prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing...

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Behavioural Brain Research 234 (2012) 255–258 Contents lists available at SciVerse ScienceDirect Behavioural Brain Research j ourna l ho mepage: www.elsevier.com/locate/bbr Short communication Prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing in rat offspring Ken-ichi Ohta 1 , Hiromi Sakata-Haga , Yoshihiro Fukui Department of Anatomy and Developmental Neurobiology, Institutes of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan h i g h l i g h t s Prenatally ethanol-exposed rats were examined for behavioral responses to fear stimuli. Elevated T-maze and elevated open platform tests was used for the assessments. The acquisition of passive avoidance was impaired in the ethanol-exposed rats. The response to unconditioned fear was facilitated in the ethanol-exposed rats. a r t i c l e i n f o Article history: Received 15 December 2011 Received in revised form 28 June 2012 Accepted 1 July 2012 Available online 7 July 2012 Keywords: Fetal alcohol Elevated T-maze Elevated open platform Passive avoidance One-way escape Unconditioned freezing a b s t r a c t Previous studies have suggested that ethanol exposure during brain development affects responses to fear and anxiety after maturity. To clarify in detail the impaired behavior related to fear and anxiety seen in rat offspring prenatally exposed to ethanol, their behaviors were observed using an elevated T-maze (ETM) test, which allows assessment of passive avoidance acquisition and one-way escape separately, and an elevated open platform (EOP) test for the assessment of unconditioned freezing against innate fear. The ETM test revealed that acquisition of passive avoidance was significantly inhibited in prena- tally ethanol-exposed rats, while their escape behavior was not altered. In the EOP test, the duration of the freezing behavior was significantly elongated in prenatally ethanol-exposed offspring. Thus, we concluded that prenatal ethanol exposure could impair acquisition of passive avoidance, while it could facilitate a response related to unconditioned fears in rat offspring. © 2012 Elsevier B.V. All rights reserved. Although abnormalities of anxiety-related behaviors have been reported in humans with a history of fetal ethanol exposure [1] and its rodent models [2–4], no conclusion about whether sen- sitivity of anxiety is increased or decreased by prenatal ethanol exposure has been reached. In some experiments that examined anxiety-related responses using an elevated plus maze (EPM) or an open field (OF) test, prenatally ethanol-exposed rats showed an increase in anxiety-like behaviors [2]. Meanwhile, other stud- ies reported that prenatal ethanol exposure was either anxiolytic or did not affect anxiety-related behaviors [3]. We have reported that anxiety-like behaviors observed in the EPM and OF tests were Corresponding author at: Department of Anatomy and Developmental Neu- robiology, Institutes of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Tel.: +81 88 633 7052; fax: +81 88 633 7053. E-mail address: [email protected] (H. Sakata-Haga). 1 Present affiliation: Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Kagawa, Japan. reduced in rat offspring prenatally exposed to ethanol [4]. Simulta- neously, however, the same rats more frequently exhibited freezing behavior, which is a sign of anxiety and fear in animals [5], on the open arms of the EPM compared to rats in the control groups [4]. Although the EPM test is one of the most frequently used behav- ioral tests for assessing anxiety in rodents, such inconsistent results have often been reported, especially in studies for serotonin (5-HT)- acting drugs [6]. These discrepancies seemed to be attributable to the fact that these anxiety models, including the EPM test, might measure a mixture of several types of anxiety; thus different envi- ronmental conditions would produce inconsistent results. To solve this problem, an elevated T-maze (ETM), which consists of one arm closed by walls that is perpendicular to two open arms elevated from the floor, has been developed [7]. In the ETM test, passive avoidance is measured by placing a rat at the end of the closed arm and the time to withdraw from this arm is recorded during three consecutive trials. Soon afterwards, the same rat is placed at the end of the open arm and the time to withdraw from this arm was also recorded as a one-way escape response. Graeff et al. have revealed that 5-HT facilitates acquisition of passive avoidance, whereas it 0166-4328/$ see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.07.001

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Page 1: Prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing in rat offspring

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Behavioural Brain Research 234 (2012) 255– 258

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

hort communication

renatal ethanol exposure impairs passive avoidance acquisition and enhancesnconditioned freezing in rat offspring

en-ichi Ohta1, Hiromi Sakata-Haga ∗, Yoshihiro Fukuiepartment of Anatomy and Developmental Neurobiology, Institutes of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan

i g h l i g h t s

Prenatally ethanol-exposed rats were examined for behavioral responses to fear stimuli.Elevated T-maze and elevated open platform tests was used for the assessments.The acquisition of passive avoidance was impaired in the ethanol-exposed rats.The response to unconditioned fear was facilitated in the ethanol-exposed rats.

r t i c l e i n f o

rticle history:eceived 15 December 2011eceived in revised form 28 June 2012ccepted 1 July 2012vailable online 7 July 2012

a b s t r a c t

Previous studies have suggested that ethanol exposure during brain development affects responses tofear and anxiety after maturity. To clarify in detail the impaired behavior related to fear and anxiety seenin rat offspring prenatally exposed to ethanol, their behaviors were observed using an elevated T-maze(ETM) test, which allows assessment of passive avoidance acquisition and one-way escape separately,and an elevated open platform (EOP) test for the assessment of unconditioned freezing against innatefear. The ETM test revealed that acquisition of passive avoidance was significantly inhibited in prena-

eywords:etal alcohollevated T-mazelevated open platformassive avoidancene-way escapenconditioned freezing

tally ethanol-exposed rats, while their escape behavior was not altered. In the EOP test, the durationof the freezing behavior was significantly elongated in prenatally ethanol-exposed offspring. Thus, weconcluded that prenatal ethanol exposure could impair acquisition of passive avoidance, while it couldfacilitate a response related to unconditioned fears in rat offspring.

© 2012 Elsevier B.V. All rights reserved.

Although abnormalities of anxiety-related behaviors have beeneported in humans with a history of fetal ethanol exposure [1]nd its rodent models [2–4], no conclusion about whether sen-itivity of anxiety is increased or decreased by prenatal ethanolxposure has been reached. In some experiments that examinednxiety-related responses using an elevated plus maze (EPM) orn open field (OF) test, prenatally ethanol-exposed rats showedn increase in anxiety-like behaviors [2]. Meanwhile, other stud-

es reported that prenatal ethanol exposure was either anxiolyticr did not affect anxiety-related behaviors [3]. We have reportedhat anxiety-like behaviors observed in the EPM and OF tests were

∗ Corresponding author at: Department of Anatomy and Developmental Neu-obiology, Institutes of Health Biosciences, The University of Tokushima Graduatechool, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Tel.: +81 88 633 7052;ax: +81 88 633 7053.

E-mail address: [email protected] (H. Sakata-Haga).1 Present affiliation: Department of Anatomy and Neurobiology, Faculty ofedicine, Kagawa University, Kagawa, Japan.

166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2012.07.001

reduced in rat offspring prenatally exposed to ethanol [4]. Simulta-neously, however, the same rats more frequently exhibited freezingbehavior, which is a sign of anxiety and fear in animals [5], on theopen arms of the EPM compared to rats in the control groups [4].Although the EPM test is one of the most frequently used behav-ioral tests for assessing anxiety in rodents, such inconsistent resultshave often been reported, especially in studies for serotonin (5-HT)-acting drugs [6]. These discrepancies seemed to be attributable tothe fact that these anxiety models, including the EPM test, mightmeasure a mixture of several types of anxiety; thus different envi-ronmental conditions would produce inconsistent results. To solvethis problem, an elevated T-maze (ETM), which consists of one armclosed by walls that is perpendicular to two open arms elevatedfrom the floor, has been developed [7]. In the ETM test, passiveavoidance is measured by placing a rat at the end of the closed armand the time to withdraw from this arm is recorded during three

consecutive trials. Soon afterwards, the same rat is placed at the endof the open arm and the time to withdraw from this arm was alsorecorded as a one-way escape response. Graeff et al. have revealedthat 5-HT facilitates acquisition of passive avoidance, whereas it
Page 2: Prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing in rat offspring

2 rain Research 234 (2012) 255– 258

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Fig. 1. Effects of prenatal ethanol exposure on inhibitory avoidance (A) and one-way escape behavior (B) in the elevated T-maze test. Each value is represented asmean ± SEM. *p < 0.05, **p < 0.01 compared to Pf and Int rats. Int: intact control; Pf:

56 K.-i. Ohta et al. / Behavioural B

nhibits one-way escape [8]. Interestingly, it is well known thatrenatal ethanol exposure induces a reduction of 5-HT neurons inhe midbrain raphe nucleus [4]. Thus we introduce the ETM test tolarify an effect of prenatal ethanol exposure on anxiety.

As previously described, our rat model for prenatal exposure tothanol more frequently showed freezing behavior, which is a signf anxiety and fear [5], on the open arms in the EPM [4]. Their exces-ive response to innate fear, i.e., the open space with high attitude,ould appear as the exaggerated freezing behavior. Recently, an

levated open-platform (EOP) has been used for the assessment ofhe behavioral response to unconditioned stress as the duration of

freezing behavior [9]. Unlike the ETM test, the freezing behav-or in the EOP is not affected by activity, exploration, and learningbility. It has been reported that which prenatal ethanol exposureffects these behavior, although our ethanol-exposed rats did nothow alteration of activity [9]. Thus, we also assessed the behav-oral response to unconditioned fear in our rat model for prenatalxposure to ethanol using the EOP test.

All animal procedures were conducted according to the Guideor the Care and Use of Laboratory Animals, and were reviewed byhe Institutional Animal Care and Use Committee of the Universityf Tokushima. Great care was taken to minimize the number ofnimals used, and their suffering.

Pregnant Sprague–Dawley rats purchased from Japan SLCHamamatsu, Japan) were divided into three groups: prenatallythanol-exposed (EtOH), pair-fed control (Pf), and intact controlInt) groups. The same procedure as described in our previoustudies [4,10] was used to prepare the rats of each experimentalroup. Briefly, pregnant rats of the EtOH group were allowed freeccess to a liquid diet (Oriental Yeast Co., Tokyo, Japan) contain-ng ethanol during gestational days (GDs) 10–21, corresponding tohe second-trimester of pregnancy in human. The ethanol concen-ration of the diet was gradually raised: 2.5% (w/v) on GDs 10–12,.0% (w/v) on GDs 13–15, and 5.0% (w/v) on GDs 16–21. The meanaily ethanol consumption of dams (n = 5) in the EtOH group was.73 ± 0.09 g during GDs 10–12, 3.55 ± 0.14 g during GDs 13–15,nd 3.66 ± 0.19 g during GDs 16–21. In our preliminary study, thisdministration regimen resulted in 145.5 ± 53.3 mg/dl of the bloodthanol concentration (n = 6, ranged from 50.8 to 178.1 mg/dl) at GD1. Pregnant rats of the Pf group were given an equivalent amountf the same liquid diet consumed by EtOH dams on a daily basis,xcept that the ethanol was replaced by isocaloric sucrose. Pregnantats of the Int group were allowed to free access laboratory chownd tap water. Pregnant rats were allowed to give birth, eight pupsrom each litter were cross-fostered to untreated dams at postnatalay (PD) 1, and male offspring from each group were used for theollowing behavioral tests at PD 60–70.

The ETM consisted of three arms (50 cm × 10 cm) of equalimensions. One of the arms had 40-cm-high walls (closed arm),nd the others only had slight lips (0.25 cm high) to prevent ani-als from falling (open arms). The three arms were connected by

square platform (10 cm× 10 cm). Each open arm was attachedcross the platform from another open arm, and the closed armas attached to one of the two remaining sides of the platform. Theaze was placed 50 cm above the floor. Prior to the experimental

ession, an experimenter gently handled rats for seven consecutiveays (5 min per day). The ETM test took place in a dimly illuminatedoom (80 lx, indirect). An ETM test session consisted of three pas-ive avoidance trials and one escape trial at 30-s intervals betweenach trial. During the interval, rats were placed in their own cages.he maze was cleaned with 70% ethanol solution after each indi-idual session. At the start of the first passive avoidance trial, a

at was placed on the end of the closed arm facing the platform,nd the time taken to leave the closed arm with the paws of bothorelimbs was recorded as the baseline latency. The same measure-

ent was subsequently repeated two times as Avoidance 1 and 2,

pair-fed control; EtOH: prenatal ethanol-exposed group.

respectively. Afterwards, the rat was placed at the end of an openarm, and the time required to enter the closed arm with all fourpaws was recorded as the escape latency. For all trials, a cutoffof 300-s was established. The rats that fell from the open armswere excluded from the data analysis. Although 12 male offspringderived from three dams in each experimental group were appliedon the ETM test, for this reason, the data of the passive avoidancetrials from 10 subjects were used for Int group, and the data ofescape latency from 9, 11, and 10 subjects were used for Int, Pf, andEtOH groups, respectively.

For the EOP test, the rats were placed on a transparent circu-lar platform (30 cm diameter) attached to the top of a 60-cm-hightransparent acrylic cylinder for 10 min in a brightly illuminatedroom (700 lx, direct), and their behavior was recorded by a videocamera. An observer blindly measured the total duration of freez-ing behavior, defined as a crouching posture with no movementexcluding respiration. Male offspring, which is different from therats applied to the ETM test and derived from three dams in eachgroup, was used for the EOP test. Although 16 rats for Int, 20 ratsfor Pf, 16 rats for EtOH were applied the test, three of Pf and fourof EtOH offsprings were eliminated from the data analysis, becausethey fell down from the apparatus during the test.

Statistical analyses were conducted using SPSS 16.1J (TokyoJapan). Two-way repeated measures for analysis of variance(ANOVA) were used to analyze passive avoidance data, with treat-ment as the independent factor and trials as the repeated measure.Moreover, each trial and the length of freezing duration were ana-lyzed using a one-way ANOVA with factor treatment. Whenever an

interaction reached significance, Post hoc Fisher’s protected leastsignificant difference (PLSD) was performed. Statistical significancewas considered at p < 0.05.
Page 3: Prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing in rat offspring

K.-i. Ohta et al. / Behavioural Brain R

Fig. 2. An effect of prenatal ethanol exposure on the duration of freezing behavior inthe elevated open platform test. Each value is represented as mean ± SEM. *p < 0.05ce

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adverse condition in the EOP test. These distinct properties of test

ompared to Pf and Int rats. Int: intact control; Pf: pair-fed control; EtOH: prenatalthanol-exposed group.

Results of the ETM test are shown in Fig. 1. As shown inig. 1 (A), acquisition of passive avoidance was impaired by pre-atal exposure to ethanol. Both Int and Pf groups took longero exit the closed arm each time, showing that they steadilycquired passive avoidance. The EtOH offspring showed no dif-erence in the latencies between the first trial (baseline) andecond trial (Avoidance 1). Two-way repeated measures ANOVAevealed the significant effect of trials [F(2, 62) = 27.256, p < 0.01],f treatment [F(2, 31) = 4.545, p < 0.05] and significant treatmenty trial interaction [F(4, 62) = 2.667, p < 0.05]. In addition, one-wayNOVA demonstrated a significant difference among the experi-ental groups at Avoidance 2 [F(2, 31) = 4.380, p < 0.05]. Post hoc

est suggested that EtOH offspring significantly decreased pas-ive avoidance latencies compared to both Pf and Int offspring atvoidance 2. However, escape behavior was not affected by treat-ents [F(2, 27) = 0.983, p = 0.387] (Fig. 1B), meaning that escape

atency was not significantly changed by prenatal ethanol expo-ure.

Total duration of freezing behavior during the 10-minute EOPest showed a significant difference among treatment groups [F(2,4) = 4.187, p < 0.05] (Fig. 2). Post hoc tests demonstrated that totaluration time of freezing behavior in EtOH offspring was signifi-antly increased compared to Pf and Int offspring. Three of EtOH,ight of PF, and nine of Int rats had no freezing during the EOP test.

In the present study, we assessed several types of anxiety-elated behavior separately, such as acquisition of passivevoidance, one-way escape, and response against unconditionedear, in prenatally ethanol-exposed rats. In the ETM test, thethanol-exposed rats showed an impairment of passive avoidancecquisition, but they had no change in one-way escape behav-or. Still these findings appear to be contradictory to each other.owever, the results can be explained by the “dual role of 5-HT”

heory. This theory against anxiety has been suggested based onehavioral assessments using ETM after some agonists or antag-nists of 5-HT receptors [8,11]. According to the theory, 5-HTacilitates a defensive response to potential threats, such as passivevoidance, whereas it inhibits defensive responses to proximal dan-ers, such as one-way escape. Indeed, administrations of buspironer ipsapirone, which are partial agonists for a 5-HT1A receptor,mpaired passive avoidance, while leaving the one-way escapenchanged and an acute administration of a 5-HT/noradrenalin

euptake inhibitor, imipramine, enhanced inhibitory avoidance12,13]. A 5-HT releaser, fenfluramine, induces facilitation of pas-ive avoidance and impairment of escape [13]. We also assessed

esearch 234 (2012) 255– 258 257

the behavioral response to an unconditioned stimulus using anEOP test, which allowed assessment of the behavioral responseto unconditioned stress as the duration of a freezing behavior.Also this behavioral indicator is affected by 5-HT. Selective 5-HT reuptake inhibitors significantly decreased the duration of thefreezing, and a 5-HT synthesis inhibitor markedly increased theduration of freezing in the EOP test [9]. In the prefrontal cor-tex of diabetic mice, which exhibit prolonged freezing in the EOPtest, EOP stress-induced 5-HT secretion was significantly decreased[14]. Previously, we demonstrated that prenatal ethanol expo-sure induces a reduction in the number of 5-HTergic neurons inthe midbrain raphe nuclei, and that persists into adulthood [4]. Areduction in the number of 5-HTergic neurons can cause an inad-equate secretion of 5-HT in their target areas. Thus, it is suspectedthat a reduced number of 5-HTergic neurons contributes to boththe impaired acquisition of passive avoidance and the enhancedresponse to unconditioned fear in our prenatally ethanol-exposedoffspring. In the present study, pregnant rats fed a liquid diet con-taining ethanol during gestational days 10–20, which is equivalentto the second trimester in humans. In rats, fetal 5-HTergic neuronswere generated and differentiated within this period, although 5-HTergic neurons would begin generation from earlier. Thus, ethanolexposure in this period could affect the development of 5-HTergicsystem.

It is known that ethanol exposure during brain developmentimpacts many brain areas and neurotransmitters, including notonly 5-HT, but also glutamate, gamma-amino butyric acid (GABA),and dopamine. Among them, also GABA is particularly suspected ofcontributing to the alterations of behavior observed in the presentstudy, although no neurotransmitter system issues were addressed.Because, just like the 5-HTergic system, the GABAergic system isclosely related to anxiety-related behaviors, it is also one of themost well-recognized targets for developmental toxicity of ethanol.Prenatal ethanol exposure reported increased extracellular GABAlevel, resulting in enhanced tangential migration of GABAergicinterneurons in fetal mice [15]. After maturity, effects of ethanolexposure during the fetal period on the GABAergic system is leftas an enhanced expression of a certain subunit of GABAA receptorin the hippocampus [16] and cerebral cortex [17] of adult guineapig. Previously it was found that the GABAergic system also regu-lates passive avoidance and escape behavior observed in the ETMtest. Microinjection of the benzodiazepine receptor agonist mida-zolam, GABAA receptor agonist muscimol, and GABAB receptoragonist baclofen impaired escape without altering inhibitory avoid-ance [18]. Thus, abnormal development of the GABAergic systemmay also have served to alter anxiety-like behavior after prenatalethanol exposure.

In the present study, the enhancement of one-way escapebehavior in the ETM test was not significant, while the freezingbehavior significantly increased in the EOP test, even though bothbehaviors were categorized as defensive behaviors attributed toexpression of unconditioned fear. One of the reasons for such a dif-ferent response is a difference in the light condition. The ETM testwas performed under dim light (80 lx, indirect), while the EOP testwas carried out in a brightly illuminated room (700 lx) by directlighting. Like higher places, bright lights can also be an adversestimulus that provokes the freezing behavior for rodents. Anotherreason is that whether rats are allowed to escape from the adversestimuli or not. The rats can escape to the closed arm from open armsin the ETM test. And so sometimes, a session to observe the one-way escape in the ETM test might be finished before the rats feelstrong anxiety. Unlike in the ETM test, rats cannot escape from the

would contribute different results between a one-way escape in theETM tests and unconditioned freezing in the EOP test. There is a lotof variation in the date of the EOP test. This could be caused by the

Page 4: Prenatal ethanol exposure impairs passive avoidance acquisition and enhances unconditioned freezing in rat offspring

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act that some offspring showed no sign of the freezing behavioruring the test.

In the present study, we attempted to explain the effects of pre-atal exposure to ethanol on anxiety and fear, an issue on which noonsensus has been reached to date. As a result, prenatal exposureo ethanol impaired acquisition of passive avoidance and enhanced

response to unconditioned fear. Therefore, the 5-HTergic and/orABAnergic system might be the key to understand these abnor-alities related to anxiety caused by fetal ethanol exposure. Father

esearch is warranted to elucidate the mechanism of the alterednxiety-like behavior after prenatal exposure to ethanol.

cknowledgments

This manuscript was supported by KAKENHI (#20790766) fromhe Japan Society for the Promotion of Science and a grant fromhe Long-range Research Initiative by the Japan Chemical Industryssociation.

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