Proc. Natl. Acad. Sci. USAVol. 93, pp. 15463–15468, December 1996Neurobiology

Arrest of neuronal migration by excitatory amino acids inhamster developing brain

(glutamateyheterotopiaylissencephalyymicrocephalyyN-methyl-D-aspartate)

STEPHANE MARRET*†‡, PIERRE GRESSENS*†, AND PHILIPPE EVRARD*†§

*Laboratoire de Neurologie du Developpement and Service de Neurologie Pediatrique, Hopital Robert-Debre, and Faculte de Medecine Xavier-Bichat,(Universite de Paris VII), 48 Boulevard Serurier, F-75019 Paris, France; and †Laboratoire de Neurologie du Developpement and Service de NeurologiePediatrique, Universite de Louvain, avenue Hippocrate 10, B-1200 Brussels, Belgium

Communicated by Jean-Pierre Changeux, Institut Pasteur, Paris, France, October 21, 1996 (received for review April 23, 1996)

ABSTRACT The influence of the excitotoxic cascade onthe developing brain was investigated using ibotenate, aglutamatergic agonist of both N-methyl-D-aspartate (NMDA)ionotropic receptors and metabotropic receptors. Injected inthe neopallium of the golden hamster at the time of productionof neurons normally destined for layers IV, III, and II,ibotenate induces arrests of migrating neurons at differentdistances from the germinative zone within the radial migra-tory corridors. The resulting cytoarchitectonic patterns in-clude periventricular nodular heterotopias, subcortical bandheterotopias, and intracortical arrests of migrating neurons.The radial glial cells and the extracellular matrix are free ofdetectable damage that could suggest a defect in their guidingrole. The migration disorders are prevented by coinjection ofDL-2-amino-7-phosphoheptanoic acid, an NMDA ionotropicantagonist, but are not prevented by coinjection of L(1)-2-amino-3-phosphonopropionic acid, a metabotropic antago-nist. This implies that an excess of ionic inf lux through theNMDA channels of neurons alters the metabolic pathwayssupporting neuronal migration. Ibotenate, a uniquemoleculartrigger of the excitotoxic cascade, produces a wide spectrumof abnormal neuronal migration patterns recognized in mam-mals, including the neocortical deviations encountered in thehuman brain.

In targeted postmigratory neurons, excitatory amino acidstrigger a calcium-dependent death (1–4). At each develop-mental step after completion of neocortical neuronal migra-tion, the excitotoxic cascade produces a timed set of laminarand multilaminar neuronal depopulations. It mimics all le-sional brain patterns described after hypoxia occurring inhuman fetuses and neonates between 20 and 40 weeks ofgestation (3–6). In contrast, no excitotoxic destructive effecthas been reported before maturation of N-methyl-D-aspartate(NMDA) receptors and before acquisition of postmigratoryneuronal aerobiosis. In seminal observations performed inorganotypic tissue cultures, Komuro and Rakic (7) reportedaccelerations and decelerations of cerebellar neuronal migra-tion under the influence of excitatory amino acids. The presentpaper reports neuronal migration disorders induced in vivo inhamster by ibotenate, a glutamatergic agonist, and uses thismodel to approach the pathophysiology of these frequentneurodevelopmental disturbances.

MATERIALS AND METHODS

Animal Handling and Injections of Ibotenate and of OtherGlutamatergic Agents. Golden hamsters were chosen for thisstudy because of the timing of brain development in this species.As regards neuronal migration in the neopallium, the newborn

hamster is at a stage similar to that of the human fetus at 15 weeksof gestation and at a stage comparable to that of the mouse at the17th embryonic day. Pregnant female golden hamsters wereallowed to deliver on day 16 of gestation. Several successive littersof newborn hamsters of both sexes were used for the experiments.Ibotenate, a glutamatergic agonist, activates both NMDA iono-tropic receptors and metabotropic receptors. Under ether anes-thesia, intracerebral injections of ibotenate (Sigma) diluted in0.02% acetic acid 0.1 M PBS were performed with a 26-gaugeneedle on a 50-ml Hamilton syringe mounted on an Oxfordmicromanipulator (3). Two 1-ml injections of ibotenate weremade at an interval of 60 sec. The injections were made 2 mmbeneath the skull surface in the frontoparietal area of the righthemisphere with coordinates of 2mm from themidline and 3mmin front of the hinder suture. After ibotenate injections, the pupswere returned to their dams. Doses of 0.001, 0.1, 10, or 20 mg ofibotenate per hamster (i.e., 0.006, 0.6, 60, and 120 nM)were used.Controls received 2 ml of 0.02% acetic acid PBS. The mortalityrate was 100% in the hamsters injected with 20 mg of ibotenate,and 10% in the hamsters injected with 10 mg of ibotenate.Hamsters injected with 20 mg of ibotenate were seizure free butwere areactive, and all died within a few hours during apneicepisodes. There was no mortality in the pups injected with thelower doses of ibotenate (i.e., 0.1 and 0.001 mg) or with PBS. TheNMDA antagonist, DL-2-amino-7-phosphoheptanoic acid (AP7;Sigma) was coinjected intracortically with ibotenate in anequimolar dose. The metabotropic receptor L-(1)-2-amino-3-phosphonopropionic acid (L-AP3) (Tocris Neuramin, Bristol,U.K.) was coinjected with ibotenate at a 50-time molar dose asdone by Schoepp et al. (8). The brain of other animalswas injectedwith 0.1, 1, 10, and 50 nM (i.e., 18.3 ng, 183 ng, 1.8 mg, and 9.15mg) of (RS)-3,5-dihydroxyphenylglycine (Tocris Neuramin), ametabotropic agonist (9).Immunohistochemical Studies of Cell Differentiation. The

pups were killed 2, 5, or 40 days after ibotenate injection. Theywere either killed by decapitation or by intracardiac perfusionwith 4% paraformaldehyde in 0.1 M PBS (pH 7.4) fixativeunder ether anesthesia. After decapitation, the brains werefrozen and 20-mm coronal sections were cut on a cryostat forthe immunohistochemical procedure. After intracardia perfu-sion with fixative, the hemispheres were removed and embed-ded in paraffin. Coronal sections (10 mm) were cut andprocessed for cresyl-violet staining and immunohistochemicalstudies. Several specific cell markers were immunohistochemi-cally identified on tissue sections: (i) microtubule-associatedproteins (MAPs) types 2 and 5, which are dendritic markerspresent in immature brain of the hamster, and MAP1, which

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: NMDA, N-methyl-D-aspartate; AP7, DL-2-amino-7-phosphoheptanoic acid; L-AP3, L-(1)-2-amino-3-phosphonopropi-onic acid;MAP,microtubule-associated protein; GFAP, glial fibrillaryacidic protein; BrdUrd, 5-bromodeoxyuridine.‡Present address: Faculte de Medecine, F-76031 Rouen Cedex,France.§To whom reprint requests should be addressed.

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is an axonal and dendritic marker mainly present in adult brain(10); (ii) glial fibrillary acidic protein (GFAP), a marker ofastrocytes; and (iii) vimentin, which labels radial glial cells. Forthe MAP and GFAP studies, paraffin-embedded sectionsadjacent to cresyl-violet stained sections displaying excitotoxicdamage were incubated with specific primary antibodies[mouse monoclonal anti-MAPs diluted 1y250-1y500 in PBS-Triton from Sigma, and rabbit polyclonal anti-GFAP diluted1y100 in PBS-Triton from Dakopatts (Glostrup, Denmark)],as described elsewhere (3). Primary antibodies were detectedand revealed using the corresponding Vectastain ABC kit(Vector Laboratories) and 3,39-diaminobenzidine chromogen.Vimentin detection was performed on cryostat sections asdescribed (11). Monoclonal antivimentin antibody from Am-ersham was diluted 1y50 in PBS. Detection of the primaryantibody was performed as above. Control sections wereimmunostained with omission of the primary antibody. In nocase was labeling observed.Study of the ExtracellularMatrix. P5 hamsters injected with

ibotenate at birth were fixed by intracardiac perfusions of 4%paraformaldehyde in 0.1 M PBS (pH 7.4) containing 0.5%cetylpyridinium, embedded in paraffin. Sections (10 mm) werestained with Alcian blue 8G-X (Sigma) at pH 2.5 to detect allthe extracellular matrix polyanions, including hyaluronan,sulfated glycosaminoglycans, and other glycoproteins. Adja-cent sections were incubated with a biotinylated sheep-brainhyaluronectin probe to detect hyaluronan as described in aprevious study (12).Study of Cell Proliferation. 5-Bromodeoxyuridine (BrdUrd)

(Sigma), an S-phase marker, was injected into pregnant ham-ster females (50 mgyg). To have BrdUrd stained cells inS-phase in the periventricular zone at the time of ibotenateinjection, we had to inject BrdUrd into pregnant mothers 24 hrbefore term. Detection was made on 10-mm embedded-paraffin sections as described by Takahashi et al. (13). Thesections were incubated with monoclonal anti-BrdUrd anti-bodies (Becton Dickinson) diluted 1y50 in PBS-Triton. De-tection of the primary antibody was performed as above. Bothweak and strong BrdUrd-positive cells were counted in theinjected and in the contralateral hemispheres. The numbers ofcells are expressed as means 6 SEM.In other ibotenate-injected hamsters, the DNA and protein

contents of each hemisphere were measured, with the methoddescribed by Burton (14) and modified byMunro (15) and withBio-Rad protein assay (16).

Terminology. The terms ‘‘band heterotopias,’’ ‘‘diffuse het-erotopias,’’ ‘‘(poly)microgyrias,’’ ‘‘pachygyrias,’’ and othersare used as defined by Friede (17), Rorke (18), and Marret etal. (3).

RESULTS

Four main types of disorders of neuronal migration wereobserved after ibotenate injections (Table 1 and Fig. 1): (i)spherical nodular heterotopias in the periventricular and in-termediate zones (Fig. 1 A and B); (ii) band and diffuseheterotopias in the white matter (Fig. 1C) [in these vastheterotopic fields, many neurons are arrested in all migratorycorridors of the entire hemisphere, between the externalsagittal stratum and the plexiform zone, producing a 1000-mm-thick heterotopic field; the resulting lamination of thecortex is made of three or four layers, mimicking the pachygy-ric pattern and the double cortex (17, 18)]; (iii) sub- andintracortical arrests of migration (Fig. 1E); and (iv) ectopias inthe molecular layer (Fig. 1E).Periventricular and band heterotopias are produced only

with high ibotenate doses; low doses produce only intracorticaland molecular layer heterotopias (Table 1). In all four types ofheterotopic neuronal arrangements, most of the heterotopiccells are small, with no sign of pyknosis in the nucleus orvacuolization in the cytoplasm.The only type of lesion of postmigratory neurons observed in

this material is the depopulation of layers V–VIa; this layeredneuronal depopulation is observed in the rostral, more mature,part of the brain, where completion of neuronal migrationoccurred before the ibotenate injection (Table 1 and Fig. 1F).Abnormal gyri appeared after ibotenate injection in two

circumstances corresponding to distinct developmental mech-anisms: (i) when the cortical plate is almost missing becausemost migrating neurons are locally arrested in rounded hete-rotopias (Fig. 1A); and (ii) when postmigratory neurons oflayers V–VIa are destroyed in the rostral regions (Fig. 1F).In 75% of the brains injected with 10 mg of ibotenate,

hypoplasia of the injected hemisphere was observed andassociated with heterotopias. No hemispheric hypoplasia wasnoted with lower doses (i.e., 0.1 and 0. 001 mg of ibotenate),even when the cortical abnormalities were conspicuous. In oneanimal injected with 10 mg of ibotenate the corpus callosumwas severely hypoplastic with generalized migration defect.Effects of Ibotenate Antagonists. Six pups were coinjected

with both ibotenate and AP7, an antagonist of the NMDA

Table 1. Neopallial dysgeneses induced by ibotenate injected in hamster brain

Doses ofibotenate,

mg

No. oftreatedanimals

No. of brainlesion, %

Meanrostro-caudaldiameter oflesions,* mm

Types of lesions (%)

Neuronalheterotopias

Rostralmicrogyric-like lesion

10 49 100 1200 Diffuse (12)Periventricular orsubcortical (84) (83)†

Intracortical (4)Ectopias (0)

0.1 6 100 600 Diffuse (0)Periventricular (0) (100)Intracortical (66)Ectopias (100)‡

0.001 6 50 200 Diffuse (0)Periventricular (0) (50)Intracortical (50)Ectopias (50)‡

*Mean rostro-caudal diameter was estimated by counting the number of 10-mm sections displaying the lesion.†Microgyric-like lesions were not observed in hamster brain with diffuse heterotopias.‡Ectopias could be associated with intracortical heterotopias.

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receptor. Three coinjected animals displayed no damage at all.In two coinjected hamsters, layers II and III were slightlydepopulated without any detectable disturbance of migration.A similar minor depopulation of layers II and III was observedin two-thirds of the six control hamsters injected with AP7

alone. A small subcortical heterotopia was observed in one outof six coinjected animals.Seven pups were coinjected with both ibotenate and with

AP3, an antagonist of the metabotropic receptor. This antag-onist was devoid of any protective effect.

FIG. 1. Neuronal migration disorders induced in the neopallium by ibotenate. Injection at birth in hamster; light microscopic study at P5. (A)Cresyl-violet stained section of a brain injected with 10 mg of ibotenate showing periventricular heterotopia (arrowhead) and hypoplasia of theinjected hemisphere. (B) Cresyl-violet stained section at a higher magnification than in A. (C) Cresyl-violet stained section of a ‘‘hemipachygyric’’brain injected with 10 mg of ibotenate showing a severe architectural disorder without any recognizable normal neuronal layer. (D) BrdUrd-stainedsection adjacent to the cresyl-violet stained section shown in B. (E) Cresyl-violet stained section of a brain injected with a low dose (0. 1 mg) ofibotenate showing ectopias in the molecular layer of the cortex. (F) Cresyl-violet stained section showing the depopulation observed in the rostralpart of the lesions (between arrowheads). (G) MAP2 stained section adjacent to the cresyl-violet stained section (B) showing no dendritic labelingin the heterotopia. (A and C, 337; B and D, 3260; E and F, 3250; and G, 3520.)

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Effects of Metabotropic Agonists. Six pups were injectedonly with (RS)-3,5-dihydroxyphenylglycine, a metabotropicagonist. These injections did not produce any migration dis-orders.Immunohistochemical Studies. No gliosis was detected in

the injected brains, either when examined 5 days after theibotenate injection or when examined at the adult age. Theabsence of gliosis was confirmed by anti-GFAP staining. Nodendritic MAP2 (Fig. 1G), MAP5, or MAP1 stainings weredetectable in the heterotopic areas or in subjacent cortex.Staining of neurofilaments was weak in the heterotopic regionsand showed an abrupt interruption of callosal connections.The radial glial cells, studied with the antivimentin antibodies2 days after ibotenate injection, appeared entirely normalthroughout the whole brains, including the dysgenetic areas(Fig. 2). Extracellular matrix, analyzed with Alcian blue 8G-Xat pH 2.5 and with a hyaluronectin probe, also appearednormal.Incorporation of BrdUrd and Estimate of DNA Content.

The total number of BrdUrd-positive neurons (Fig. 1D)counted in 10 injected hemispheres (i.e., BrdUrd-positiveneurons of heterotopias plus BrdUrd-positive normally lo-cated neurons) on adjacent sections to cresyl-violet stainedsections showing heterotopias was similar to the total numberof BrdUrd positive neurons counted in the 10 contralateralintact hemispheres (128 6 43 neurons per vertical neopallialcolumn of 400 mm in diameter in the heterotopic hemispheres,versus 121 6 38 neurons per similar column in the normalhemispheres, a statistically nonsignificant difference). Thenumber of BrdUrd-positive neural cells found in heterotopiasreached ,15% of the number of neural cells counted in thesame heterotopias on cresyl-violet sections.Dosages of DNA and protein contents were also similar

between 10 injected hemispheres versus 10 control hemi-spheres (DNA content, 16 0.167mg per ml versus 0.9666 0.15mg per ml, a statistically nonsignificant difference; proteincontent, 15.4 6 2.97 mg per ml versus 14.5 6 2.24 mg per ml,a statistically nonsignificant difference).

DISCUSSION

The salient feature of this study is the induction of disordersof neuronal migration in hamsters injected with an NMDAglutamatergic agonist before the end of neuronal migration.This animal model permits an analysis of a whole spectrum oftopographic and topological types of disorders of neuronalmigration and provides data concerning the complement ofneurons reaching the neocortex along radial migratory corri-dors (19). In all neocortical fields with migration disorders, thelateral scattering of neurons above the heterotopic area is verymodest, an argument against a conspicuous tangential migra-tion in the mammalian brain. In the most affected specimens(Fig. 1B), where the radial migration arrest seems to becomplete, the number of neurons reaching the very thin‘‘cortical plate’’ does not exceed a fifth of the neuronalcomplement destined to the neocortex, combining in thisestimate of the residual neocortex, neurons of the very firstradial wave that reached the plate and tangentially migratingneurons. In this study, we did not obtain data concerning apossible interference of ibotenate with tangential migration.Migration defects are often focal (88%) and of the nodularheterotopic type. In 12% of the animals, the migration deficitscover the entire hemisphere and result in a band or diffuseheterotopic neocortical pattern exactly mimicking hemi-pachygyric brains. The injection procedure is fairly well re-producible and has no influence on the type and topographyof migration disorders. As shown in Table 1, the type of deviantarchitectural pattern is dose-dependent. The focalynodularversus hemisphericyband migration disturbances produced byhigh ibotenate doses seem to be dependent upon the devel-opmental age. Indeed, the postconceptional age at deliverycannot be estimated with a better than 12-hr accuracy; at thisdevelopmental epoch, such a time interval can reduce thewater content of extracellular matrix and influence the migra-tion mechanisms and the level of migratory completion (19–21). The nodular heterotopias coexist in the anterior neocor-tical regions with areas of laminar depopulation of installed

FIG. 2. Radial glial fibers and extracellular matrix in neuronal heterotopias induced by ibotenate. (A) Injection of 10 mg in hamster neopalliumat birth; killed at P2. Vimentin stained section. In the nodular periventricular heterotopia, radial glial cells are morphogically normal. Arrowheadsindicate the limits of heterotopia (H); B, normal brain. (B) Injection of 10 mg in hamster neopallium at birth; killed at P5. Hyaluronanimmunostained section of a nodular heterotopia reveals a morphogically normal extracellular matrix. H, heterotopia; L, limit of heterotopia; B,normal brain. (A, 3220; B, 3840.)

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layers V–VIa, a postmigratory event extensively described inperfusion failuresyhypoxias in the human (22, 23), and inexcitotoxic hypoxic-like lesions described in the mouse (3).This topographic distribution can be attributed to an earliercompletion of neuronal migration and to an earlier acquiredpostmigratory maturation (i.e., aerobic metabolism andNMDA receptor stabilization) in the rostral rather than in thecaudal regions in the hamster at the moment of injection (3, 21,23, 24). Seventy-five percent of the injected animals are‘‘hemimicrocephalic;’’ the brain asymmetry being more pro-nounced in the ‘‘pachygyric’’ type but often present also in the‘‘nodular’’ type. The complement of neural cells, estimated bythe DNA content, the protein content, and with the BrdUrdmethod, is similar in both hemispheres in all specimens, evenin the most asymmetric brains. Defective cell proliferation is,therefore, unlikely. The reduction of size of the injectedhemisphere is probably due to defective development of theneuropile following the migration defect. The absence ofMAP2, MAP5, andMAP1 stainings in all heterotopic neuronsreflects disturbances of dendritic outgrowth, which is consis-tent with this hypothesis. As an alternative explanation of the‘‘hemimicrocephaly,’’ however, we cannot exclude the role ofan increased proliferation followed by an excessive apoptosis(25). Nor can we completely exclude the possibility of in-creased cell death in installed postmigratory neuronal layersV–VIa, which are associated with migrational disorders in thismodel. But in a previous excitotoxic model developed in themouse at birth after the end of neuronal migration, weobserved isolated depopulation of layers V–VIa without anymigrational disorder and without any hemimicrocephaly (3).The NMDA glutamatergic agonist arrests neurons at all

levels of the radial migratory corridors (germinative zone,white matter, cortical plate, molecular layer) resulting in aspectrum of architectonic patterns: nodular heterotopias in theperiventricular and intermediate zones, band and diffuseheterotopias in the white matter, sub- and intracortical arrestsof migration, and ectopias in the molecular layer. The periven-tricular heterotopias induced by ibotenate injection at P0 inhamster are similar to the neuronal ectopic nodules encoun-tered in Aicardi and other human syndromes (for a review, seerefs. 17, 18, 23, and 26). The subcortical band heterotopias and

the three- to four-layered cortex mimick the spectrum ofneocortical patterns encountered in the subcortical, cortico-subcortical, and intracortical migration blockades of the hu-man double cortex and pachygyriasylissencephalies (27, 28)and of the rat after prenatal irradiation (29). Ectopias andprotrusions of neurons noted in layer I (obtained with ibote-nate at low doses) display some similarities with the disordersof neuronal migration produced by cryolesions in newborn ratby Dvorak et al. (30, 31) and with status verrucosus deformanobserved in the human (17).This study demonstrates that excessive stimulation of

NMDA receptors by the excitotoxin ibotenate disturbs neu-ronal migration. An antagonist of the NMDA receptor ro-bustly prevents neopallial dysplasias while an antagonist of themetabotropic receptor is devoid of any protective effect and aselective metabotropic agonist does not interfere with neuro-nal migration. This implies that an excess of ionic influxthrough the NMDA channels of neurons alters the metabolicpathways supporting neuronal movement. The calcium over-f lux could act directly or through genetic alterations on thecontrol of neuronal excursion (32). The NMDA-mediatedaction of ibotenate could be due to an effect on some phasesof the cell cycle, on pre- or permigratory phases after the endof the cell cycle, or on the environment of the cell. Anibotenate effect on the environment of migrating cell could notbe totally excluded, even if extracellular matrix and the radialglial guiding system are everywhere normal, including in theareas where neuronal migration is the most defective. In theirin vitro study, Komuro and Rakic (7) demonstrated that theNMDA receptor is implicated in migration of granule cells inslices of rat cerebellum. Alternatively, McConnell and Kaz-nowski (33) demonstrated that environmental factors duringthe late S-phase are determinants of the ultimate position inthe neocortex. In the severely affected areas, most migratingneurons are arrested by ibotenate injection. This could be dueto the presence of NMDA receptors on all the migratingneurons. It could also be due to the activation of a selectedpopulation of neurons with NMDA receptors on their mem-brane; a secondary alteration of calcium concentration in theenvironment could therefore modify the migration of other

Table 2. Deviant neopallial patterns induced by ibotenate in rodent brains

Developmentalsteps of CNS

NMDA*receptor

Ibotenate-inducedlesions

Disorders ofneuronalmigration

Neuronalexcitotoxicity

White mattersensitivity

Preparation ofthe neuralepithelium

ND No lesion 2 2 2

Migration ofinfragranularlayers

1 ND ND ND ND

Migration ofsupragranularlayers

1 Heterotopia 1 1 2(layers V–VI)

Maturation ofinfragranularlayers

1 Microgyria 1 1 2(layers V–VI)

Maturation ofsupragranularlayers plusfull activity ofwhite matter

1Corticoysubcorticaldamage

2 1 1

PVL (layers II–VI)

This table is the synthesis of all the data we obtained in different animal models of excitotoxicity using ibotenate as glutamateagonist. The step of the preparation of the neuroepithelium was investigated in vitro by whole-embryo cultures (41, 42); thestep of migration supragranular layers was investigated in vivo by intracerebral injection in hamster (present study); the stepsof maturation of neuronal layers were investigated in vivo by intracerebral injection in mice at P0 and P5 (3). ND, not done;1, present; 2, absent; PVL, periventricular leukomalacia; CNS, central nervous system.*Data shown are derived from Watanabe et al. (38).

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non-NMDA neurons through the activation of other receptorycalcium channels (34).In addition to the major neuronal migration disturbances,

destructions of postmigratory neurons mimicking the micro-gyric brains are also observed in the rostral part of the brain,a phenomenon we have previously described in detail at laterdevelopmental stages (3, 4). These staged, different, andconsecutive effects of the ibotenate excitotoxin depend onneuronal maturation and can be explained by a developmentalchange of the NMDA receptor (Table 2) (35–40).In the present study, we used an excitotoxin to disturb

neuronal migration and produce neuronal arrests at all levelsof the migratory corridors. At later developmental stages, thisexcitotoxin has quite different dysmorphogenetic properties,producing hypoxic-like neuronal death. This study also stressesthe interactions between environmentalyepigenetic and ge-netic factors in the pathogenesis of cytoarchitectonic disorders.

We thank Annie Delpech from the University of Rouen for hersupport, BertrandDelpech from theUniversity of Rouen for his adviceand for the gift of sheep brain hyaluronectin, and Anne-Marie Ronafor her excellent technical assistance. This work was partly supportedby the Fonds National de la Recherche Scientifique of Belgium, by theFondation de France, by the Fondation de la Recherche Medicale(France), and by the Centre Hospitalier Universitaire of Rouen,France.

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