-exposed animals resemble animals treated with anticholinergic drugs on a number o f behaviors [21]. This suggests that prenatal alcohol exposure may result in alterations in central cholinergic systems. Neurochemical data provide some indirect support for this. Rawat [24] found decreased levels of acetylcholine in alcohol-exposed rat
Physostigmine
fetuses and neonates. However, there is a paucity of work examining acetylcholine levels at other stages of development following fetal alcohol exposure. Some investigators have attempted to clarify the functional significance of putative changes in cholinergic systems resulting from alcohol exposure in utero through the use of psychopharmacological techniques, and have reported altered responsiveness to the reversible acetylcholinesterase inhibitor physostigmine. Riley et al. [27] treated 25-day-old alcohol-exposed rats with physostigmine, and at the doses used (0.1 and 0.2 mg/kg), physostigmine decreased activity of alcohol-exposed animals while control animals showed no change. In contrast, Bond [6] found that activity in 22-dayold alcohol-exposed rats was unaffected following physostigmine treatment, while activity levels in controls decreased. Differential responsiveness to the cholinergic antagonist scopolamine has been observed in 22-day-old alcohol-exposed animals [7]. To extend these findings to another behavior and to a different stage of development, we examined acquisition of a shuttle avoidance task following treatment with physostigmine in adult rats exposed prenatally to alcohol. In normal animals, physostigmine administered just prior to acquisition of this task reduces the number of avoidances made [ 14,31]. If fetal alcohol-exposext animals are more responsive in adulthood to the behavioral effects of physostigmine, as they were at 25 days in the Riley et al. study [27], then those
1Requests for reprints should be addressed to Dr. E. P. Riley.
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B L A N C H A R I ) AND RILEY Females 0.1 mg/kg physostigmine
saline
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0.2 mg/kg physostigmine
LC © O%EDC o 35% EDC •
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saline
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physostigmine
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FIG. I. Mean number of avoidances made on each day of training by male and female rats from the three prenatal treatment groups for each dose of physostigmine (0.0.0.1 or 0.2). LC rats are represented with circles, 0% EDC animals with squares, and 35% EDC animals with closed triangles. animals should show a greater physostigmine-induced impairment than controls. An alternative outcome was also possible. The poorer performance on an active avoidance task by fetal alcoholexposed rats may reflect a disruption of attentional processes, which may be related to cholinergic function. For example, in normal animals, increasing cholinergic activity with small doses of physostigmine improves performance on some types o f discrimination tasks [15, 16, 18, 25], while blocking cholinergic receptors with atropine or scopolamine decreases performance [22, 23, 25]. If the alcohol-induced deficit on a signalled shuttle avoidance task is a consequence of an inability to attend to salient cues, then physostigrnine may actually improve performance of alcohol-exposed animals. METHOD Male and female Long Evans hooded rats (Blue Spruce Farms, Altamont, NY) were mated each night until a vaginal plug was detected (designated Day 1 of pregnancy). Pregnant animals were housed individually in clear breeding cages with ad lib lab chow and water available, and assigned randomly 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 o f pregnancy. Animals in a second group were matched individually to 35% EDC animals and pair-fed a liquid diet with sucrose substituted isocalorically for ethanol (0% EDC). This group served as a nutritional control for the effects of liquid diet. A third group 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.
Liquid diets provided approximately 1.3 kcal/ml and were the sole source of nutrition during the period they were administered. Animals were weighed every five days during pregnancy to monitor weight gain and maintain more precise control of pair-feeding, which was done on an intake volume/body weight basis. Just prior to the expected parturition date cages were inspected twice daily for births. Pups were weighed on the day following birth (Day 1), 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 pair-housed with same sex littermates and maintained on a 12-hr light-dark cycle with free access to food and water.
Behavioral Testing Male and female offspring from each prenatal treatment group were tested in squads of 4 animals, beginning at 85 to 100 days o f age and continuing for 4 days ( n ' s = 5 to 7 per cell). Only one animal per litter was included in any cell of the d©sign to control for possible litter effects. On Day 1, animals were weighed and injected with 0.1 or 0.2 mg/kg physostigmine sulfate (Sigma Chemical Co. No. E8625) or with an equivalent volume (t ml/k8) o f 0.9% saline vehicle immediately prior to testing. Testing was conducted in 4 shuttle boxes measuring 45.7x21.6×24.5 era. The $rid floor of the shuttle box consisted o f steel rods which measured 0.63 cm in diameter, placed 1.9 c m apart. Each box was comprised of two equal sized c o m p e a ~ t , z ~ s dli~ded by an aluminum wall with a centered opening (5.1x10.2 era) beginning at floor level through which the animals could pass. Lights (28 V) on the upper portion of the far wall in each
PHYSOSTIGMINE AND F E T A L A L C O H O L E X P O S U R E compartment and a tone (80 dB) served as the compound conditioned stimulus. Electrical shock (0.9 mA) generated from a Coulbourn Instruments Shock Generator (No. F1316) served as the unconditioned stimulus. Photocells (3.8 cm above floor level) monitored crossings between the two compartments. Each shuttle box was enclosed in a soundattenuating chamber which contained a ventilation fan and a house light. At the beginning of each session, the animal was placed into one side of the shuttle box. The animal received a 5-sec presentation of the CS. If it crossed over to the other side of the apparatus during this 5-sec period, shock was avoided. If it did not cross over, shock was administered and continued until the animal crossed over or 30 sec elapsed. Animals received 50 trials/day, and trials were separated by a 40-sec intertrial interval. After testing animals were returned to their home cages. Testing procedures were identical on Days 2 through 4. The dose of physostigmine each animal received daily remained the same over training. Dependent measures included number of avoidances per 10-trial block per day, mean escape latency on each day and number of intertrial crossings on each day. Testing was conducted between 1200 and 1600 hr. RESULTS
Dam and Litter Characteristics An ongoing breeding program in our laboratory generates animals from which several investigators select subjects. Dam and litter characteristics are reported for the larger group of animals from which the test subjects were randomly selected. Mothers in the 35% EDC group consumed an average of 13.44_+0.16 g/kg ethanol daily. Maternal data were analyzed by one-way analyses of variance, with Treatment as the factor. There were no differences in mean length of pregnancy. Analysis revealed a significant effect of Treatment on percent weight gain during pregnancy, F(2,90)= 11.54, p<0.0001. Comparisons with Fisher's Least Significant Difference (LSD) test (p<0.05) indicated that dams in the two liquid diet groups showed a significantly smaller percent weight gain over the course of pregnancy (34.40% and 33.11% for 0% and 35% EDC dams, respectively) than LC dams (39.85%). There were no group differences in litter size. Pup data were analyzed by two-way analyses of variance, with Prenatal Treatment and Sex as factors. There were no significant effects of Treatment on sex ratio. Analysis of pup body weight at birth indicated that Treatment was a significant source of variance, F(2,180) = 12.48, p <0.0001. Further LSD comparisons (p<0.05) indicated that LC pups weighed significantly more than both 0% EDC and 35% EDC pups, who did not differ from each other. There was also a significant effect of Sex on pup body weight, F(1,180)--5.45, p<0.05. Prenatal Treatment did not interact with Sex. Behavioral Testing Due to a computer interface problem that went undetected during the study, some data points were missing (approximately 2%). These missing data, which were distributed randomly, were estimated by a computer program using a regression technique. Because such a technique minimizes within-group variance, only those main effects and interactions for which the probability was less than 0.01 were examined. The number of avoidances per five 10-trial blocks on each
29 test day was analyzed by mixed analysis of variance ( 3 × 2 x 3 × 4 x 5 ) with Prenatal Treatment, Sex and Dose of physostigmine as between-group factors, and Day and Block as within-group factors. Significant interactions and main effects were examined further with Fisher's LSD comparisons (p <0.05 for all comparisons). Analysis revealed a significant Treatment × Day interaction, F(6,282)=5.07, p<0.0005. LSD comparisons indicated that although there were no group differences in performance on Day 1, 35% EDC animals made fewer avoidances than 0% EDC controls on Day 2, and fewer than both LC and 0% EDC animals on Days 3 and 4. In addition, LC and 0% EDC animals showed significant improvement over the previous day's performance on Days 2 and 3, while alcohol-exposed animals showed improvement only on Day 2, but not thereafter (see Fig. 1). The Treatment main effect was significant as well, F(2,94)=8.06, p<0.001, due to 35% EDC animals making fewer avoidances than LC and 0% EDC controls over the entire training period. LC and 0% EDC rats did not differ from each other. There was a significant Dose x Day interaction, F(6,282)=8.59, p<0.0001, which comparisons indicated was due to the fact that animals that received 0.1 or 0.2 mg/kg physostigmine prior to training made fewer avoidances than those that received saline on each day of training. In addition, on Day 3, animals that received 0.2 mg/kg made fewer avoidances than those that received 0.1 mg/kg physostigmine. At all other times there were no differences between the two doses of physostigmine. A significant main effect of Dose, F(2,94)=24.77, p<0.0001, was due to physostigminetreated animals making fewer avoidances than saline controls over the entire training period. Sex x Day was a significant source of variance, F(3,282)=8.29, p<0.0001. Comparisons indicated that on Day 1, females made fewer avoidances than males, but were not different on Day 2. On Days 3 and 4, females made more avoidances than males. Mean inverse escape latencies (speed) for each day of training were analyzed by mixed analysis of variance ( 3 x 2 × 3 x 4 ) , with Prenatal Treatment, Sex and Dose of physostigmine as between-group factors, and Day as a within-group factor. Sex x Day was a significant source of variance, F(3,282)=5.29, p<0.005. Further comparisons indicated that females had faster speeds than males on Days 3 and 4. In addition, physostigmine reduced speed in a dose-dependent fashion, as indicated by a significant main effect of Dose, F(2,94)= 11.04, p<0.0001, followed by LSD comparisons. The number of intertrial crossings on each day of training was analyzed as described for speed. With the exception of a day effect (means=12.2, 13.8, 16.0, and 19.5 for Days 1-4, respectively) there were no other significant main effects or interactions. This suggests that neither Prenatal Treatment nor Dose of physostigmine affected this measure of activity in the shuttle avoidance apparatus. Furthermore, only 8 animals consistently failed to show escape or avoidance behaviors and this was not affected by either prenatal treatment or physostigmine. Alcohol-exposed animals did not appear to be differentially affected by physostigmine, since Treatment did not interact with Dose on any dependent measure. Treatment also failed to interact with Sex. DISCUSSION
The results replicate earlier findings that prenatal alcohol
30
B L A N C H A R D AND RII~EY
exposure disrupts two-way active avoidance learning in adulthood [1, 10, 30]. Both males and females exposed prenatally to alcohol made fewer avoidances than controls regardless of dose of physostigmine. The poorer performance of animals treated with physostigmine prior to training also replicates earlier findings [14,31]. The deficit in performance following physostigmine does not appear to be due to motor impairment, since activity (as measured by intertrial crossings) was not affected by physostigmine. Fetal alcohol-exposed animals did not appear to respond differentially to physostigmine. Because differential responsiveness to physostigmine's effects on activity has been demonstrated in younger alcohol-exposed animals [6,27], the absence of a Treatment × Dose interaction in the present study could be due to some recovery of cholinergic function by 35% EDC animals. This is consistent with the findings of Rockwood and Riley [30] who demonstrated that adult fetal alcohol-exposed rats responded normally (i.e., similarly to controls) when treated with the anticholinergic drug scopolamine prior to training on an active avoidance task. We have also examined the effects of cholinergic drugs on activity in fetal alcohol-exposed adult rats, and have observed no differential responding to arecoline or to scopolamine (manuscript in preparation). Neurochemical data provide some indirect support for the recovery of cholinergic function hypothesis, although a systematic examination of the development of cholinergic systems following fetal alcohol exposure has not been made. Decreased levels of acetylcholine have been observed in rat fetuses and neonates whose mothers received alcohol during pregnancy [24]. However, Chan and Abel [13] examined cholinergic receptor binding in fetal alcohol-exposed adult rats and found no differences from controls. It is possible also that the failure to observe differential responsiveness to physostigmine by alcohol-exposed rats was due to the choice of dose range. Differences may have been detected had lower doses been used. The second, alternative prediction of physostigmineinduced enhancement of performance in alcohol-exposed offspring was not borne out. Several factors could account for this. The conditions under which physostigmine improves cue discrimination may be very specific, and may not exist in the context of shuttle avoidance training. Using a signal detection analysis, Milar [22] found that scopolamine and physostigmine affected cue discrimination only when the
difference between the stimulus that signalled reward and the stimulus that signalled nonreward was small. When the difference was large and discriminability was high, scopolamine and physostigmine had no effect on discrimination per se, although physostigmine reduced overall responding. In the present study, the difference between the CS (light and tone) and no CS was quite large and discriminability was presumably high. Under these conditions, physostigmine's effects on response variables may outweigh its effects on sensory variables. Alternatively, the doses of physostigmine which improve cue discrimination may be lower than those employed in the present study. Cox [15] and Cox and Tye [16] reported that small doses (0.02-0.06 mg/kg) enhanced performance on a cue discrimination task while higher doses (0. t mg/kg) disrupted performance. Other studies, however, have reported enhanced discrimination at doses of physostigmine comparable to ours (e.g., [18]). Finally, the failure to detect differential responsiveness to physostigmine on shuttle avoidance may have been due to an abbreviated training period. When treated with physostigmine prior to training, controls exhibited some improvement in performance over days, while alcohol-exposed animals showed no improvement. However, the interaction between prenatal treatment and dose of physostigmine was not significant. Had the training period been extended beyond 4 days, differences in the effects of physostigmine may have been revealed. In summary, alcohol-exposed animals made fewer avoidances than controls during acquisition of an active avoidance task. Treatment with physostigmine prior to training reduced the number of avoidances made, but did so similarly for all three prenatal treatment groups. In contrast to younger animals, alcohol-exposed adults were not differentially responsive to the behavioral effects of physostigmine, suggesting that some recovery of cholinergic function had occurred in those animals. These findings suggest also that underlying cholinergic dysfunction may not be responsible for the active avoidance deficits in alcohol-exposed animals. ACKNOWLEDGEMENTS This investigation was supported in part by Research Scientist Development Award AA00077, and grant AA03249 from the National Institute on Alcohol Abuse and Alcoholism.
REFERENCES
1. Abel, E. L. Prenatal effects of alcohol on adult learning in rats. Pharmacol Biochem Behav 10: 239-243, 1979. 2. Abel, E. L. The behavioral teratology of alcohol. Psychol Bull 90: 564--581, 1981. 3. Abel, E. L. In utero alcohol exposure and developmental delay of response inhibition. Alcohol: Clin Exp Res 6: 369-376, 1982. 4. Abel, E. L. Fetal Alcohol Syndrome and Fetal Alcohol Effects. New York: Plenum Press, 1984. 5. Barron, S., L. S. Meyer and E. P. Riley. Prenatal alcohol exposure alters appetitive learning in three-day-old rat pups. Alcohol: Clin Exp Res 10: 106, 1986. 6. Bond, N, W. Behavioural teratology: Fetal alcohol exposure and hyperactivity. In: Animal Models in Psychopathology, edited by N. W. Bond. Sydney: Academic Press, 1984, pp. 279-311. 7. Bond, N. W. Prenatal alcohol exposure and offspring hyperactivity: Effects of scopolamine and methylscopolamine. Neurobehav Toxicol Teratol 8: 287-292, 1986.
8. Bond. N. W. and E. L. DiGiusto. Effects of prenatal alcohol consumption on open-field behaviour and alcohol preference in rats. Psychopharmacologia 46: 163-165. 1976. 9. Bond, N. W. and E. L. DiGiusto. Prenatal alcohol consumption and open-field behaviour in rats: Effects of age at time of testing. Psychopharmacology (Berlin) 52: 311-312, 1977. 10. Bond, N. W. and E. L. DiGiusto. Avoidance conditioning and Hebb-Williamsmaze performance in rats treated prenatally with alcohol. Psychopharmacology (Berlin) SS: 69-7 l, 1978. 11. Carlton, P. L. Brain aeetylcholiae and inhibition. In: Reinforcement and Behavior, edited by J. T. Tapp. New York: Academic Press, 1969, pp. 286-327. 12. Carlton. P. L. and B. Markieweicz. Behavioral effects of atropine and scopolamine. In: Pharmacological and Biophysical Agents and Behavior, edited by E. Furehgott. New York: Academic Press, 1971. pp. 354-373.
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13. Chan, A. W. and E. L. Abel. Absence of long-lasting effects on brain receptors for neurotransmitters in rats prenatally exposed to alcohol. Res Commun Subst Abuse 3: 219-224, 1982. 14. Cox, T. The effects of physostigmine during the acquisition of avoidance behaviour as a function of the intersession interval. Q J Exp Psychol 26: 387-394, 1974. 15. Cox, T. Effects of physostigmine on the accuracy and activity of discrimination behavior in rats. Neuropharmacology 13: 701705, 1974. 16. Cox, T. and N. Tye. Effects of physostigmine on the maintenance of discrimination behavior in rats. Neuropharmacology 13: 205-221, 1974. 17. Jones, K. L. and D. W. Smith. Recognition of the fetal alcohol syndrome in early infancy. Lancet 2: 999-1001, 1973. 18. Leaton, R. N. and M. Kreindler. Effects of physostigmine and scopolamine on operant brightness discrimination in the rat. Physiol Behav 9: 121-123, 1972. 19. Lemoine, P., H. Harousseau, J.-P. Borteryu and J.-C. Menuet. Les enfants de parents alcooliques: Anomalies observees a propos de 127 cas. Quest Med 21" 476--482, 1968. 20. Lochry, E. A. and E. P. Riley. Retention of passive avoidance and T-maze escape in rats exposed to alcohol prenatally. Neurobehav Toxicol 2: 107-115, 1980. 21. Meyer, L. S. and E. P. Riley. Behavioral teratology of alcohol. In: Handbook of Behavioral Teratology, edited by E. P. Riley and C. Voorhees. New York: Plenum Press, 1986, pp. 101-140. 22. Milar, K. S. Cholinergic drug effects on visual discrimination: a signal detection analysis. Psychopharmacology (Berlin) 74: 383-388, 1981. 23. Milar, K. S., C. R. Halgren and G. A. Heise. A reappraisal of scopolamine effects on inhibition. Pharmacol Biochem Behav 9: 307-313, 1978. 24. Rawat, A. K. Developmental changes in the brain levels of neurotransmitters as influenced by maternal alcohol consumption in the rat. J Neurochem 28: 1175-1182, 1977.
25. Rick, J. T., K. L. Whittle and S. H. Cross. Disruption and facilitation of cue discrimination in the rat by cholinergic agents. Neuropharmacology 20: 747-752, 1981. 26. Riley, E. P., S. Barron, C. D. Driscoll and J.-S. Chen. Taste aversion learning in preweanling rats exposed to alcohol prenatally. Teratology 29: 325-331, 1984. 27. Riley, E. P., S. Barron, C. D. Driscoll and R. Hamlin. The effects of physostigmine on open-field behavior in rats exposed to alcohol prenatally. Alcohol: Clin Exp Res 10: 50-53, 1986. 28. Riley, E. P., S. Barron and J. H. Hannigan. Response inhibition deficits following prenatal alcohol exposure: A comparison to the effects of hippocampal lesions in rats. In: Alcohol and Brain Development, edited by J. R. West. New York: Oxford University Press, 1986, pp. 71-102. 29. Riley, E. P., E. A. Lochry and N. R. Shapiro. Lack of response inhibition in rats prenatally exposed to alcohol. Psychopharmacology (Berlin) 62: 47-52, 1979. 30. Rockwood, G. A. and E. P. Riley. Effects of scopolamine on spontaneous alternation and shuttle avoidance in rats exposed to alcohol in utero. Alcohol 2: 575-579, 1985. 31. Rosic, N. and G. Bignami. Depression of two-way avoidance learning and enhancement of passive avoidance learning by small doses of physostigmine. Neuropharmacology 9:311-316, 1970. 32. Shaywitz, B. A., G. G. Griffieth and J. B. Warshaw. Hyperactivity and cognitive defects in developing rats pups born to alcoholic mothers: An expanded fetal alcohol syndrome (EFAS). Neurobehav Toxicol 1: 113-122, 1979. 33. Streissguth, A. P. The behavioral teratology of alcohol: Performance, behavioral, and intellectual deficits in prenatally exposed children. In: Alcohol and Brain Development, edited by J. R. West. New York: Oxford University Press, 1986, pp. 3--44. 34. Streissguth, A., S. Landesman-Dwyer, J. Martin and D. Smith. Teratogenic effects of alcohol in humans and laboratory animals. Science 209: 353-367, 1980.
0741-8329/88$3.00 + .00
Effects of Physostigmine on Shuttle Avoidance in Rats Exposed Prenatally to Ethanol B. A. B L A N C H A R D
A N D E. P. R I L E Y 1
Center f o r Behavioral Teratology and Department o f Psychology State University o f N e w York at Albany, Albany, N Y 12222 R e c e i v e d 6 M a y 1987; A c c e p t e d 3 A u g u s t 1987 BLANCHARD, B. A. AND E. P. RILEY. Effects of physostigmine on shuttle avoidance in rats exposed prenatally to ethanol. ALCOHOL $(1) 27-31, 1988.--Shuttle avoidance performance following pretreatment with physostigmine was assessed in 85- to 100-day-old rats whose mothers consumed a liquid diet consisting of 35% ethanol-derived calories (EDC) during pregnancy. Offspring of pair-fed (0% El)C) and ad lib lab chow (LC) dams served as controls. Animals received either 0, 0.1 or 0.2 mg/kg physostigmine sulfate prior to acquisition training in a shuttle avoidance apparatus. Training consisted of 50 trials/day for 4 days. Thirty-five percent EDC rats made fewer avoidances than controls during acquisition training. Treatment with physostigmine reduced the number of avoidances made, and did so similarly for all prenatal treatment groups. Escape latencies were not affected by prenatal treatment, although they were increased by physostigmine administration prior to training. Neither prenatal treatment nor physostigmine treatment affected activity as measured by the number of intertrial crossings while in the apparatus. These data indicate that alcohol-exposed animals did not respond differentially to physostigmine relative to controls, suggesting that cholinergic dysfunction may not underlie the prenatal alcohol-induced deficit in active avoidance. Prenatal alcohol exposure
Shuttle avoidance
Cholinergic
F E T A L Alcohol Sydrome is comprised of a number of characteristic morphological, physiological and neurobehavioral abnormalities resulting from chronic maternal alcohol con. sumption during pregnancy [4, 17, 19, 34]. While the full range of symptoms is primarily seen following high levels of prenatal alcohol exposure, behavioral deficits in the absence of morphological anomalies may be associated with lower levels of exposure [2, 4, 33, 34]. The effects reported in humans have been demonstrated in laboratory animals as well (for reviews, see [2, 4, 21, 34]), providing models with which to explore the mechanisms of alcohol damage to the developing organism. Behavioral deficits produced in rats by fetal ethanol exposure include hyperactivity in an open field [3, 8, 9, 32] and poorer performance on a number of learning tasks, including passive [20,29] and active [1, 10, 30, 32] avoidance, taste aversion [26] and appetitive odor conditioning [5]. A number of the behavioral deficits seen following fetal ethanol exposure are consistent with a response inhibition deficit hypothesis [3, 21, 28, 29]. Central cholinergic systems appear to be involved in response inhibition [11,12], and alcohol-exposed animals resemble animals treated with anticholinergic drugs on a number o f behaviors [21]. This suggests that prenatal alcohol exposure may result in alterations in central cholinergic systems. Neurochemical data provide some indirect support for this. Rawat [24] found decreased levels of acetylcholine in alcohol-exposed rat
Physostigmine
fetuses and neonates. However, there is a paucity of work examining acetylcholine levels at other stages of development following fetal alcohol exposure. Some investigators have attempted to clarify the functional significance of putative changes in cholinergic systems resulting from alcohol exposure in utero through the use of psychopharmacological techniques, and have reported altered responsiveness to the reversible acetylcholinesterase inhibitor physostigmine. Riley et al. [27] treated 25-day-old alcohol-exposed rats with physostigmine, and at the doses used (0.1 and 0.2 mg/kg), physostigmine decreased activity of alcohol-exposed animals while control animals showed no change. In contrast, Bond [6] found that activity in 22-dayold alcohol-exposed rats was unaffected following physostigmine treatment, while activity levels in controls decreased. Differential responsiveness to the cholinergic antagonist scopolamine has been observed in 22-day-old alcohol-exposed animals [7]. To extend these findings to another behavior and to a different stage of development, we examined acquisition of a shuttle avoidance task following treatment with physostigmine in adult rats exposed prenatally to alcohol. In normal animals, physostigmine administered just prior to acquisition of this task reduces the number of avoidances made [ 14,31]. If fetal alcohol-exposext animals are more responsive in adulthood to the behavioral effects of physostigmine, as they were at 25 days in the Riley et al. study [27], then those
1Requests for reprints should be addressed to Dr. E. P. Riley.
27
28
B L A N C H A R I ) AND RILEY Females 0.1 mg/kg physostigmine
saline
40
0.2 mg/kg physostigmine
LC © O%EDC o 35% EDC •
30
20
10 ~P 0f . ~o
N
i
i
i
I
0
Males
E g 0
40
t
saline
0.1 mg/kg
0.2 m g / k g physostigmine
physostigmine
3°[ 10 I
I
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2
3
4
1
2
3
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Days
FIG. I. Mean number of avoidances made on each day of training by male and female rats from the three prenatal treatment groups for each dose of physostigmine (0.0.0.1 or 0.2). LC rats are represented with circles, 0% EDC animals with squares, and 35% EDC animals with closed triangles. animals should show a greater physostigmine-induced impairment than controls. An alternative outcome was also possible. The poorer performance on an active avoidance task by fetal alcoholexposed rats may reflect a disruption of attentional processes, which may be related to cholinergic function. For example, in normal animals, increasing cholinergic activity with small doses of physostigmine improves performance on some types o f discrimination tasks [15, 16, 18, 25], while blocking cholinergic receptors with atropine or scopolamine decreases performance [22, 23, 25]. If the alcohol-induced deficit on a signalled shuttle avoidance task is a consequence of an inability to attend to salient cues, then physostigrnine may actually improve performance of alcohol-exposed animals. METHOD Male and female Long Evans hooded rats (Blue Spruce Farms, Altamont, NY) were mated each night until a vaginal plug was detected (designated Day 1 of pregnancy). Pregnant animals were housed individually in clear breeding cages with ad lib lab chow and water available, and assigned randomly 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 o f pregnancy. Animals in a second group were matched individually to 35% EDC animals and pair-fed a liquid diet with sucrose substituted isocalorically for ethanol (0% EDC). This group served as a nutritional control for the effects of liquid diet. A third group 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.
Liquid diets provided approximately 1.3 kcal/ml and were the sole source of nutrition during the period they were administered. Animals were weighed every five days during pregnancy to monitor weight gain and maintain more precise control of pair-feeding, which was done on an intake volume/body weight basis. Just prior to the expected parturition date cages were inspected twice daily for births. Pups were weighed on the day following birth (Day 1), 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 pair-housed with same sex littermates and maintained on a 12-hr light-dark cycle with free access to food and water.
Behavioral Testing Male and female offspring from each prenatal treatment group were tested in squads of 4 animals, beginning at 85 to 100 days o f age and continuing for 4 days ( n ' s = 5 to 7 per cell). Only one animal per litter was included in any cell of the d©sign to control for possible litter effects. On Day 1, animals were weighed and injected with 0.1 or 0.2 mg/kg physostigmine sulfate (Sigma Chemical Co. No. E8625) or with an equivalent volume (t ml/k8) o f 0.9% saline vehicle immediately prior to testing. Testing was conducted in 4 shuttle boxes measuring 45.7x21.6×24.5 era. The $rid floor of the shuttle box consisted o f steel rods which measured 0.63 cm in diameter, placed 1.9 c m apart. Each box was comprised of two equal sized c o m p e a ~ t , z ~ s dli~ded by an aluminum wall with a centered opening (5.1x10.2 era) beginning at floor level through which the animals could pass. Lights (28 V) on the upper portion of the far wall in each
PHYSOSTIGMINE AND F E T A L A L C O H O L E X P O S U R E compartment and a tone (80 dB) served as the compound conditioned stimulus. Electrical shock (0.9 mA) generated from a Coulbourn Instruments Shock Generator (No. F1316) served as the unconditioned stimulus. Photocells (3.8 cm above floor level) monitored crossings between the two compartments. Each shuttle box was enclosed in a soundattenuating chamber which contained a ventilation fan and a house light. At the beginning of each session, the animal was placed into one side of the shuttle box. The animal received a 5-sec presentation of the CS. If it crossed over to the other side of the apparatus during this 5-sec period, shock was avoided. If it did not cross over, shock was administered and continued until the animal crossed over or 30 sec elapsed. Animals received 50 trials/day, and trials were separated by a 40-sec intertrial interval. After testing animals were returned to their home cages. Testing procedures were identical on Days 2 through 4. The dose of physostigmine each animal received daily remained the same over training. Dependent measures included number of avoidances per 10-trial block per day, mean escape latency on each day and number of intertrial crossings on each day. Testing was conducted between 1200 and 1600 hr. RESULTS
Dam and Litter Characteristics An ongoing breeding program in our laboratory generates animals from which several investigators select subjects. Dam and litter characteristics are reported for the larger group of animals from which the test subjects were randomly selected. Mothers in the 35% EDC group consumed an average of 13.44_+0.16 g/kg ethanol daily. Maternal data were analyzed by one-way analyses of variance, with Treatment as the factor. There were no differences in mean length of pregnancy. Analysis revealed a significant effect of Treatment on percent weight gain during pregnancy, F(2,90)= 11.54, p<0.0001. Comparisons with Fisher's Least Significant Difference (LSD) test (p<0.05) indicated that dams in the two liquid diet groups showed a significantly smaller percent weight gain over the course of pregnancy (34.40% and 33.11% for 0% and 35% EDC dams, respectively) than LC dams (39.85%). There were no group differences in litter size. Pup data were analyzed by two-way analyses of variance, with Prenatal Treatment and Sex as factors. There were no significant effects of Treatment on sex ratio. Analysis of pup body weight at birth indicated that Treatment was a significant source of variance, F(2,180) = 12.48, p <0.0001. Further LSD comparisons (p<0.05) indicated that LC pups weighed significantly more than both 0% EDC and 35% EDC pups, who did not differ from each other. There was also a significant effect of Sex on pup body weight, F(1,180)--5.45, p<0.05. Prenatal Treatment did not interact with Sex. Behavioral Testing Due to a computer interface problem that went undetected during the study, some data points were missing (approximately 2%). These missing data, which were distributed randomly, were estimated by a computer program using a regression technique. Because such a technique minimizes within-group variance, only those main effects and interactions for which the probability was less than 0.01 were examined. The number of avoidances per five 10-trial blocks on each
29 test day was analyzed by mixed analysis of variance ( 3 × 2 x 3 × 4 x 5 ) with Prenatal Treatment, Sex and Dose of physostigmine as between-group factors, and Day and Block as within-group factors. Significant interactions and main effects were examined further with Fisher's LSD comparisons (p <0.05 for all comparisons). Analysis revealed a significant Treatment × Day interaction, F(6,282)=5.07, p<0.0005. LSD comparisons indicated that although there were no group differences in performance on Day 1, 35% EDC animals made fewer avoidances than 0% EDC controls on Day 2, and fewer than both LC and 0% EDC animals on Days 3 and 4. In addition, LC and 0% EDC animals showed significant improvement over the previous day's performance on Days 2 and 3, while alcohol-exposed animals showed improvement only on Day 2, but not thereafter (see Fig. 1). The Treatment main effect was significant as well, F(2,94)=8.06, p<0.001, due to 35% EDC animals making fewer avoidances than LC and 0% EDC controls over the entire training period. LC and 0% EDC rats did not differ from each other. There was a significant Dose x Day interaction, F(6,282)=8.59, p<0.0001, which comparisons indicated was due to the fact that animals that received 0.1 or 0.2 mg/kg physostigmine prior to training made fewer avoidances than those that received saline on each day of training. In addition, on Day 3, animals that received 0.2 mg/kg made fewer avoidances than those that received 0.1 mg/kg physostigmine. At all other times there were no differences between the two doses of physostigmine. A significant main effect of Dose, F(2,94)=24.77, p<0.0001, was due to physostigminetreated animals making fewer avoidances than saline controls over the entire training period. Sex x Day was a significant source of variance, F(3,282)=8.29, p<0.0001. Comparisons indicated that on Day 1, females made fewer avoidances than males, but were not different on Day 2. On Days 3 and 4, females made more avoidances than males. Mean inverse escape latencies (speed) for each day of training were analyzed by mixed analysis of variance ( 3 x 2 × 3 x 4 ) , with Prenatal Treatment, Sex and Dose of physostigmine as between-group factors, and Day as a within-group factor. Sex x Day was a significant source of variance, F(3,282)=5.29, p<0.005. Further comparisons indicated that females had faster speeds than males on Days 3 and 4. In addition, physostigmine reduced speed in a dose-dependent fashion, as indicated by a significant main effect of Dose, F(2,94)= 11.04, p<0.0001, followed by LSD comparisons. The number of intertrial crossings on each day of training was analyzed as described for speed. With the exception of a day effect (means=12.2, 13.8, 16.0, and 19.5 for Days 1-4, respectively) there were no other significant main effects or interactions. This suggests that neither Prenatal Treatment nor Dose of physostigmine affected this measure of activity in the shuttle avoidance apparatus. Furthermore, only 8 animals consistently failed to show escape or avoidance behaviors and this was not affected by either prenatal treatment or physostigmine. Alcohol-exposed animals did not appear to be differentially affected by physostigmine, since Treatment did not interact with Dose on any dependent measure. Treatment also failed to interact with Sex. DISCUSSION
The results replicate earlier findings that prenatal alcohol
30
B L A N C H A R D AND RII~EY
exposure disrupts two-way active avoidance learning in adulthood [1, 10, 30]. Both males and females exposed prenatally to alcohol made fewer avoidances than controls regardless of dose of physostigmine. The poorer performance of animals treated with physostigmine prior to training also replicates earlier findings [14,31]. The deficit in performance following physostigmine does not appear to be due to motor impairment, since activity (as measured by intertrial crossings) was not affected by physostigmine. Fetal alcohol-exposed animals did not appear to respond differentially to physostigmine. Because differential responsiveness to physostigmine's effects on activity has been demonstrated in younger alcohol-exposed animals [6,27], the absence of a Treatment × Dose interaction in the present study could be due to some recovery of cholinergic function by 35% EDC animals. This is consistent with the findings of Rockwood and Riley [30] who demonstrated that adult fetal alcohol-exposed rats responded normally (i.e., similarly to controls) when treated with the anticholinergic drug scopolamine prior to training on an active avoidance task. We have also examined the effects of cholinergic drugs on activity in fetal alcohol-exposed adult rats, and have observed no differential responding to arecoline or to scopolamine (manuscript in preparation). Neurochemical data provide some indirect support for the recovery of cholinergic function hypothesis, although a systematic examination of the development of cholinergic systems following fetal alcohol exposure has not been made. Decreased levels of acetylcholine have been observed in rat fetuses and neonates whose mothers received alcohol during pregnancy [24]. However, Chan and Abel [13] examined cholinergic receptor binding in fetal alcohol-exposed adult rats and found no differences from controls. It is possible also that the failure to observe differential responsiveness to physostigmine by alcohol-exposed rats was due to the choice of dose range. Differences may have been detected had lower doses been used. The second, alternative prediction of physostigmineinduced enhancement of performance in alcohol-exposed offspring was not borne out. Several factors could account for this. The conditions under which physostigmine improves cue discrimination may be very specific, and may not exist in the context of shuttle avoidance training. Using a signal detection analysis, Milar [22] found that scopolamine and physostigmine affected cue discrimination only when the
difference between the stimulus that signalled reward and the stimulus that signalled nonreward was small. When the difference was large and discriminability was high, scopolamine and physostigmine had no effect on discrimination per se, although physostigmine reduced overall responding. In the present study, the difference between the CS (light and tone) and no CS was quite large and discriminability was presumably high. Under these conditions, physostigmine's effects on response variables may outweigh its effects on sensory variables. Alternatively, the doses of physostigmine which improve cue discrimination may be lower than those employed in the present study. Cox [15] and Cox and Tye [16] reported that small doses (0.02-0.06 mg/kg) enhanced performance on a cue discrimination task while higher doses (0. t mg/kg) disrupted performance. Other studies, however, have reported enhanced discrimination at doses of physostigmine comparable to ours (e.g., [18]). Finally, the failure to detect differential responsiveness to physostigmine on shuttle avoidance may have been due to an abbreviated training period. When treated with physostigmine prior to training, controls exhibited some improvement in performance over days, while alcohol-exposed animals showed no improvement. However, the interaction between prenatal treatment and dose of physostigmine was not significant. Had the training period been extended beyond 4 days, differences in the effects of physostigmine may have been revealed. In summary, alcohol-exposed animals made fewer avoidances than controls during acquisition of an active avoidance task. Treatment with physostigmine prior to training reduced the number of avoidances made, but did so similarly for all three prenatal treatment groups. In contrast to younger animals, alcohol-exposed adults were not differentially responsive to the behavioral effects of physostigmine, suggesting that some recovery of cholinergic function had occurred in those animals. These findings suggest also that underlying cholinergic dysfunction may not be responsible for the active avoidance deficits in alcohol-exposed animals. ACKNOWLEDGEMENTS This investigation was supported in part by Research Scientist Development Award AA00077, and grant AA03249 from the National Institute on Alcohol Abuse and Alcoholism.
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