Neurogenesis in Cerebral Heterotopia Induced in Rats by Prenatal Methylazoxymethanol Treatment

We have previously demonstrated that the antiproliferative agentmethylazoxymethanol acetate (MAM) is able to induce in ratscerebral heterotopia that share striking similarities with thoseobserved in human periventricular nodular heterotopia (PNH), acerebral dysgenesis frequently observed in human patients affectedby drug-resistant focal epilepsy. In this study, we investigated thetime-course of neurogenesis in the cerebral heterotopia of MAM-treated rats, with the idea of understanding why PNH develop inhuman patients. For these goals, we analyzed the cytoarchitecturalfeatures, the time of neurogenesis and the cellular phenotype of theheterotopia, by means of BrdU immunocytochemistry and confocalimmunofluorescence experiments. Our data demonstrate that thedifferent types of heterotopia in MAM-treated rats are formedthrough the same altered neurogenetic process, which follows quiteorganized neurogenetic gradients. The MAM-induced ablation of anearly wave of cortical neurons is sufficient to alter per se themigration and differentiation of subsequently generated neurons,which in turn set the base for the formation of the differentheterotopic structures. The neurogenesis of MAM-inducedheterotopia may explain the origin and intrinsic epileptogenicity ofperiventricular nodular heterotopia in human patients.

IntroductionCerebral dysgeneses are developmental malformations of thebrain structure determined by impairment of the spatiallyorganized and time-regulated processes of neurogenesis,migration, neuron/glia interaction and cell differentiation,which take place in the developing brain. In recent years, it hasbeen increasingly recognized that cerebral dysgeneses are arelevant cause of mental and neurological deficits in humans,including epilepsy (Galaburda et al., 1985; Kuzniecky et al.,1993; Barkovich et al., 1994). In particular, human patients withdrug-resistant focal epilepsy are frequently (in ∼20–40% of cases)affected by developmental malformations of the cortex and theyfrequently undergo epilepsy surgery for the relief of theirintractable seizures (Hardiman et al., 1988; Meencke and Veith,1992; Mischel et al., 1995).

The mechanisms leading to the genesis of the mature brainhave been extensively investigated and the processes ofneurogenesis, radial and tangential migration, gliogenesis andaxonogenesis have been studied in detail in the normaldeveloping brain (Rakic, 1972, 1995; Hatten, 1990; O’Rourke et

al., 1992; Bayer and Altman, 1995; Zerlin et al., 1995; Tamamakiet al., 1997; Tan et al., 1998; Lavdas et al., 1999). In contrast tothis, with a few exceptions of rare genetically proven casesdetermined by impairment of genes involved in the processes ofneuronal migration (des Portes et al., 1998; Fox et al., 1998),the etiology and pathogenic mechanisms responsible for thedevelopment of most cerebral dysgeneses in humans are largelyunknown.

The present study is part of a larger project investigating themechanisms underlying the genesis and the hyperexcitability of

neurons of cerebral heterotopia induced by the pre-nataladministration of the antimitotic agent methylazoxymethanolacetate (MAM) (Colacitti et al., 1998, 1999; Sancini et al., 1998;Battaglia et al., 2002). MAM is an alkylating agent that has longbeen used to induce developmental brain dysfunction in rodents(Singh, 1977; Cattabeni and Di Luca, 1997). In vivo, MAM israpidly converted to methyl-diazonium, which damages DNA bymethylating the O6 or N7 positions of guanine nucleic acids(Matsumoto et al., 1972). Actively dividing neuroepithelial cellsduring the S-phase are affected, whereas post-mitotic neurons orneuroblasts in the G0 phase are spared (Johnston and Coyle,1979). The narrow time-window of biological activity of MAM(2–24 h, with maximal activity at 12 h after administration)affects the proliferation of specific neuronal cell populations(Cattaneo et al., 1995). We have previously demonstrated that adouble MAM administration in rats on embryonic day 15 is ableto induce cerebral heterotopia made up by hyperexcitableneurons (Sancini et al., 1998; Colacitti et al., 1999) that sharestriking similarities with those observed in human peri-ventricular nodular heterotopia (PNH), a cerebral dysgenesischaracterized by nodular masses of gray matter located in closeapposition to the periventricular germinative neuroepithelium(Battaglia et al., 1996, 1997). In this study, we investigatedneurogenesis in the cerebral heterotopia of MAM-treated rats,with the idea of understanding why periventricular heterotopiadevelop in human patients. For these goals, we have employed acombined approach, by analyzing: (i) the cytoarchitecturalfeatures of the heterotopia in the early post-natal period; (ii) thebirthdating of cells within the heterotopia, by means of theincorporation of the thymidine analog bromodeoxyuridine(BrdU); and (iii) the cellular phenotypes of the heterotopia, bymeans of confocal immunof luorescence experiments. Our datademonstrate that all heterotopia in MAM-treated rats share acommon neurogenesis and suggest that MAM-induced ablationof early generated waves of neurons is sufficient to deeply altermigration and differentiation of the subsequent waves of newlygenerated neurons, leading to the formation of the differenttypes of heterotopia.

Materials and Methods

Animal Handling and Cerebral Tissue ProcessingAll experimental procedures were carried out with care to minimizediscomfort and pain to treated rats, in accordance with the guidelines ofthe European Communities Council (Directive of 24 November 1986,86/609/EEC). Pregnant Sprague–Dawley rats received two MAM acetatedoses (15 mg/kg maternal body wt, i.p. in sterile saline) on E15, the firstinjection at 12.00 a.m. and the second at 12.00 p.m., as previouslyreported (Colacitti et al., 1999). On the same day, control pregnant ratswere sham injected with the vehicle alone. A group of MAM-treated andsham-operated pregnant rats were then subjected to injections of5-bromo,2′-deoxy-uridine (BrdU, 50 mg/kg maternal body wt, i.p. insterile saline) at different embryonic ages (see below). The day after

Neurogenesis in Cerebral HeterotopiaInduced in Rats by PrenatalMethylazoxymethanol Treatment

Giorgio Battaglia, Silvia Pagliardini, Laura Saglietti, FlaminioCattabeni1, Monica Di Luca1, Stefania Bassanini and VeronicaSetola

Molecular Neuroanatomy Laboratory, ExperimentalNeurophysiology Department, Neurological Institute ‘C. Besta’,via Celoria 11 and 1Department of Pharmacological Sciences,University of Milano, Via Balzaretti 9, 20133 Milano, Italy

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conception (as determined by vaginal smear) was designated embryonicday 1 (E1). Litters were born on day 22 or 23 of gestation and the day ofbirth was designated post-natal day 1 (P1). The pups were housed understandard conditions, as previously reported (Colacitti et al., 1999).

At various post-natal ages (1 day to 4 months of age), MAM/BrdU-treated and sham-operated control rats were deeply anesthetizedwith ice (from P1 to P7) or chloral hydrate (from P15 to adulthood;1 ml/100 g body wt of a 4% solution) and perfused with 1%paraformaldehyde followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at pH 7.2. Brains were removed from the skull,post-fixed overnight in 4% paraformaldehyde and cut with a vibratomeinto 40–50 µm thick coronal sections. Sections were collected in PBS andsodium azide (0.01%) in serial order. One out of three sections (fordeveloping rats) or five sections (for adult rats) were reacted forimmunocytochemistry (ICC) as outlined below. The sections adjacent tothe immunoreacted ones were counterstained with 0.1% thionine.

BrdU ImmunocytochemistryTo investigate the time of neurogenesis of the MAM-induced heterotopicneurons, seven different time points of BrdU injection were chosen, at 24,36, 48, 60, 84, 108 and 132 h after the last MAM administration, referredto as E16/24, 17/12, 17/24, 18/12, 19/12, 20/12 and 21/12, respectively.Earlier BrdU administrations were not performed, given the alreadyreported MAM-induced ablation of neuroblasts generated at the time ofMAM administration or shortly thereafter (Gillies and Price, 1993; Noctoret al., 1999).

To optimize BrdU immunocytochemistry, we performed several pilotexperiments according to protocols reported in the literature (Miller andNowakowsky, 1988; Soriano and Del Rio, 1991; DeDiego et al., 1994). Weobtained best results with the following protocol. Free-f loating sectionswere initially pre-treated with 2 N HCl in PBS for 60 min to separate DNAstrands and then with sodium borate 0.1 M for 10 min to neutralize theacid. Sections were then treated with 1% H2O2 in PBS for 20 min toneutralize the endogenous peroxidase activity, rinsed in PBS andincubated with 10% normal goat serum (NGS) and 0.2% Triton-X100for 60 min, to mask non-specific adsorption sites and to increase thepenetration of the reagents. Sections were then incubated overnight withanti-BrdU monoclonal antibodies (Becton-Dickinson, San José, CA; orBoehringer-Mannheim GmbH, Germany) diluted 1:75. After rinsing inPBS, the sections were incubated with biotinylated goat anti-mouse IgG(GAM, diluted 1:200; Jackson, PA), rinsed in PBS and then incubated withExtravidin (1:5000; Sigma-Aldrich, Milano, Italy). All immunoreagentswere diluted in 1% NGS in PBS. Peroxidase staining was obtained byincubating the sections either in DAB (0.075%) and H2O2 (0.002%) or inDAB (1.25 mg/ml), NAS (0.04%), NH4Cl (0.004%), glucose (0.2%) andglucose oxidase (1.2 U/ml) in 0.05 M Tris–HCl at pH 7.6. The immuno-reacted sections were mounted onto gelatine-coated glass slides, air-dried,dehydrated and coverslipped with DPX. Slides were then analyzed andphotographed with a Nikon Microphot FXA microscope.

Immunof luorescence Double-labelingTo evaluate the cellular phenotype of BrdU-labeled cells, sequentialdouble-labeling immunof luorescence experiments were performed withantibodies against BrdU and (i) the monoclonal antibody against glialfibrillary acidic protein (GFAP, diluted 1:1000; Swant, Bellinzona,Switzerland) as astroglial marker, or (ii) the monoclonal antibody againstmicrotubule associated protein 2 (MAP2, diluted 1:1000; SternbergerMonoclonals Inc., Lutherville, MA), as neuronal marker. For BrdU/GFAPexperiments, the tyramide signal amplification protocol (NEN LifeScience, Boston, MA) was employed, following the manufacturer’sinstructions and using biotinylated GAM (1:200), streptavidin-HRP(1:200) and Cy3-conjugated tyramide (1:100) to reveal BrdU-labelednuclei and then FITC-conjugated GAM (1:200) to reveal the glial cellularphenotype. For BrdU/MAP2 experiments, the Alexa FluorR 546 GAM(diluted 1:2000; Molecular Probes, Eugene, OR) was used to revealBrdU-labeled nuclei and the Alexa FluorR 488 GAM (diluted 1:2000;Molecular Probes) was subsequently used to demonstrate MAP2-positivecell bodies.

To evaluate the cellular phenotype of the white matter bands close tothe heterotopia in early post-natal rats, double-labeling experiments wereperformed with antibodies against neuronal and glial cellular markers and

the f luorescent Nissl stain NeuroTrace™ (Molecular Probes, Eugene, OR).Free-f loating sections from P1–P7 MAM-treated rats were pre-treatedwith H2O2 and then with NGS/Triton before overnight incubation withthe primary antibodies: (i) monoclonal anti-vimentin (Dako, Glostrup,Denmark), diluted 1:200, as an early glial marker (Zerlin et al., 1995);(ii) monoclonal anti-MAP2, diluted 1:1000 and anti-SMI 32, against non-phosphorylated neurofilaments, diluted 1:500 (Sternberger MonoclonalsInc., Lutherville, MA), as specific markers for early-generated corticalneurons (Del Rio et al., 2000). The sections were then incubated for 2 hwith Cy2-conjugated GAM (1:200) and then in NeuroTrace™ 530/615 redf luorescent Nissl stain, according to the manufacturer’s instructions.After all experiments, sections were repeatedly rinsed, mounted onslides, coverslipped with Fluorsave (Calbiochem, Darmstadt, Germany)and examined on a Radiance 2100 confocal microscope (Bio-Rad). Imageswere saved in TIFF format and then elaborated by means of AdobePhotoshop software.

Quantification of BrdU ImmunocytochemistryTo determine the percentage of BrdU labeled neurons after BrdUinjections at different time points, double labeling BrdU/MAP2 confocalimmunof luorescence experiments were used. BrdU-labeled, MAP2-labeled and BrdU/MAP2-double-labeled neurons were counted in threenon-adjacent immunoreacted sections through different periventricularheterotopia at different rostro-caudal levels from five rats after E17/12,E18/12, E19/12, E20/12 and E21/12 BrdU injections. Labeled cells werecounted without correction, since only estimates of ratios were sought —BrdU/MAP2-double-labeled neurons versus the total number of MAP2-labeled neurons (Saper, 1996). Only MAP2-positive neurons displayingthe nucleus surrounded by immunoreactive cell body in the plane of theconfocal sections were considered. Neurons with partially but clearlyBrdU-labeled nuclei, as well as neurons with fully labeled nuclei wereconsidered as BrdU-positive. The outer borders of the periventricularheterotopia were always easily recognizable from the surrounding whitematter and lateral ventricle. Data from cell counts were obtained by usingMicrosoft Excel. Results were averaged and expressed as percentages ofBrdU/MAP2-double-labeled neurons (±standard deviation) on the totalnumber of MAP2-labeled neurons.

ResultsOur protocol of MAM administration consistently induced in theoffspring of treated dams cortical hypoplasia with disruption ofcortical layering and clusters of heterotopic neurons within theneocortex (cortical heterotopia), in the dorso-lateral part ofthe lateral ventricles (periventricular heterotopia) and withinthe hippocampal CA1 and CA2 regions [intra-hippocampalheterotopia; see also Colacitti et al. (Colacitti et al., 1999)]. Theperiventricular heterotopia were frequently in anatomicalcontinuity with the lateral hippocampal border. In contrast, nocytoarchitectural abnormalities were ever found, either insham-operated animals injected with saline or in rats treatedwith BrdU alone, given the low administered dose of BrdU(Miller and Nowakowski, 1988; Kolb et al., 1999). In the presentstudy, we have specifically addressed the following issues:(i) the development of the heterotopia in the first two post-natalweeks; (ii) the neurogenesis of the heterotopia, by means ofBrdU immunocytochemistry; and (iii) the cellular phenotypeswithin the heterotopia, by means of double-labeling immuno-f luorescence.

Early Post-natal Development of the MAM-inducedHeterotopiaOne of the main cytoarchitectural features of the MAM-treatedrats during early post-natal development was the presence ofelongated cellular bands of intensely thionine-stained cellswithin the subcortical white matter (Figs 1 and 2D, arrows).These bands were most probably clusters of young migratingneurons similar to those observed pre-natally in the intermediate

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and subventricular zones of the developing white matter (Bayerand Altman, 1995). In MAM-treated rats, however, numerouscellular bands were still present in the white matter of the earlypost-natal period, intermingled with many elongated cells withmorphological features typical of young migrating neurons(Fig. 1B, arrowheads). More importantly, in MAM-treated rats

the bands were located in close anatomical relationship to theheterotopia, i.e. in the white matter immediately beneath thecortical heterotopia (Fig. 1A–C), or overlying the periventricularand intra-hippocampal heterotopia (Figs 1D,E and 2D). Theyremained conspicuous during the first post-natal week, butprogressively disappeared during the second post-natal week.

Figure 1. Cortical and periventricular heterotopia of MAM-treated rats during early post-natal development. Thionine stain. (A–C) Cortical heterotopia (c). Note in (A) the elongatednodules of immature neurons extending throughout the cortical layers of the fronto-parietal cortex (left is medial and right is lateral), the reduction of cellular density (asterisks in A)in the cortical layers overlying the heterotopia and the close anatomical relationship between the heterotopia and the white matter cellular bands (arrows in A–C). The white matterbands are intermingled with numerous elongated migrating neurons (arrowheads in B). Neurons at the borders of the heterotopia progressively acquire a less immature morphologyduring the first post-natal week (arrowheads in C). (D–F) The periventricular heterotopia, also already present at birth, are located dorsal to the germinative neuroepithelium andbeneath the white matter cellular bands (arrows in D–F). Note their increase in size (cf. P1 in D and P3 in E), and the thin space at P5 (asterisks in F) dividing the darkly stained smallcells at the borders from the round and densely packed neurons in the core of the heterotopia. Scale bars: 100 µm (A, D, E); 50 µm in (B, C, F).

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The cortical heterotopia overlying the white matter bandswere already present at birth (P1), as round or elongated nodulesof densely packed neurons of small size and ill-definedmorphology (Fig. 1A). The neocortex around the heterotopiacontained many small neurons of uniform morphology and thenormal cortical layering was not recognizable. A clear reductionof cellular density was evident above the heterotopia (Fig. 1A,asterisk). During the first post-natal week the corticalheterotopia enlarged in size; the neurons at the borders of theheterotopia progressively displayed a less immature morphology(Fig. 1C, arrowheads), whereas the neurons within the hetero-topia were still more densely packed than the surroundingneocortical neurons and intermingled with darkly stained smallcells (Fig. 1C). At P15, the cortical heterotopia displayed theanatomical features already described for the adult rats (Colacittiet al., 1999).

The periventricular heterotopia were also already present atbirth (Fig. 1D). They were located just dorsally to thegerminative neuroepithelium and immediately beneath thebands of darkly stained cells (Fig. 1D,E). They progressivelyincreased their size during the first post-natal week (cf. Fig. 1D

and E). The core of the nodules was made up of densely packedround cells with ill-defined borders, whereas elongated neuronswere marginally placed at the border of the nodules inter-mingled with more darkly stained small cells (Fig. 1F). At P5, athin space, possibly tangentially running fibers, divided theborder from the core of the nodules (Fig. 1F, asterisks). In

contrast, the intra-hippocampal heterotopia were neverpresent at birth (Fig. 2A,B). At P1, sectors of the germinativeneuroepithelium dorsal to the developing hippocampus wereparticularly thick (Fig. 2A), with thin columns of dark migratingneurons extending to the already formed pyramidal cell layerof CA1 and CA2 (Fig. 2B, arrowheads). At P3, wedge-shapedmasses of neurons filled the stratum oriens between theneuroepithelium and the pyramidal cell layer (Fig. 2C), whereasat P5 the intra-hippocampal heterotopia consistently disruptedthe CA1 and CA2 pyramidal cell layer and invaded the stratum

radiatum (Fig. 2D). Thick cellular bands were present in thesubcortical white matter overlying the intra-hippocampalheterotopia (Fig. 2D, arrows). At P15, the subcortical as well asthe intra-hippocampal nodules displayed the anatomical featuresof the adult rat (Colacitti et al., 1999).

BrdU LabelingData from BrdU labeling in MAM-treated rats are illustrated inFigures 3–7 and summarized in the following, by groupingtogether E16/24–E17/12–E17/24 BrdU injections (i.e. from 24 to48 h after the last MAM administration), E18/12–E19/12 BrdUinjections (from 60 to 84 h after MAM) and E20/12–E21/12BrdU injections (from 108 to 132 h after MAM). In general, theneocortical and hippocampal areas surrounding the heterotopiafollowed the previously reported time-course of cortical andhippocampal neurogenesis (Hicks and D’Amato, 1968; Bayer,1980; Bayer and Altman, 1995). Indeed, inside-out, latero-medial

Figure 2. Progressive genesis of intra-hippocampal heterotopia during early post-natal development. Thionine stain. (A–B) At P1, thin columns of migrating neurons (arrowheads inB) extend from the thick germinative neuroepithelium (asterisks in A and B) to the CA2 pyramidal cell layer. (C) At P3, the developing heterotopia invade the stratum oriens (or). (D)At P5, the heterotopia disrupts the pyramidal cell layer (Py). Note the dense cellular cluster in the white matter overlying the heterotopia (arrows). The boxed area in (A) is representedat higher magnification in (B). Scale bars: 200 µm (A); 50 µm (B); 100 µm (C–D).




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and rostro-caudal gradients of neurogenesis were observed inthe MAM neocortex after BrdU injections from E16 to E21. In thehippocampus, CA3 pyramidal neurons were generated beforeCA1 and CA2 neurons and granule cells in the dentate gyrus, anda superficial to deep (or external to internal) gradient ofneurogenesis was clearly present (not shown). By contrast, thetime of generation of neurons within the heterotopia andadjacent white matter bands was extended and it roughlyoverlapped that of superficial neocortical neurons. Indeed, asillustrated in Figure 3, after E18/19 BrdU injections labeledneurons were located in the more superficial cortical layers andin the cortical (Fig. 3A,B) and periventricular (Fig. 3B)heterotopia. In contrast, neurons in the deep cortical layerssurrounding the heterotopia were mostly unlabeled (Fig. 3A,B).

After E16/24–E17/12–E17/24 BrdU injections, the cellularbands within the white matter were BrdU-negative, even if rarecells were BrdU-positive after E17/24 injections (not shown). Incontrast, BrdU-positive neurons were found in the cortical andsubcortical heterotopia (Figs 4–6) and their number increasedprogressively from E16/24 to E17/24 injections. They weremainly located at the periphery of cortical (Fig. 4A,B) andperiventricular (Fig. 5A,C) heterotopia. The numbers of labeledcells and the intensity of labeling tended to decrease movinginside the core of the heterotopia. In the intra-hippocampalheterotopia, labeled cells were more evident dorsally and thenumbers of labeled cells and intensity of labeling decreasedmoving ventrally (Fig. 6A,B).

After E18/12–E19/12 administration, BrdU-positive cells werenumerous between P1 and P7 in the white matter bands close tothe cortical and heterotopia (Fig. 7A) and overlying thesubcortical heterotopia (Fig. 7B,D). They progressively dis-appeared after the first post-natal week, but some BrdU-positiveneurons were still present in adulthood in the white matter closeto the heterotopia (Fig. 7C). Within the heterotopia, manyneurons were BrdU-positive during the post-natal developmentand in adult stages (Figs 4–6). Labeled heterotopic neurons werelocated throughout the extension of the heterotopia, in boththe periphery and the core of the cortical (Fig. 4C,D) andperiventricular (Fig. 5B,D) heterotopia and in both the dorsaland ventral aspects of the intra-hippocampal heterotopia(Fig. 6C,D).

After E20/12–E21/12 BrdU injections, the white matter bandsclose to the heterotopia were still BrdU-positive in P1–P7MAM-treated rats, but less conspicuously if compared to those

labeled after E18/19 injections. Some BrdU-positive neuronswere still present in adult rats in the white matter close to theheterotopic nodules (not shown). In both developing and adultrats, labeled heterotopic neurons were confined inside thecortical (Fig. 4E,F) and periventricular (Fig. 5E,F) heterotopiaand in the more ventral aspects of the intra-hippocampalheterotopia (Fig. 6E,F). Thus, taken together, these data indicatethat the white matter bands begin to form after the heterotopiaand that the heterotopia are formed through an outside-in (forcortical and periventricular heterotopia) and dorso-ventral (forintra-hippocampal heterotopia) neurogenetic gradient.

Most BrdU-labeled Heterotopic Cells are NeuronsTo verify the cellular phenotype of the BrdU-labeled cells in theMAM-induced heterotopia, we performed double-labelingimmunof luorescence experiments by combining BrdU stainingwith labeling for either GFAP, a well-known marker forastrocytes, or MAP2, a specific neuronal marker. At all con-sidered time-points of BrdU administration, the vast majorityof BrdU-positive cells within the heterotopia were characterizedby large nuclei not surrounded by GFAP positive processes(Fig. 8A,B, arrowheads). In particular, double-labeled BrdU andGFAP-positive glial cells were very rare in the heterotopia afterE17/12–E18/12 injections and only slightly more numerous afterlater BrdU injections (Fig. 8A). BrdU/GFAP-positive glial cellswere present after E19/12 and later BrdU injections in the whitematter adjacent to the heterotopic structures (Fig. 8B, arrows).By contrast, most BrdU-positive nuclei were surrounded byMAP2-positive cell bodies after BrdU injections at all consideredtime-points (Fig. 8C,D, arrows). After E19/12–E21/12 BrdUinjections, some BrdU labeled nuclei not surrounded by MAP2staining were present within the heterotopia and more con-spicuously in the adjacent white matter (not shown). Thesefindings clearly demonstrate that most BrdU-labeled cells withinthe heterotopia at the different considered time-points possess aneuronal phenotype and they also suggest that gliogenesisfollows neurogenesis in the MAM-induced heterotopia.

Cells within the White Matter Bands Possess a NeuronalPhenotypeTo investigate the cellular phenotype of the white matter bandsclose to the heterotopia, we then performed double-labelingexperiments with the f luorescent Nissl stain NeuroTrace™ andantibodies against neuronal and glial markers. In the first

Figure 3. Pattern of BrdU labeling of heterotopia and surrounding cerebral areas. BrdU immunocytochemistry at P5 (A) and P15 (B). Note that after relatively late BrdU injections (atE18 in A and E19 in B), labeled cells are confined to the cortical and periventricular heterotopia and superficial cortical layers. Neurons in the deep cortical layers surrounding theheterotopia are mostly unlabeled. Scale bars: 200 µm.




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post-natal week, the white matter bands were made up of cellswith a relatively large nucleus, intensely stained by NeuroTrace™(Fig. 8H–L) and a thin rim of cytoplasm, immunoreactive for theanti-MAP2 (Fig. 8E) and anti-SMI 32 antibodies (Fig. 8F). Incontrast, the bands were crossed by vimentin-positive fibers butthey did not contain vimentin-immunoreactive cell bodies(Fig. 8G,L). Rare vimentin-positive cells were occasionally foundat the borders of the white matter bands (Fig. 8G,L, arrow).Neurons within the cortical and periventricular heterotopiadisplayed a more evident MAP2 cytoplasmic staining ifcompared to neurons within the nearby white matter bands(Fig. 8E,H). Taken together, these double-labeling experimentsdemonstrate that the white matter bands close to the heterotopiaare mostly made up by neurons.

Quantification of BrdU ImmunocytochemistryFinally, we used double labeling BrdU/MAP2 experiments toquantify the birthdating at different embryonic ages of neuronscommitted to form the heterotopia. We selected periventricularheterotopia for this analysis, since their anatomical borderswere always easily distinguishable and BrdU/MAP2 immuno-f luorescence since single- and double-labeled neurons wererecognizable with certainty when analyzed at the confocalmicroscope (Fig. 8C,D). Data are illustrated in Figure 9 as meanratios (±standard deviations) of BrdU/MAP2 double-labeledneurons on the total number of MAP2-positive neurons withinthe heterotopia. Neurogenesis began in periventricularheterotopia soon after the last MAM administration (27.27% ofdouble-labeled neurons after E17/12 BrdU injections) and itrapidly progressed to reach a neurogenetic peak around the 18th

Figure 4. BrdU labeling in cortical heterotopia. BrdU immunocytochemistry (A, B, D, F) and thionine stain (C, E). Note that at E16/24 (A, B) BrdU-labeled cells are located at the ventraland dorsal borders of the heterotopia, whereas they are progressively located inside the heterotopia at E19/12 (D) and E21/12 (F). The boxed area in (A) is represented at highermagnification in (B). (C, D) and (E, F), respectively, are taken from consecutive adjacent sections. Scale bars: 250 µm (A); 50 µm (B); 80 µm (C–F).




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Figure 5. BrdU labeling in periventricular heterotopia. Thionine stain (A, B) and BrdU immunocytochemistry (C–F). Note the preferential BrdU labeling at the periphery of theheterotopia at E17/24 (C), and the progressive staining of the core of the heterotopia at E19/12 (D) and E21/12 (E). The preferential labeling of the core of the heterotopia after E21/12BrdU injection is shown at higher magnification in (F). (A, C) and (B, D), respectively, are taken from consecutive adjacent sections. lv, lateral ventricle. Scale bars: 100 µm (A–E);40 µm (F).




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Figure 6. BrdU labeling in intra-hippocampal heterotopia. Thionine stain (A, C, E) and BrdU immunocytochemistry (B, D, F). Note the preferential BrdU labeling in the dorsal part ofthe heterotopia at E17/24 (B) and the progressive staining in more ventral sectors at E19/12 (D) and E21/12 (F). (A, B), (C, D) and (E, F), respectively, are taken from consecutiveadjacent sections. The boxed area in (A) is represented at higher magnification in (B). Scale bars: 80 µm (A,C–F); 50 µm (B).




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embryonic day for periventricular heterotopia (52.09% ofdouble-labeled neurons). Interestingly, neurogenesis continuedon the 19th and 20th embryonic days and still 10.55% of neuronsforming periventricular heterotopia were generated at the 21stembryonic day (Fig. 8).

DiscussionIn the present study, by combining cytoarchitectural, BrdU andimmunof luorescence analyses we have provided an assessmentof the altered neurogenesis which underlies the formation ofcerebral heterotopia in MAM-treated rats. We believe that thepresent data should provide insight not only into the process of

cortical development, but also into the possible way by whichperiventricular nodular heterotopia develop in human patients.

Ontogenesis of Cerebral Heterotopia in MAM-treatedRatsNumerous cellular bands of migrating neurons are present in thewhite matter of MAM-treated rats during the first post-natalweek, in close anatomical relationship with the newly formingheterotopia (Figs 1 and 2). During the process of migration in thenormal rat brain, cells committed to the neocortex sojournbetween E15 and E21 in cellular bands within the intermediateand subventricular zones before reaching their final cortical

Figure 8. Most cells in the heterotopia and adjacent white matter cellular bands possess a neuronal phenotype. (A–B) Confocal immunofluorescence images combining BrdU (red)and GFAP (green) immunostaining. At all considered time-points of BrdU administration, the vast majority of BrdU-positive cells within the heterotopia were characterized by large rednuclei not surrounded by GFAP positive green processes (arrowheads). Rare double-stained glial cells with small nuclei surrounded by GFAP positive processes (arrows in A) werepresent from E19 onwards. By contrast, double-labeled BrdU/GFAP glial cells were more numerous in the white matter adjacent to the heterotopia (arrows in B) from E19 onwards.(C–D) Confocal immunofluorescence images combining BrdU (red) and MAP2 (green) immunostaining. Most BrdU-positive red nuclei within the heterotopia were surrounded byMAP2-positive green perikarya at all considered time-points of BrdU administration. Note that the nuclei of double-labeled BrdU/MAP2 neurons can be fully (arrows) or partially(broken arrows) stained by BrdU immunofluorescence. Arrowheads mark single-labeled MAP2-positive neurons. (E–L) Confocal single-labeled immunofluorescence images of MAP2(E), SMI32 (F) and vimentin (G) immunostaining, merged with the fluorescent Nissl staining NeuroTrace™ (H–L). At P3, the white matter cellular bands were made up by cells withlarge nuclei, labeled by NeuroTrace™ in red (H, I, L), surrounded by thin rims of MAP2 (E) or SMI32 (F) positive cytoplasm (green). In contrast, the cellular bands were crossed byvimentin-positive green fibers, but they did not contain vimentin immunoreactive cell bodies (G, L). Note in (E) that the neurons within the periventricular heterotopia (PV) displayeda more evident MAP2 cytoplasmic staining if compared to neurons within the nearby white matter band (marked by arrows in E and H). Rare vimentin-positive cells (arrow in G andL) were occasionally found at the borders of some white matter bands. PV, periventricular heterotopia; wm, white matter. Scale bars: 20 µm.

Figure 7. BrdU labeling in the white matter during development and in adulthood. BrdU immunocytochemistry (A–C) and thionine stain (D). After BrdU administration at E18/12,many cells in the white matter cellular bands are BrdU immunoreactive (arrows) both at P1 (A) and P5 (B). Note in (B) and (D) the close anatomical relationship between theBrdU-positive white matter cellular band (arrows) and intra-hippocampal heterotopia (arrowheads). After BrdU administration at E19/12 (C), some BrdU-immunoreactive cells werestill present in adult MAM-treated rats in the white matter (arrows) close to cortical heterotopia (arrowheads). (B) and (D) are taken from adjacent sections. wm, white matter. Scalebars: 100 µm.




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destination (Bayer and Altman, 1995). The white matter bandsdescribed here in the post-natal brain of MAM-treated rats likelycorrespond to the superior bands of the pre-natal brain of normal

rats (Bayer and Altman, 1995), indicating that the MAMtreatment determines a delayed post-natal maturation of theheterotopia. The bands are made up mostly of neurons, as




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demonstrated by their nuclear size (Robertson et al., 2000) andtheir perikaryal staining by anti-MAP2 and anti-SMI 32, but notby anti-vimentin antibodies (Fig. 8). MAP2-positive neuronswere reported in the developing white matter of newbornMAM-treated ferrets (Noctor et al., 1999), but the neuronalbands were not described, possibly for the fact that the stage ofcortical development in neonatal ferrets is more advanced incomparison to that in neonatal rats. Indeed, the MAP2-positiveneurons within the white matter bands reported here arestill-differentiating young neurons, since they display less intenseMAP2 immunoreactivity than neurons already located withinthe heterotopia (Fig. 8). Their morphology and staining pattern,together with the close anatomical relationship with the differ-ent types of heterotopia, suggest that these bands function in theearly post-natal period as a reservoir of young migrating neuronsspecifically committed to the newly forming heterotopia. Theylater disappear, possibly for the combination of migration intothe heterotopia and apoptotic cell death (Del Rio et al., 2000),but quite a few neurons are still present within the white matterclose to the heterotopia during adulthood (Fig. 7C), as describedalso for human PNH (Battaglia et al., 2002).

The close anatomical relationship between these bands andthe newly forming heterotopia suggests that the heterotopiathemselves are able to inf luence and direct the migration of latergenerated waves of neurons. The reduction of cellular densityconsistently observed just above the cortical heterotopia (Fig.1A) also suggests that the absence of superficial neurons impairsthe proper migration of later generated cortical neurons. Takentogether, these data support the idea that early generated andproperly migrated neurons are of key importance for themigration and differentiation of subsequent waves of neurons. Asimilar hypothesis has been recently put forward in a paper inwhich MAM was specifically administered in ferrets to affect thegeneration of subplate neurons (Noctor et al., 1999). In thatpaper, the MAM-induced dramatic alteration of cortical layeringwas taken to support the hypothesis that early generated corticallayers provided environmental factors necessary for thesubsequent formation of cortical layers (Noctor et al., 1999). Inkeeping with this view, we hypothesized that the MAM-inducedablation of early generated cortical neurons determines thepresence of heterotopic cortical neurons (Chevassus et al.,1998b; Colacitti et al., 1999) in the deep cortical layers andperiventricular white matter and that, in turn, these heterotopic

neurons inf luence the migration of later generated neurons todetermine the genesis of the heterotopia.

Our birth-dating BrdU analysis has revealed a clearneurogenetic gradient in all MAM-induced heterotopia. Corticaland periventricular heterotopia are formed by progressivesettling of neurons from the outside to the inside, whereas theintra-hippocampal heterotopia are progressively formedthroughout a dorsal to ventral migration of neurons. Adorso-ventral neurogenetic gradient has been already reported inrats for the intra-hippocampal heterotopia induced by a singleMAM dose (Chevassus et al., 1998b). Our data extend thosefindings in clearly demonstrating that in MAM-treated rats theneurogenesis is not chaotic nor rudimentary, as previouslysuggested (Chevassus et al., 1998b). Rather, it seems to followthe general ontogenetic rules of cortical layering formation. InMAM-treated rats, however, as a consequence of the MAM-induced ablation of early generated neuroblasts, heterotopicallylocated neurons in the periphery of the cortical andperiventricular heterotopia and in the dorsal part of theintra-hippocampal heterotopia set the base for the subsequentmigration of neurons into the heterotopia.

Our morphologic analysis also demonstrate that intra-hippocampal heterotopia are not present at birth, butprogressively formed in the early post-natal period, as alreadysuggested (Singh, 1977; Chevassus et al., 1998b; Castro et al.,2001). In addition, our BrdU experiments demonstrate thatneurons within the intra-hippocampal heterotopia display thesame neurogenetic profile as neurons forming the corticalheterotopia, thus lending further support to the idea that theseneurons are neurons committed to superficial cortical layersgrowing into the hippocampus (Chevassus et al., 1998b;Colacitti et al., 1999). Why do these heterotopia develop into thehippocampus and why do they develop post-natally? If alreadymigrated neurons are able to direct subsequent migration of latergenerated neurons, we can hypothesize that hippocampalneurons may attract the young migrating neurons sojourning inthe white matter bands toward the pyramidal cell layer. Acommon ontogenesis for all types of heterotopia is furthersupported by the fact that in rats deeply affected by MAMtreatment it is not uncommon to find large cortical, periven-tricular and intra-hippocampal heterotopia fused together (datanot shown). The post-natal development of intra-hippocampalheterotopia could be related to the pre-natal physical separationbetween the hippocampus and the subcortical white matter,which would make the migration into the hippocampusimpossible (Chevassus et al., 1998b). Finally, these data togetherwith the already reported anatomical and electrophysiologicalfeatures of MAM-induced heterotopia (Chevassus et al., 1998a;Sancini et al., 1998; Colacitti et al., 1999) further demonstratethat heterotopic neurons maintain cortical features even ifpositioned outside the borders of the neocortex or withinthe hippocampus, thus supporting an early commitment ofneocortical neurons (Tan et al., 1998).

The neuronal phenotype of most cells in both heterotopia andwhite matter bands and the double-labeling GFAP/BrdUexperiments (Fig. 8) clearly demonstrate that in MAM-inducedheterotopia, neurogenesis precedes gliogenesis. These findingsref lect the general rule in CNS development that astrocytes aremostly generated after neurons. In addition, they suggest that theabnormalities of glial cells described in other papers dealingwith the prenatal effect of MAM (Collier and Ashwell, 1993;Zhang et al., 1995; Noctor et al., 1999) are most probably notthe cause of the genesis of heterotopia, but more simply a

Figure 9. Histograms showing percentages of BrdU-labeled neurons in periventricularheterotopia after BrdU injections at different embryonic ages. For each time point ofBrdU administration [from E17/12 (far left) to E21/12 (far right)], the percentage oflabeled cells was calculated as ratios of BrdU/MAP2 double-labeled neurons on the totalnumber of MAP2-positive neurons within the heterotopia. Note that neurogenesisbegins in all heterotopia soon after the last MAM administration, to reach aneurogenetic peak around the 18th embryonic day. Data show means and standarddeviations.




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consequence of the altered neuronal neurogenesis reportedhere.

Relevance of the MAM Model for the Ontogenesis ofHeterotopia in HumansWe have previously demonstrated that MAM-induced hetero-topia are characterized by anatomical features similar to thoseobserved in human PNH (Colacitti et al., 1999). Similaritiesbetween MAM induced heterotopia and human PNH are alsosupported by the fact that heterotopic neurons in MAM- treatedrats are hyperexcitable (Baraban and Schwartzkroin, 1995;Sancini et al., 1998; Baraban et al., 2000; Castro et al., 2001)and heterotopic nodules in human PNH can give rise to epi-leptic discharges (Dubeau et al., 1995; Kothare et al., 1998). Inaddition, recent data from our group suggest that heterotopia inboth MAM-treated rats and human PNH are characterized byreduced expression and altered function of NMDA receptorcomplex and the alpha subunit of Ca2+/calmodulin-dependentprotein kinase II (αCaMKII) (Battaglia et al., 2002; Gardoniet al., 2003). Therefore, anatomical, electrophysiological andmolecular analyses indicate that heterotopia in MAM-treated ratsand heterotopia in human PNH share common features. Thediscussion of the molecular and electrophysiologic similaritiesbetween MAM-induced heterotopia and PNH in humans areobviously beyond the scope of the present paper. However,these similarities can be taken to further validate MAM-treatedrats as an experimental model for human PNH, thus allowing theuse of the developmental profile of heterotopia in MAM-treatedrats as clues to inform speculation about the origin and intrinsicepileptogenicity of human PNH.

If it is true, as suggested by the evidence provided in thispaper, that ablation of early waves of neuroblasts is sufficient toalter the structure of selected sectors of the brain, it can bespeculated that in humans noxious events around the 7th–8thweek of gestation (Bayer et al., 1993), such as ischemic insults inlimited parts of the germinative neuroepithelium, are sufficientto determine the genesis of periventricular nodules typical ofPNH. The existence of these nodules may, in turn, determinealtered axonogenesis, since, during their development, theheterotopic neurons send and receive axonal projections to andfrom the cortex. The ‘misplaced’ axonogenesis sets the base forthe establishment of altered connections between heterotopiaand neocortical and archicortical areas (Chevassus et al., 1998b;Colacitti et al., 1999; Hannan et al., 1999). In addition, even ifthe heterotopic neurons display normal expression of manyneurochemical markers (Chevassus et al., 1998b; Colacitti et al.,1999), the process of neuronal differentiation is probablyaltered. Indeed, neurons in MAM-induced intra-hippocampalheterotopia are characterized by reduced expression of Kv4.2A-type potassium channel subunits (Castro et al., 2001) and byaltered GABA-mediated synaptic inputs (Calcagnotto et al.,2002) and neurons in both MAM-induced heterotopia andhuman PNH are characterized by reduced expression of theNMDA receptor complex and αCaMKII (Gardoni et al.,submitted for publication). Even if the molecular mechanismsunderlying the increased excitability are still unexploited,human PNH are likely characterized by hyperexcitable neuronsinserted in a redundant neocortical/archicortical network thatmay facilitate the diffusion of single interictal discharges anddetermine the genesis of sustained epileptiform discharges.

NotesThis study was partially supported by grants ICS 030.3/RF98.36 andRF162/02 from the Italian Ministry of Health.

Address correspondence to Giorgio Battaglia, Molecular Neuro-anatomy Laboratory, Department of Experimental Neurophysiology,Istituto Neurologico ‘C. Besta’, Via Celoria 11, 20133 Milano, Italy. Email:[email protected].

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