- Email: [email protected]
6J mice
Pharmacology Biochemistry & Behavior, Vol. 3, pp. 1137--1139. Copyright © 1975 by ANKHO International Inc. .All rights of reproduction in any form reserved. Printed in the U.S.A.
BRIEF COMMUNICATION Phenobarbital During Pregnancy Alters Operant Behavior of Offspring in C57BL/6J Mice I LAWRENCE D. MIDDAUGH, C A R R O L L A. SANTOS, III AND JOHN W. ZEMP
Departments o f Biochemistry, and Psychiatry and Behavioral Science Medical University o f South Carolina, Charleston, SC 29401 (Received 12 May 1975) MIDDAUGH, L. D.~ C. A. SANTOS, III, AND J. W. ZEMP. Phenobarbital during pregnancy alters operant behavior of offspring in C57BL/6J mice. PHARMAC. BIOCHEM. BEHAV. 3(6) 1137-1139, 1975. - Offspring of C57BL/6J injected daily with phenobarbital for the last third of pregnancy responded less than control animals when maintained on various fixed ratio schedules of reinforcement. The response decrement became more pronounced as the schedule demands were increased and was noted in offspring of both sexes. The highest dose (80 mg/kg) was less effective than the 2 lower doses (20 mg and 40 mg/kg) in producing the decrement which may reflect a selection factor due to high neonatal mortality previously reported at this dose. The study provides no evidence of the mechanism mediating the long term behavioral abnormality but does clearly extend the finding of such changes to doses which do not produce increased neonatal mortality or noticeable morphological changes. Prenatal maternal phenobarbital
Operant behavior in C57BL/6J mice
PHENOBARBITAL is being ingested by a substantial r u m b e r of pregnant women. Epidemiological studies suggest that from 24 to 32 percent of pregnant women ingest some type of sedative, frequently phenobarbital, generally during the last trimester [4,5]. There are also a number of women with epilepsy using phenobarbital as an anticonvulsant throughout pregnancy [3,5]. In addition, the drug is used by members of the drug subculture including use during pregnancy [3]. Finally, phenobarbital has been administered to gravid women for 2 week periods prior to parturition as a prophylaxis for neonatal hyperbilirubinemia [16]. In spite of the frequent use of phenobarbital during pregnancy, there is relatively little information regarding potential effects of the drug on offspring, particularly long-term effects. Although administering the drug to pregnant women as a prophylaxis for neonatal hyperbilirubinemia has been reported to produce no detrimental side effects [16], a thorough investigation for possible effects is absent [15]. Withdrawal symptoms have been noted in neonates addicted to the drug during pregnancy [31 and high doses of phenobarbital injected into pregnant rats can increase fetal death and produce morphological abnormalities in surviving offspring [ 8 ]. There is also some evidence that mature offspring of rats [ 1,7] and o f mice [9, 10, 17] injected with the drug during pregnancy have altered brain function as evidenced by behavior abnormalities.
We have previously reported the results of experiments on offspring of C57BL/6J mice injected daily with phenobarbital for the last third of pregnancy [9, 10, 17]. Increased neonatal mortality and reduced body weight in mature surviving offspring were noted for animals injected with either 80 mg or 40 mg/kg of the drug [171. A 20 mg/kg dose had no significant effect on these measures. The high dose of the drug produced reductions in offspring brain weights accompanied by reduced levels of nucleic acids and protein. We have also reported that offspring of mice injected with a 40 mg/kg dose of the drug for the same time period were retarded in the development of several motor reflexes [9] and differed from controls on 3 behavioral tests conducted after maturity [10]. A comprehensive study varying drug dose and assessing the behavioral affects on mature offspring of both sexes, however, is currently absent from the literature. The purpose of the study reported here was to assess the effects of phenobarbital ( 2 0 - , 4 0 - , and 80 mg/kg) injected into pregnant mice for the last third of pregnancy on lever responding by offspring maintained on increasing fixed ratio schedules of reinforcement. This task was previously effective in differentiating female offspring of animals injected with 40 mg/kg phenobarbital from control mice. METHOD Animals
for
the
study
were C57BL/6J
mice (Mus
1This project was supported by Public Health Service Grant MH 17455 and by the South Carolina State Appropriation to the Medical University of South Carolina Biomedical Research. 1137
1138
M I D D A U G H , S A N T O S A N D ZEMP
musculus) o b t a i n e d from o u r b r e e d e r colony. T h e y were •
.
.
o
+
1800-
o
m a i n t a i n e d m a t e m p e r a t u r e regulated r o o m (23 - 2 C) on 12 h r l i g h t : d a r k cycle a n d had f o o d and w a t e r available ad lib until testing w h i c h required food deprivation. Pregnancies were d e t e r m i n e d by i n s p e c t i o n for sperm plugs each m o r n i n g a n d were verified b y weight gains ( > 4 g) 12 days a f t e r plug d e t e c t i o n . Animals in the drug groups were i n j e c t e d s u b c u t a n e o u s l y each m o r n i n g w i t h 2 0 - , 4 0 - , or 80 mg p h e n o b a r b i t a l s o d i u m per kg b o d y w e i g h t (P20, P40, P80) for t h e last 6 or 7 days of p r e g n a n c y . Saline c o n t r o l (SC) animals were injected for the same time period w i t h 0.9 p e r c e n t saline. In each case, t h e a n i m a l was i n j e c t e d with a 0.008 ml s o l u t i o n / g b o d y weight. On the day of delivery, t h e litter was culled to 6 and offspring were reared by t h e i r biological m o t h e r u n t i l weaning at 21 days of age. A f t e r weaning, animals were caged a c c o r d i n g to sex w i t h 2 6 animals per cage u n t i l behavioral assessment w h e n t h e y were m a i n t a i n e d in individual cages. At 90 days of age, the b e h a v i o r of t h e pups f r o m the various t r e a t m e n t s was assessed using an o p e r a n t task requiring the animal to make an increasing n u m b e r of responses per unit of r e i n f o r c e m e n t across days. T h e animals were tested in 1 of 6 o p e r a n t c h a m b e r s e n c l o s e d in s o u n d a t t e n u a t e d boxes. T h e c h a m b e r s (16 × 16 x 11.4 cm) were c o n s t r u c t e d o f Plexiglas w i t h stainless steel grid floors. A food tray w i t h a 1.9 x 2.5 cm o p e n i n g was centrally located on one wall at floor level. A Lehigh Valley Model No. 1 2 1 - 0 3 r o d e n t lever located 4 cm to one side of t h e food tray a n d 3.0 cm above the floor served as t h e response indicator. Eight grams dead weight on the lever a c t i v a t e d a m i c r o s w i t c h w h i c h i n i t i a t e d p r o g r a m m i n g equipm e n t . A f t e r one week on a food d e p r i v a t i o n schedule w h i c h reduced b o d y weight to 7 5 - - 8 5 p e r c e n t of ad lib levels, the animals were familiarized with the test c h a m b e r a n d self-trained to press the lever for food r e i n f o r c e m e n t ( 2 0 m g N o y e s pellets). A f t e r this, animals were r u n 30 min per day for 5 days on each of the following schedules of reinforcem e n t : C R F (1 response per r e i n f o r c e m e n t ) , F R 5 (5 responses per r e i n f o r c e m e n t ) , F R 2 0 , a n d F R 4 0 . Male animals were a d d i t i o n a l l y run t h r e e days each on F R 8 0 , FR120, and FR160. RESULTS
I n j e c t i n g p r e g n a n t mice with p h e n o b a r b i t a l did n o t alter the ability of offspring to acquire the lever response p r o d u c i n g food r e i n f o r c e m e n t • A p p r o x i m a t e l y 90 p e r c e n t of the animals in each group a c q u i r e d the response to a criterion of > 10 responses per session d u r i n g t w o 15 rain self shaping sessions. The r e m a i n i n g animals m e t this criterion a f t e r t w o a d d i t i o n a l 30 m i n sessions. The effect of these t r e a t m e n t s on b e h a v i o r m a i n t a i n e d by t h e various fixed ratio schedules of r e i n f o r c e m e n t was assessed by c o m p a r i n g t h e drug groups of each sex w i t h t h e i r respective SC groups• Mean responses g e n e r a t e d per daily session u n d e r each s c h e d u l e o f r e i n f o r c e m e n t (i.e., over a 5 day test period) were calculated for e a c h animal and served as an i n d e x of its p e r f o r m a n c e on the particular schedule• Since t h e r e was a great deal of individual v a r i a t i o n within e a c h group, particularly on the h i g h e r fixed r a t i o schedules, group t e n d e n c i e s are r e p o r t e d as medians. These data are s u m m a r i z e d in Fig. 1. C o n t r o l animals s h o w e d t h e e x p e c t e d increased res p o n d i n g with increasing d e m a n d s of the r e i n f o r c e m e n t schedule. F o r males, statistical c o m p a r i s o n of individual
600 "
~400, Male
£ 1200" r: o
I000-
800Q. 600-
400-
200
-
T~ea,me0t ~ Schedule
CRF
151 o
o
o
FR5
FR20
o
1
FR40
o
o
FRSO
o FRI20
o
FR(60
800Female ® £c 6 0 0 o
a m 400c ec ~
g ",5
o 2oo-
L3 Treatment Schedule
0._ c° CRF
FR5
FR20
FR40
FIG. 1. Effect of phenobarbital during pregnancy on lever responding of mature offspring maintained under various fixed ratio (FR) schedules of reinforcement. Animals were 90-day-old offspring of C57BL/6J mice injected daily with saline (SC) or phenobarbital at doses of 20 mg (P20), 40 mg (P40), or 80 mg (P80) per kg body weight for the last third of pregnancy. See text for statistical analysis. The bars represent medians determined from Ns of 6 - 1 1 per group. Upper and lower graphs summarize performance of male and female offspring respectively. drug g r o u p s w i t h saline c o n t r o l s via M a n n - W h i t n e y U-Tests [ 13] e s t a b l i s h e d significant r e d u c t i o n s in r e s p o n d i n g at the 95 p e r c e n t level o f c o n f i d e n c e for the P40 and P20 groups b e g i n n i n g at F R 4 0 and e x t e n d i n g t h r o u g h the highest schedule tested. The m e d i a n score for the P80 g r o u p does n o t differ significantly f r o m t h a t of the SC group u n t i l F R 160. The p a t t e r n o f r e s p o n d i n g for SC females was similar to t h a t of males b u t at a r e d u c e d level. F e m a l e animals of the P80 g r o u p r e s p o n d e d at rates s o m e w h a t higher t h a n the SC group on all s c h e d u l e s tested, however, n o n e of these differences are significant. A significant r e d u c t i o n in r e s p o n d i n g b y t h e P40 group c o m p a r e d to e i t h e r the SC or P80 groups was n o t e d for all schedules tested e x c e p t CRF. DISCUSSION
This s t u d y clearly establishes t h a t injecting mice with
MATERNAL PHENOBARBITAL AND O F F S PRI N G BEHAVIOR phenobarbital for the last third of pregnancy produced long lasting behavioral changes in offspring of both sexes. In this ,;tudy, responding maintained by various fixed ratio schedules of reinforcement was reduced. The reduction became more pronounced as the animal was required to increase its response output per unit of reinforcement (i.e., on the higher ratio schedules). We have previously reported [17] that the two higher doses of phenobarbital (80 mg and 40 mg/kg) caused a weight reduction in mature surviving offspring and it could be argued that the reduced weight might contribute to the reduction in response output due lo the increased effort required of the lighter animals to depress the lever. This however, is certainly not a complete explanation since the highest dose produced the greatest weight reduction [17] yet in the current study had the least effect on response output. In addition, female offspring of animals injected with 40 mg/kg of phenobarbital have previously been reported to show a response decrement on a similar task differing only in that the iesponse was merely contact with a metal disc which presumably would be independent of body weight [ 10]. In the same study, it was also shown that the response decrement was due to the schedule and not to the length of time on food deprivation. The reduced effect of the higher drug dose in the current study was certainly unexpected and we currently have no explanation for this finding. Reverse dose-response effects have been reported in other studies on the effects of maternally administered amphetamine or morphine on offspring [2, 12, 17]. It should also be noted that the 80 mg/kg dose of phenobarbital produces a large increase in neonatal mortality [17] hence, selection factor might
1139
account for the reduced behavioral effect noted in surviving offspring. In addition, we have found that 80 mg/kg of this drug injected into adult animals produced behavioral effects different from that of the two lower doses used in this experiment (L. D. Middaugh, unpublished observations). Whereas the 80 mg/kg dose depressed responding maintained by a variable interval schedule of reinforcement, 20 mg and 40 mg/kg doses produced an increased response output. It is thus probable that the different doses are producing different physiological effects on either the pregnant animal or her fetuses. The mechanism mediating these long-term behavioral changes is currently unknown. Possibly, changes in motherinfant interaction essential for normal development [6] may have contributed to the observed behavioral changes in adult offspring. Either abrupt withdrawal from the drug following parturition or the previously reported [17] changes in neonates of animals injected with the drug would be capable of altering this interaction. It is also known that phenobarbital can cross the placenta in both animals [14] and humans I l l ] ; and is reported to be highly concentrated in fetal liver, adrenal, and brain stem tissue. Hence, it is possible that the drug may be interfering with the development of the brain and/or pituitary adrenal system to produce the long-term behavioral effects. Although this study provides no information regarding the mechanism by which phenobarbital injected into pregnant animals produces the long lasting changes in offspring behavior, it does clearly establish the existence of such alterations and should prompt further investigation of the extent and mechanism of the change.
REFERENCES [. Armitage, S. G. The effects of barbiturates on the behavior of the rat offspring as measured in learning and reasoning situations. J. comp. physiol. Psychol. 45: 146-152, 1952. 2. Buchenauer, D., M. Turnbow and M. A. Peters. Effect of chronic methadone administration on pregnant rats and their offspring. J. PharmacoL exp. Ther. 189: 66-71, 1974. 3. Desmond, M. M., R. P. Schwanecke, G. S. Wilson, S. Yasunaga and I. Burgdorff. Maternal barbiturate utilization and neonatal withdrawal symptomatology. J. Pediat. 80: 190-197, 1972. 4. Forfar, J. O. and M. N. Nelson. Epidemiology of drugs taken by pregnant women: Drugs that may affect the fetus adversely. Clin. Pharmac. Ther. 14: 632-642, 1973. 5;. Hill, R. M, Drugs ingested by pregnant women. Clin. Pharmac. Ther. 14: 654-659, 1973. 6. Joffe, J. M. Prenatal Determinants o f Behaviour. New York: Pergamon, 1969. 7. Mauri, N. Effect of maternal medication during pregnancy upon behavioral development of offspring. Tohoku J. exp. Med. 89: 265-272, 1966. 8. McColl, J. D., M. Globus and S. Robinson. Drug induced skeletal malformations in the rat. Experientia 19: 183-184, 1963. 9. Middaugh, L. D., S. T, Drake, J. E. Fraley and J. W. Zemp. Effects of subanesthetic doses of phenobarbital administered to pregnant mice on behavior of offspring. Fedn Proc. 31: 2147, 1972.
10. Middaugh, L. D., C. A. Santos, III and J. W. Zemp. Effects of phenobarbital given to pregnant mice on behavior of mature offspring. Devel. Psychobiol. 18: 305-313, 1975. 11. Ploman, L. and B. H. Persson. On the transfer of barbiturates to the human fetus and their "accumulation in some of its vital organs. J. Obset. Gynaec. Br. Commonw. 64: 706-711, 1957. 12. Seliger, D. L. Effect of prenatal maternal administration of d-amphetamine on rat offspring activity and passive avoidance learning. Physiol. Psychol. 1: 273-280, 1973. 13. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956, pp. 116-127. 14. Villee, C. A. Placental transfer of drugs. Ann. N. Y. Acad. Sci. 123: 237-242, 1965. 15. Wilson, J. T. Caution with phenobarbital. Clin. Pediat. I0: 684-687, 1971. 16. Yeung, C. Y., L. S. Tam, A. Chan and K. H. Lee. Phenobarbitone prophylaxis for neonatal hyperbilirubinemia. Pediatrics 48: 372-376, 1971. 17. Zemp, J. W. and L. D. Middaugh. Some effects of prenatal exposure to d-amphetamine sulfate and phenobarbital on developmental neurochemistry and on behavior. Addict. Dis., 2: 307-331, 1975.
BRIEF COMMUNICATION Phenobarbital During Pregnancy Alters Operant Behavior of Offspring in C57BL/6J Mice I LAWRENCE D. MIDDAUGH, C A R R O L L A. SANTOS, III AND JOHN W. ZEMP
Departments o f Biochemistry, and Psychiatry and Behavioral Science Medical University o f South Carolina, Charleston, SC 29401 (Received 12 May 1975) MIDDAUGH, L. D.~ C. A. SANTOS, III, AND J. W. ZEMP. Phenobarbital during pregnancy alters operant behavior of offspring in C57BL/6J mice. PHARMAC. BIOCHEM. BEHAV. 3(6) 1137-1139, 1975. - Offspring of C57BL/6J injected daily with phenobarbital for the last third of pregnancy responded less than control animals when maintained on various fixed ratio schedules of reinforcement. The response decrement became more pronounced as the schedule demands were increased and was noted in offspring of both sexes. The highest dose (80 mg/kg) was less effective than the 2 lower doses (20 mg and 40 mg/kg) in producing the decrement which may reflect a selection factor due to high neonatal mortality previously reported at this dose. The study provides no evidence of the mechanism mediating the long term behavioral abnormality but does clearly extend the finding of such changes to doses which do not produce increased neonatal mortality or noticeable morphological changes. Prenatal maternal phenobarbital
Operant behavior in C57BL/6J mice
PHENOBARBITAL is being ingested by a substantial r u m b e r of pregnant women. Epidemiological studies suggest that from 24 to 32 percent of pregnant women ingest some type of sedative, frequently phenobarbital, generally during the last trimester [4,5]. There are also a number of women with epilepsy using phenobarbital as an anticonvulsant throughout pregnancy [3,5]. In addition, the drug is used by members of the drug subculture including use during pregnancy [3]. Finally, phenobarbital has been administered to gravid women for 2 week periods prior to parturition as a prophylaxis for neonatal hyperbilirubinemia [16]. In spite of the frequent use of phenobarbital during pregnancy, there is relatively little information regarding potential effects of the drug on offspring, particularly long-term effects. Although administering the drug to pregnant women as a prophylaxis for neonatal hyperbilirubinemia has been reported to produce no detrimental side effects [16], a thorough investigation for possible effects is absent [15]. Withdrawal symptoms have been noted in neonates addicted to the drug during pregnancy [31 and high doses of phenobarbital injected into pregnant rats can increase fetal death and produce morphological abnormalities in surviving offspring [ 8 ]. There is also some evidence that mature offspring of rats [ 1,7] and o f mice [9, 10, 17] injected with the drug during pregnancy have altered brain function as evidenced by behavior abnormalities.
We have previously reported the results of experiments on offspring of C57BL/6J mice injected daily with phenobarbital for the last third of pregnancy [9, 10, 17]. Increased neonatal mortality and reduced body weight in mature surviving offspring were noted for animals injected with either 80 mg or 40 mg/kg of the drug [171. A 20 mg/kg dose had no significant effect on these measures. The high dose of the drug produced reductions in offspring brain weights accompanied by reduced levels of nucleic acids and protein. We have also reported that offspring of mice injected with a 40 mg/kg dose of the drug for the same time period were retarded in the development of several motor reflexes [9] and differed from controls on 3 behavioral tests conducted after maturity [10]. A comprehensive study varying drug dose and assessing the behavioral affects on mature offspring of both sexes, however, is currently absent from the literature. The purpose of the study reported here was to assess the effects of phenobarbital ( 2 0 - , 4 0 - , and 80 mg/kg) injected into pregnant mice for the last third of pregnancy on lever responding by offspring maintained on increasing fixed ratio schedules of reinforcement. This task was previously effective in differentiating female offspring of animals injected with 40 mg/kg phenobarbital from control mice. METHOD Animals
for
the
study
were C57BL/6J
mice (Mus
1This project was supported by Public Health Service Grant MH 17455 and by the South Carolina State Appropriation to the Medical University of South Carolina Biomedical Research. 1137
1138
M I D D A U G H , S A N T O S A N D ZEMP
musculus) o b t a i n e d from o u r b r e e d e r colony. T h e y were •
.
.
o
+
1800-
o
m a i n t a i n e d m a t e m p e r a t u r e regulated r o o m (23 - 2 C) on 12 h r l i g h t : d a r k cycle a n d had f o o d and w a t e r available ad lib until testing w h i c h required food deprivation. Pregnancies were d e t e r m i n e d by i n s p e c t i o n for sperm plugs each m o r n i n g a n d were verified b y weight gains ( > 4 g) 12 days a f t e r plug d e t e c t i o n . Animals in the drug groups were i n j e c t e d s u b c u t a n e o u s l y each m o r n i n g w i t h 2 0 - , 4 0 - , or 80 mg p h e n o b a r b i t a l s o d i u m per kg b o d y w e i g h t (P20, P40, P80) for t h e last 6 or 7 days of p r e g n a n c y . Saline c o n t r o l (SC) animals were injected for the same time period w i t h 0.9 p e r c e n t saline. In each case, t h e a n i m a l was i n j e c t e d with a 0.008 ml s o l u t i o n / g b o d y weight. On the day of delivery, t h e litter was culled to 6 and offspring were reared by t h e i r biological m o t h e r u n t i l weaning at 21 days of age. A f t e r weaning, animals were caged a c c o r d i n g to sex w i t h 2 6 animals per cage u n t i l behavioral assessment w h e n t h e y were m a i n t a i n e d in individual cages. At 90 days of age, the b e h a v i o r of t h e pups f r o m the various t r e a t m e n t s was assessed using an o p e r a n t task requiring the animal to make an increasing n u m b e r of responses per unit of r e i n f o r c e m e n t across days. T h e animals were tested in 1 of 6 o p e r a n t c h a m b e r s e n c l o s e d in s o u n d a t t e n u a t e d boxes. T h e c h a m b e r s (16 × 16 x 11.4 cm) were c o n s t r u c t e d o f Plexiglas w i t h stainless steel grid floors. A food tray w i t h a 1.9 x 2.5 cm o p e n i n g was centrally located on one wall at floor level. A Lehigh Valley Model No. 1 2 1 - 0 3 r o d e n t lever located 4 cm to one side of t h e food tray a n d 3.0 cm above the floor served as t h e response indicator. Eight grams dead weight on the lever a c t i v a t e d a m i c r o s w i t c h w h i c h i n i t i a t e d p r o g r a m m i n g equipm e n t . A f t e r one week on a food d e p r i v a t i o n schedule w h i c h reduced b o d y weight to 7 5 - - 8 5 p e r c e n t of ad lib levels, the animals were familiarized with the test c h a m b e r a n d self-trained to press the lever for food r e i n f o r c e m e n t ( 2 0 m g N o y e s pellets). A f t e r this, animals were r u n 30 min per day for 5 days on each of the following schedules of reinforcem e n t : C R F (1 response per r e i n f o r c e m e n t ) , F R 5 (5 responses per r e i n f o r c e m e n t ) , F R 2 0 , a n d F R 4 0 . Male animals were a d d i t i o n a l l y run t h r e e days each on F R 8 0 , FR120, and FR160. RESULTS
I n j e c t i n g p r e g n a n t mice with p h e n o b a r b i t a l did n o t alter the ability of offspring to acquire the lever response p r o d u c i n g food r e i n f o r c e m e n t • A p p r o x i m a t e l y 90 p e r c e n t of the animals in each group a c q u i r e d the response to a criterion of > 10 responses per session d u r i n g t w o 15 rain self shaping sessions. The r e m a i n i n g animals m e t this criterion a f t e r t w o a d d i t i o n a l 30 m i n sessions. The effect of these t r e a t m e n t s on b e h a v i o r m a i n t a i n e d by t h e various fixed ratio schedules of r e i n f o r c e m e n t was assessed by c o m p a r i n g t h e drug groups of each sex w i t h t h e i r respective SC groups• Mean responses g e n e r a t e d per daily session u n d e r each s c h e d u l e o f r e i n f o r c e m e n t (i.e., over a 5 day test period) were calculated for e a c h animal and served as an i n d e x of its p e r f o r m a n c e on the particular schedule• Since t h e r e was a great deal of individual v a r i a t i o n within e a c h group, particularly on the h i g h e r fixed r a t i o schedules, group t e n d e n c i e s are r e p o r t e d as medians. These data are s u m m a r i z e d in Fig. 1. C o n t r o l animals s h o w e d t h e e x p e c t e d increased res p o n d i n g with increasing d e m a n d s of the r e i n f o r c e m e n t schedule. F o r males, statistical c o m p a r i s o n of individual
600 "
~400, Male
£ 1200" r: o
I000-
800Q. 600-
400-
200
-
T~ea,me0t ~ Schedule
CRF
151 o
o
o
FR5
FR20
o
1
FR40
o
o
FRSO
o FRI20
o
FR(60
800Female ® £c 6 0 0 o
a m 400c ec ~
g ",5
o 2oo-
L3 Treatment Schedule
0._ c° CRF
FR5
FR20
FR40
FIG. 1. Effect of phenobarbital during pregnancy on lever responding of mature offspring maintained under various fixed ratio (FR) schedules of reinforcement. Animals were 90-day-old offspring of C57BL/6J mice injected daily with saline (SC) or phenobarbital at doses of 20 mg (P20), 40 mg (P40), or 80 mg (P80) per kg body weight for the last third of pregnancy. See text for statistical analysis. The bars represent medians determined from Ns of 6 - 1 1 per group. Upper and lower graphs summarize performance of male and female offspring respectively. drug g r o u p s w i t h saline c o n t r o l s via M a n n - W h i t n e y U-Tests [ 13] e s t a b l i s h e d significant r e d u c t i o n s in r e s p o n d i n g at the 95 p e r c e n t level o f c o n f i d e n c e for the P40 and P20 groups b e g i n n i n g at F R 4 0 and e x t e n d i n g t h r o u g h the highest schedule tested. The m e d i a n score for the P80 g r o u p does n o t differ significantly f r o m t h a t of the SC group u n t i l F R 160. The p a t t e r n o f r e s p o n d i n g for SC females was similar to t h a t of males b u t at a r e d u c e d level. F e m a l e animals of the P80 g r o u p r e s p o n d e d at rates s o m e w h a t higher t h a n the SC group on all s c h e d u l e s tested, however, n o n e of these differences are significant. A significant r e d u c t i o n in r e s p o n d i n g b y t h e P40 group c o m p a r e d to e i t h e r the SC or P80 groups was n o t e d for all schedules tested e x c e p t CRF. DISCUSSION
This s t u d y clearly establishes t h a t injecting mice with
MATERNAL PHENOBARBITAL AND O F F S PRI N G BEHAVIOR phenobarbital for the last third of pregnancy produced long lasting behavioral changes in offspring of both sexes. In this ,;tudy, responding maintained by various fixed ratio schedules of reinforcement was reduced. The reduction became more pronounced as the animal was required to increase its response output per unit of reinforcement (i.e., on the higher ratio schedules). We have previously reported [17] that the two higher doses of phenobarbital (80 mg and 40 mg/kg) caused a weight reduction in mature surviving offspring and it could be argued that the reduced weight might contribute to the reduction in response output due lo the increased effort required of the lighter animals to depress the lever. This however, is certainly not a complete explanation since the highest dose produced the greatest weight reduction [17] yet in the current study had the least effect on response output. In addition, female offspring of animals injected with 40 mg/kg of phenobarbital have previously been reported to show a response decrement on a similar task differing only in that the iesponse was merely contact with a metal disc which presumably would be independent of body weight [ 10]. In the same study, it was also shown that the response decrement was due to the schedule and not to the length of time on food deprivation. The reduced effect of the higher drug dose in the current study was certainly unexpected and we currently have no explanation for this finding. Reverse dose-response effects have been reported in other studies on the effects of maternally administered amphetamine or morphine on offspring [2, 12, 17]. It should also be noted that the 80 mg/kg dose of phenobarbital produces a large increase in neonatal mortality [17] hence, selection factor might
1139
account for the reduced behavioral effect noted in surviving offspring. In addition, we have found that 80 mg/kg of this drug injected into adult animals produced behavioral effects different from that of the two lower doses used in this experiment (L. D. Middaugh, unpublished observations). Whereas the 80 mg/kg dose depressed responding maintained by a variable interval schedule of reinforcement, 20 mg and 40 mg/kg doses produced an increased response output. It is thus probable that the different doses are producing different physiological effects on either the pregnant animal or her fetuses. The mechanism mediating these long-term behavioral changes is currently unknown. Possibly, changes in motherinfant interaction essential for normal development [6] may have contributed to the observed behavioral changes in adult offspring. Either abrupt withdrawal from the drug following parturition or the previously reported [17] changes in neonates of animals injected with the drug would be capable of altering this interaction. It is also known that phenobarbital can cross the placenta in both animals [14] and humans I l l ] ; and is reported to be highly concentrated in fetal liver, adrenal, and brain stem tissue. Hence, it is possible that the drug may be interfering with the development of the brain and/or pituitary adrenal system to produce the long-term behavioral effects. Although this study provides no information regarding the mechanism by which phenobarbital injected into pregnant animals produces the long lasting changes in offspring behavior, it does clearly establish the existence of such alterations and should prompt further investigation of the extent and mechanism of the change.
REFERENCES [. Armitage, S. G. The effects of barbiturates on the behavior of the rat offspring as measured in learning and reasoning situations. J. comp. physiol. Psychol. 45: 146-152, 1952. 2. Buchenauer, D., M. Turnbow and M. A. Peters. Effect of chronic methadone administration on pregnant rats and their offspring. J. PharmacoL exp. Ther. 189: 66-71, 1974. 3. Desmond, M. M., R. P. Schwanecke, G. S. Wilson, S. Yasunaga and I. Burgdorff. Maternal barbiturate utilization and neonatal withdrawal symptomatology. J. Pediat. 80: 190-197, 1972. 4. Forfar, J. O. and M. N. Nelson. Epidemiology of drugs taken by pregnant women: Drugs that may affect the fetus adversely. Clin. Pharmac. Ther. 14: 632-642, 1973. 5;. Hill, R. M, Drugs ingested by pregnant women. Clin. Pharmac. Ther. 14: 654-659, 1973. 6. Joffe, J. M. Prenatal Determinants o f Behaviour. New York: Pergamon, 1969. 7. Mauri, N. Effect of maternal medication during pregnancy upon behavioral development of offspring. Tohoku J. exp. Med. 89: 265-272, 1966. 8. McColl, J. D., M. Globus and S. Robinson. Drug induced skeletal malformations in the rat. Experientia 19: 183-184, 1963. 9. Middaugh, L. D., S. T, Drake, J. E. Fraley and J. W. Zemp. Effects of subanesthetic doses of phenobarbital administered to pregnant mice on behavior of offspring. Fedn Proc. 31: 2147, 1972.
10. Middaugh, L. D., C. A. Santos, III and J. W. Zemp. Effects of phenobarbital given to pregnant mice on behavior of mature offspring. Devel. Psychobiol. 18: 305-313, 1975. 11. Ploman, L. and B. H. Persson. On the transfer of barbiturates to the human fetus and their "accumulation in some of its vital organs. J. Obset. Gynaec. Br. Commonw. 64: 706-711, 1957. 12. Seliger, D. L. Effect of prenatal maternal administration of d-amphetamine on rat offspring activity and passive avoidance learning. Physiol. Psychol. 1: 273-280, 1973. 13. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956, pp. 116-127. 14. Villee, C. A. Placental transfer of drugs. Ann. N. Y. Acad. Sci. 123: 237-242, 1965. 15. Wilson, J. T. Caution with phenobarbital. Clin. Pediat. I0: 684-687, 1971. 16. Yeung, C. Y., L. S. Tam, A. Chan and K. H. Lee. Phenobarbitone prophylaxis for neonatal hyperbilirubinemia. Pediatrics 48: 372-376, 1971. 17. Zemp, J. W. and L. D. Middaugh. Some effects of prenatal exposure to d-amphetamine sulfate and phenobarbital on developmental neurochemistry and on behavior. Addict. Dis., 2: 307-331, 1975.