Alcohol 41 (2007) 433e439

Effect of gestational ethanol exposure on long-term memoryformation in newborn chicks

Venugopal Raoa, Joydeep D. Chaudhurib,*aDepartment of Anatomy, University Malaysia Sarawak, Sarawak, Malaysia

bDepartment of Anatomy, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India

Received 1 February 2007; received in revised form 23 April 2007; accepted 24 April 2007

Abstract

Fetal alcohol syndrome (FAS), a condition occurring in some children of mothers who have consumed alcohol during pregnancy, ischaracterized by craniofacial malformations, and physical and mental retardation. It is significant that even children with history of gesta-tional ethanol exposure but relatively unaffected overall IQ performance, often exhibit learning difficulties and behavioral problems, sug-gestive of impaired memory formation. Hence, the specific aim of this study was to examine memory formation in chicks exposed toethanol during early gestation toward the understanding of neurobehavioral disturbances in FAS. Chicks were exposed to alcohol on ges-tational days 1e3 by injection of ethanol into the airspace of freshly fertilized eggs. The effects of prenatal ethanol on physical growth anddevelopment, and memory formation were studied. The one-trial passive avoidance learning paradigm in 1-day-old chicks was used to studymemory formation in these chicks. It was observed that chick embryos exposed to 10% ethanol on gestational days 1e3 had significantreduction in all body parameters when compared with appropriate controls. Further, ethanol-exposed chick embryos had significantly im-paired (P ! .05) long-term memory (LTM) formation after training, though short-term or intermediate-term memory formation was unim-paired. Thus, the findings of the current study demonstrate the detrimental effects of ethanol exposure during early pregnancy on developingchick embryos in general and on memory formation in particular. Hence, it is suggested that impairment in LTM could be a fundamentalmechanism for learning disorders and neurobehavioral abnormalities observed in FAS. � 2007 Elsevier Inc. All rights reserved.

Keywords: Memory impairment; Fetal alcohol syndrome; FAS

Introduction

Gestational ethanol exposure can cause physical growthretardation, neurodevelopmental anomalies, and character-istic craniofacial anomalies that were collectively termedas Fetal Alcohol Syndrome (FAS) by Jones and Smith(1973). Exposure to alcohol in utero has been implicatedas the most common cause of preventable mental retarda-tion in the United States (Abel and Sokol, 1991), andoverall an estimated 1% of all newborns are affected bygestational ethanol exposure (Sampson et al., 1997).Though no safe levels of alcohol consumption duringpregnancy have yet been demonstrated, conservative esti-mates have shown that approximately 13e20% of womenconsume alcohol during pregnancy (Tough et al., 2006),and health warnings to avoid alcohol consumption during

* Corresponding author. Department of Anatomy, Midwestern Univer-

sity, Agave 201F, 19555 N, 59th Avenue, Glendale, AZ 35303, USA. Tel.:

þ1-623-572-3332; fax: þ1-623-572-3679.

E-mail address: jchaud@midwestern.edu (J.D. Chaudhuri).

0741-8329/07/$ e see front matter � 2007 Elsevier Inc. All rights reserved

doi: 10.1016/j.alcohol.2007.04.012

pregnancy are often disregarded (Kaskutas, 2000). Further,many women continue to consume alcohol during earlypregnancy without realizing that they are pregnant (Ed-wards and Werler, 2006; Floyd et al., 1999; Toughet al., 2006). The detrimental effects of drinking duringthe early gestational period have been demonstrated instudies by Streissguth et al. (1980) who have observeda stronger negative effect induced by drinking duringearly pregnancy as compared to drinking in the fifthmonth of pregnancy.

Children exposed to alcohol in the gestational period of-ten demonstrate learning, memory, and behavioral prob-lems without apparent physical deformities (Rasmussen,2005), and a relatively unaffected overall IQ performance(Mattson et al., 1996). The reported neurobehavioral defi-cits include impairment in acquisition of verbal informa-tion, word comprehension, and naming abilities anddeficits in visuospatial processing (Riley and McGee,2005), suggesting that defects in memory formation andconsolidation could be a fundamental mechanism in learn-ing disorders in FAS.

.

434 V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

Impaired learning and memory formation has also beenreported in animal models of FAS. Spatial learning wasfound to be significantly impaired following a single in-stance of gestational ethanol exposure in early pregnancy(Matthews and Simson, 1998; Summers et al., 2006) andethanol exposure during the entire pregnancy (Gianoulakis,1990). However, in all these models, learning and memoryfunctions were tested only in adulthood when other con-founding influences such as influences of environment en-richment are also operative (Ickes et al., 2000; Loskutovaand Dubrovina, 2004). There are no reports of study ofthe effect of gestational ethanol exposure on memory for-mation immediately after birth, and hence the goal of thisstudy was to investigate memory formation in 1-day-oldchicks following early ethanol exposure.

The chick is a suitable model for studies on FAS, since itallows for the determination of direct effects of ethanol inearly pregnancy (when administered before day 10), with-out the influences of maternal undernutrition, concurrentnicotine or drug use, acetaldehyde formation, or impairedplacental function (Bupp Becker and Shibley, 1998; Wilsonet al., 1984). Further, studies on early neural growth and de-velopment in chick embryos are feasible due to a relativelylow mortality rate, and ethanol-exposed chicks demonstrategrowth suppression early in development (Pennington,1991), and experience learning deficits after hatching evenwithout evident growth suppression (Means et al., 1989).

The day-old chick is also a powerful model system forthe study of mechanisms of early memory formation, sincetraining of these chicks accurately replicates events paralle-ling that in human brain such as early gene expression, andmorphological, biochemical, and pharmacological changes(Rose and Stewart, 1999). In this study, it is proposed to usethe one-trial passive avoidance learning (PAL) paradigm fortesting of early memory formation in chicks. Since in thePAL paradigm, training is administered in 1-day-old birds,it takes only a single brief trial for memory formation, andobservations are not confounded by other variables inducedby alternate learning mechanisms that occur during normalbehavioral development and establishment of learning(Stewart and Banks, 2006). This model has proven to beuseful in the study of mechanisms of memory formationfollowing prenatal hypoxia and malnutrition (Rodrickset al., 2004), and administration of morphine (Che et al.,2005). It has also been used to study the pharmacologicaleffects of drug administration on memory formation (Steeleet al., 1996; Stewart and Banks, 2006).

The PAL paradigm in chicks is based on the spontaneoustendency of chicks to peck at objects in their immediate en-vironment, and uses a distasteful stimulus to investigateneural mechanisms underlying learning and memory for-mation (Richard and Davies, 2000). In this task, a chickis presented with a small visually conspicuous bead thatit normally pecks spontaneously. If a similar bead is coatedwith water, the chick will continue to peck at it on subse-quent presentation. However, if the bead is coated with

a distasteful substance such as methyl anthranilate (MeA),the chick will learn in a single trial to avoid a visually sim-ilar bead at a future presentation even when uncoated witha distasteful substance (Richard and Davies, 2000). ThePAL paradigm proposed by Ng and Gibbs (1991), consistsof three sequentially dependent stages of memory forma-tion that are designated as short-term memory (STM; lastsup to 15 min), intermediate-term memory (ITM; lasting upto 15e55 min), and long-term memory (LTM; lasting morethan 55 min).

The specific aim of this study was to examine the effectsof ethanol exposure, while minimizing embryo morbidityand mortality, during early pregnancy on physical develop-ment in general and on the different stages of memory for-mation in particular in a chick model of FAS.

Material and methods

Handling of eggs

Freshly fertilized White Leg Horn chick eggs, type Sin-gle Comb were obtained from the local hatchery and di-vided into three Groups I, II, and III. The weight of theeggs ranged from 0.051 to 0.057 kg with a mean weightof 0.053 kg. There was no variability in the weight of theeggs, and 45 eggs each were randomly assigned to GroupsI, II, and III.

The fertilized eggs were obtained within 6 h of beinglaid, and cleaned with 70% ethanol. Eggs in Group I wereleft untouched and constituted the absolute control group,in which no experimental manipulations were carried out.In the eggs in Group II, a small hole was made at the bluntend of each egg, under a laminar flow hood, and 250 ml ofchick Ringer’s solution (sodium chloride [123 mM], potas-sium chloride [5 mM], and calcium chloride [1.6 mM], pH7.2) was injected directly into the air sac of these eggs.Eggs in Group III were similarly injected with a 10% solu-tion of ethanol in chick Ringer’s solution. The opening inthe eggshells was sealed with wax. The injections were car-ried out on embryonic days 1e3 (E1eE3), the day the eggswere laid being considered as E0. All injections were car-ried out in a horizontal position, so that ethanol exposurein the chick embryos was diffusion mediated. The ethanolused for injection was the one used in a previous study inthis laboratory (Chaudhuri, 2004), and conformed to the re-quirements of Absolute (Anhydrous) Alcohol, Indian Stan-dard 321 of 1964, and was obtained from a commercialsupplier (Ishika International, Mumbai, India).

Eggs were incubated in an egg incubator (Model EI1999, Capital Engineering Corporation, New Delhi, India)in a horizontal position at 37�C and relative humidity of70% for 3 weeks, and the eggs were manually rotated twicea day. The guidelines set up by the All India Institute ofMedical Sciences Animal Care and Use Committee wasfollowed concerning the treatment and disposal of embryos.

435V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

Examination of chicks

Chicks from all groups (Groups I, II, and III) were recov-ered after full gestational period of 21 days. The eggshellswere manually cracked, and the chicks were allowed toemerge from their shells. The chicks were examined for grossdeformities, and body weight, crown rump length, and headcircumference were measured, according to the proceduredescribed by Pennington and Kalmus (1987).

Training and testing of memory formation in day-old chicks

After hatching, chicks were housed in pairs for 24 h ina standard housing pen 45� 40� 60 cm in size, maintainedat 23e30�C and illuminated by an overhead 25-W light-bulb. Twenty-four hours after hatching, chicks were trainedand tested based on a modification of protocols describedby Che et al. (2005), and Richard and Davies (2000).Chicks were placed in 20� 20� 25-cm training pens andleft for 30 min to settle down, and the training and testingwere carried out in three phases namely, pretraining, train-ing, and retention.

At the pretraining phase all the chicks were presentedwith a glass bead, 2 mm in diameter and dipped in water,for 120 s to encourage the natural pecking response. An an-gled mirror placed above the arena allowed the experi-menter to position the bead a few millimeters in front ofthe chick’s beak, without permitting the chick to see the ex-perimenter. The second pretraining was carried out after20 min when the chicks were again presented for 120 s witha similar red bead soaked in water. The number of pecks byeach chick was recorded, and chicks that failed to peck atthe training bead within 30 s were excluded from subse-quent analysis. The training phase involved presentationof a red bead visually identical to that used in pretraining,but dipped in 100% concentration of bitter tasting MeA, for60 s. The number of pecks was again recorded and the pres-ence or absence of a disgust response (head shaking, eyeclosure, and beak wiping) was noted. If a chick pecked ata MeA-coated bead but failed to show a disgust response,it was not subjected to subsequent investigation.

After training, the chicks were coded so that the investiga-tor conducting the retention was blinded to the nature of thetreatment to which the chicks had been exposed. The reten-tion trial was conducted for 15 (STM), 30 (ITM), and120 min (LTM) after the training phase, and chicks were pre-sented with a red bead visually identical to the one in thetraining procedure for 60 s. The normal response of chicksis to avoid pecking at the red bead on subsequent presenta-tions. The number of pecks of each chick in each groupwas recorded. An avoidance ratio (AR) for each chick wascalculated as described by Che et al. (2005). The AR was cal-culated as follows: the number of pecks at the red pretrainingbead divided by the number of pecks at the red retention trialbead plus the number of pecks at the red pretraining bead(i.e., AR 5 pecks pre-/pecks pre-þ pecks retention trial).

Statistical analysis

All data were analyzed using a statistical package for so-cial sciences (SPSS 10.0). A one-factor ANOVA was per-formed on data regarding the body parameters and on themean number of pecks at the red pretraining bead of chicksin control chicks, chicks injected with chick Ringer’s solu-tion and 10% ethanol. A two-way mixed ANOVA was usedto analyze results of memory testing at 15, 30, and 120 minin all the three groups. Post hoc analysis was done using theConferring test. A P value of !.05 was considered statisti-cally significant. Data are presented as mean 6 S.D.

Results

Gross anomalies and body parameters

After hatching, all the chicks were examined for grossanomalies, and dry body weight, crown rump length, andhead circumference were recorded. Infertility was notedin the control group (Group I), and also in chicks injectedwith chick Ringer’s solution (Group II) and 10% ethanol(Group III), and a total of 42, 43, and 43 live chicks wererecovered from Groups I, II, and III, respectively. Therewere no statistically significant differences in body param-eters among individual chicks in each of the separategroups. Hence, a single representative mean of the mea-surements of each body parameter for each group is pre-sented in Table 1.

A significant reduction (P ! .05) in mean body parame-ters was noted in chicks exposed to ethanol, as compared tochicks in the control group and chicks exposed to chickRinger’s solution. There was no difference in body param-eters between control chicks and chicks exposed to chickRinger’s solution.

Memory formation

The training for memory formation commenced with 42,43, and 43 live chicks in Groups I, II, and III, respectively.

Table 1

Body parameters (mean 6 standard deviation), infertility, and mortality of

chick embryos exposed to ethanol

Groups I (n 5 36) II (n 5 36) III (n 5 38)

Head circumference (cm) 4.6 6 0.2 4.8 6 0.3 3.8 6 0.2*

Body weight (g) 22.6 6 0.3 23.1 6 0.4 18.4 6 0.3*

Crown rump length 8.1 6 0.1 8.3 6 0.3 5.7 6 0.2*

Percentage of unfertile eggs 8.8 11.1 11.1

Percent mortality 0 0 0

Groups I, II, and III represent chicks in the control group (no manipu-

lations carried out), chicks exposed to chick Ringer’s solution (E1eE3),

and chicks injected with 10% ethanol (E1eE3), respectively. *Signifi-

cantly different (P ! .05) from values for chicks in Groups I and II in

which no manipulations were carried out and in those exposed to equivo-

lume chick Ringer’s solution. There were no significant differences in body

parameters in chicks in Groups I and II.

436 V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

During the pretraining phase, four chicks each in Groups Iand II, and two chicks in Group III failed to peck the redbead within 30 s and were subsequently excluded fromthe study. During the training phase, two chicks in GroupI, and three chicks each in Groups II and III failed to showan adequate disgust response after pecking at MeA-coatedbead and were subsequently excluded from the retentiontrial. Thus, the results reported are from 36 live chicks eachin Groups I and II, and 38 chicks in Group III.

There was no significant difference (P O .05) in thenumber of pecks at the red bead during pretraining amongthe chicks in the control group, and also in chicks injectedwith chick Ringer’s solution and 10% ethanol (Fig. 1).Hence, the spontaneous tendency of young chicks to peckat objects was not affected by prenatal ethanol exposure.

Using a two-way mixed ANOVA, the two main effectsstudied were time of testing after training and ethanol expo-sure. The dependent variable was the AR in all analyses.The within-factor repeated measures variable was time oftesting, which showed a statistically significant main effect[F(2, 214) 5 142.0, P ! .05]. The between-factor variablewas ethanol exposure, and a statistically significant maineffect [F(2, 107) 5 134.7, P ! .05] was also observed. Inaddition, the interaction effect between time of testingand ethanol exposure was statistically significant [F(4,214) 5 127.4 P ! .05].

Post hoc analysis using the Bonferroni test revealed sig-nificant differences in AR at different times following train-ing for the chicks injected with 10% ethanol, but not for thecontrol chicks or chicks injected with chick Ringer’s solu-tion. Ethanol-exposed chicks, when tested at 120 min fol-lowing training had statistically significantly lower ARthan they did at 15 and 30 min posttraining (P ! .05 forboth).

Thus, the three groups of chicks were able to retain in-formation regarding the distasteful taste of the red bead af-ter 15 (STM) and at 30 min (ITM) and avoided it. However,chicks injected with ethanol when tested after 120 min

Fig. 1. Mean number of pecks at the red pretraining bead of chicks in

control chicks (n 5 36), chicks injected with chick Ringer’s solution

(n 5 36), and with 10% ethanol (n 5 38). There were no significant differ-

ences (P ! .05) in the number of pecks at the red pretraining bead between

the groups. T-bars represent S.D.

(LTM) failed to retain this information, as assessed by themean AR, in contrast to control chicks or those injectedwith chick Ringer’s solution (Fig. 2).

Discussion

The results of this study demonstrate that ethanol expo-sure during early pregnancy causes growth retardation, andare similar to previous reports in other experimental modelsof FAS (Boyd et al., 1984; Chaudhuri, 2004; Penningtonet al., 1983) and in humans (Halvorsen et al., 2006), andhence will not be discussed further. The striking observa-tion in this study is that LTM formation is significantly im-paired in chicks following prenatal ethanol exposure duringearly gestation, while STM and ITM formation isunaffected.

Ethanol is an amphiphathic compound that distributesuniformly in both the membrane and the cytoplasm of thecell (Wilson et al., 1984). Similar to a previous study (Cart-wright and Smith, 1995), chick embryos exposed to 10%ethanol on the first 3 days of gestation, demonstrated signif-icant growth retardation without an increase in mortality.Further, eggs were injected during the first 3 days of gesta-tion, which has been reported to be the period of active neu-rogenesis in the developing chick embryos (Freeman andVince, 1974). Hence, this chick model of FAS is represen-tative of the current human scenario where many womenconsume alcohol during early gestation without realizingthat they are pregnant.

The effect of ethanol exposure during early gestation onmemory formation in chicks was the particular focus of thisstudy. The PAL paradigm was used in this study, and ittakes advantage of the innate tendency that chicks that have

Fig. 2. Mean avoidance ratio (AR) for the passive avoidance learning

tasks at 15 (STM), 30 (ITM), and 120 min (LTM) training. After 15 and

30 min following training, there were no significant differences in AR

among the control groups (n 5 36), chicks injected with chick Ringer’s so-

lution (n 5 36), and chicks injected with 10% ethanol (n 5 38). At 120 min

after training, chicks injected with 10% ethanol exhibited significantly de-

creased (P ! .05) AR than control chicks, but there were no significant dif-

ferences of AR between chicks injected with chick Ringer’s solution and

control chicks (P ! .05). T-bars represent S.D. An asterisk denotes a signif-

icant difference (P ! .05) when compared with other groups.

437V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

pecked at bead coated with a distasteful substance willavoid a similar dry bead for at least 24 h subsequently(Rose, 2000). This learning paradigm has the advantageof being rapid, sharply timed, and well defined, and an ac-curate response also controls for effects of ethanol on atten-tion and visual and motor processes. To ensure an adequatestimulus for LTM formation, a high concentration (100%)of MeA was used since it has been reported that low con-centrations of MeA are at times only able to induce STMformation and low levels of memory-associated gene ex-pression (Edelheit and Meiri, 2004).

In the PAL paradigm, the first stage of memory forma-tion, termed STM, is formed 5 min after training anddecays after 15 min (Bennett et al., 2002). Although theprecise mechanism of STM formation has not been de-scribed, it has been suggested that STM may require neuro-nal hyperpolarization resulting from increased Kþ

conductance following neural input (Delord et al., 2000).Following the decay of STM, a second stage of memory,ITM, emerges that is evident after 20 min of training thatsubsequently decays after 50 min (Malleret et al., 2001).ITM formation appears to depend on hyperpolarization re-sulting from Naþ/Kþ/ATPase activity (Stough et al., 2006).The final stage, termed LTM, involves an irreversible pas-sage via labile phases to the stable form that has been dem-onstrated to persist for at least 24 h following exposure toa distasteful stimulus (Rose, 2000). It has been suggestedto involve a closely regulated cascade of events that hasbeen postulated to be initiated by glutamate release and en-gages a series of synaptic transients including increasedcalcium and upregulation of NMDA-glutamate receptors(Che et al., 2005). A significant event in LTM formationin the PAL paradigm involves two successive waves of pro-tein synthesis in selected brain areas of the chick. The firstwave occurring within the first 2 h is associated withincreased neuronal expression of fos and jun proteins fol-lowing activation of the Intermediate Early Genes, c-fos,and c-jun (Rose, 2000). This is followed by a second waveof protein synthesis that characteristically occurs after5e6 h and involves the synthesis of Neural Cell AdhesionMolecule and neuron/glial Cell Adhesion Molecule(Scholey et al., 1993, 1995). Together these events arethought to be necessary for induction of permanent struc-tural changes associated with the consolidation of LTM(Patel et al., 1988; Patel and Stewart, 1988; Rose, 1991).Most of these processes have also been observed in otherlearning paradigms in mammals, and are identical withthe induction phase of NMDA-dependent LTM formation(Ng and Gibbs, 1991; Rose, 2000).

The optimal functioning of LTM involves the ability toretain and consolidate memory, and accurately retrievethe stored information after a certain time period. In thePAL paradigm, these functions are characteristically testedat 5e6 h (Campbell and Edwards, 2006; Freeman andRose, 1999; Salinska, 2006), and 24 h after training (Hornet al., 1985; Mileusnic et al., 2005; Patel et al., 1988; Patel

and Stewart, 1988; Rose, 1991), that correspond to a secondwave of protein synthesis and morphological alterations.However, in this study memory function was tested onlyat 120 min, which represents the earliest phase in LTM for-mation (Rose, 2000). Hence, based on the current results itis only possible to comment that prenatal ethanol exposurecauses impairment in LTM formation. An accurate insightinto specific mechanisms of ethanol induce impairment ofLTM would require investigations into patterns of proteinexpression (Rose, 2000) and synaptic morphology (Patelet al., 1988; Patel and Stewart, 1988), which was not theaim of the study.

Thus, while this study demonstrates impaired LTM for-mation following prenatal ethanol exposure, the specificmechanisms of impairment following prenatal ethanol ex-posure cannot be examined by the design of this study.However, of particular relevance, is that expression ofc-fos and c-jun genes, a characteristic feature of LTM for-mation in the PAL paradigm (Ambalavanar et al., 1999;Anokhin et al., 1991; Freeman and Rose, 1999; Rose andStewart, 1999), has been reported to be significantly re-duced in rats exposed to ethanol during gestation (Nagaharaand Handa, 1995; Poggi et al., 2003). Moreover, consider-ing the interrelationship between intracellular calciumstores and LTM formation (Salinska et al., 2001), it is sig-nificant that prenatal ethanol exposure has been demon-strated to cause disruption of intracellular calcium levels(Garic-Stankovic et al., 2005). Further, functions of gluta-mate receptors, another critical mechanism for memoryconsolidation (Salinska, 2006), have also been shown tobe blocked by prenatal ethanol exposure (Olney et al.,2002). Thus, the cumulative evidence suggests that prenatalethanol exposure during early pregnancy could causeimpairment of LTM formation by one or a multitude of pro-cesses, and defects in memory formation and consolidationcould be a fundamental mechanism in learning disorders inFAS.

In summary, the results of the present study demonstratethe effects of drinking during early pregnancy on the devel-oping fetus in general and memory formation in particular.It also contradicts the general belief among some womenthat drinking during early pregnancy is not detrimental tothe developing fetus (Whitlock et al., 2004). Thus, the pres-ent study may provide an important model to investigate thecellular and molecular mechanisms underlying cognitivedeficits resulting from prenatal ethanol exposure.

References

Abel, E. L., and Sokol, R. J. (1991). A revised conservative estimate of the

incidence of FAS and its economic impact. Alcohol. Clin. Exp. Res.

15, 514–524.

Ambalavanar, R., McCabe, B. J., Potter, K. N., and Horn, G. (1999).

Learning-related fos-like immunoreactivity in the chick brain: time-

course and co-localization with GABA and parvalbumin. Neuroscience

93, 1515–1524.

438 V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

Anokhin, K. V., Mileusnic, R., Shamakina, I. Y., and Rose, S. P. (1991).

Effects of early experience on c-fos gene expression in the chick fore-

brain. Brain Res. 544, 101–107.

Bennett, P. C., Schmidt, L., Lawen, A., Moutsoulas, P., and Ng, K. T.

(2002). Cyclosporin A, FK506 and rapamycin produce multiple, tem-

porally distinct, effects on memory following single-trial, passive

avoidance training in the chick. Brain Res. 927, 180–194.

Boyd, J. W., Kalmus, G. W., and Pennington, S. N. (1984). Ethanol-

induced inhibition of chick brain growth. Alcohol. Clin. Exp. Res. 8,

343–346.

Bupp Becker, S. R., and Shibley, I. A. Jr. (1998). Teratogenicity of ethanol

in different chicken strains. Alcohol Alcohol. 33, 457–464.

Campbell, E., and Edwards, T. (2006). Zaprinast consolidates long-term

memory when administered to neonate chicks trained using a weakly

reinforced single trial passive avoidance task. Behav. Brain Res. 169,

181–185.

Cartwright, M. M., and Smith, S. M. (1995). Increased cell death and re-

duced neural crest cell numbers in ethanol-exposed embryos: partial

basis for the fetal alcohol syndrome phenotype. Alcohol. Clin. Exp.

Res. 19, 378–386.

Chaudhuri, J. D. (2004). Effect of a single dose of ethanol on developing

skeletal muscle of chick embryos. Alcohol 34, 279–283.

Che, Y., Sun, H., Tan, H., Peng, Y., Zeng, T., and Ma, Y. (2005). The effect

of prenatal morphine exposure on memory consolidation in the chick.

Neurosci. Lett. 380, 300–304.

Delord, B., Baraduc, P., Costalat, R., Burnod, Y., and Guigon, E. (2000). A

model study of cellular short-term memory produced by slowly inacti-

vating potassium conductances. J. Comput. Neurosci. 8, 251–273.

Edelheit, S., and Meiri, N. (2004). Cyclin S: a new member of the cyclin

family plays a role in long-term memory. Eur. J. Neurosci. 19, 365–

375.

Edwards, E. M., and Werler, M. M. (2006). Alcohol consumption and time

to recognition of pregnancy. Matern. Child Health J. 10, 467–472.

Floyd, R. L., Decoufle, P., and Hungerford, D. W. (1999). Alcohol use

prior to pregnancy recognition. Am. J. Prev. Med. 17, 101–107.

Freeman, B. M., and Vince, M. A. (1974). Development of the Avian Em-

bryo: A Behavioral and Physiological Study. London, England: Chap-

man and Hall.

Freeman, F. M., and Rose, S. P. (1999). Expression of fos and jun proteins

following passive avoidance training in the day-old chick. Learn. Mem.

6, 389–397.

Garic-Stankovic, A., Hernandez, M. R., Chiang, P. J., Debelak-

Kragtorp, K. A., Flentke, G. R., Armant, D. R., and Smith, S. M.

(2005). Ethanol triggers neural crest apoptosis through the selective ac-

tivation of a pertussis toxin-sensitive G protein and a phospholipase Cbe-

ta-dependent Ca2þ transient. Alcohol. Clin. Exp. Res. 29, 1237–1246.

Gianoulakis, C. (1990). Rats exposed prenatally to alcohol exhibit impair-

ment in spatial navigation test. Behav. Brain Res. 36, 217–228.

Halvorsen, C. P., Andolf, E., Hu, J., Pilo, C., Winbladh, B., and

Norman, M. (2006). Discordant twin growth in utero and differences

in blood pressure and endothelial function at 8 years of age. J. Intern.

Med. 259, 155–163.

Horn, G., Bradley, P., and McCabe, B. J. (1985). Changes in the structure

of synapses associated with learning. J. Neurosci. 5, 3161–3168.

Ickes, B. R., Pham, T. M., Sanders, L. A., Albeck, D. S.,

Mohammed, A. H., and Granholm, A. C. (2000). Long-term environ-

mental enrichment leads to regional increases in neurotrophin levels

in rat brain. Exp. Neurol. 164, 45–52.

Jones, K. L., and Smith, D. W. (1973). Recognition of the fetal alcohol

syndrome in early infancy. Lancet 2, 999–1001.

Kaskutas, L. A. (2000). Understanding drinking during pregnancy among

urban American Indians and African Americans: health messages, risk

beliefs, and how we measure consumption. Alcohol. Clin. Exp. Res.

24, 1241–1250.

Loskutova, L. V., and Dubrovina, N. I. (2004). The effects of the duration

of adaptation to laboratory conditions on the formation of a passive

avoidance reflex in rats. Neurosci. Behav. Physiol. 34, 189–192.

Malleret, G., Haditsch, U., Genoux, D., Jones, M. W., Bliss, T. V.,

Vanhoose, A. M., Weitlauf, C., Kandel, E. R., Winder, D. G., and

Mansuy, I. M. (2001). Inducible and reversible enhancement of learn-

ing, memory, and long-term potentiation by genetic inhibition of calci-

neurin. Cell 104, 675–686.

Matthews, D. B., and Simson, P. E. (1998). Prenatal exposure to ethanol

disrupts spatial memory: effect of the training-testing delay period.

Physiol. Behav. 64, 63–67.

Mattson, S. N., Riley, E. P., Delis, D. C., Stern, C., and Jones, K. L. (1996).

Verbal learning and memory in children with fetal alcohol syndrome.

Alcohol. Clin. Exp. Res. 20, 810–816.

Means, L. W., McDaniel, K., and Pennington, S. N. (1989). Embryonic

ethanol exposure impairs detour learning in chicks. Alcohol 6, 327–

330.

Mileusnic, R., Lancashire, C. L., and Rose, S. P. (2005). Recalling an aver-

sive experience by day-old chicks is not dependent on somatic protein

synthesis. Learn. Mem. 12, 615–619.

Nagahara, A. H., and Handa, R. J. (1995). Fetal alcohol exposure alters

the induction of immediate early gene mRNA in the rat prefrontal

cortex after an alternation task. Alcohol. Clin. Exp. Res. 19,

1389–1397.

Ng, K. T., and Gibbs, M. E. (1991). Stages of memory formation: a review.

In R. J. Andrew (Ed.), Neural and Behavioral Plasticity: The Use of the

Domestic Chick as a Model (pp. 351–369). Oxford: Oxford University

Press.

Olney, J. W., Wozniak, D. F., Jevtovic-Todorovic, V., Farber, N. B.,

Bittigau, P., and Ikonomidou, C. (2002). Glutamate and GABA recep-

tor dysfunction in the fetal alcohol syndrome. Neurotox. Res. 4, 315–

325.

Patel, S. N., Rose, S. P., and Stewart, M. G. (1988). Training induced

dendritic spine density changes are specifically related to memory for-

mation processes in the chick, Gallus domesticus. Brain Res. 463, 168–

173.

Patel, S. N., and Stewart, M. G. (1988). Changes in the number and struc-

ture of dendritic spines 25 hours after passive avoidance training in the

domestic chick, Gallus domesticus. Brain Res. 449, 34–46.

Pennington, S. N. (1991). Molecular changes associated with ethanol-

induced growth suppression in the chick embryo. Alcohol. Clin. Exp.

Res. 14, 832–837.

Pennington, S. N., Boyd, J. W., Kalmus, G. W., and Wilson, R. W. (1983).

The molecular mechanism of fetal alcohol syndrome (FAS). I. Ethanol-

induced growth suppression. Neurobehav. Toxicol. Teratol. 5, 259–262.

Pennington, S. N., and Kalmus, G. (1987). Brain growth during ethanol-in-

duced hypoplasia. Drug Alcohol Depend. 20, 279–286.

Poggi, S. H., Goodwin, K. M., Hill, J. M., Brenneman, D. E., Tendi, E.,

Schninelli, S., and Spong, C. Y. (2003). Differential expression of c-

fos in a mouse model of fetal alcohol syndrome. Am. J. Obstet. Gyne-

col. 189, 786–789.

Rasmussen, C. (2005). Executive functioning and working memory in fetal

alcohol spectrum disorder. Alcohol. Clin. Exp. Res. 29, 1359–1367.

Richard, S., and Davies, D. C. (2000). Comparison of methyl anthranilate

and denatonium benzoate as aversants for learning in chicks. Physiol.

Behav. 70, 521–525.

Riley, E. P., and McGee, C. L. (2005). Fetal alcohol spectrum disorders: an

overview with emphasis on changes in brain and behavior. Exp. Biol.

Med. (Maywood) 230, 357–365.

Rodricks, C. L., Rose, I. A., Camm, E. J., Jenkin, G., Miller, S. L., and

Gibbs, M. E. (2004). The effect of prenatal hypoxia and malnutrition

on memory consolidation in the chick. Brain Res. Dev. Brain Res.

148, 113–119.

Rose, S. P. (1991). How chicks make memories: the cellular cascade from

c-fos to dendritic remodelling. Trends Neurosci. 14, 390–397.

Rose, S. P. (2000). God’s organism? The chick as a model system for mem-

ory studies. Learn. Mem. 7, 1–17.

Rose, S. P., and Stewart, M. G. (1999). Cellular correlates of stages of

memory formation in the chick following passive avoidance training.

Behav. Brain Res. 98, 237–243.

439V. Rao, J.D. Chaudhuri / Alcohol 41 (2007) 433e439

Salinska, E. (2006). The role of group I metabotropic glutamate receptors

in memory consolidation and reconsolidation in the passive avoidance

task in 1-day-old chicks. Neurochem. Int. 48, 447–452.

Salinska, E. J., Bourne, R. C., and Rose, S. P. (2001). Long-term memory

formation in the chick requires mobilization of ryanodine-sensitive in-

tracellular calcium stores. Neurobiol. Learn. Mem. 75, 293–302.

Sampson, P. D., Streissguth, A. P., Bookstein, F. L., Little, R. E.,

Clarren, S. K., Dehaene, P., Hanson, J. W., and Graham, J. M. Jr.

(1997). Incidence of fetal alcohol syndrome and prevalence of alco-

hol-related neurodevelopmental disorder. Teratology 56, 317–326.

Scholey, A. B., Mileusnic, R., Schachner, M., and Rose, S. P. (1995). A

role for a chicken homolog of the neural cell adhesion molecule L1

in consolidation of memory for a passive avoidance task in the chick.

Learn. Mem. 2, 17–25.

Scholey, A. B., Rose, S. P., Zamani, M. R., Bock, E., and

Schachner, M. (1993). A role for the neural cell adhesion molecule

in a late, consolidating phase of glycoprotein synthesis six hours fol-

lowing passive avoidance training of the young chick. Neuroscience

55, 499–509.

Steele, R. J., Dermon, C. R., and Stewart, M. G. (1996). D-cycloserine

causes transient enhancement of memory for a weak aversive stimulus

in day-old chicks (Gallus domesticus). Neurobiol. Learn. Mem. 66,

236–240.

Stewart, M. G., and Banks, D. (2006). Enhancement of long-term memory

retention by Colostrinin in one-day-old chicks trained on a weak pas-

sive avoidance learning paradigm. Neurobiol. Learn. Mem. 86, 66–71.

Stough, S., Shobe, J. L., and Carew, T. J. (2006). Intermediate-term pro-

cesses in memory formation. Curr. Opin. Neurobiol. 16, 672–678.

Streissguth, A. P., Barr, H. M., Martin, D. C., and Herman, C. S. (1980).

Effects of maternal alcohol, nicotine, and caffeine use during preg-

nancy on infant mental and motor development at eight months. Alco-

hol. Clin. Exp. Res. 4, 152–164.

Summers, B. L., Rofe, A. M., and Coyle, P. (2006). Prenatal zinc treatment

at the time of acute ethanol exposure limits spatial memory impair-

ments in mouse offspring. Pediatr. Res. 59, 66–71.

Tough, S., Tofflemire, K., Clarke, M., and Newburn-Cook, C. (2006). Do

women change their drinking behaviors while trying to conceive? An

opportunity for preconception counseling. Clin. Med. Res. 4, 97–105.

Whitlock, E. P., Polen, M. R., Green, C. A., Orleans, T., and Klein, J., U.S.

Preventive Services Task Force (2004). Behavioral counseling inter-

ventions in primary care to reduce risky/harmful alcohol use by adults:

a summary of the evidence for the U.S. Preventive Services Task Force.

Ann. Intern. Med 140, 557–568.

Wilson, R. W., Kalmus, G. W., and Pennington, S. N. (1984). Alcohol de-

hydrogenase activity in the developing chick embryo. Comp. Biochem.

Physiol. B 77, 191–196.