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Effects of ethanol, given during pregnancy, on the offspring dopaminergic system
Phurma, ,,I,,~,~ Bi,,( hcm:~trx & Behavior. Vol. I% pp. 567-570. 1983. Ankho International Inc. Printed in the U.S.A.
Effects of Ethanol, Given During Pregnancy, on the Offspring Dopaminergic System LAURA LUCCHI, l V1TO COVELLI, ~ VESSELIN V. P E T K O V , PIER-FRANCO SPANO AND MARCO TRABUCCHI
Department
of t)harmac'ology and Pharmaco,enosy. Received
23 J u n e
a
University of Milan, Milan. Italy
1983
I.UCCHI, L.. V. COVEI.LI, V. V. PE'I"K()V. P.-F. SPAN(') AND M. TRABUCCHI. E~li'+t~ +{l'etham,I..eiv(,n durin.t,, l, rc~,,nan( v. ,m the ,{tt?~'princ d,,pamincr,,ic s.v.~tcm. PHARMA(_'()I, BIf)(.'HEM BEHAV 19(4) 567-570. 1983.--The fetal alcohol syndrome is characterized by a number of abnormalities consisting of a pre- and post-natal growth deficiency. microcephaly, areas of abnormal nerve cell migration in the brain, mental and psychomotor retardation in children of alcoholic v, omen. These findings may be referred as a tcratogenic effect of ethanol on the central nervous system. In order to investigate the above ethanol-neurotoxic effect the striatal dopaminergic transmission was studied. The dopaminergic turnover "~,as measured b.v 3,4-dihydroxyphcnilacetic acid content and :+H-Spipcrone binding has been carried out to determine dopaminergic receptor alterations induced by chronic ethanol consumption during pregnancy. Our work demonstrates long-lasting modifications of dopaminergic neuromd function after exposure of the experimental animal to ethanol during fetal life. In particular, a decreased receptor function has been observed in rats exposed to ethanol only during the perinatal period. In the same group of rats. diminished receptor activity leads to an enhancement in D()PA(_" content still detectable after a long period from cessation of ethanol treatment. Ncurochemical data arc reinforced by behavioral obscrvatitms. In fact, a significant decrease of spontaneous Ioconmtor activity in the rats chronically treated ,a. ith ethanol during fetal life was observed. ]n addition, the altered response of locomotor activity after drug administration may be ascribed to the modified dopaminergic function. With this experimental approach '.~,c assume that the action of ethanol on the central nervous system may be a marker of its tcratogenic effect.
Fetal alcohol syndrome
Behavior
Rat brain
Dopaminergic transmission
e v e n t s arc possible m a r k e r s o f the t e r a t o g e n i c effect o f ethanol.
I'H[:~ t e r a t o g e n i c h y o f alcohol has b e e n d e m o n s t r a t e d in h u m a n s by clinical o b s e r v a t i o n s a n d b e h a v i o r a l studies 121, 24, 29]. ()n the o t h e r h a n d . several e x p e r i m e n t a l studies on a n i m a l m o d e l s h a v e e x a m i n e d the polential h a z a r d s for the fetus reduced by m a t e r n a l ingestion o f alcohol during pregn a n c y [I, 3 . 9 , 12, 15, 18, 19]. M o r e o v e r , d u r i n g lactation the pups suckled milk c o n t a i n i n g e t h a n o l at the s a m e c o n c e n t r a tion as that o f m a t e r n a l blood [14]. [ - x p e r i m e n t a l d a t a indicate that e t h a n o l m o v e s freely a c r o s s the p l a c e n t a l b a r r i e r w h e r e a s a c e t a l d e h y d e d o e s not [3.13]. In fact, b i o c h e m i c a l o b s e r v a t i o n s s h o w a direct action o f e t h a n o l on the d e v e l o p ing ner~,ous s y s t e m c a u s i n g an i m p a i r m e n t of e m b r y o n i c cellular proliferation [3]. In relation with t h e s e o u t c o m e s , several studies h a v e been d e m o n s t r a t e d that e t h a n o l c o n s u m p tion d u r i n g p r e g n a n c y affects various n e u r o c h e m i c a l par a m e t e r s in offspring brain 14. 10, 20, 28]. T h e p u r p o s e of o u r work is to p r o v i d e f u r t h e r data on Ihe long-lasting a l t e r a t i o n s i n d u c e d by e t h a n o l a d m i n i s t e r e d during p r e g n a n c y at n e u r o n a l level, in particuhtr on brain d o p a m i n c r g i c t r a n s m i s s i o n . In the light of t h e s e results, it m a y be a s s u m e d that c e r t a i n n e u r o c h e m i c a l and b e h a v i o r a l
MErH()D Pregnant Sprague Dav. ley rats were supplied from Charles Rivet'. Calco Italy. Rats ,.,,ere individually caged and e t h a n o l was a d m i n i s t e r e d as the,only available liquid in a 6 ~ a q u e o u s solution (viv), using R i c h t e r tubes. T h e 40 p r e g n a n t rats used were s e p a r a t e d in four g r o u p s (10 a n i m a l s per group). T h e first g r o u p r e c e i v e d e t h a n o l from thc fourth day of p r e g n a n c y to deliver}': the pups were not t r e a t e d with e t h a n o l . A n o t h e r g r o u p of d a m s r e c e i v e d the s a m e solution from the fourth day o f p r e g n a n c y to delivery, and subseqt.ently for t h r e e w e e k s during lactation. A third g r o u p of d a m s was not treated with e t h a n o l during p r e g n a n c y but e t h a n o l was a d m i n i s t e r e d to the m o t h e r s during lactation for t h r e e w e e k s a f t e r birth. T h e body weight o f d a m s from the g r o u p s r e c e i v i n g e t h a n o l r e m a i n e d u n a h e r e d during the t r e a t m e n t v. ith respect to control d a m s receiving an e q u i c a l o r i c s u c r o s e - s o l u t i o n (230+_20, 235--_-_24. 238=18, 2 2 9 ± 2 0 g, respectively).
'Requests for reprints should be addressed to l,aura l,ucchi, Via A. Del Sarto. 21. 20129 Milan. Italy. ella ('linica Neurologica, University of Bari. Bari. Italy. :qnstitute of Ph~,siolt)g). Bulgarian Academy of Sciences. Sofia. Bulgaria.
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FIG. I. Rats locomotor activity (counts/30 rain) after exposure to ethanol during pregnancy (P). during pregnancy and 3 weeks ;trier birth (P+3). and 3 weeks after birth (ABI. The bars represent the ±S.E.M. of 3 determinations each of ,,shich was carried out at the basis o f 5 animals which were individually tested. Statistical significance was calculated by one way A N O V A followed by the tv,.otailed Student's t-test. *p,:0.01 in relation to control values.
The daily intake of liquid was measured and the quantity of ethanol c o n s u m e d was in the range of 9 g kg ' per day for each dam belonged to the three ethanol groups. No weight changes were o b s e r v e d in the treated offspring groups in respect to control pups receiving equicaloric sucrose. At eight weeks of age the spontaneous and drug induced l o c o m o t o r activities of male rats in electronic activity cages were tested. Only the male rats from each treatment group <6 animalsl were included in the behavioral studies. An elecIronic activity cage ( A N I M E X . I+KB F A R A D ) has been used. The A N I M E X sensing unit consists of an oscillator operating at a frequency of 1.2 M H z and a resonance circuit tuned to this frequency. Each rat m o v e m e n t providing a sigmtl large enough to e x c e e d the Schmitt-trigger threshold ( 10/.tA), produces one count. The total counts derived from the rat m o v e m e n t s done have becn considered. After adaptation to the cages, (30 rain) the spontaneous activities of the rats were recorded e v e r y 15 minutes for a period of two hours. In order to determine the l o c o m o t o r activity response to drugs, the first group of rats were removed from the cages and treated acutely with d - a m p h e t a m i n e (3 mg/kg, IP) and the l o c o m o t o r activity was recorded again for one hour after drug administration. A second rat group was treated with haloperidol (0.05 mg/kg IP) acutely after basal l o c o m o t o r activity recording. The durg-induced motility was recorded again for about two hours. Each l o c o m o t o r activity determination was carried out for the four rat groups with the same experimental
protocol. The offspring were killed by decapitation at the end of the 9th week of age (63rd day). Brains were rapidly r e m o v e d and striata dissected as indicated by Glowinski and lversen [ III. In o r d e r to investigate the biochemical modifications, we measured dopamine (DA) metabolism in the striata derived from control and ethanol treated rats. The striatal 3,4d i h y d r o x y p h e n l y a c e t i c acid (D()PAC) concentration was as-
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FIG. 2. Effect of d-amphetamine (3 mglkg, IP) on the locomotor activity (countsll5 rain) of rats exposed to ethanol during fetal life (PI. during pregnancy plus 3 v, eeks after birth ( P * 3 ) and onl~. after birth tAB). Each point represents the mean "-S.E.M. of 3 determinations run v. ith 5 animals v, hich were individually tested. ()no v,a)
AN()VA was used For statistical significance then followed by the two-tailed Student's/-teM. *p<0.01 in respect It) controls.
sayed according to the radioenzymatic micromethod described by Argiolas ('t ,/. 121. :'H-Spiroperidol specific Binding was curried out following the method of Burr (,t ,/. 161 with minor modifications. A tissue suspension corresponding to 200 ~,g/protein was put in each incubation tube containing various concentrations of radioligand. Specific binding was recast, red as the difference in binding obtained after incubation in presence or absence of 10 '+ M haloperidol. :'H( - )-Sulpiride specific binding, as indicated by Spano ct al. [231. was determined in crude synaptic m e m b r a n e fractions corresponding to 15 mg of original tissue per incubation tube (400/.tg/protein). Stereospecific binding was meast,red as the difference in binding obtained in presence or in absence of 10- ~ M( ) sulpiride. K,~ and B,,:,, values are expressed ax nM and fmol/mg protein, respectively. The protein content ssas determined according to Lowry (,t a/. [16]. The statistical significance was calculated by one way A N ( ) V A l\~llowed by Student's/-test. RtSUI.IS
Results on rut spontaneous locomotor activity are summarized in Fig. I showing a significant d e c r e a s e in the basal l o c o m o t o r activity only in rats which had received ethanol during fetal life. In an attempt to understand the neuronal mechanism inducing the decrease in l o c o m o t o r activity, each group of rats was treated with d-amphetamine. In Fig. 2 it ix shown that d-amphetamine increased l o c o m o t o r activity by 140c4 in controls. 85G~ in prenatal period. 87%~ in prenatal period plus 3 weeks after birth and 15r/r in after birth. ()n the other hand. haloperidol (Fig. 3) induced a more pronounced d e c r e a s e in l o c o m o t o r activity in the prenatal period and the prenatal period plus 3 weeks groups (9()'?~). while the effect in the after birth group is not significantly different f r o m that of c o n t r o l s . In order to investigate the biochemical basis of these Be-
EFFI(CTS ()F I-THAN()L GIVEN DURING PREGNANCY
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STRIAI'AI. DOPAC I.EV|-I S (ngmg IlSSU|".) IN RA'IS EXPOSED T() ETHANOL DURING PREGNANCY. I)URING PREGNAN('Y AND IHREE WEt-KS AF'I'ER BIRTH. AN[)'IHREI- WEEKS AFTER BIRIH
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havioral r e s p o n s e s . ,,~e m e a s u r e d I)A m e t a b o l i s m in the striata o f rats r e c e i v i n g e t h a n o l . T a b l e I d e m o n s t r a t e s that an i m p a i r m e n t of d o p a m i n c r g i c activity only o c c u r s after exposure to e t h a n o l d u r i n g the fetal period. In fact. the I)()PA(" levels, v`hich arc a m a r k e r of DA t u r n o v e r , are i n c r e a s e d in relation It) the values of the control g r o u p and to a n i m a l s receiving e t h a n o l only d u r i n g lactation. F u r t h e r r e s e a r c h v`as d e v e l o p e d on the m e c h a n i s m s i n v o l v e d in d o p a m i n e r g i c t r a n s m i s s i o n by s t u d y i n g d o p a m i n e r e c e p t o r f u n c t i o n by mean.,, of various radioligands, such as :~H-Spirt)peridol and :~H( )Sulpiride. T h e results r e p o r t e d in Fig. 4 s h o w that r e c e p t o r i m p a i r m e n t c a u s e d by e t h a n o l only d e v e l o p s d u r i n g the fetal period. In particular. S c a t c h a r d ' s analysis d e m o n s t r a i t s a d e c r e a s e d r e c e p t o r p o p u l a t i o n o f the binding sites characterized by :~H-gpiroperidol. In addition, the d o p a m i n c r g i c r e c o g n i t i o n sites, labelled by : q ' t ( - ) Sulpiride. s e e m to be modified by e t h a n o l e x p o s u r e . In fact. the B ...... values decre;tsed in rats e x p o s e d to e t h a n o l in p r e n a t a l period, w h e r e a s the K,~ values i n c r e a s e d in the case o f high affinity and d e c r e a s e d for the It)v, affinity c o m p o n e n t s only a f t e r t r e a t m e n t dttring the first t h r e e v`ecks o f age.
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I)IS(t'SSlON The consumption of alcohol by pregnant mothers is k n o w n to impair fetal development. The molecular mechanisms by v`hich alcohol causes fetal dysmorphogenesis are not yet u n d e r s t o o d . ()ne o f the c o n s e q u e n t pathological c o n d i t i o n s is the Fetal Alcohol S y n d r o m e v`hich is distinct from o t h e r genetic or nutritionally related fetal d e f e c t s , as d e m o n s t r a t e d in animal studies 171. In fact. v`c h a v e s h o w n that the d e r a n g e m e n t of d o p a m i n e r g i c t r a n s m i s s i o n in rats is still e v i d e n t ~,~hen the food intake is normal. O u r results clearly r e i n f o r c e the h y p o t h e s i s that e t h a n o l has l e r a t o g e n i c effects dttring the d e v e l o p m e n t o f the c e n t r a l n e r v o u s s y s t e m . P r e v i o u s studies on the o r g a n o g e n e s i s c , f the c e n t r a l n e r v o u s s y s t e m [8.221 lead us to the c o n c l u s i o n that e t h a n o l e x e r t s its toxic action on brain d o p a m i n e r g i c n e u r o n s during this time. F o r e x a m p l e , the rat e m b r y o s exp o s e d to e t h a n o l d u r i n g o r g a n o g e n s i s h a v e s h o w n retardation of g r o w t h in vitro [5] and bch;.tvioral i m p a i r m e n t [17]. T h e d e c r e a s e d s p o n t a n e o u s l o c o m o t o r activity in the rats and the a h e r c d r e s p o n s e a f t e r drug a d m i n i s t r a t i o n m a y be c o r r e l a t e d with the b i o c h e m i c a l o b s e r v a t i o n s o n the modified d o p a m i n c r g i c ftmctitm. In particular, the effect o f the
i n c r e a s e d release o f dop.'unine induced by d - a m p h e t a m i n e d e t e c t e d in c o n t r o l rats. is t o w e r in rats treated during |Z~tal life with e t h a n o l , suggesting that e t h a n o l m a y r e d u c e the availability of specific r e c e p t o r proteins. T h e s a m e conclusions m a y also bc r e a c h e d in the case o f haloperidol. It has b e e n s h o w n that w h e n the n u m b e r o f d o p a m i n c r g i c r e c e p t o r s is r e d u c e d . Iov, d o s e s o f h a l o p e r i d o l are sufficient to block l o c o m o t o r activity. T h e c h a n g e s of r e c e p t o r f u n c t i o n after e t h a n o l c o n s u m p tion m a y bc due to a d e r a n g e m e n t in the b i o s y n t h e s i s of the r e c e p t o r s . E t h a n o l a d m i n i s t r a t i o n in the post-natal period i n d u c e s an alteration in the a M n i t y of the d o p a m i n c r g i c rec e p t o r r e c o g n i t i o n sites, labelled by :~H(- )Sulpiride, w h i c h may he d u c to a modification in r e c e p t o r protein c o n f o r m a lion. Various studies h a v e led to the c o n c l u s i o n that e t h a n o l interferes with the genetic code transcription and protein synthesis 125. 26. 271. R e c e p t o r units might bc i m p a i r e d a n d / o r modified in t h e i r o~,~,n protein s t r u c t u r e . T h e d e c r e a s e d rec e p t o r activity leads to an i n c r e a s e in I ) ( ) P A C c o n t e n t that is still d e t e c t a b l e a f t e r a long period from t r e a t m e n t s u s p e n s i o n as a c o m p e n s a t o r y r e s p o n s e It) the loss o f r e c e p t o r function.
I.UCCHI
570
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REFERENCES
I. Abel. E. L. Effects of ethanol on pregnant rats and their offspring, l~syc'hopharma~oh~,,y {Berlin) 57: 5- I I. 1978. 2. Argiolas, A., F. Fadda. E. Stet:anini and G. I,. Gessa. A simple radioenzymatic method for determination of picogram a m o u n t s of 3.4-dihydroxyphenylacetic acid (DOPAC) in rat brain. J .N'~',rochcm 29: 599-601. 1977. 3. Borges, S. and P. D. Lewis. Effects of alcohol on the developing n e r v o u s s y s t e m . "l)'cnds N u m s c i 4: 13-16. 1981. 4. Branchey. I.. and A. J. Friedhoff. The influence of ethanol administred to pregnant rats on tyroxine h y d r o x y l a s e activity of their offspring. /'.sy~ ht~pharmac,lm,,ia 3 2 : 1 5 I- 156, 1973. 5. Brown. N. A., E. H. Goulding and S. Fabro. I:~thanol embryotoxicity: direct effects on m a m m a l i a n e m b r y o s in vitro. .S'ciellte 20~: 573-575. 1979. 6. Burt. D. R.. J. Creese and S. H. Snyder. Antischizophrenic drugs: chronic treatment elevates dopaminc receptors binding in brain. 5"cicn¢ e 206: 854-856. 1979. 7. Chernoff. G. F. The fetal alcohol s y n d r o m e in mice: An animal model. lcr~mdogy 15: 223-229, 1977. 8. Coyle, J. T. and D. Henry. Catecholamines in fetal and newborn rat brain..I Num'ochem 21: 61-67, 1973. 9. Da Silva, A. V., M. J. Ribeiro and J. Masur. Dcvelopmental. behavioral and pharmacological characteristics of rat offspring from m o t h e r s receiving ethanol during gestation or lactation. Din" I~.~'ychohiol 13: 653-660. 1980. 10. Detering, N.. R. M. Collins, R. I... Hawkins. P. T. ()zand and A. K a r a h a s a n . Comparative effects of ethanol and malnutrition tm the d e v e l o p m e n t of catecholamine neurons: changes in neurotransmitter levels../ N e a r , , hem 34:1587-1593, 198(I. 1 I. Glowinski, J. and L. L. lversen. Regional studies of catecholamines in rat brain../ Neurot h e m 13: 655-663, 1966. 12. H e n d e r s o n . G. 1.. R. V. Patwardhan, A. M. H o y u m p a and S. Schender. Fetal alcohol s y n d r o m e : ()verview of pathogcnesis. .'%'cttrobehm" 7",,xicol 7cratol 3: 73-80, 1981. 13. Kes~iniemi, Y. A. and H. W. Sippcl. Placental and foetal metabolism of acetaldehyde in rat. I. Contents of ethanol and acetaldehyde in placenta and foetus of the pregnant rat during ethanol oxidation. Acttt I'harnla~ ~,1 7,,~i~ ol 37: 43-48, 1975. 14. K u m a r , S. and A. K. Rawat. Prolonged ethanol c o n s u m p t i o n by pregmmt and lactating rats amt its effect on cerebral dopamine in the fetus and neonate. Re~ ("OIIIDI Ps)'uhol t'.~v~/ti~th'v I?,~'h(tv 2: 259-278, 1977. 15. Kronick, J. B. Teratogenic effects oteth3,1 alcohol administered to pregnant mice. A m J Ohstct (;vm,~ ol 124: 677-680. 1976.
16. L o w r y . (). H.. N. J. Rosebrough. A. I.. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent..I Biol ("hum 193: 265-27.% 1951. 17. Morrissey, R. E. and N. K. Mottet. Neural tube defects and brain anomalies: a review of selected teratogens and their possible models of a c t i o n . . \ c u r , t o . t h ioh~.~y 2:125-162. 1980. I~. RandalI. C. L. and W. J. raylor. Prenatal ethanol exposure in mice: teratogenic effects, lcr,mh~.~,,v 19:305-31 I. 1979. 19. Randall. ('. 1... W. J. Taylor and D. W. Walker. I-'lhanol induced malformations in mice..,lit ,d~,d ( l i n t'..~p .~l¢'d I: _ 17-__4, 1977. 20. Slotkin. 1. A., S. M. Shanberg and C. M. Kuhn. S)naptic de,.clopmcnt in brains of rats exposed perinatally to ethanol. I:~t,c,'iu,~ti~l .16: I(X)S-I(X)7. 1980. 21. Sokol. R. J. Alchol and abnormal o u t c o m e s of pregnant,,. ( a n 31cdA~so~ .I 125: 143-148, 1981. 22. Sp:.mo. P. F.. K. K u m a k u r a . S. (iovoni and M. Trahncchi. ()ntogenic development of neostriatal dopaminc receptors in the rat. ,I ..\'curo~/win 27: 621-624. 1976. 23. Spano, P. F., M..\'lem,.~. I'. Slefanini. P. Frcsia and M. Trahucchi. Detection of multiple receplors for dopamine. In: Rc~ cp,',, ~ .Ji~r :\~'lll'oll'llll'~lllillcY', HIlt/ I'cplidc II~Jrmonu~. edited b.~, (L Pcpeu, M. J. Kt,har and S. J. tmna. Nov, York: Ra',en Press. 1980, pp. _ 4 _ - . . I 24. Streissguth, P. A.. S. I.andesman-I)xv,,.cr. J. ('. Martin and I). W. Smith. l'cratogenic effects of alcoht~l in h u m a n and laboratory animals..~, icm c 2.119: 353-361. 19~(). 25. Tcv, ari. S. and E. P. Nobel. I'-ffects of ethanol on cerebral protein and ribonucleic acid synthesis. In: Ht,,hcmi.~trv m~d Plmrmcit rdok, v ,~l /:than ,I ',ol I, edited h', E. Majchov, icz and E. P. Noble. New York: Plenum Press. 1979. pp. 541 54~. 26. Tewari, S. and I:.. P. Noble. Ethanol and bran protein -~ nthe,,i,,. lcrai¢z /¢c~ 26: 469-473. 1971. 27. Tewari. N.. I-'.. W. Fleming and E. P. Noble. Aheratiom, m brain RNA metabolism following chronic ethanol ingestion../ :\c,r,,t h e m 24: 561-589. 1975. 2~. Thadani. P. V., ('. l.au. T. A. Slotkin and S. M. Schanbcrg. l-~l'fects of maternal ethanol ingestion on amine uptake into synaptoson1,:s of fetal and neonatal rat brain..I l'h(irnlat ,,l l.~p l h c r 20t1: _)_-_97. 1977. ~t _:). Weiss. (;. and 1.. H c c h l m a n . The h~,peracti,,c child s~ndromc. .~, i c m c 2115: 134~-1354. 197~L
Effects of Ethanol, Given During Pregnancy, on the Offspring Dopaminergic System LAURA LUCCHI, l V1TO COVELLI, ~ VESSELIN V. P E T K O V , PIER-FRANCO SPANO AND MARCO TRABUCCHI
Department
of t)harmac'ology and Pharmaco,enosy. Received
23 J u n e
a
University of Milan, Milan. Italy
1983
I.UCCHI, L.. V. COVEI.LI, V. V. PE'I"K()V. P.-F. SPAN(') AND M. TRABUCCHI. E~li'+t~ +{l'etham,I..eiv(,n durin.t,, l, rc~,,nan( v. ,m the ,{tt?~'princ d,,pamincr,,ic s.v.~tcm. PHARMA(_'()I, BIf)(.'HEM BEHAV 19(4) 567-570. 1983.--The fetal alcohol syndrome is characterized by a number of abnormalities consisting of a pre- and post-natal growth deficiency. microcephaly, areas of abnormal nerve cell migration in the brain, mental and psychomotor retardation in children of alcoholic v, omen. These findings may be referred as a tcratogenic effect of ethanol on the central nervous system. In order to investigate the above ethanol-neurotoxic effect the striatal dopaminergic transmission was studied. The dopaminergic turnover "~,as measured b.v 3,4-dihydroxyphcnilacetic acid content and :+H-Spipcrone binding has been carried out to determine dopaminergic receptor alterations induced by chronic ethanol consumption during pregnancy. Our work demonstrates long-lasting modifications of dopaminergic neuromd function after exposure of the experimental animal to ethanol during fetal life. In particular, a decreased receptor function has been observed in rats exposed to ethanol only during the perinatal period. In the same group of rats. diminished receptor activity leads to an enhancement in D()PA(_" content still detectable after a long period from cessation of ethanol treatment. Ncurochemical data arc reinforced by behavioral obscrvatitms. In fact, a significant decrease of spontaneous Ioconmtor activity in the rats chronically treated ,a. ith ethanol during fetal life was observed. ]n addition, the altered response of locomotor activity after drug administration may be ascribed to the modified dopaminergic function. With this experimental approach '.~,c assume that the action of ethanol on the central nervous system may be a marker of its tcratogenic effect.
Fetal alcohol syndrome
Behavior
Rat brain
Dopaminergic transmission
e v e n t s arc possible m a r k e r s o f the t e r a t o g e n i c effect o f ethanol.
I'H[:~ t e r a t o g e n i c h y o f alcohol has b e e n d e m o n s t r a t e d in h u m a n s by clinical o b s e r v a t i o n s a n d b e h a v i o r a l studies 121, 24, 29]. ()n the o t h e r h a n d . several e x p e r i m e n t a l studies on a n i m a l m o d e l s h a v e e x a m i n e d the polential h a z a r d s for the fetus reduced by m a t e r n a l ingestion o f alcohol during pregn a n c y [I, 3 . 9 , 12, 15, 18, 19]. M o r e o v e r , d u r i n g lactation the pups suckled milk c o n t a i n i n g e t h a n o l at the s a m e c o n c e n t r a tion as that o f m a t e r n a l blood [14]. [ - x p e r i m e n t a l d a t a indicate that e t h a n o l m o v e s freely a c r o s s the p l a c e n t a l b a r r i e r w h e r e a s a c e t a l d e h y d e d o e s not [3.13]. In fact, b i o c h e m i c a l o b s e r v a t i o n s s h o w a direct action o f e t h a n o l on the d e v e l o p ing ner~,ous s y s t e m c a u s i n g an i m p a i r m e n t of e m b r y o n i c cellular proliferation [3]. In relation with t h e s e o u t c o m e s , several studies h a v e been d e m o n s t r a t e d that e t h a n o l c o n s u m p tion d u r i n g p r e g n a n c y affects various n e u r o c h e m i c a l par a m e t e r s in offspring brain 14. 10, 20, 28]. T h e p u r p o s e of o u r work is to p r o v i d e f u r t h e r data on Ihe long-lasting a l t e r a t i o n s i n d u c e d by e t h a n o l a d m i n i s t e r e d during p r e g n a n c y at n e u r o n a l level, in particuhtr on brain d o p a m i n c r g i c t r a n s m i s s i o n . In the light of t h e s e results, it m a y be a s s u m e d that c e r t a i n n e u r o c h e m i c a l and b e h a v i o r a l
MErH()D Pregnant Sprague Dav. ley rats were supplied from Charles Rivet'. Calco Italy. Rats ,.,,ere individually caged and e t h a n o l was a d m i n i s t e r e d as the,only available liquid in a 6 ~ a q u e o u s solution (viv), using R i c h t e r tubes. T h e 40 p r e g n a n t rats used were s e p a r a t e d in four g r o u p s (10 a n i m a l s per group). T h e first g r o u p r e c e i v e d e t h a n o l from thc fourth day of p r e g n a n c y to deliver}': the pups were not t r e a t e d with e t h a n o l . A n o t h e r g r o u p of d a m s r e c e i v e d the s a m e solution from the fourth day o f p r e g n a n c y to delivery, and subseqt.ently for t h r e e w e e k s during lactation. A third g r o u p of d a m s was not treated with e t h a n o l during p r e g n a n c y but e t h a n o l was a d m i n i s t e r e d to the m o t h e r s during lactation for t h r e e w e e k s a f t e r birth. T h e body weight o f d a m s from the g r o u p s r e c e i v i n g e t h a n o l r e m a i n e d u n a h e r e d during the t r e a t m e n t v. ith respect to control d a m s receiving an e q u i c a l o r i c s u c r o s e - s o l u t i o n (230+_20, 235--_-_24. 238=18, 2 2 9 ± 2 0 g, respectively).
'Requests for reprints should be addressed to l,aura l,ucchi, Via A. Del Sarto. 21. 20129 Milan. Italy. ella ('linica Neurologica, University of Bari. Bari. Italy. :qnstitute of Ph~,siolt)g). Bulgarian Academy of Sciences. Sofia. Bulgaria.
567
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FIG. I. Rats locomotor activity (counts/30 rain) after exposure to ethanol during pregnancy (P). during pregnancy and 3 weeks ;trier birth (P+3). and 3 weeks after birth (ABI. The bars represent the ±S.E.M. of 3 determinations each of ,,shich was carried out at the basis o f 5 animals which were individually tested. Statistical significance was calculated by one way A N O V A followed by the tv,.otailed Student's t-test. *p,:0.01 in relation to control values.
The daily intake of liquid was measured and the quantity of ethanol c o n s u m e d was in the range of 9 g kg ' per day for each dam belonged to the three ethanol groups. No weight changes were o b s e r v e d in the treated offspring groups in respect to control pups receiving equicaloric sucrose. At eight weeks of age the spontaneous and drug induced l o c o m o t o r activities of male rats in electronic activity cages were tested. Only the male rats from each treatment group <6 animalsl were included in the behavioral studies. An elecIronic activity cage ( A N I M E X . I+KB F A R A D ) has been used. The A N I M E X sensing unit consists of an oscillator operating at a frequency of 1.2 M H z and a resonance circuit tuned to this frequency. Each rat m o v e m e n t providing a sigmtl large enough to e x c e e d the Schmitt-trigger threshold ( 10/.tA), produces one count. The total counts derived from the rat m o v e m e n t s done have becn considered. After adaptation to the cages, (30 rain) the spontaneous activities of the rats were recorded e v e r y 15 minutes for a period of two hours. In order to determine the l o c o m o t o r activity response to drugs, the first group of rats were removed from the cages and treated acutely with d - a m p h e t a m i n e (3 mg/kg, IP) and the l o c o m o t o r activity was recorded again for one hour after drug administration. A second rat group was treated with haloperidol (0.05 mg/kg IP) acutely after basal l o c o m o t o r activity recording. The durg-induced motility was recorded again for about two hours. Each l o c o m o t o r activity determination was carried out for the four rat groups with the same experimental
protocol. The offspring were killed by decapitation at the end of the 9th week of age (63rd day). Brains were rapidly r e m o v e d and striata dissected as indicated by Glowinski and lversen [ III. In o r d e r to investigate the biochemical modifications, we measured dopamine (DA) metabolism in the striata derived from control and ethanol treated rats. The striatal 3,4d i h y d r o x y p h e n l y a c e t i c acid (D()PAC) concentration was as-
4'5
6'0 Min.
FIG. 2. Effect of d-amphetamine (3 mglkg, IP) on the locomotor activity (countsll5 rain) of rats exposed to ethanol during fetal life (PI. during pregnancy plus 3 v, eeks after birth ( P * 3 ) and onl~. after birth tAB). Each point represents the mean "-S.E.M. of 3 determinations run v. ith 5 animals v, hich were individually tested. ()no v,a)
AN()VA was used For statistical significance then followed by the two-tailed Student's/-teM. *p<0.01 in respect It) controls.
sayed according to the radioenzymatic micromethod described by Argiolas ('t ,/. 121. :'H-Spiroperidol specific Binding was curried out following the method of Burr (,t ,/. 161 with minor modifications. A tissue suspension corresponding to 200 ~,g/protein was put in each incubation tube containing various concentrations of radioligand. Specific binding was recast, red as the difference in binding obtained after incubation in presence or absence of 10 '+ M haloperidol. :'H( - )-Sulpiride specific binding, as indicated by Spano ct al. [231. was determined in crude synaptic m e m b r a n e fractions corresponding to 15 mg of original tissue per incubation tube (400/.tg/protein). Stereospecific binding was meast,red as the difference in binding obtained in presence or in absence of 10- ~ M( ) sulpiride. K,~ and B,,:,, values are expressed ax nM and fmol/mg protein, respectively. The protein content ssas determined according to Lowry (,t a/. [16]. The statistical significance was calculated by one way A N ( ) V A l\~llowed by Student's/-test. RtSUI.IS
Results on rut spontaneous locomotor activity are summarized in Fig. I showing a significant d e c r e a s e in the basal l o c o m o t o r activity only in rats which had received ethanol during fetal life. In an attempt to understand the neuronal mechanism inducing the decrease in l o c o m o t o r activity, each group of rats was treated with d-amphetamine. In Fig. 2 it ix shown that d-amphetamine increased l o c o m o t o r activity by 140c4 in controls. 85G~ in prenatal period. 87%~ in prenatal period plus 3 weeks after birth and 15r/r in after birth. ()n the other hand. haloperidol (Fig. 3) induced a more pronounced d e c r e a s e in l o c o m o t o r activity in the prenatal period and the prenatal period plus 3 weeks groups (9()'?~). while the effect in the after birth group is not significantly different f r o m that of c o n t r o l s . In order to investigate the biochemical basis of these Be-
EFFI(CTS ()F I-THAN()L GIVEN DURING PREGNANCY
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havioral r e s p o n s e s . ,,~e m e a s u r e d I)A m e t a b o l i s m in the striata o f rats r e c e i v i n g e t h a n o l . T a b l e I d e m o n s t r a t e s that an i m p a i r m e n t of d o p a m i n c r g i c activity only o c c u r s after exposure to e t h a n o l d u r i n g the fetal period. In fact. the I)()PA(" levels, v`hich arc a m a r k e r of DA t u r n o v e r , are i n c r e a s e d in relation It) the values of the control g r o u p and to a n i m a l s receiving e t h a n o l only d u r i n g lactation. F u r t h e r r e s e a r c h v`as d e v e l o p e d on the m e c h a n i s m s i n v o l v e d in d o p a m i n e r g i c t r a n s m i s s i o n by s t u d y i n g d o p a m i n e r e c e p t o r f u n c t i o n by mean.,, of various radioligands, such as :~H-Spirt)peridol and :~H( )Sulpiride. T h e results r e p o r t e d in Fig. 4 s h o w that r e c e p t o r i m p a i r m e n t c a u s e d by e t h a n o l only d e v e l o p s d u r i n g the fetal period. In particular. S c a t c h a r d ' s analysis d e m o n s t r a i t s a d e c r e a s e d r e c e p t o r p o p u l a t i o n o f the binding sites characterized by :~H-gpiroperidol. In addition, the d o p a m i n c r g i c r e c o g n i t i o n sites, labelled by : q ' t ( - ) Sulpiride. s e e m to be modified by e t h a n o l e x p o s u r e . In fact. the B ...... values decre;tsed in rats e x p o s e d to e t h a n o l in p r e n a t a l period, w h e r e a s the K,~ values i n c r e a s e d in the case o f high affinity and d e c r e a s e d for the It)v, affinity c o m p o n e n t s only a f t e r t r e a t m e n t dttring the first t h r e e v`ecks o f age.
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FI(L 4. Scatchard anal3,xis of (,1 'H-Spiropcridol and (b):q-tl-I Sulpiridc specific bindingx in striatal membranes obtained from control rats IO--O) and ethanol exposed rats during pregnancy (/.--._".L during pregnant,, and three weeks after birlh ( ; - - ,) or three weeks after birlh IA--_A). Pointx :ire the mean-' S.I':.M of three experiments run in triplicate in v.hich 5 ,mimalx per group wcrc used.
I)IS(t'SSlON The consumption of alcohol by pregnant mothers is k n o w n to impair fetal development. The molecular mechanisms by v`hich alcohol causes fetal dysmorphogenesis are not yet u n d e r s t o o d . ()ne o f the c o n s e q u e n t pathological c o n d i t i o n s is the Fetal Alcohol S y n d r o m e v`hich is distinct from o t h e r genetic or nutritionally related fetal d e f e c t s , as d e m o n s t r a t e d in animal studies 171. In fact. v`c h a v e s h o w n that the d e r a n g e m e n t of d o p a m i n e r g i c t r a n s m i s s i o n in rats is still e v i d e n t ~,~hen the food intake is normal. O u r results clearly r e i n f o r c e the h y p o t h e s i s that e t h a n o l has l e r a t o g e n i c effects dttring the d e v e l o p m e n t o f the c e n t r a l n e r v o u s s y s t e m . P r e v i o u s studies on the o r g a n o g e n e s i s c , f the c e n t r a l n e r v o u s s y s t e m [8.221 lead us to the c o n c l u s i o n that e t h a n o l e x e r t s its toxic action on brain d o p a m i n e r g i c n e u r o n s during this time. F o r e x a m p l e , the rat e m b r y o s exp o s e d to e t h a n o l d u r i n g o r g a n o g e n s i s h a v e s h o w n retardation of g r o w t h in vitro [5] and bch;.tvioral i m p a i r m e n t [17]. T h e d e c r e a s e d s p o n t a n e o u s l o c o m o t o r activity in the rats and the a h e r c d r e s p o n s e a f t e r drug a d m i n i s t r a t i o n m a y be c o r r e l a t e d with the b i o c h e m i c a l o b s e r v a t i o n s o n the modified d o p a m i n c r g i c ftmctitm. In particular, the effect o f the
i n c r e a s e d release o f dop.'unine induced by d - a m p h e t a m i n e d e t e c t e d in c o n t r o l rats. is t o w e r in rats treated during |Z~tal life with e t h a n o l , suggesting that e t h a n o l m a y r e d u c e the availability of specific r e c e p t o r proteins. T h e s a m e conclusions m a y also bc r e a c h e d in the case o f haloperidol. It has b e e n s h o w n that w h e n the n u m b e r o f d o p a m i n c r g i c r e c e p t o r s is r e d u c e d . Iov, d o s e s o f h a l o p e r i d o l are sufficient to block l o c o m o t o r activity. T h e c h a n g e s of r e c e p t o r f u n c t i o n after e t h a n o l c o n s u m p tion m a y bc due to a d e r a n g e m e n t in the b i o s y n t h e s i s of the r e c e p t o r s . E t h a n o l a d m i n i s t r a t i o n in the post-natal period i n d u c e s an alteration in the a M n i t y of the d o p a m i n c r g i c rec e p t o r r e c o g n i t i o n sites, labelled by :~H(- )Sulpiride, w h i c h may he d u c to a modification in r e c e p t o r protein c o n f o r m a lion. Various studies h a v e led to the c o n c l u s i o n that e t h a n o l interferes with the genetic code transcription and protein synthesis 125. 26. 271. R e c e p t o r units might bc i m p a i r e d a n d / o r modified in t h e i r o~,~,n protein s t r u c t u r e . T h e d e c r e a s e d rec e p t o r activity leads to an i n c r e a s e in I ) ( ) P A C c o n t e n t that is still d e t e c t a b l e a f t e r a long period from t r e a t m e n t s u s p e n s i o n as a c o m p e n s a t o r y r e s p o n s e It) the loss o f r e c e p t o r function.
I.UCCHI
570
1-7 A I . .
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