THE ANATOMICAL RECORD 292:1922–1939 (2009)

Impact of Ethanol on the DevelopingGABAergic System

RICARDO NOBORO ISAYAMA,1 PAULO EMILIO CORREA LEITE,2

JEAN PIERRE MENDES LIMA,1,3 DANIELA UZIEL,1*

AND EDNA NANAMI YAMASAKI3,4*1Institute of Biomedical Sciences, Federal University of Rio de Janeiro,

Rio de Janeiro, Brazil2Laboratorio de Patologia Celular e Molecular, Institute of Biology,

Federal Fluminense University, Rio de Janeiro, Brazil3Program in Neurobiology, Institute of Biophysics Carlos Chagas Filho,

Federal University of Rio de Janeiro, Rio de Janeiro, Brazil4Department of Life and Health Sciences, School of Sciences,

University of Nicosia, Nicosia, Cyprus

ABSTRACTAlcohol intake during pregnancy has a tremendous impact on the

developing brain. Embryonic and early postnatal alcohol exposures havebeen investigated experimentally to elucidate the fetal alcohol spectrumdisorders’ (FASD) milieu, and new data have emerged to support a devas-tating effect on the GABAergic system in the adult and developing nervoussystem. GABA is a predominantly inhibitory neurotransmitter that duringdevelopment excites neurons and orchestrates several developmental proc-esses such as proliferation, migration, differentiation, and synaptogenesis.This review summarizes and brings new data on neurodevelopmentalaspects of the GABAergic system with FASD in experimental telencephalicmodels. Anat Rec, 292:1922–1939, 2009. VVC 2009 Wiley-Liss, Inc.

Keywords: GABA; developing brain; prenatal ethanol expo-sure; cortical neurons; ganglionic eminence

THE DEVELOPING TELENCEPHALON

The central nervous system develops in a programmedorder, following subsequent steps. Because of the cellulardiversity and complexity throughout its laminar andareal structure, the cerebral cortex is a good model forstudying developmental events. After closure of the neu-

ral tube, progenitor cells located near its lumen (ven-tricular zones) start to proliferate intensely, first toincrease progenitor pool and then to originate postmi-totic cells (Rakic, 1988). The destination of proliferatingcells is largely influenced by their mitotic spindle rota-tion during metaphase and their cleavage plane during

Abbreviations used: ARBD ¼ alcohol-related birth defects;ARND ¼ alcohol-related neurodevelopmental disorders; BDNF ¼brain-derived neurotrophic factor; BrdU ¼ bromodeoxyuridine;CAMs ¼ cell adhesion molecules; CGE ¼ caudal ganglioniceminence; CNS ¼ central nervous system; CON ¼ control; CP ¼cortical plate; CTX ¼ cortex; E ¼ embryonic day; ETOH ¼ethanol; FAS ¼ fetal alcohol syndrome; FASD ¼ fetal alcoholspectrum disorder; GABA ¼ gamma amino butyric acid; GAD ¼glutamic acid decarboxylase; IZ ¼ intermediate zone; LGE ¼lateral ganglionic eminence; MGE ¼ medial ganglionic eminence;NMDA ¼ N-methyl-D-aspartate; P ¼ postnatal day; SVZ ¼subventricular zone; VZ ¼ ventricular zone.

Grant sponsors: FAPERJ, CNPq.

*Correspondence to: E.N. Yamasaki, School of Sciences, 46Makedonitissas Ave, PO Box 24005, Nicosia 1700, Cyprus. Phone:357-22 841743. E-mail: Yamasaki.e@unic.ac.cy or D. Uziel, Insti-tute of Biomedical Sciences, Federal University of Rio de Janeiro,Rio de Janeiro, Brazil. Phone: 55-21-25626469.E-mail: daniuzi@anato.ufrj.br

Received 18 February 2009; Accepted 10 June 2009

DOI 10.1002/ar.20966Published online in Wiley InterScience (www.interscience.wiley.com).

anaphase in the ventricular zone: cleavage planes verti-cal to the ventricle yield two daughter cells that remainconfined to the ventricular zone, whereas horizontalcleavages result in cells that migrate away from the ven-tricle (Haydar et al., 2003). Although some differencesbetween proliferative behaviors in the ventricular andsubventricular zones exist (Noctor et al., 2004), it is gen-erally accepted that postmitotic cells leave the prolifera-tive zones and migrate to their final destination toestablish connections that ultimately lead to function.Neurons are generated through cell proliferation at earlyembryonic stages, whereas appearance of glial cellsoccurs at later embryonic and early postnatal periods(Qian et al., 2000).

In the cortex, neuronal populations can be divided andsubdivided based on their morphological, biochemical,and functional features. One main division is based onthe pattern of neurotransmitter expression: glutamater-gic cells usually have a pyramidal soma, long axons, andare born in the cortical ventricular zone (reviewed byMolnar and Cheung, 2006). In contrast, GABAergic cellsare local circuit neurons (interneurons) with a smallsoma and originate in the ventricular zones of the mu-rine ganglionic eminences in the basal telencephalon.Following proliferation, postmitotic GABAergic cellsmigrate tangentially to reach the developing corticalplate (De Carlos et al., 1996; Marin and Rubenstein,2001), where they settle in the same layers as synchroni-cally generated glutamatergic projection neurons (Miller,1985; Valcanis and Tan, 2003). In vitro experimentsreveal that interneurons present a large range of behav-iors before arriving and settling in the neocortex(Tanaka et al., 2003, 2006). Along their tangential jour-ney to the cortex, migrating GABAergic cells are sub-jected to local and long distance cues that contribute toguide their way to the cortex, including the influence ofneurotransmitters (Marin et al., 2001, 2003; Tamamakiet al., 2003; Flames et al., 2004; Andrews et al., 2006,2008; Pla et al., 2006; Heng et al., 2007; Lopez-Benditoet al., 2008). Glutamatergic neurons born in the dorsaltelencephalon migrate radially in the cortex using theprocess of radial glia progenitors as scaffold and are alsoinfluenced by environmental signals (Komuro and Rakic,1998; Lambert de Rouvroit and Goffinet, 2001; Noctor etal., 2004; Metin et al., 2008).

Even before finishing radial migration, neurons ex-hibit neurites that actively seek their targets (Schwartzet al., 1991; Auladell et al., 1995). Projection neurons inthe cortex can connect to cortical and subcortical struc-tures, and this choice of target appears to be related togenetic and epigenetic factors (recently reviewed by Mol-nar and Cheung, 2006; Leone et al., 2008). Guidancemolecules, such as Netrins, Semaphorins, and Ephrins,are common to several regions of the nervous systemand to different cellular processes and are evolutionarilyconserved, being responsible for axonal pathfinding dur-ing late embryonic and early postnatal development(Serafini et al., 1994; Drescher et al., 1995; Puschel etal., 1995; Bagnard et al., 1998; Uziel et al., 2006).

When growing axons finally reach their targets, syn-aptogenesis starts involving reciprocal interactionsbetween pre and postsynaptic cells (for review see Sali-nas and Zou, 2008). The next and long last step in nerv-ous system development is myelination of axons, aprocess that extends throughout the first 2 months of

rodent life and the first two decades of human life (Bau-mann and Pham-Dinh, 2001).

Late developmental steps also comprehend regressivephenomena, when remodeling of axonal and dendriticarbors and removal of nonstable synapses take place(Price and Blakemore, 1985; Heffner et al., 1990;Elberger, 1994; Aggoun-Aouaoui et al., 1996; Cesa andStrata, 2005). During prenatal and early postnatal de-velopment, programmed cell death defines the final neu-ral population in the brain as a physiological geneticallyand epigenetically regulated phenomenon (Finlay andSlattery, 1983; Heumann and Leuba, 1983; Ferrer et al.,1992; Blaschke et al., 1996, 1998; Lossi and Merighi,2003). According to Blaschke et al. (1996), there is anincrease in cell death in the developing cortex betweenE10 and E18, with a majority of dying cells within theproliferative zones rather than in postmitotic layers.Cell death occurs either to compensate for the significantsupernumerary cell production or to partially correctchromosomal abnormalities (Rehen et al., 2001; Kings-bury et al., 2005).

EXPERIMENTAL MODELS OF PRENATALETHANOL EXPOSURE

One of the most severe effects of drinking during preg-nancy is the fetal alcohol syndrome (FAS), which is theleading known preventable cause of mental retardationand birth defects (reviewed by Bailey and Sokol, 2008).The criteria to define FAS include confirmed maternalalcohol exposure, evidence of characteristic facial anoma-lies, of prenatal or postnatal growth retardation, andof CNS neurodevelopmental abnormalities (Adamset al., 2002). CNS and neurobehavioral abnormalitiesinclude microcephaly, intellectual (mild-to-moderatemental retardation) and cognitive impairment, develop-mental delay, irritability in infancy, hyperactivity inchildhood or attention deficit/hyperactivity disorder(ADHD), seizures, delayed or deficient myelination,agenesis or hypoplasia of the corpus callosum.

When the diagnosis of FAS cannot be established, butin case of documented maternal alcohol intake whereother pathologies can be excluded, the terms alcohol-related neurodevelopmental disorder (ARND) and alco-hol-related birth defects (ARBD) are used. Fetal alcoholspectrum disorder (FASD) is an umbrella term thatdescribes the range of effects (functional or structural)that can occur in an individual whose mother drankalcohol during pregnancy and includes FAS, ARND, andARBD. Serum levels of ethanol that result in FAS arenot established, and until now only chronic ethanol useis associated with FAS (Clarren et al., 1978; Hansonet al., 1978; Davis et al., 1982; Galea and Goel, 1989;Hoyseth and Jones, 1989; Leonard et al., 1991; Sampsonet al., 2000).

Most experimental models of prenatal ethanol expo-sure use chronic intoxication to simulate the outcome ofFAS and report several brain abnormalities (Miller,1985, 1986, 1988, 1989, 1993, 1995a,b, 2006; Miller andPotempa, 1990; Miller and Nowakowski, 1991; Millerand Robertson, 1993; Mooney and Miller, 2001, 2003).Just a few studies use acute or short-term exposure toaddress the role of ethanol abuse on specific cell popula-tions (Miller, 1996; Obernier et al., 2002; Li et al., 2005;Mooney and Miller, 2007; Oyedele and Kramer, 2008).

ETHANOL AND THE DEVELOPING GABAergic SYSTEM 1923

MATERIALS AND METHODSAnimal Handling and Blood Ethanol Levels

All procedures involving the use of living animalswere approved by the Committee on Animal Care of theFederal University of Rio de Janeiro (protocol numberDAHEICB 035). All efforts were made to minimize painand the number of animals involved in this study. Swisspregnant female mice were kept in individual cages, pro-vided with food and water ad libitum under constantroom temperature (23�C–25�C) and light–dark cycle 12/12 hr. The presence of a plug, the day after mating wasdefined as embryonic day 1 (E1).

Pregnant female mice between E11 and E14 in theethanol (ETOH) and control (CTR) groups were givenethanol (2 g/kg, wt/wt) and 0.1 M phosphate-buffered sa-line (PBS; pH 7.4), respectively, by intragastric gavage.Blood samples were collected from the tail at E13, at 10,45, 60, 120, and 180 min after ethanol administration.Serum ethanol levels were measured using the AlcoholReagent Kit (Point Scientific), as indicated by the manu-facturer. ETOH and CTR-treated animals were anesthe-tized with ether inhalation on E14 and embryosextracted by caesarean section. The encephalon was dis-sected in ice-cooled PBS. The medial (MGE) and lateral(LGE) ganglionic eminences as well as the dorsal corticalanlage (CTX) were dissected for Western blot analysis.Whole brains were fixed in freshly prepared 4% para-formaldehyde (PFA) þ 1% glutaraldehyde in PBS forimmunohistochemistry.

Western Blot

The MGE, LGE, and CTX harvested from E14 brainswere immersed in a solution containing protease inhibi-tors (Protease inhibitor cocktail, P8340; Sigma, St Louis,MI), homogenized, electrophoresed in a 10% polyacryl-amide gel, and transferred to a PVDF membrane(Hybond-P; Amersham Biosciences, Fairfield, CT). Awhole litter (8–10 animals, n ¼ 3) from both ETOH- orCTR-treated groups was pooled together for the proteinassay. The membrane was blocked with 5% low fat milkin Tris-saline buffer and 0.05% Tween 20 (TTBS) for 2hr at room temperature and incubated overnight withthe primary antibodies (sheep anti-GAD65/67, 1:10,000;Oertel et al., 1981; goat anti-b actin conjugated to HRP,1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA)diluted in the same solution at 4�C. Following a rinsewith TTBS, a secondary antibody conjugated to HRP(anti-sheep; 1:5,000; Vector Laboratories, Burlingame,CA) was incubated for 2 hr at room temperature. Bandsfor both markers were revealed by chemiluminescence(ECL Plus, Amersham).

Exposed films were scanned in a PC for densitometricanalysis using the Scion-Image software (5th version,available at http://www.scioncorp.com/pages/scion_image_windows.htm).

Immunohistochemistry

Harvested whole brains at E14 (n ¼ 10, from 5 litters),P10 (n ¼ 2, from 2 litters), and adult (n ¼ 3, from 3 lit-ters) were fixed in freshly prepared 4% PFA and 1% glu-taraldehyde in PBS for 4 hr, cryoprotected in 30%sucrose in PBS (wt/vol) for 5 hr and blocked in an tissue

freezing medium (OCT, Tissue Tek, Elkhard, IN), andfrozen in liquid nitrogen. Coronal sections (7–12 lm)were cut in a Leica cryostat and collected on 2.5% gela-tin-1% poly-L-lysine-treated slides.

Slides were preincubated for 10 min with PBS (0.1 M,pH 7.2) containing 3% hydrogen peroxide, rinsed twicein PBS for 10 min, followed by a 40-min incubation in10% normal goat serum (NGS) and Triton X-100(Reagen, 0.03% and 0.25% for embryonic and postnataltissues, respectively) in PBS to block nonspecific bindingsites. After brief rinses with PBS, the slides were incu-bated overnight with a rabbit anti-GABA antibody(1:5,000, Sigma) and the next day with secondary anti-body (Vector, biotinylated anti-rabbit antibody; 1:200) for1.5 hr. After three rinses of 10 min with PBS, slideswere incubated with an avidin-biotin-peroxidase complex(ABC kit, Vector) for 1 hr following the manufacturer’sinstructions and revealed using a chromogen for the per-oxidase reaction (Vector SG Substrate Kit, SK-4700) for10 min. To minimize differential staining for GABA orslight differences on reagents, all ETOH brain sectionswere processed along with its control in the same slide.Negative controls were obtained by omitting incubationof primary antibody for each set of experiments.

Data Analysis

Tissue was analyzed using a Zeiss microscopeequipped with camera and Axioplan software (AxioVi-sion, LE) coupled to a computer. For quantification, ana-tomical landmarks were identified according to theChemoarchitectonic Atlas of the Developing MouseBrain (Jacobowitz and Abbott, 1998) and the Allen BrainAtlas–Developing Mouse Brain (Allen Institute for BrainScience VC 2008. Available at: http://www.brain-map.org).GABAergic cells in the parietal cortex of postnatal andadult brains were quantified in two random fields of twodifferent nonadjacent coronal (25 lm) brain sections,using always the left hemisphere. For migrating cells inthe E14 cortex, the cortical anlage was divided in 3thirds and labeled cells situated in the first proximalthird from the corticostriatal angle were counted. Anocular reticule of 120 lm2 was used for cell counting, usinga 10� objective (1.562 mm2). Two fields per slide werecounted for each animal analyzed. Cortical thickness didnot differ between control and ethanol-treated animals.

TUNEL Labeling

Programmed cell death was measured using a TUNELapoptosis detection kit (DNA Fragmentation/Fluores-cence staining; Upstate cell signaling solution) followingthe manufacturer’s specification. Protease K step wasreplaced by a short incubation in 0.5% Triton X-100 inPBS for 15 min due to fragility of embryonic tissues.Slides were then rinsed in PBS (0.1 M, pH 7.4) and incu-bated with terminal deoxynucleotidyl transferase (TdT)buffer 1� (TdTBuffer) for 5 min at room temperature.This step was followed by incubation with TdT enzymeand the reaction buffer at 37�C for 1 hr in a dark humidcamera. Reaction was stopped by two rinses with stopsolution for 5 min each at room temperature and a PBSrinse for 10 min. Thereafter, incubation with anti-digoxi-genin conjugated to FITC for 1 hr at room temperature.Finally, the slides were rinsed in PBS for 10 minutes,

1924 ISAYAMA ET AL.

briefly incubated with 40-6-diamidino-2-phenylindole(DAPI; Molecular Probes), and mounted on slides using5% N-propyl-gallate in 80% glycerin diluted in phos-phate buffer 0.1 M, pH 8. Quantification of TUNEL-posi-tive nuclei density per area was performed in the MGE,LGE, CTX as well as the primordium of the hippocam-pus (PH) for both CON and ETOH tissues. Paired brainsections in both groups were mounted in the same slideand reacted at the same time. Proof-staining was per-formed using a DNAse provided by the TUNEL kit.

Statistical Analysis

Data statistical analysis was performed using Prisma(3.0) and Biostat (3.1) software. The mean values werecompared using t-test and ANOVA as well as Kruskal-Wallis with statistical significance set at 5% (P < 0.05).

THE DEVELOPING TELENCEPHALON IN THESCOPE OF PRENATAL ETHANOL EXPOSURE

Prenatal ethanol affects the developing brain in atime-, age-, and dose-dependent manner (Sakata-Hagaet al., 2002). When administered throughout gestation,ethanol compromises growth and viability of rat embryos(Ghimire et al., 2008), although shorter treatment peri-ods or lower concentrations of ethanol may not stronglyharm brain structures (Sanchis et al., 1986; Oyedele andKramer, 2008). In fact, all brain neurodevelopmentalevents such as proliferation, migration, differentiation,synaptogenesis, myelination, and natural occurringneuronal death can potentially be impacted by ethanolexposure (Levitt, 1998). During the past 20 years, exper-imental chronic models of prenatal ethanol exposurehave uncovered some of the ethanol effects on the devel-oping telencephalic vesicles, as described later.

Early experimental studies conducted by Miller (1986,1987, 1988, 1993) using [3H] thymidine or bromodeox-yuridine (BrdU) incorporation demonstrate an increasedtelencephalic subventricular zone (SVZ), probably due toa more prolonged permanency of postmitotic cells intheir proliferative zones in chronically intoxicated rats(from embryonic day 5, E5). Paradoxically, despite theincreased size of the SVZ, no changes in the migrationrate of SVZ neurons are observed (Miller, 1989). Towardthe end of the early gestational phase (E12–E15) in rats,cell cycle duration of developing cortical neurons in ven-tricular zone (VZ) extends under prenatal ethanol expo-sure (Miller and Nowakowski, 1991; Miller, 1996). Atlater stages (E18–E21), ethanol inhibits proliferation inSVZ rather than in VZ (Miller, 1989; Miller and Nowa-kowski, 1991). Experimental models using cortical stemcells in culture have shown that ethanol exposurereduces stem cell population, without committing themto neuronal differentiation (Crews et al., 2003; Santil-lano et al., 2005; Camarillo and Miranda, 2008; Mirandaet al., 2008). Thus, a reduction of telencephalic struc-tures (Sanchis et al., 1986; Miller and Potempa, 1990;Miller, 1995a, 2006), such as in microcephaly (Miller,1995b; Sari and Gozes, 2006) or miswired large fibersbundles, are likely to be impacted by the neuronal prolif-erative status and viability (Qiang et al., 2002; Livy andElberger, 2008). Concentration of ethanol, duration oftreatment, and developmental period of prenatal intoxi-cation play an important role on the structures affected

(Miller, 1995b; Maier and West, 2001; Miranda et al.,2008).

Prenatal ethanol exposure also impairs cell migrationin the developing telencephalon. Indeed, gross abnormal-ities due to inefficient migration, such as lissencephalyand heterotopias, are described after prenatal ethanolexposure (Miller, 1986; Anton et al., 1999; Andermann,2000; Pilz et al., 2002; Kato and Dobyns, 2003). In thepresence of ethanol, neurons do not reach superficialcortical targets (layers II/III) and remain in deeperlayers (layers IV–VI) of the cerebral cortex (Miller, 1986,1997). In some cases, migrating cells pass beyond thepial surface and form suprapial heterotopias (Miller,1986; Eksioglu et al., 1996; Takeda et al., 2003; Mooneyet al., 2004). Ethanol may impair migration by acting ei-ther on the substrate or in cell autonomous mechanismsof cell locomotion (Miller, 1993; Siegenthaler and Miller,2004). Radial glial cells, a scaffold for migrating neu-rons, are affected by prenatal ethanol exposure,although it is still a matter of controversy if ethanolaccelerates or delays their differentiation into astrocytes(Miller and Robertson, 1993; Valles et al., 1996, 1997).

Cell migration also relies on cellular interactionsmediated by cell adhesion proteins and are influenced bygrowth factors (Miura et al., 1992). TGFb, for example,expressed in developing cortical neurons and radial glia(Flanders et al., 1991; Tomoda et al., 1996; Miller, 2003)modulates the expression of cell adhesion molecules(CAMs) and modulates migration in the cerebral cortexin a dose-dependent fashion (Siegenhalter and Miller,2004). In vivo, prenatal ethanol exposure alters both theamount of TGFb ligand and receptor in the developingcortex (Miller, 2003) blocking the TGFb stimulatoryeffect on migration (Hirai et al., 1999; Siegenhalter andMiller, 2004). A deficient glial substratum, combinedwith impaired cell-autonomous migration mechanism orwith misexpression of guiding signals, is likely to play asignificant role in the final positioning of corticalneurons.

L1, a well-studied cell adhesion molecule, has alsobeen considered to play a role in the pathogenesis ofFAS (reviewed in Bearer, 2001; Goodlett et al., 2005). L1has been implicated in targeting, axonal fasciculation,cell migration, and synaptic plasticity in the developingbrain. Mutations of the L1 are associated with the samestructural morphological anomalies in FASD (such ashydrocephalus, agenesis, or hypoplasia of the corpus cal-losum and cerebella dysplasia). At low concentrations,ethanol disrupts L1 function, interfering in neurite out-growth (Bearer et al., 1999), and blockade of ethanoleffects on L1 signaling prevents teratogenic effects (Wil-kemeyer et al., 2000; Chen et al., 2001, 2005). Further-more, L1 has been shown to have a neuroprotectiveeffect by rescuing neurons from ethanol-induced celldeath (Gubitosi-Klug et al., 2007). Although the precisemechanism of interaction between L1 and ethanol is notclear, Arevalo et al. (2008) have recently described abinding pocket for ethanol in the L1 molecule.

Pathway abnormalities due to ethanol are also presentin large fiber bundles such as the pyramidal tract andcorpus callosum. Miller (1987) reports that after prena-tal exposure, the number of neurons in cortical layer Vis reduced by 30%, but the fraction of remaining neuronsin the somatomotor cortex that have spinal projections isincreased by 40% (Miller and Al-Rabiai, 1994). This

ETHANOL AND THE DEVELOPING GABAergic SYSTEM 1925

effect is probably due to a number of axon collateralsthat were not removed during the period of normalaxonal pruning, retaining their exuberant projection(O’Leary and Terashima, 1988; Ramoa et al., 1988; Heff-ner et al., 1990). Similarly, ethanol delays the develop-ment and migration of callosal cells long enough tooverlap with the elimination of transitory axons, leadingto a delayed or nonexistent regressive phenomena andconsequently abnormal positioning of the callosal popu-lation (Miller, 1997; Livy and Elberberger, 2008). Thereis increasing evidence that ethanol can disrupt not onlygrowth-cone motility associated with axon growth butalso motility associated with axon guidance leading toabnormal bundle formation. In vitro ethanol exposure ofrat hippocampal pyramidal neurons delays initial axonoutgrowth to the establishment of polarity but acceler-ates subsequent axon growth rate by reducing retrac-tions during periodic pauses in elongation (Lindsley etal., 2003). Hippocampal axons are attracted to brain-derived neurotrophic factor (BDNF), but when neuronsare kept in ethanol-containing medium and exposed to agradient of BDNF, nearly all axons exhibit marked re-pulsive turning, suggesting that ethanol disrupts theway axons respond to guidance cues (Lohof et al., 1992).

The ability of ethanol to induce widespread apoptosisin the telencephalon strongly contributes to the reducedbrain mass and neurobehavioral disturbances associatedwith FASD. An increasing number of studies have shownreduced neuronal density in somatosensory cortical areasof rodents (Bailey et al., 2004; Medina and Krahe, 2008)and primates prenatally exposed to ethanol (Miller,2006), which may be due to an increased cell death ratiocombined with reduced cycling activity (Jacobs andMiller, 2001). Developmental cell death is affected by thematurational state of neural tissues and by the type ofneuron targeted (Wood et al., 1992; Miller, 1995b).In vitro and in vivo ethanol exposures induce corticalneuronal death (Miller, 1988; Miller and Potempa, 1990;Mooney and Miller, 2001, 2003; Jiang et al., 2007),chiefly between E11 and E21 in rats, similar to thatobserved in the beginning of synaptogenesis (between P4and P10; Miller, 1988; Miller and Potempa, 1990). Morerecently, it has been acknowledged that embryonic corti-cal neurons are more vulnerable to ethanol-related celldeath than tissues taken postnatally (Miller andPotempa, 1990; Miller, 1996; Mooney and Miller, 2007).The hippocampus can also be affected by ethanol expo-sure in both prenatal and postnatal stages in rodents ina time- and area-dependent manner (for review see Mikiet al., 2008). Anatomical studies demonstrate thatchronic prenatal ethanol exposure decreases the totalnumber of neurons in CA1 of rats with low impact on theneuronal number in the dentate gyrus. In contrast, whenethanol is continued during the early postnatal phase,the total number of neurons in the dentate gyrusincreases with no change in CA1 (Miller, 1995a). Ethanolaction is probably related to NMDA antagonism andGABA mimetic patterns, since comparing the pattern ofneurodegeneration induced by ethanol with that inducedby NMDA antagonists or GABAmimetics revealed simi-lar results (Olney, 2002). An inhibition of phosphatidyl-serine (PS) accumulation has also been related toethanol-induced cell death (Akbar et al., 2006).

Cognitive impairment in children prenatally andchronically exposed to ethanol has been related to abnor-

mal formation of the white matter (Lancaster, 1994).Miller and al Rabiai (1994) demonstrated that the over-all size of the pyramidal tract is reduced, diameter ofmyelinated axons is smaller, and the myelin is thinnerin ethanol-treated rats. The effect of ethanol is probablydirectly or indirectly related to the inhibition of myelin-specific proteins (Chiappelli et al., 1991; Kojima et al.,1994; Zoeller et al., 1994; Miller et al., 1995). Iron isrequired for cholesterol and lipid synthesis, and Milleret al. (1995) related myelin alterations to abnormal ironmetabolism in ethanol-treated rats. Two key componentsof myelin, transferrin and ferritin, are found predomi-nantly in oligodendrocytes (Connor and Menzies, 1996),and the peak in iron uptake is concurrent with the peakof myelin formation (Taylor and Morgan, 1990). Bichen-kov and Ellingson (2001, 2002) demonstrate that ethanolmodulates the expression of myelin basic protein (MBP)through protein kinase C activation, but not of 20,30-cyclic nucleotide 30-phosphodiesterase (CNP) in CG-4oligodendrocytes.

DEVELOPMENTAL ASPECTS OFTELENCEPHALIC GABAergic CELLS

GABA is the main inhibitory neurotransmitter in thecentral nervous system, and in the cortex is synthesizedin 20%–30% of all neurons and present in approximately20% of all synapses, counterbalancing the excessive ac-tivity of excitatory systems (Hendry et al., 1987; Beau-lieu et al., 1992; a review on the general aspects of theGABAergic system can be found in Varju et al., 2001).

GABA is synthesized from glutamate by two isoformsof the enzyme glutamic acid decarboxylase (GAD-65 andGAD-67; reviewed by Martin and Rimvall, 1993), and itexerts its action through binding to ionotropic (GABAA

and GABAC) and metabotropic (GABAB) GABA recep-tors. GABA action is terminated by neurotransmitter re-uptake through GABA transporters (GAT) to the cyto-plasm where it is metabolized by GABA transaminases.Upon GABA receptor activation, an influx of chloride orefflux of potassium ions hyperpolarize the membrane,preventing cell depolarization and activation.

GAD 65 and 67 are products of two genes and differfrom each other in sequence, molecular size, subcellulardistribution, and interactions with the cofactor pyridoxalphosphate. GAD 65 and 67 mRNAs in mice are detectedearly in development, already at E10.5 at comparablelevels (Katarova et al., 2000). In the rat, GAD mRNA ispresent at E17 in the developing cortex (Ma and Barker,1998), and GAD 67 mRNA and protein is present atbirth in the somatosensory barrel cortex (Golshani et al.,1997; Kiser et al., 1998). The appearance of GAD 65 lagsslightly behind it. The first GABA immunoreactive neu-rons are observed in the cortical anlage at E12 in theprimitive plexiform layer of the mouse. In the followingdays, GABA-positive neurons are present in the mar-ginal zone, subplate, and subventricular zone (Del Rio etal., 1992).

Our perception of the GABAergic system as just an in-hibitory neurotransmitter system with defined syntheticpathways has been changing (review in Owens andKriegstein, 2002). In the early stages of development,GABA can originate from putrescine (Seiler and Al-Therib, 1974; De Mello et al., 1976; Hokoc et al., 1990;Yamasaki et al., 1999; Sequerra et al., 2007), bypassing

1926 ISAYAMA ET AL.

the synthesizing enzyme GAD, and constituting a sec-ond, alternative pathway for GABA synthesis. GABA isalso excitatory during development. The lack of expres-sion of KCC2, a chloride transporter, results in anincreased intracellular chloride concentration, generat-ing depolarization upon GABA receptor activation(reviewed by Ben-Ari, 2007). This excitatory action isalso found in adult neural progenitors and immatureneurons (Lo Turco et al., 1995; Liu et al., 2005; reviewedin Ge et al., 2006). The developmental shift of GABAaction from excitatory to inhibitory occurs between post-natal days 5 and 7 in the hippocampal formation of rats(Ben-Ari et al., 1989; reviewed in Ben-Ari et al., 2007)and occurs transiently during delivery, stimulated by asurge in oxytocin (Khazipov et al., 2008). The commentsby Ganguly et al. (2001, see also comments by Staleyand Smith [2001]) show that this functional reversal isactually dependent on GABAA receptor activation, butBDNF (brain derived neurotrophic factor), tyrosine ki-nase receptors, and cholinergic activity may also beinvolved in this developmental switch (Liu et al., 2006;reviewed in Ben-Ari et al., 2007).

Nonconventional (nonsynaptic) actions of GABA havebeen described in the earliest stages of corticogenesis,influencing key processes such as proliferation, differen-tiation, migration, and morphological maturation(reviewed in Owens and Kriegstein, 2002; Represa andBen-Ari, 2005; Cancedda et al., 2007). These develop-mental aspects related to the GABAergic system seem tobe better correlated with the activation of GABA recep-tors. For example, GABA-induced membrane depolariza-tion opens calcium channels and increases intracellularconcentration of calcium ions, facilitating the opening ofNMDA receptors (Owens and Kriegstein, 2002). Suchobservations suggest that GABA could influence cell mo-tility and neurite growth through calcium transients inmigrating neurons (Barbin et al., 1993; Marty et al.,1996; Komuro and Rakic, 1998; Maric et al., 2001; Tapiaet al., 2001; Borodinsky et al., 2003).

Cortical GABAergic neuron generation extendsthrough a lengthy period, roughly between E10.5 andE18 in mice. These neurons migrate tangentially to theneocortex and hippocampus from the subpallial telen-cephalon (the ganglionic eminences), initially from themedial ganglionic eminence, and then from the lateraland caudal ganglionic eminences and the entopeduncu-lar region (Anderson et al., 2001; Corbin et al., 2001; Heet al., 2001; Nery et al., 2002; Xu et al., 2004; Butt etal., 2005; reviews in Marin and Rubenstein, 2001; Won-ders and Anderson, 2006; Petanjek et al., 2008). Devel-opmental studies have shown that the origin of specificsubpopulations of GABAergic cells can be traced to spe-cific niches of gene expression in the ventral telencepha-lon (Flames et al., 2007; Fogarty et al., 2007). As thesecells leave the proliferative zone and initiate theirmigration toward their final targets they start express-ing GABA and their synthetic enzyme GAD 67 (Denaxaet al., 2001; Tanaka et al., 2003, 2006). Three spatiallyand temporally distinct streams of tangentially migrat-ing interneurons originate from the subpallial telenceph-alon. The population that originates GABAergic neuronsin the telencephalon follows a lateral-caudal pattern ofmigration. Migration pathways used by MGE cellschange through development: early (E12) migrating neu-rons assume more superficial routes near the developing

marginal zone, whereas later migrating neurons navi-gate through deeper pathways at the border of the sub-ventricular zone (Anderson et al., 2001; Marin andRubenstein, 2001; Metin et al., 2006). Once in the cere-bral cortex, interneurons penetrate the cortical plate byturning and migrating radially to their final positions(Ang et al., 2003; Tanaka et al., 2003). Radial glia fibersare probably involved in this final phase of GABAergiccell migration (Nadarajah et al., 2002).

Along their way to the cortex, several molecules regu-late the migration of GABAergic cells. To exit the ven-tricular zones, newborn interneurons are repelled byephrins expressed in these layers (Zimmer et al., 2008).The ventral to dorsal direction of interneuron migrationappears to be the result of simultaneous activity of che-morepulsive (ventral) and chemoattractive (dorsal) fac-tors produced, respectively, by the preoptic area (POa)and the cortex (Marin et al., 2003; Wichterle et al.,2003), but some other guidance molecules expressed enroute to the cortex also influence their trajectory. Slit-1or -2, two large extracellular molecules, that possess che-morepulsive activity for growing axons and migratingcells may be one of the molecules involved in this pro-cess (Zhu et al., 1999), although cortical interneuronmigration is not affected in the Slit1/2 double knockoutmice (Marin et al., 2003). Migrating interneurons com-mitted to populating the cortex express Npn1/2 and aretherefore repelled away from the striatum, where theirrepulsive ligands Semaphorin 3A and 3F are expressed(Marin et al., 2001). Many other factors, such as differ-ent isoforms of Neuregulin-1 (Flames et al., 2004) andglial cell line-derived neurotrophic factor (GDNF; Pozasand Ibanez, 2005) were shown to guide cells to the cor-tex, whereas BDNF and neurotrophin-4 (NT4, Polleux etal., 2002), hepatocyte growth factor (Powell et al., 2001)were shown to stimulate tangential migration of MGE-derived cells to the cortex. Also, the CXC chemokine re-ceptor 4 (CXCR4) is expressed in tangentially migratinginterneurons and its ligand stromal cell-derived factor-1(SDF-1 or CXCL12), a potent chemoattractant for thesemigrating interneurons, is secreted by embryonicmeninges (Stumm et al., 2003). Although the data aboutsubstrates that support the tangential migration arescattered and no evidence for the involvement of largeextracellular molecules is described, it has been sug-gested that growing corticofugal axons act as a scaffoldfor migrating cells, which involve TAG-1 adhesion mole-cule (Metin and Godement, 1996; Denaxa et al., 2001).

Migrating cells do not have a fully developed synapticapparatus but are strongly influenced by extracellularneurotransmitter molecules. Neurotransmitters areindeed part of the cortical milieu since early stages ofbrain development (Benitez-Diaz et al., 2003), and sev-eral classes of neurotransmitter receptors are expressedby telencephalic radially and tangentially migrating cells(Nguyen et al., 2001, 2004; Lujan et al., 2005). Growingevidence points to the role of GABA, glutamate, glycine,and dopamine during the migration process (Behar etal., 1996, 1998, 2000; Flint et al., 1998; Metin et al.,2000; Ohtani et al., 2003; Yozu et al., 2008). Behar et al.(1996, 1998) reported a concentration-dependent modu-lation of cellular movements in vitro by GABA and dif-ferent GABA receptors (GABAR) are involved in theprocesses of chemotaxis and chemokinesis. A blockade ofGABAA–C receptors impairs radial migration from the

ETHANOL AND THE DEVELOPING GABAergic SYSTEM 1927

ventricular to the intermediate zone (IZ) and a specificblockade of GABAB receptors (GABABR) impairs migra-tion from the IZ to the cortical plate (Behar et al., 2000).Tangentially migrating interneurons also express GABARand while GABABR influences the route of migration,GABAAR signaling is essential for interneurons to crossthe corticostriatal junction and to settle in the cortex(Lopez-Bendito et al., 2003; Cuzon et al., 2006).

ETHANOL AND THE DEVELOPINGGABAergic SYSTEM

In the past years, an increasing interest in exploringthe teratogenic effects of ethanol in the developingGABAergic system has emerged. Prenatal ethanol expo-sure specifically reduces the density of cortical GABAer-gic population chiefly in the mature somatosensorycortex of rodents (Bailey et al., 2004) and primates(Miller, 2006). These data suggest that local circuitryinterneurons in a given cortical layer seem to be moresusceptible to ethanol-related cell death than projectionneurons in corresponding cortical lamination (Miller,2006).

In contrast, recent in vivo and in vitro data demon-strate that moderate ethanol intoxication between E1and E14 can increase both the number and density oftelencephalic GABAergic interneurons at E14 as well asthe ambient GABA (Cuzon et al., 2008). These authorsattribute this rise in GABAergic cell number not to anincreased proliferative activity of GABAergic precursors

cells in the medial ganglionic eminence but to enhancedtangential migration of MGE cells. There is an apparentdisagreement between in vivo (Bailey et al., 2004; Miller,2006) and in vitro (Cuzon et al., 2008) aspects of ethanolinfluence on GABAergic cells that must be furtherinvestigated.

In our model of binge-like ethanol treatment duringthe peak of GABAergic cell generation (E11–E14), etha-nol does not induce major changes in the pattern ofGABA immunoreactivity (Fig. 1). Ethanol-treated andcontrol animals show an intense labeling in the mantlezone of the ventral telencephalon that extend to the an-terior entopeduncular area. However, increased immuno-reactivity is observed in the LGE, without clear changesin the medial ganglionic eminence (MGE) and the cere-bral cortical anlage (CTX) of treated embryos (Fig. 1),which is substantiated by GAD protein analysis for botheminences (Fig. 2).

GABAergic cell generation also follows an inside-outgradient, with cells destined for cortical layers V-VIpeaking at E16, whereas those destined for layer IVbeing mostly generated on E17, and GABAergic precur-sors that populate the superficial layers II-III at the endof the gestational period (Del Rio et al., 1992; Rymarand Sadikot, 2007). Hence, by looking at which corticalGABAergic cell layer is affected one could possibly definethe timing of ethanol toxicity. Chronic prenatal ethanolexposure reduces the number of cortical neurons mostlyin superficial layers II-III (Miller, 1986, 2006) anddecreases the density of GABAergic cells in cortical

Fig. 1. Low-power photomicrographs of GABA-immunohistochem-istry in coronal telencephalic slices of E14 ethanol-treated (ETOH, A)and control (B, CON) groups. (C, D) Higher magnification of theregions are depicted in A and B. Note the increased immunoreactivityin the mantle zone of the ganglionic eminences when compared withthe proliferative zones in both groups. GABA staining is more intense

in the lateral ganglionic eminence (LGE) than in the medial ganglioniceminence (MGE) and CTX (cortex). (C, D) GABAergic neurons can beseen exiting the ventral telencephalon and migrating toward thecortex (arrows). Abbreviations: H ¼ hippocampus; AEP ¼ anteriorentopeduncular area; CSJ ¼ corticostriatal junction. Scale bar repre-sents 100 lm.

1928 ISAYAMA ET AL.

layers II-III of guinea pigs exposed to ethanol during thedevelopment (Bailey et al., 2004). These results suggestthat chronic prenatal ethanol treatment seems to affectmostly the late-generated GABAergic cells. Ikonomidouet al. (2000) found that layer II neurons are particularlysensitive to binge-like ethanol administration in theearly postnatal period that corresponded to synaptogene-sis, similar to the findings observed following chronictreatment.

In our model, ethanol exposure between E11 and E14produces a change in the pattern of GABA immunolabel-ing in the somatosensory (parietal) cortex at P10, duringthe period of synaptogenesis. An isotropic pattern of amosaic distribution is found throughout cortical laminae(Fig. 3A) in the ETOH group, in contrast with GABApositive cells more densely localized in layers II-III andV-VI of the CON group (Fig. 3B). On the other hand, thepattern of GABA immunoreactivity in the parietal cortexof P180 mice does not seem to be different from controlanimals (Fig. 3C,D), despite a slight but not significantincrease in the number of GABA positive cells in theETOH group. The changes observed at P10 seem to be

transient since no differences are later observed in theadult cortex of ethanol and control groups. These resultscould be explained by (1) GABA mimetic effects of etha-nol promoting survival or affording specific neuropro-tection to GABAergic precursors, or inducing GADexpression (Zink and Spanagel, 2005); or (2) an acuteand direct neuroapoptotic effect, followed by a later andperhaps indirect compensatory mechanism that allowsfor maintenance of final numbers and/or function (Yama-saki et al., 1997; Mitchell et al., 2000; Williams et al.,2001). Surviving extranumerous GABAergic neuronscould still maintain or reverse to their immature proper-ties, such as being excitatory upon depolarization, andtherefore be responsible for the occurrence of seizures(Cossart et al., 2005; Ben-Ari and Holmes, 2006), one ofthe characteristic findings in children with FAS. Thelack of significant changes between the two groups atP180 could also be due to the small sample analyzed.

Ethanol treatment could also affect individual corticallayers in a manner unrelated to the time of generationof neurons. GABA can have a selective action at individ-ual layers, being able to generate action potentials inlayers 5/6 but not in layers 2/3 (Rheims et al., 2008).Ethanol mimicking GABA action through the activa-tion of GABAR could target preferentially those layers,perhaps promoting selective survival of those neurons(Redburn and Schousboe, 1987; Abraham et al., 1993;Owens and Kriegstein, 2002).

The type of exposure (chronic vs. acute) and the periodduring which the treatment occurred (prenatal vs. earlypostnatal) could account for the differences observed invarious models in the literature. Moreover, several im-portant developmental processes are still underway. Pro-liferation of GABAergic progenitors is still occurring, aswell as the process of natural cell death. Synaptic anddendritic remodeling is yet to occur.

One of the most important targets of ethanol in thecentral nervous system is the GABAA receptor, a mem-ber of the ligand-gated ion channel superfamily of recep-tors that have a binding site for ethanol (Iqbal et al.,2004; Lee et al., 2007). It has been proposed that ethanolacts by a dual mechanism involving GABAA receptorpotentiation and NMDA receptor inhibition, whichcauses excessive neuronal inhibition (Ikonomidou et al.,2000; Olney et al., 2002b). Calcium elevations secondaryto GABAAR activation have been shown to trigger celldeath in developing neurons (Nunez et al., 2003).

Ethanol seems to modulate GABAR (GABAAR andGABABR) in a cell type- and subunit composition-de-pendent manner (Aguayo, 1990; Wafford et al., 1990;Nakahiro et al., 1991; Tatebayashi et al., 1998; Mori etal., 2000; Li et al., 2005; Lee et al., 2007; Korpi et al.,2007). Activation of subtypes of GABAergic receptors areinvolved in several aspects of brain development (reviewin Lujan et al., 2005). In the developing telencephalon,GABAAR influences DNA synthesis (LoTurco et al.,1995; Haydar et al., 2000), cell cycle of VZ progenitors(Haydar et al., 2000), provides stop signals for radiallymigrating neurons as they approach their target desti-nations in the cortical plate (Behar et al., 1998), whereasGABABR and GABACR stimulate migration of neuronsfrom the VZ and IZ, respectively. In the ventral telen-cephalon, GABAAR influences the migration of inter-neurons and GABABR are thought to influencetangential migration (Lopez-Bendito et al., 2003).

Fig. 2. Level of expression of the two isoforms of GAD (GAD 65/67)in the ganglionic eminences of control and ethanol-treated animals. b-Actin levels were used as loading control. Both GAD isoforms werepresent in the LGE and MGE at E14. GAD levels were increased in theLGE but not in the MGE. Densitometric analysis with average arbitraryvalues and standard deviation (1 litter/group, n ¼ 3 for each group)revealed a significant increase in GAD expression in the LGE (*P ¼0.0068).

ETHANOL AND THE DEVELOPING GABAergic SYSTEM 1929

Prenatal ethanol exposure leads to an increase inmuscimol binding sites and to a corresponding increasein the amount of GABAAR a1 and b2/3 subunit proteins.Prenatal exposure also modulates the expression ofGABABR mRNA in embryonic rat brain in an age- andarea-dependent manner (Li et al., 2005). In situ and invitro studies have shown that GABAB receptors modu-late ethanol effects at cellular level, including PKA anddopamine D1 receptor activities (Lee et al., 2008).

Concerning GABA synthesis, acute postnatal ethanolintoxication has been shown to reduce GAD67 proteinlevels in the cerebellum, but not in the medial septum ordiagonal band (Hsiao et al., 2002). In organotypic tissuecultures from ethanol-treated newborn rats, Zink andSpanagel (2005) report an induced gene expression ofGAD67 and the vesicular GABA transmembrane trans-porter (VGAT) that could contribute to potentiateGABAAR hyperactivation.

PRENATAL ETHANOL EXPOSURE ANDGABAERGIC CELL MIGRATION

The rodent embryonic GABAergic system is vulnerableto several agents (Kato and Dobyns, 2003; Crandall etal., 2004; Vutskits et al., 2006; Cuzon et al., 2008), andprenatal ethanol exposure impacts GABAergic cells dur-ing development, being associated with gross morpholog-ical changes. Chronic prenatal ethanol treatment causesheterotopias, disorganization of cortical and hippocampallayers, and hydrocephalus (Kotkoskie and Norton, 1988;Sakata-Haga et al., 2001, 2004; Mooney et al., 2004;Camarillo and Miranda, 2008). However, GABAergiccells are also affected in other brain dysmorphologies,such as in cortical dysplasia and lissencephaly (Kato andDobyns, 2003; Moroni et al., 2008). The overall effect isthought to result from impaired migration of interneur-ons because of the changes in reelin function (indirectly)

Fig. 3. GABA immunoreactivity in the cerebral cortex of P10 (A, B)and P180 (C, D) mice prenatally exposed to ethanol (2.0 g/kg). AtP10, GABA positive cells in the ETOH group showed an isotropic pat-tern of a mosaic distribution throughout cortical laminae (A), in con-trast with GABA positive cells more densely localized in layers II-IIIand V-VI of the CON group (B). This difference was not observed in

the parietal cortex of P180 mice (C, D), although we did find a slightincreased number of GABA positive cells in the ETOH group (CON89.63 � 17.4 cells/mm2, n ¼ 2, and ETOH 101.66 � 17.54 cells/mm2,n ¼ 3, P ¼ 0.45). Indicated: I-VI (cortical layers), WM (white matter).Scale bar: 100 lm.

1930 ISAYAMA ET AL.

or BDNF levels that influence positioning of migratingneurons (Climent et al., 2002; Hevner et al., 2004;Alcantara et al., 2006; Pla et al., 2006; Yabut et al.,2007). Ambient GABA is also thought to influence finalneuronal destination (Heck et al., 2007) and neuronalproduction (Weissman et al., 2004). These factors associ-ated with increased availability of GABA (effect on GADexpression) or indirectly through GABAA receptor activa-tion by ethanol (Cancedda et al., 2007; Cuzon et al.,2008) could lead GABAergic cells to settle at inappropri-ate layers or cortical columns resulting in abnormal mat-uration of cortical circuitries.

Earlier effects of ethanol could be due to the involve-ment of transcription factors in neurodevelopmentalprocesses, including Pax6, Xbra, Xnot2, FoxA4b, Shh,Otx2, and several members of the Sox family (Peng etal., 2004; Kashyap et al., 2007; Yelin et al., 2007). Sonichedgehog (Shh) is an important ventral gradient morph-ogen (Chiang et al., 1996; Rallu et al., 2002) that modu-lates the maturation of cortical GABAergic neuronallineage (Yung et al., 2002; Gulacsi and Lillien, 2003;Watanabe et al., 2005). Loss of Shh signaling around E9results in the absence of cortical interneurons (Fuccilloet al., 2004) and induces holoprosencephaly in mice fol-lowing a single dose of ethanol at E7 (Aoto et al., 2008).A pilot study using a higher dose of ethanol (3.5 g/kg)induced holoprosencephaly, limb malformation, andincreased incidence of abortion in mice (Isayama et al.,unpublished results).

Between E14 and E17, GABAergic neurons migratemainly into the lower SVZ/IZ (Marin and Rubenstein,2001, 2003). GABAA receptors are thought to be an im-portant signal for interneurons to cross the corticostria-tal junction and therefore to enter the cortex, whereasGABAB receptors seem to be involved in the final loca-tion of GABAergic interneurons in the cortical plate.

Cuzon et al. (2008) showed that ethanol in uterus doesnot affect tangential migratory routes but it does alterthe characteristics of tangentially migrating interneur-ons through cell-extrinsic and -intrinsic mechanisms.Under ethanol exposure, the MGE-derived cells migratefaster with an increased migration rate throughout theirmigratory path, which can be reversed by suppressingGABAA receptor activity or by reducing ambient GABA.Furthermore, this study reports an increased ratio ofNeuN-immunopositive neurons originating from MGEfrom alcoholized embryos, suggesting that ethanol drivesMGE-GABAergic interneurons toward differentiation(Cuzon, et al., 2008). Differently, our model of binge-likeethanol treatment during the peak of GABAergic cellgeneration neither compromise tangential migratoryroutes nor block the entry of interneurons in the corticalplate (Figs. 1C,D and 4). GABA-immunopositive neuronsdisplay typical bipolar morphology along the dorsal tel-encephalon following superficial and deep routes alongthe cortical plate (Fig. 4), and the number of GABAergiccells in the CTX, proximal to the cortico-striatal junctiondoes not differ from those in the control group (CON 119� 30.66 cells/field, n ¼ 6; ETOH 123 � 27.54 cells/field,n ¼ 6, P ¼ 0.68). It is possible that the absence of anethanol effect on migration lies on the type of treatment(acute vs. chronic), where the chronic treatment affectsearlier progenitor cells, influencing their response toETOH from earlier stages, or on the ETOH concentra-tions used in both studies, or in a difference in the

expression of GABA receptors at these developmentalstages, or a combination of all factors.

In the postnatal brain, GABA decreased the rate ofcell migration (Bolteus and Bordey, 2004) in the SVZand in the rostral migratory stream via GABAA recep-tors activation, the opposite effect observed by Cuzon etal. (2006, 2008) for tangentially migrating MGE-derivedcells in the prenatal brain. This differential effect couldbe age- (postnatal vs. prenatal) or region-related (LGEvs. MGE). Eckardt et al. (1998) noticed that ethanoldoes not stimulate GABAA receptors ubiquitously indifferent brain regions. Additionally, in embryonic andearly postnatal neocortex, GABAA receptors expressedby CP neurons have a lower affinity for GABA and arerelatively sensitive to receptor desensitization whencompared with neurons in the VZ (Owens et al., 1999).

Although this review focuses in the GABAergic sys-tem, it is important to note that the glutamatergic sys-tem is involved in several developmental aspects of thebrain (Heng et al., 2007) and also constitutes a targetfor ethanol. Interaction between these two systemsshould also be considered for the final effects of ethanolin the developing brain, since at these early stagesGABA receptor activation generates an excitatoryresponse and is able to generate intracellular calciumwaves and regulate NMDA receptor activation, whichcould then impart changes in the cell machinery (Weiss-man et al., 2004; Kriegstein, 2005; Wang and Kriegstein,2008). Additionally, other neurochemical and neuromo-dulatory systems have been implicated in the effects ofethanol in the developing brain such as cAMP, adeno-sine, and dopamine (Mailliard and Diamond, 2004; Maaset al., 2005; Conti et al., 2009).

PRENATAL ETHANOL EXPOSUREAND CELL DEATH

Ethanol triggers a widespread apoptotic neurodegener-ation during the brain growth spurt period in rat and

Fig. 4. Coronal section obtained from an ETOH-treated corticalanlage at E14 immunoreacted for GABA. Note tangentially migratingGABA-immunopositive cells along their superficial and deep migratoryroutes. Indicated: ventricular zone (VZ), marginal zone (MZ), corticalplate (CP), subventricular zone (SVZ), and lateral ventricle (LV). Scalebar: 50 lm.

ETHANOL AND THE DEVELOPING GABAergic SYSTEM 1931

mice models (Olney et al., 2002a,b,c) in the cerebral cor-tex, cerebellum, and hippocampus (Samson and Diaz,1981; West et al., 1989; Miller, 1995a; Liesi, 1997; Lightet al., 2002; Iqbal et al., 2004). In the cerebral cortex, ap-optosis varies between 10% and 60% in rats (Miller,1995, 2006) and humans (Ramachandran et al., 2001).

Recent reports have shown that isolated binge-likeepisodes of ethanol administration (mimicking heavyepisodes of drinking) induce apoptosis in the developingbrain, especially during the period of synaptogenesis(Ikonomidou et al., 2000; Olney et al., 2001; Olney,2004). Quantitative TUNEL analysis of E11–E14 etha-nol-treated embryos at E14 shows a significant increasein apoptotic cells in the cerebral cortex and LGE, butnot in other brain regions (Table 1). These findings couldhave clinical implications and could explain microce-phaly and cognitive and behavioral disturbances associ-ated with FAS.

Olney et al. (2002b) proposed that a dual mecha-nism—blockade of NMDA glutamate receptors andhyperactivation of GABAA receptors—was responsiblefor mediating ethanol-induced apoptosis, supported byfindings that ethanol displayed both NMDA antagonisticand GABA mimetic properties, and application of ago-nists and antagonists produced similar effects (Ikonomi-dou et al., 2001). Ethanol’s effect on apoptosis could alsooccur at a genomic level, affecting the levels of Bax andBcl-2 in an age- and area-dependent manner (Young etal., 2003; Lee et al., 2008) or caspase 3 (Pompeiano etal., 2000; Young et al., 2005), among others.

More recently Young and Olney (2006) found that arise in blood ethanol levels in the range of 50 mg/dL for30 to 45 min was sufficient to trigger a significant apo-ptotic response. These values are commonly achieved ina social drinking context, and a woman with only a mod-erate drinking habit might expose her fetus to such ele-vations on multiple occasions during pregnancy. In ourmodel of binge-like administration of ethanol during thepeak of GABAergic cell generation, the dam’s peak ofblood alcohol concentration at approximately 130 mg/dL(127 � 2.1 mg/dL, n ¼ 3, 45 min postadministration) isabove the corresponding state in humans (at 100 mg/dL)of slight slurring of speech, loss of control of fine motormovements, confusion and emotionally instability, butwithout reaching lethargy, loss of balance and coordina-tion and memory loss at 200 mg/dL. Besides the data ofYoung and Olney (2006), Tenkova et al. (2003) showedthat a transient elevation of blood alcohol to approxi-mately 120 mg/dL was sufficient to activate the celldeath program in visual neurons.

Not all neurons are susceptible to cell death followingethanol administration. It is possible that surviving

neurons are affected by ethanol at the cellular and func-tional levels, contributing to the FAS or FASD character-istics. Ethanol has been shown to impair long-termdepression in the cerebellum (Belmeguenai et al., 2008),and surviving cerebellar Purkinje cells prenatallyexposed to ethanol showed decreased expression of anisoform of protein kinase C, leading to decreased volt-age-gated calcium currents and changes in LTD (Servaiset al., 2007). Ethanol effects on calcium and cyclic nucle-otide signaling also affects migration of cerebellar gran-ule cells, resulting in their abnormal function andpositioning in the cerebellar cortex (Kumada et al.,2007).

CONCLUDING REMARKS

FAS and FASD are multifactorial entities with func-tional and structural abnormalities affecting not onlythe nervous system but also the whole organism. Assuch, we cannot conclusively establish which of the dif-ferent factors (neurotransmitters, adhesion molecules,cell death, transcription factors, trophic factors, etc.)reported in the literature are the major players in thesyndrome. More likely, the final result is a complexinteraction, with some of the players being up or down-regulated to compensate for the loss of function. Thesecompensatory changes can be maladaptative, resultingin FAS or FASD.

We present here a review on the effects of ethanol onthe GABAergic system. GABA is the major inhibitoryneurotransmitter in the nervous system, with the peculi-arity of being an excitatory molecule during early stagesof development. GABA, as with other neurotransmitters,also has nonsynaptic functions, being involved in severaldevelopmental steps such as proliferation, migration, dif-ferentiation, and synaptogenesis. The GABAergic systemseems to respond to the ethanol insult with compensa-tory changes, but we do not know if the compensatorymechanisms in place allow for normal function. Animbalance in the precise anatomical and functional in-hibitory connectivity will lead to physiological (Henschand Stryker, 2004; Ben-Ari, 2006) and cognitive (Lohrand Bracha, 1989; Barr et al., 2006; Gonzalez-Burgosand Lewis, 2008; Lewis et al., 2008) deficits that couldcontribute to the findings in FAS.

ACKNOWLEDGMENTS

The authors thank Adiel Batista do Nascimento, Lud-mila Ribeiro, and Rosilane T. Silva for their invaluabletechnical support in handling and caring for the animalsand with the different techniques that were used in this

TABLE 1. Density of TUNEL1 nuclei in control and ethanol groups

LGE MGE CTX H

CON 1.79 � 1.81 4.47 � 1.08 3.69 � 2.64 7.75 � 6.58ETOH 4.33 � 1.32* 4.97 � 2.29 8.25 � 1.75** 11.16 � 4.37

Density values of TUNEL þ nuclei (number of apoptotic nuclei per mm2, average � SD) in con-trol (CON, n ¼ 6) and ethanol (ETOH, n ¼ 6) groups in the medial (MGE), lateral ganglioniceminence (LGE), cortical (CTX), and hippocampus (H) at E14.Increase in the number of apoptotic nuclei reached statistical significance in the LGE (*P ¼0.035) and CTX (**P ¼ 0.016), but not in the hippocampus or MGE, suggesting a region-specificor a developmentally-related cell death effect.

1932 ISAYAMA ET AL.

study. P.E.C. Leite is a recipient of a graduate fellowshipfrom CAPES, and J.P.M. Lima and R.N. Isayama arerecipients of graduate fellowships from CNPq.

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