Experimental Neurology 229 (2011) 364–371

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Experimental Neurology

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Effects of prenatal ethanol exposure on rat brain radial glia and neuroblast migration

María Paula Aronne, Tamara Guadagnoli, Paula Fontanet, Sergio Gustavo Evrard, Alicia Brusco ⁎Instituto de Biología Celular y Neurociencias "Prof. Eduardo De Robertis", Facultad de Medicina, Universidad de Buenos Aires (UBA), Paraguay 2155 3rd fl.,(C1121ABG) Buenos Aires, Argentina

⁎ Corresponding author. Fax: +54 11 5950 9626.E-mail address: hbrusco@fmed.uba.ar (A. Brusco).

0014-4886/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.expneurol.2011.03.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 March 2010Revised 4 January 2011Accepted 7 March 2011Available online 14 March 2011

Keywords:Prenatal ethanol exposurePax6Neuronal migrationCorticogenesisCortical dysplasiaFetal alcohol syndrome

Prenatal ethanol exposure (PEE) induces morphologic and functional alterations in the developing centralnervous system. The orderly migration of neuroblasts is a key process in the development of a layeredstructure such as the cerebral cortex (CC). From initial stages of corticogenesis, the transcription factor Pax6 isintensely expressed in neuroepithelial and radial glia cells (RGCs) and is involved in continual regulation ofcell surface properties responsible for both cellular identity and radial migration. In the present work, onemonth before mating, during pregnancy and lactation, a group of female Wistar rats were fed a liquid dietwith 5.9% (w/w) ethanol (EtOH), rendering moderate blood EtOH concentrations. Maternal gestationalweight progression and fetal CC thickness were measured. CC from E12-P3 rats were examined for expressionof vimentin, nestin, S-100b, Pax6 and doublecortin using immunohistochemical assays. RGCs expressingvimentin, nestin, S-100b and Pax6 had abnormal morphologies. The migration distance through the CC andthe number of doublecortin-ir neuroblasts in germinative zones were decreased. We found significantmorphologic defects on RGCs, a marked delay in neuronal migration, decreased numbers of neuroblasts, anddecreased numbers of Pax6-ir cells in the CC as a consequence of exposure to ethanol during development.These observations suggest a sequence of toxic events that contribute to cortical dysplasia in offspringexposed to EtOH during gestation.

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Introduction

Fetal alcohol syndrome (FAS) represents a public health issueassociated with maternal alcohol abuse during pregnancy. Centralnervous system (CNS) developmental defects, which can lead tomicrocephaly and mental retardation, are among the mostsignificant characteristics of FAS. Different types of corticaldysplasia occur after prenatal ethanol exposure (PEE) due to,among other factors, disorders in neuroblasts migration (Miller,1986). Although maturation of the cerebral cortex (CC) iscompleted during postnatal period, most CC gross architecture isestablished prenatally and crucially involves the orderly migrationof neuroblasts from the proliferative ventricular zone (VZ), at theinner surface of the telencephalon, through the overlying interme-diate zone to the cortical plate (Angevine and Sidman, 1961; Rakic,1974). Neuroblasts, the precursors of neurons, migrate mainlyattached to the cellular processes of radial glia cells (RGCs) by usingthem as living scaffolding.

During corticogenesis, RGCs perform a dual function: theybehave as precursor cells (Choi and Lapham, 1978; Levitt et al.,1981, 1983; Misson et al., 1991) and as migratory substrates forneuroblasts (Rakic, 1972; Gadisseux et al., 1990; Hatten andMason,

1990). RGCs are ultrastructurally similar to glial cells, e.g., theyshow bundles of filaments and glycogen accumulation (Rakic,1972). They express nestin, a cytoskeletal type VI intermediatefilament commonly used as a marker for immature cells in thedeveloping CNS (Alberts et al., 2007; Hockfield and McKay, 1985);they also express vimentin, another cytoskeletal protein, a type IIIintermediate filament (Alberts et al., 2007; Pixley and DeVellis,1984). Moreover, by the end of neurogenesis and during neuroblastmigration, RGCs differentiate into astrocytes in some regions suchas the CC (Pixley and DeVellis, 1984; Voigt, 1989). Upondifferentiation into astrocytes, RGCs stop expressing vimentinand upregulate glial fibrillary acidic protein (GFAP; another type IIIintermediate filament) with an overlapping period when theyexpress both (Bignami and Dahl, 1974; Levitt et al., 1983; Voigt,1989). As mature astrocytes, RGCs also express S-100b protein, adimeric neurotrophic and neurite outgrowth-promoting proteinwith many important actions during both pre- and postnatal life.Among these multiple functions, S-100b acts as a promoter ofcytoskeletal stabilization (Donato, 2003; Donato et al., 2009;Huttunen et al., 2000). The correct specification of RGCs is thereforeessential for normal corticogenesis (Pinto-Lord et al., 1982).

Pax6, a paired box family transcription factor, plays animportant role in development of the brain and other organs (Chiand Epstein, 2002; Buckingham and Relaix, 2007). Pax6 isspecifically expressed by RGCs and controls their differentiationin the CC (Götz, 1998; Götz et al, 1998; Simpson and Price, 2002). In

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the absence of a functional Pax6, cortical RGCs exhibit alteredmorphology, cell number and cell-cycle kinetics (Götz et al, 1998).We previously showed that high blood ethanol concentrations (BECbetween 119 and 300 mg/dl) alter Pax6 expression during prenatalcortical development (Aronne et al., 2008).

Neuroblasts migrating through RGC processes express doublecor-tin (DCX), a type of microtubule-associated protein (MAP) thatregulates microtubule stability and influences the cytoskeletondynamic state (Francis et al., 1999; Horesh et al., 1999). Doublecortinis widely expressed in migrating neuroblasts during prenatal andpostnatal development in the CNS and during adult neurogenesis(Gleeson et al., 1999).

The cytoskeleton of nerve tissue cells is substantially altered byPEE (see Evrard and Brusco, 2011 for a comprehensive review). Weshowed that cytoskeletal proteins in neurons and glia are changedby exposure to both low (Ramos et al., 2002; Evrard et al., 2003,2006) and high EtOH concentrations (Aronne et al., 2008). Thiscurrent study examines the consequences of moderate BECs onPax6 expression, RGCs andmigratory neuroblasts in the developingCC.

Materials and methods

Animals were fed with a Liquid Diet LD 102 and LD 102A (PMIAlcohol Rodent Liquid Diet, USA). Ethanol (EtOH) (Sorialco S.A.C.I.F.,Buenos Aires, Argentina) administered to the animals was analyticalgrade. For the study of the nuclear morphology we used Hoechst33,342 stain (Sigma, St. Louis, Mo). Primary antibodies were: (a)mouse monoclonal vimentin (lot 071H4837, Sigma); (b) mousemonoclonal S-100b (lot 086H4864, Sigma); (c) rabbit polyclonal Pax6(lot 0608036825; Chemicon International, Temecula, Ca); (d) guineapig polyclonal doublecortin (DCX; lot 0605030194; Chemicon) and(e) mouse monoclonal nestin (lot 0704058033, Chemicon). Second-ary antibodies were (a) goat anti-rabbit biotin-conjugate (lot078H9160; Sigma); (b) goat anti-mouse-biotin conjugate (lot078H9060; Sigma); (c) Texas Red anti-rabbit (lot T1002; VectorLaboratories, Burlingame, CA); and (d) Fluorescein anti-guinea pig(lot MFCD 00164607, Sigma). An avidin-peroxidase complex (Extra-vidin-Peroxidase®, lot 103K4840, Sigma), 3,3′-diaminobenzidinetetrahydrochloride (lot 65H0170, Sigma), nickel ammonium sulfate(Carlo Erba Reagenti; Milano, Italy) and hydrogen peroxide (H2O2;Merck KgaA; Darmstadt, Germany) were used for immunoperoxidase

Fig. 1. Maternal weight gain showed that EtOH-exposed rats gained significantly less weight d**Pb0.01; ***Pb0.0001.

technique. All other chemical substances used in the experimentswere analytical grade.

Animal treatment

Fifty adult nulliparous female Wistar rats (Rattus norvegicus; Hsd:WI) initially weighing 250–300 g were mated with 10 adult Wistarmales. One month before mating (metabolic and taste acclimatizationperiod) during pregnancy and lactation a group of females assigned tothe treated group (T) were fed with the LD 102A liquid dietsupplemented with 5.9% (w/w) EtOH. The control group (C) was fedthe LD 102 control liquid diet. LD 102A powder is mixed with EtOH ormaltodextrin to normalize calories between groups. All animals werekept in the same environment throughout the experimental period:12/12 h light–dark cycle (lights off at 06:00 PM), controlled relativehumidity (50–70%) and temperature (20±2 °C), well-ventilatedroom and free access to liquid diets. Females were housed in groupsof five and weighed each morning. Gestational day 0 (G0) wasdeterminedwhen spermwere found in vaginal smears, and thereafterthe pregnant rats were housed singly, and assigned to five groups(n=10/group). The five groups were: collection of E12, E14, E16 andE18 or P3 offspring.

The animal care for this experimental protocol was in accor-dance with the NIH guidelines for the Care and Use of LaboratoryAnimals and the principles presented in the Guidelines for the Useof Animals in Neuroscience Research by the Society for Neurosci-ence andwith the CICUAL Animal Protocol of the School ofMedicine(University of Buenos Aires, UBA).

Cesarean operation and specimen preparation

Pregnant rats were deeply anesthetized with ketamine (10 mg/kg)and xylazine (75 mg/kg) intaperitoneally. Uteri were extracted andimmediately submerged in cold saline. Embryos were extracted intheir membranes. Simultaneously, blood was collected in heparinizedtubes from the maternal heart for determination of BEC. Heads ofembryos were submerged in cold 4% paraformaldehyde (w/v) in0.1 M phosphate buffer (PBS) pH 7.4 for 4 h at 4 °C. The fixed headswere dissected and isolated from the surrounding skull tissues undera Wild Heerbrugg M5 stereomicroscope. Each brain was thensubmerged in 5% w/v sucrose in 0.1 M PBS, pH 7.4 for 12–24 h andthen in a cryoprotective solution (25% sucrose w/v in 0.1 M PBS, pH

uring gestation, starting at day 2, than control rats. Graph symbols represent mean±SD.

Fig. 2. Cortical thickness wasmeasured from the inner (ilm) to the outer (olm) limiting membrane from E12 to E18, considering as such the total thickness of the entire telencephalicwall in both C (A–D) and T (E–H) fetuses. Cortical thickness was similar between T and C offspring until E16 when it began to be thinner in EtOH-exposed animals. Scale bar in Erepresents 100 μm (valid for A–H).

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7.4). Coronal 50-μm-thick brain sections were cut with a cryostat andmounted on glass slides. Sections were stored at −20 °C.

Determination of blood ethanol concentration

BECs were determined in a spectrophotometer by means of anenzymatic method with a specific kit QuantiChrom™ Ethanol Assay Kit(BioAssay Systems Hayward, CA, USA).

Toluidine blue and Hoechst 33,342 staining

Brain sections from the various groups were processed fortoluidine blue or Hoechst staining following the same procedure aspreviously described (Aronne et al., 2008).

Immunohistochemistry

Brain sections from the various groups were processed simulta-neously for each IHC assay following the same procedure aspreviously described (Aronne et al., 2008). In this study, primaryantibodies directed to vimentin (diluted 1:800), S-100b (1:800) ornestin (diluted 1:1000); biotinylated secondary antibody (1:200), and

Fig. 3. Quantification of cortical thickness, from E12 to E18, in C and T fetuses. Graphsymbols represent mean±SD. ***Pb0.001.

extravidin–peroxidase complex (1:400) were used. No backgroundstaining was observed.

Immunofluorescence

Brain sections from the various groups were processed byimmunofluorescence technique similarly as in the IHC experiments(Aronne et al., 2008). However, Pax-6 (diluted 1:500) and DCX(diluted 1:3000) primary antibodies and the corresponding second-ary fluorescent antibody (diluted 1:500) were used.

Morphometric digital image analysis:

All measurements were performed on coded slides under blindedand standardized conditions. The dorsolateral region of the CCwas thefocus ofmorphometric studies. Digital images from the toluidine blue-stained and IHC sections were taken through with a Zeiss Axiophotmicroscope.

CC thickness was measured in toluidine blue-stained sections withthe ImageJ software by means of a line drawn between the inner andouter limiting membranes, traced through the middle zone of thetelencephalic vesicle.

In order to quantify IHC studies, the total area of the immunore-active fiber-like structures was compared to the total area of themicroscopic field (Ramos et al., 2000) using Image-Pro Plus software.In order to evaluate the Pax6-ir and DCX-ir positive cells, the totalnumber of the immunolabeled cells was related to the total area of thecorresponding microscopic field with Optimas 6.0 software. In orderto quantify migration, a relative migration index was calculated bymeasuring the migration distance from the inner through the outerlimiting membrane relative to the total CC, using Image J software.

Statistical analysis

Four to six separate IHC experiments were run for eachimmunomarker. Individual experiments were composed of 6 to 10tissue sections of each animal from each group. Five to ten fields weremeasured from each brain area in each section of each animal.Differences between animals within each group were not statisticallysignificant. Reported values represent the mean±SD of experimentsperformed for each marker and each brain area. Differences amongthemeans C and T groupswere statistically analyzed by a “two-tailed”test. Statistical significance was set to a value of Pb0.05. For the

Fig. 4. Vimentin expression in the CC of C (A–E) and T (F–J) offspring showed a predominantly parallel pattern throughout the CC, corresponding to RGCs processes. Vimentinexpression was reduced at all ages (F–J) in T offspring. Scale bar in J represents 25 μm (valid for A–J). VZ denotes the direction toward the subjacent ventricular zone.

Fig. 5. From E12 to P3, the temporal curve of vimentin expression in the developing CCshowed a similar profile in both C and T offspring. However, T offspring showed asignificantly reduced level of expression at all ages. Graph symbols represent mean±SD.***Pb0.001.

Fig. 6. S-100b expression in the CC of C (A–E) and T (F–J) offspring showed a fiber-like patterat all ages (F–J) in T offspring. Scale bar in J represents 25 μm (valid for A–J). VZ denotes th

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statistical analysis, the GraphPad Prism 4.00 software (GraphPadSoftware Inc.) was used.

Results

Blood ethanol concentration

Measured BECs were of 89.34±6.42 mg/dl. This value wasobtained from 15 different randomly chosen T mothers. As expected,C BECs were undetectable.

Evolution of maternal gestational weight

Pre-pregnancy weights did not differ between groups. However,starting at gestational day 2, the EtOH-treated rats gained significantlyless weight than controls (Fig. 1).

Cerebral cortex thickness

CC thickness was similar early in development in T and C offspring,but CC was thinner as a function of EtOH exposure in E16 (C:1.55±0.07×10−3 μm versus T: 1.0±0.06×10−3 μm; Pb0.0001;

n throughout the CC, corresponding to RGCs processes. S-100b expression was reducede direction towards the subjacent ventricular zone.

Fig. 7. From E12 to P3, the temporal curve of S-100b expression in the developing CCshowed a similar profile in both C and T offspring. However, T offspring showed asignificantly reduced level of expression at all ages. Graph symbols represent mean±SD.***Pb0.001.

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Figs. 2 and 3) and E18 fetuses (C: 1.57±0.27×10−3 μm versusT:1.25±0.19×10−3 μm; Pb0.0001; Figs. 2 and 3).

Immunohistochemical experiments

VimentinVimentin expressionwas concentrated in the RGC cellular processes

extending from the inner to the outer limitingmembrane (Fig. 4). In allstudied ages, there was a significant consistent reduction in the relativearea of tissue covered by vimentin-ir fibers in the CC of T respective tothat of the C group (Fig. 5). On E12 a 57.66% decrease was observed (C:0.27±0.03 versus T: 0.11±0.04; Pb0.0001). On E14 a 47.11% decreasewas observed (C: 0.19±0.03 versus T: 0.1±0.03; Pb0.0001). On E16 a40.64% decrease was observed (C: 0.21±0.02 versus T: 0.12±0.02;Pb0.0001). OnE18 a 59.5% decreasewas observed (C: 0.16±0.03 versusT: 0.06±0.01; Pb0.0001). Finally, on P3 a 60.8% decrease was observed(C: 0.12±0.01 versus T: 0.04±0.005; Pb0.0001).

S-100b

S-100b distribution was limited to the cell body located in theventricular zone, near the inner limiting membrane, and to the radialprocesses of the RGCs (Fig. 6). Distribution of S-100b throughout theCC was similar in the two groups, but EtOH-exposed brains showedconsistently less intensity of cytoplasmic staining than the controls

Fig. 8. Nestin expression in the CC of C (A-E) and T (F-J) offspring showed a fiber-like patternall ages (F–J) in T offspring. Scale bar in J represents 25 μm (valid for A–J). VZ denotes the

(Fig. 7). On E12 a 73.5% immunolabeling decrease was observed(C: 0.13±0.01 versus T: 0.03±0.01; Pb0.0001). On E14 a 46.4%decrease was observed (C: 0.15±0.02 versus T: 0.08±0.02;Pb0.0001). On E16 a 47.67% decrease was observed (C: 0.15±0.02versus T: 0.08±0.08; Pb0.0001). On E18 a 51% decrease was observed(C: 0.13±0.03 versus T: 0.06±0.03; Pb0.0001). Finally, on P3 a47.24% decrease was observed (C: 0.21±0.05, T: 0.11±0.05;Pb0.0001).

Nestin

Nestin staining in a fibrillary pattern was observed in RGCs cellularprocesses traversing the telencephalic wall. The intensity of nestin-ir(Fig. 8) was consistently lower in the developing CC of T respective tothat of C (Fig. 9). On E12 a 41.3% decrease was observed (C: 0.27±0.06versus T: 0.16±0.04; Pb0.0001). On E14 a 51.76% decrease wasobserved (C: 0.25±0.03 versus T: 0.12±0.02; Pb0.0001). On E16 a49.08% decrease was observed (C: 0.23±0.02 versus T: 0.12±0.01;Pb0.0001). On E18 a 48.93% decrease was observed (C: 0.28±0.09versus T: 0.14±0.04; Pb0.0001). Finally, on P3 a 72.06% decrease wasobserved (C: 0.25±0.005 versus T: 0.07±0.01; Pb0.0001).

Pax6

Pax6 is expressed early in the gestational period in differentregions of the developing CNS but only from E8 to E14.5 in the CC. Asshown in Figs. 10 and 11, PEE induced a significant decrease in thenumber of Pax6-ir cells per microscopic field (40×) in the ventricularzone (VZ) of the CC of T brains respective to C ones only on E14(Fig. 6B) when a 55% decrease was observed (C: 15.7±3.5 versusT: 7.5±2.4; Pb0.0001).

Doublecortin

As shown in Figs. 12 and 13, PEE induced a significant delay in themigrationprocessof neuroblasts through theCCof T brains respective toC ones only on E14where a 17.6% decreasewas observed (C: 0.91±0.01versus T: 0.75±0.07; Pb0.0001).

We also quantified the total number of DCX-ir cells located in theVZ and SVZ at ages E12, E14, E16 and E18 (Fig. 14). We observed astatistically significant reduction on ages E12 and E14 in the brains of Trespective to C. On E12 a 51.92% decreasewas observed (C: 20.8±6.51versus T: 10±1.78; Pb0.0009) and, on E14 a 39.44% decrease wasobserved (C: 12±2.91 versus T: 7.26±1.67; Pb0.0001). However, on

throughout the CC, corresponding to RGCs processes. Nestin expression was reduced atdirection towards the subjacent ventricular zone.

Fig. 9. From E12 to P3, the temporal curve of nestin expression in the developing CCshowed a similar profile in both C and T offspring. However, T offspring showed asignificantly reduced level of expression at all ages. Graph symbols represent mean±SD.***Pb0.001.

Fig. 11. In the ventricular zone of the developing CC of EtOH-exposed fetuses, thequantification of the number of Pax-6-ir cells showed a significant 55% reduction, onE14 but not on E12. Graph symbols represent mean±SD. ***Pb0.001.

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E16we observed a significant 68.76% increase of the number of DCX-ircells in the brains of T respective to C (C: 7.87±0.99 versusT: 13.29 ±2.36; Pb0.0001) (Fig. 15). On E18 we could not observedDCX-ir cells (data not shown).

Hoechst 33,342

In Hoechst-stained sections, pyknotic nuclei were distinguished bytheir characteristic nuclear morphology (reduced nuclear size andhigh fluorescence intensity due to chromatin condensation). We alsoobserved nuclear debris consistent with apoptosis. We countedpyknotic nuclei and debris, and found no differences between EtOH-exposed and control groups at E12 and E14 (data not shown).

Fig. 10. In the developing CC of EtOH-exposed fetuses, the number of Pax-6-ir cells wasreduced at the ventricular zone on E14 (D) but not on E12 (B) relative to control fetuses(C and A, respectively). Scale bar in A represents 25 μm (valid for A–D). VZ denotes thedirection to the subjacent ventricular zone.

Discussion

Cortical dysplasia due to PEE can result from abnormalities inneuronal and glial proliferation and apoptosis, as well as delayed orblunted migration of neurons to the cortical plate, resulting in asmaller and disordered laminar and columnar patterning (Miller,1986; Mischel and Vinters, 1998). Our present results suggest thatthese pathogenic factors may start early in pregnancy when mothersare exposed to EtOH.

Underdeveloped CC is characteristic of FAS (Roebuck et al., 1996).A thinner CC was a later developmental finding of exposed offspring,apparent only at E16 when animals were exposed to moderate BECs.Our present results suggest that corticogenesis could be altered as aresult of several mechanisms, such as: i) impaired neuroblastsgeneration, ii) delayed neuroblasts migration, and iii) impairment of

Fig. 12. Doublecortin-ir cells in the CC on ages E12 and E14 of C (A, B) and T (C, D)fetuses corresponded to migrating neuroblasts. Relative to C fetuses, DCX-ir cells inEtOH-exposed ones were arrested in their migration through the CC on E14 (D) but noton E12 (B). Scale bar in D represents 20 μm (valid for A–D). MG: marginal zone; PP:preplate; CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; bVZ: basalventricular zone; VZ: ventricular zone.

Fig. 13. A significant decreased in the relative migrated distance of doublecortin-ir cellswas observed in EtOH-exposed offspring on E14 but not on E12, both relative to C.Graph symbols represent mean±SD. ***Pb0.001.

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the scaffolding (i.e., RGCs) through which neuroblasts migrate. Theyalso suggest that Pax6 is critically involved in these processes.

The decreased number of DCX-ir neuroblasts (E12 to E14) and thereduction in their relative migrated distance through the telence-phalic wall (E14) suggest that the thinner CC may be, partially, a laterresult of the reduction in the number of generated neuroblasts. Thisreduction could be due to a minor generation from precursor cells or,alternatively, to an increased number of them disappeared afternecrotic or apoptotic death. Since our evaluation of Hoechst-stainedsections (E12 and E14) did not show any differences, between C and Tanimals, for these types of cell death, we are prone to consider that aminor number of neuroblasts were effectively generated. Thisconclusion is consistent with the decreased number of Pax6-ir cellsfound at E14. Pax6 is involved in both neuroblasts and RGCsgeneration (Simpson and Price, 2002). Evidence exists suggestingthat Pax6 is essential for correct cell migration (Matsuo et al., 1993)and that it could be involved in the pathogenesis of certain types ofcortical dysplasia not related with EtOH exposure (Fitzgerald et al.,2011; Fukuda et al, 2000). In EtOH-exposed Xenopus laevis embryos,Peng et al. (2004) found a dose-dependent related microcephaly,growth retardation and Pax6 decreased expression. Except for thereduced number of Pax6-ir cells found in a previous report (Aronne etal., 2008), there are no data available regarding Pax6 in EtOH-exposedbrain development in mammals. As well as in that report with highBECs, we found here with moderate BECs, a decreased number ofPax6-ir cells in the CC of rat embryos at E14 and cortical dysplasia.Since Pax6 is a transcriptional factor that regulates downstream the

Fig. 14. On E16, the number of DCX-ir cells seemed to be increased in the developing CC of Tplate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.

expression of many important molecules involved in neurogenesisand neuroblasts migration (for example, cytoskeletal and celladhesion molecules) (Osumi et al., 2008), further experiments areneeded in order to fully elucidate its contribution to the alteredneuroblasts migration in PEE.

Of note here, less neuroblasts were available to migrate. Theyshowed a delayed migration and slowed rate of arrival to thedeveloping cortical plate. On the other hand, the increased numberof DCX-ir neuroblasts at E16 in the VZ and SVZ of EtOH-exposedembryos suggest a delayed generation and/or beginning of migrationin a place and at a moment when neuroblasts are normally no longerbeing generated. These findings are in full agreement with previousreports on cortical dysplasia after PEE (Miller, 1986). Our results arealso in accordance to the suggestion that DCX is required for all radialmigrating neocortical neuroblasts to enter the cortical plate from theVZ (Bai et al., 2003).

During normal corticogenesis, once the RGCs scaffolding iscorrectly established, neuroblasts begin to migrate attached to theRGCs processes until they reach their final position in all those brainstructures exhibiting a layered or stratified adult appearance, such asthe CC (Rakic, 2003). Since vimentin and nestin are expressed not onlyby glial precursors and RGCs but also by neural progenitors, theirimpaired expression levels consistently found from E12 to P3, suggestthat a global neurogenetic impairment took place in EtOH-exposedoffspring. Unlike the in vivo and in vitro findings of previous reports(Eriksen et al., 2002), we found a decreased expression of S-100bprotein. This protein display neurotrophic and neurite outgrowthpromoting activities and is strongly involved in the development ofseveral neurotransmitter systems (Donato et al., 2009). The alteredexpression of S-100b that we observed could further be related withthe known impaired development of these systems observed in theoffspring of alcoholic mothers.

Thus, in this study moderate BECs altered not only the migratingneuroblasts pertaining to the first migratory wave (that lately shouldgive rise to cortical pyramidal neurons) but also those cells servingthem as migration scaffolding (i.e., RGCs). We showed that, byaltering only these two corticogenetic mechanisms, moderate PEEwas able to induce cortical dysplasia in the offspring rat brains andthat the reduction in Pax6-ir cells may be an important pathogenicfactor.

Acknowledgments

We thank Mrs Emérita Jorge Vilela for her expertise technicalassistance. We also greatly thank the anonymous reviewers for theirvery helpful remarks in the improvement of the final text of ourpresent work. This work was supported by grants PIP-00774 from the

fetuses relative to C ones. Scale bar represents 100 μm. MZ: marginal zone; CP: cortical

Fig. 15. Relative to C fetuses, the quantified number of doublecortin-ir cells of EtOH-exposed ones was significantly decreased on E12 and E14 but increased on E16. Graphsymbols represent mean±SD. ***Pb0.001.

371M.P. Aronne et al. / Experimental Neurology 229 (2011) 364–371

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)and UBACyT M-004 from the Universidad de Buenos Aires (UBA) toProf. Dr Brusco.

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