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It is widely thought that neurons of the cerebral cortex are gen-erated in the ventricular zone (VZ) that lines the dorsal telen-cephalic ventricles1,2. Normal development of the cortex requiresthe orchestrated migration of postmitotic neurons from the ger-minal VZ to the overlying cortical plate (CP). Cell birth–datingstudies have shown that the generation of the neurons of the CPfollows an ‘inside-out’ sequence, such that early-generated cellsform the deeper layers whereas later-born cells migrate past theexisting layers to reside more superficially3–5. Upon completionof migration, neurons become organized in six layers, each con-taining a complement of pyramidal cells, the excitatory projec-tion neurons, and nonpyramidal cells, the inhibitoryinterneurons. Although radial migration is the predominantmode of movement by cortical neurons6, a substantial propor-tion migrate tangentially7–11. A direct association between neu-ronal phenotype and dispersion pattern within the cortex hasbeen seen, with pyramidal neurons arranged radially and GABA-containing interneurons dispersed tangentially12.

The majority of cortical interneurons are generated in theventral telencephalon. Different experimental approaches haveshown that cells arising in the ganglionic eminence (GE), theprimordium of the basal ganglia, transgress the corticostriatalboundary and follow tangential migratory routes to take uppositions in the developing cerebral wall13–17. Work on mutantswith genetic deletions of Dlx1 and Dlx2 has shown that bothgenes are required for the migration of these cells from the ven-tral to the dorsal telencephalon14,18. In addition, experimentson slice cultures have indicated that the neural adhesion mole-cule TAG-1, expressed on developing corticofugal axons, medi-ates this process11,19. To investigate how interneurons migrateand integrate within the developing cortical circuitry, we usedtime-lapse imaging of acute brain slices to follow the migrato-ry behavior of neurons traversing tangentially through the cor-tical anlage. These experiments showed that populations of

Ventricle-directed migration in thedeveloping cerebral cortex

Bagirathy Nadarajah1, Pavlos Alifragis1, Rachel O. L. Wong2 and John G. Parnavelas1

1Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK2Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA

Correspondence should be addressed to J.G.P. (j.parnavelas@ucl.ac.uk)

Published online: 19 February 2002, DOI: 10.1038/nn813

It is believed that postmitotic neurons migrate away from their sites of origin in the germinal zonesto populate distant targets. Contrary to this notion, we found, using time-lapse imaging of brainslices, populations of neurons positioned at various levels of the developing neocortex that migratetowards the cortical ventricular zone. After a pause in this proliferative zone, they migrate radially inthe direction of the pial surface to take up positions in the cortical plate. Immunohistochemicalanalysis together with tracer labeling in brain slices showed that cells showing ventricle-directedmigration in the developing cortex are GABAergic interneurons originating in the ganglioniceminence in the ventral telencephalon. We speculate that combinations of chemoattractant andchemorepellent molecules are involved in this ventricle-directed migration and that interneuronsmay seek the cortical ventricular zone to receive layer information.

postmitotic neurons, located at various zones of the develop-ing cortex, migrate towards the cortical VZ. We have definedthis mode of movement as ‘ventricle-directed migration’, andhave shown that neurons that enter the cortical proliferativezone subsequently migrate towards the pial surface. Further,using tracer-labeling techniques and immunohistochemistry,we have shown that neurons showing ventricle-directed migra-tion are indeed GABAergic interneurons arising in the ventraltelencephalon. These observations indicate that the VZ con-tains permissive cues for cells of noncortical origin, and mayprovide layer information not only to pyramidal neurons20,21

but also to interneurons.

RESULTSVentricle-directed migration: time-lapse imagingAcute brain slices taken from mouse or rat embryos and labeledwith Oregon Green BAPTA 488 were used to follow the migra-tory behavior of cortical cell types. Labeled cells were observedin all layers of the developing cortex after a 2-hour incubationin the dye. They were labeled in their entirety, such that tips ofgrowth cones and thin trailing processes were clearly visible(Figs. 1 and 2). Similar to earlier reports7,22, cells migrating inthe direction of the pia were oriented radially or at an angle(Fig. 1a). There were also labeled cells migrating tangentially(parallel to the pial surface) within the VZ, intermediate zone(IZ) (Fig. 1a) and CP. In these acute slice preparations, the VZmay also include portions of the subventricular zone, as thesetwo zones cannot be clearly delineated. In addition to radialand tangential modes of movement, we also observed a popu-lation of labeled cells actively migrating in the direction of theventricle from various levels of the developing cortex (Fig. 1a).We refer to this pattern of movement as ‘ventricle-directed migra-tion’. Examination of acute brain slices taken from mice at embry-onic day (E) 13–16 (n = 65 slices, 40 embryos, 320 moving cells)

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Fig. 2. Time-lapse sequence of a cell undergoing ventricle-directed migra-tion in an acute mouse cortical slice labeled with Oregon Green BAPTA 488.The arrows point to the tip of the leading process that advances rapidlytowards the ventricular surface (bottom). As soon as it contacts the ventri-cle (t = 66, t = 70), the cell ‘waits’ before rapidly retracting the process(arrows, t = 72) to resume movement in the opposite direction. The arrow-heads point to the trailing process of the cell as it moves initially towards theventricle; it subsequently grows into the leading process when the cellchanges direction and moves towards the pial surface. CP, cortical plate; IZ,intermediate zone; VZ, ventricular zone. Scale bar, 20 µm. (For a time-lapseview of ventricle-directed migration, see Web Movie 2 on the supplemen-tary information page of Nature Neuroscience online.)

Fig. 1. Time-lapse imaging illustrating the typical patterns of cell movement inan acute mouse cortical slice labeled with Oregon Green BAPTA 488. (a) A–Dare labeled cells in the IZ migrating in the direction of the pial surface (A), tan-gentially through the IZ (B) and towards the ventricle (C, D) over a period of70 min. The cell bodies are highlighted with asterisks, and the arrows point tothe direction of the leading processes of cells C and D. (b) Illustration of thetrajectories and direction (arrows) of cells A–D; time interval between pointsis 10 min. (c) Somal displacement, plotted as a function of time, shows thesaltatory pattern of movement of cells C and D. CP, cortical plate; IZ, inter-mediate zone; VZ, ventricular zone. Scale bar, 15 µm. (For a time-lapse view ofneuronal migration, see Web Movie 1 on the supplementary informationpage of Nature Neuroscience online.)

showed that the earliest age at which ventricle-directed migra-tion took place was E14 (17% of moving cells). Such movementwas more prevalent, however, in the later stages of corticogen-esis (22% at E15 and 26% at E16). Cells that underwent ven-tricle-directed migration had distinct morphological features.Their somata were located in the CP, IZ or VZ, and their lead-ing processes were oriented in the direction of the ventricle(Figs. 1a and 2). The leading processes were often branched andshowed growth cone–like structures at the tips, indicating activemovement. In addition, a short, thin trailing process pointingtowards the pial surface was sometimes evident in the courseof migration (Fig. 2).

Time-lapse sequences showed that cells that migratetowards the ventricle move at average speeds of 50 µm/hour.We often observed cells that had unbranched leading process-es at the start of imaging, but gave rise to branches with time.In such cases, the soma moved rapidly (1–3 µm/min) up tothe branch point, and paused for an extended period to retractone of the processes before resuming movement in the direc-tion of the remaining branch (Fig. 1a, cell C, t = 20 min).Thus, ventricle-directed migration seemed to be saltatory, withrapid movements punctuated by short periods of relativelyslow advancement or stationary phases (Fig. 1c). Previousstudies in a variety of culture systems have indicated that cellsshowing saltatory patterns of movement are closely associated

with radial glia7,23,24. Notably, our time-lapse sequences haveshown that cells that undergo ventricle-directed migrationmove faster than do those glial-guided neurons that migratetowards the pia (Fig. 1b; compare the displacement of cell Awith cells C, D).

To further characterize the behavior of cells that undergoventricle-directed migration, we followed the movement ofthose that were located in the lower IZ at the start of imaging.These recordings showed that some cells actively migratedtowards the VZ until their leading processes, with a growthcone–like structure at the tip, reached the ventricular surface.The soma of these cells then paused for an extended period(∼ 45 min) while a thin trailing process appeared (Fig. 2). Withtime, the trailing process became thicker and extended in thedirection of the pia to become the new leading process. Sub-sequently, the old leading process retracted from the ventric-ular surface (Fig. 2; t = 70 min) and eventually disappeared asthe soma resumed movement in the direction of the pia.

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Fig. 3. Characterization of cells with ventricle-directedprocesses in fixed brain sections. (a and b) Section taken froman E17 rat brain and double immunostained for Tuj1 (red) andGABA (green). A high proportion of Tuj1-positive neurons inthe VZ of the LCX that have their leading processes orientedtowards the ventricular surface show strong expression ofGABA (arrows). A weakly labeled cell is indicated with anarrowhead. (c and d) Section from an E16 rat brain that hadreceived a single pulse of BrdU and was fixed 12 h later. It showsthat Tuj1-positive neurons with ventricle-directed morphologies(green, arrows) in the VZ of the MCX do not contain BrdU(red). (e and f) Section from an E16 rat brain that had receivedthree pulses of BrdU and fixed after 18 h shows that GABAergicneurons (green, arrows) with ventricle-directed features in theVZ of the MCX are not positive for BrdU (red). (g) Illustrationof calbindin-positive neurons in the CP that have their leadingprocesses oriented in the direction of the ventricle (arrows) in asection taken from an E18 rat brain. (h) Schematic illustration ofthe spatio-temporal pattern of Tuj1- or GABA-positive neuronsthat show ventricle-directed morphologies in the cortical VZ.The earliest cohort was observed in the LCX at E15 and contin-ued towards the MCX over time, maintaining a lateral to medialgradient. VZ, ventricular zone; CP, cortical plate; IZ, intermedi-ate zone; MCX, medial cortex; LCX, lateral cortex. Scale bars:(a–f), 25 µm; (g), 12 µm.

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Interneurons show ventricle-directed morphologyTo investigate whether cells with leading processes orient-ed in the direction of the ventricle were present in thedeveloping brain, we stained sections of fixed embryonicmouse (E13–16) and rat (E16–18) brains with a panel ofantibodies that label early neuronal populations. Theseexperiments showed the presence of Tuj1-positive cells inthe VZ whose features were similar to those of cells thatunderwent ventricle-directed migration in real-time imag-ing (compare Figs. 3a and b with Fig. 2). Further, a numberof cells with ventricle-directed features in the VZ, IZ andCP were also immunopositive for GABA or calbindin,markers of cortical interneurons. To determine the pro-portion of neurons with ventricle-directed processes in the cor-tical VZ that are GABAergic, we double immunolabeled sectionsfor both Tuj1 and GABA. Our analysis showed that, at all agesexamined, nearly all (90%) Tuj1-positive neurons with ventri-

cle-directed processes also expressed GABA (Figs. 3a and b).Notably, although the intensity of Tuj1 labeling was fairly con-stant in these neurons (Fig. 3b), the intensity of immunostain-ing for GABA varied considerably (Fig. 3a). Further, to determinethe spatio-temporal pattern of distribution of neurons with ven-tricle-directed features, we examined brain sections that werestained for Tuj1 or GABA. The earliest group of ventricle-direct-ed cells appeared in the lateral cortex of rats around E15 (E13 in

Fig. 4. Cells that undergo ventricle-directed migration arise in ven-tral telencephalon. Placement of CMTMR-coated particles in the LGEof slices obtained from embryonic rat brains showed dye-labeled cellsin the cortex after 1–2 DIV. (a) Horizontally and radially orientedlabeled cells present in the VZ, IZ and CP of an E17 slice after 2 DIV.(b, c) Labeled cells with leading process oriented towards the ventri-cle were present in the VZ of an E16 cortical slice (b) and in the CPand IZ of an E18 slice (c; arrows) after 1 DIV. In addition, cells thathad leading processes oriented in the direction of the pial surfacewere also observed in the VZ (indicated by * in c). (d) Schematic dia-gram illustrating the distribution of dye-labeled cells that had ventri-cle-directed processes in regions of the cortical VZ along therostro-caudal axis of the brain. These cells were more prevalent inthe VZ of slices that were obtained from middle regions of the brain.VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; LV, lat-eral ventricle. Scale bars: (a, c), 100 µm; (b), 15 µm.

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mouse) and continued to progress towards the medial cortex(Fig. 3h). To ascertain whether this population of cells alsoincluded migrating neurons that were generated in the corticalVZ, we carried out birth-dating experiments with bromod-eoxyuridine (BrdU). For these experiments, pregnant rats (E16)were given a single injection of BrdU and the embryos harvested12 hours or, in some cases, 18 or 24 hours after three pulses ofBrdU (at 2-hour intervals). Examination of brain sections thatwere double immunolabeled for Tuj1 or GABA with BrdU showedthat none of the Tuj1- or GABA-positive neurons in the VZ, eitherpositioned horizontally or with ventricle-directed processes, hadincorporated BrdU at 12-, 18- (Figs. 3c to f) or 24-hour time-points. These observations indicate that neurons that lay scatteredin the VZ with ventricle-directed processes must have becomepostmitotic at least 12 hours before to the injection of BrdU and,hence, probably are not cortical VZ–derived neurons. Takentogether, these observations corroborate our time-lapse data andillustrate that neurons with ventricle-directed features are a sub-set of interneurons that are probably of noncortical origin.

Ventricle-directed neurons arise in the basal forebrainRecent studies indicate that the vast majority of cortical interneu-rons migrate into the cortex from the GE in the ventral telen-cephalon14,16. To confirm the origin of interneurons that showventricle-directed migration, tungsten particles coated withCMTMR were placed in the subventricular zone of the lateral gan-glionic eminence (LGE) of slices taken from embryonic rat ormouse brains. Examination of slices after 2 days in vitro (DIV)showed that dye-labeled cells had migrated across the corticos-

triatal boundary, with the earliest cohort observed inthis region 12 hours after dye placement. The greatmajority of labeled cells had leading processes orient-ed horizontally, indicating active tangential movement.In addition, dye-labeled cells with ventricle-directedmorphology were observed in the VZ, IZ and CP at allembryonic ages examined (Figs. 4b and c). Further, inthe VZ, cells with such morphology often had leadingprocesses that contacted the ventricular surface in amanner similar to that seen in time-lapse sequences(compare Fig. 4b with Fig. 2). Immunohistochemicalanalysis of cultured slices obtained from E16–18 ratbrains showed that the majority of dye-labeled cellswith ventricle-directed morphology were GABA posi-tive (Figs. 5a and b). Cell counts in slices (30 slices from10 embryos) taken from E17 brains and maintained for2 DIV showed that ∼ 70% of dye-labeled cells (n = 190cells) located in the VZ that had ventricle-directedprocesses were strongly immunoreactive for GABA. Theremainder were either unlabeled or weakly labeled.

Recent reports have indicated that interneuronsarising in the MGE and LGE show temporal differencesin their migratory patterns25. To investigate whetherneurons that emanate from both eminences undergoventricle-directed migration, the subventricular zonesof the two regions were labeled each with different flu-orescent dyes in slices taken from E17 rat embryos andmaintained for 2 DIV. These experiments indicatedthat neurons emanating from both the MGE and LGEundergo ventricle-directed migration (Figs. 5c to e).

Previous experiments in the rostral migratorystream have indicated that migrating neurons have theability to divide in situ after being committed to theneuronal phenotype26. To investigate whether some

GABAergic neurons seek the cortical VZ for mitosis, slices fromE16 rat embryos were incubated with BrdU after the applicationof dye for 24–48 hours. Immunohistochemical analysis showedthat none of the dye-labeled cells in the neocortex had incorpo-rated BrdU (Figs. 6a and b). To further investigate the time ofterminal division of these cells, slices were prepared from E17rat embryos that had received a single injection of BrdU 24 hoursbefore the fetuses were harvested. Examination of slices that weremaintained for 1–3 DIV showed that, although some dye-labeledcells were positive for BrdU after 1 DIV, most, including thosewith ventricle-directed processes, were positive only after 2–3DIV (Figs. 6c and d). Thus, these ventrally derived neurons musthave become postmitotic during the 24-hour period after BrdUinjection in vivo. Taken together, these observations show thatcells undergoing ventricle-directed migration are postmitoticand committed to the neuronal phenotype at the time of enter-ing the dorsal telencephalon.

Prevalence of ventricle-directed migrationTo investigate whether the ventricle-directed mode of movementis prevalent in all regions of the cortical VZ, we placed CMTMRin the rostral or caudal portions of the ganglionic eminences inslices of E16–18 rat brains. Slices obtained from the anteriorregions of brains (sectioned at the level of the septum) and main-tained for 2 DIV showed rostral GE cells with ventricle-directedprocesses in the cortical VZ. Similarly, slices taken from caudalparts of brains (sectioned at the level of the ventral thalamus)contained cells from the caudal GE with ventricle-directedprocesses. Comparison of slices from rostral, middle and caudal

Fig. 5. Characterization of labeled cells with ventricle-directed features in vitro. (aand b) Immunohistochemical characterization of an E17 cortical slice after 2 DIVshowed that the majority of dye-labeled cells (a) were positive for GABA (greenstaining in b; colocalization appears yellow, as indicated by arrows). (c–e) Placementof red- and green-dye-coated particles in the subventricular zones of the LGE andMGE, respectively (c), showed that neurons emanating from both eminences undergoventricle-directed migration (d, e); large arrows point to the green cells from MGE,whereas the small arrows indicate the red cells that emanated from LGE. VZ, ventric-ular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 60 µm.

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labeled neurons with their main processes oriented towards theventricle in sections of fixed embryonic cortex lends support tothese in vitro observations. Further, our double immunolabelingexperiments have shown that the vast majority of Tuj1-positiveneurons in the cortical VZ with horizontal- or ventricle-directedleading processes also contain GABA. An earlier study, usingcumulative BrdU labeling, had shown that neurons migratingtangentially through the cortical VZ are postmitotic27. The lack ofBrdU in Tuj1- or GABA-positive neurons in our experimentsindicates that these neurons must have become postmitotic atleast 12 hours (the shortest survival time analyzed) before theembryos were harvested and, hence, are probably not generatedin the cortical VZ. In this context, it is noteworthy that neuronsgenerated in the cortical VZ show pial-directed leading process-es during their radial ascent7.

Our CMTMR tracer-labeling experiments have shown that asubstantial proportion of all dye-labeled cells (30–40%) thatmigrated into the cortex had ventricle-directed leading process-

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cortical regions showed, however, that neurons withventricle-directed features were more prevalent in thecortex halfway in the rostro-caudal dimension (Fig. 4d).Immunohistochemical analysis indicated that themajority of dye-labeled cells from the rostral and caudalGE that had populated the anterior and posterior cor-tical regions, respectively, were also GABA positive(data not shown). To investigate whether ventricle-directed migration is widespread during corticogen-esis, slices were prepared from E16–18 rat brains andthe LGE labeled with CMTMR. Examination of slicesafter 2 DIV showed that a substantial percentage ofall labeled cells in the cortex of these slices had mor-phologies suggestive of ventricle-directed migration(30%, 33%, 40% at E16, E17 and E18, respectively)(Fig. 7). Although the fraction of labeled cells with ven-tricle-directed processes in the VZ and IZ did notchange significantly, the population of neurons thatappeared to descend from the CP increased markedlywith the progression of cortical development (Fig. 7).In addition to cells with ventricle-directed processes,the VZ also contained populations of labeled cells withother orientations. Characterization of these dye-labeled cells indicated that, although a substantialnumber (30–50%) were positioned horizontally, oth-ers had their leading processes oriented radially in thedirection of the pia (Fig. 4c; 8%, 21%, 26% at E16, E17and E18, respectively).

DISCUSSIONOur time-lapse study provides the first direct evidencethat postmitotic cells in different layers of the devel-oping neocortex actively seek the proliferative zone.Using tracer labeling together with immunohistochemistry, wehave identified the cells that undergo ventricle-directed migra-tion as populations of interneurons that arise in the ventral telen-cephalon. We suggest that these neurons actively enter the VZ toreceive layer information that is essential for their correct inte-gration into the developing cortex.

Recent studies have shown that cortical interneurons arisingin the GE follow tangential migratory routes to the developingcortex14,16. On entering the cortex, they show a tangential ori-entation and appear predominantly in the IZ but also in otherlayers of the cortical anlage. Our tracer-labeling experiments andtime-lapse recordings presented here have shown that a subsetof interneurons coursing through the IZ move initially towardsthe ventricular wall before migrating radially to their destina-tions in the CP. The presence of Tuj1-, GABA- or calbindin-

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Fig. 7. Neurons with ventricle-directed features are present at all stagesof corticogenesis. Analysis of E16–18 rat cortical slices after 2 DIVshowed CMTMR-labeled cells with ventricle-directed features in theVZ, IZ and CP. The number of cells with ventricle-directed processes isplotted as a fraction of all labeled cells in a given region and after nor-malizing for the thickness of the corresponding region. The total popula-tion of CMTMR-labeled cells in a given region includes those withventricle-directed features, those that were oriented horizontally, cellsthat were radially with pial-directed features, and those that could notbe classified with certainty due to insufficient labeling. Numbers refer tothe total number of labeled cells.

Fig. 6. Cells with ventricle-directed features are postmitotic. (a and b) E16 rat brainslices were treated with BrdU for 24 h after placement of CMTMR-coated particles inthe LGE. Examination of sections that were stained for BrdU (green) showed that thedye-labeled cells (a) were negative for BrdU (b, arrows), indicating that these cells werepostmitotic. (c and d) Cortical slices obtained from E17 rat embryos that had receiveda single injection of BrdU 24 h before the fetuses were harvested. Examination of slicesafter 2 DIV showed that a number of CMTMR-labeled neurons (c) were positive forBrdU (green staining in d; arrows), indicating that these cells became postmitotic duringthe 24-h period after injection. VZ, ventricular zone. Scale bar, 30 µm.

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es at all embryonic ages examined. These cells closely resembledthose seen in our time-lapse imaging, strongly suggesting thatthe cells showing ventricle-directed migration in our time-lapserecordings were indeed cortical interneurons. Although the frac-tion of dye-labeled cells with ventricle-directed processes in theVZ and IZ did not vary with time, their proportion in the CPincreased significantly. It is pertinent to note that calbindin-pos-itive cells with ventricle-directed processes were more abundantin the CP of fixed brain sections in late neurogenesis (Fig. 3g).

In addition to neurons with ventricle-directed processes inthe VZ, we also saw a substantial number of dye-labeled cells ori-ented parallel to the ventricular wall. We speculate that althoughsome of the labeled cells oriented tangentially may have descend-ed from the IZ, others may have transgressed the LGE-cortex ven-tricular zones, particularly during mid- and late corticogenesis.An earlier report28 indicated that cell movements between telen-cephalic proliferative zones are restricted; however, we have seenin fixed brain sections GABA- or Tuj1-stained neurons with lead-ing process oriented tangentially in the VZ of the cortico-striatalboundary. It is possible that GE neurons continue to migrate tan-gentially through the cortical VZ as reported earlier27, but theappearance of a subset of labeled cells with pial-directed mor-phologies indicates that these cells probably do exit the VZ.

Little is known about the molecular mechanisms that guideinterneurons or the cellular elements that may provide a sub-stratum for their migration from ventral to dorsal telencephalon.It has been suggested that axons may provide a substratum fornon-radial neuronal migration29,30, and there is recent evidencethat cortical interneurons migrate along axonal bundles of thecorticofugal fiber system to reach the developing cortex11,19. Ourtime-lapse recordings have shown that interneurons that under-go ventricle-directed migration show a saltatory pattern of move-ment. Such movement suggests the involvement of glia in themigration of these cells, as previous studies23 have associatedsaltatory motion with glial guidance. Nonetheless, the possibili-ty of neurophilic interactions between axonal fibers and descend-ing interneurons cannot be ruled out.

Why ventricle-directed migration?Earlier birth-dating studies have shown that pyramidal neuronsare disposed in an ‘inside-out’ pattern within the CP. According tothe protomap hypothesis, the cortical VZ contains intrinsic posi-tional information and serves as a blueprint for the organizationof the developing cerebral cortex20. In agreement with this hypoth-esis, transplantation studies have shown that cortical neurons—presumptive pyramidal cells—obtain their laminar informationfrom the VZ before their terminal division21. Despite accumulat-ing evidence that the majority of cortical interneurons are gener-ated in the ventral telencephalon, relatively little is known abouthow these neurons integrate into specific cortical layers. Earlierthymidine autoradiography studies have shown that corticalinterneurons also have an inside-out pattern of disposition with-in the CP31,32. It is, therefore, likely that cortical interneurons mayalso require layer information that enables them to integrate with-in the CP in an inside-out gradient. Based on these earlier obser-vations, we hypothesize that a subset of cortical interneuronsenters the proliferative zone, guided by a combination of chemoat-tractant and chemorepellent molecules, in order to acquire layerinformation. Our finding that ventricle-directed migration isprevalent in all regions of the cortical VZ at all stages of cortico-genesis lends support to this notion. It is possible that theseinterneurons obtain cues from the local environment or frompyramidal cells through neural–neural interactions.

The present finding of ventricle-directed migration, togeth-er with the recent demonstration of two modes of radial migra-tion, locomotion and somal translocation22, indicates that youngneurons may use different distinct modes of cell movement toreach their positions in the developing cortex.

METHODSPreparation of brain slices. Brain slices were prepared from embryonicmice (E13–16, where E1 = day vaginal plug was found) or rats (E16–19)as described previously22. Briefly, brains embedded in 3% low-melting-point agarose (Sigma, London, UK), were sectioned in ice-cold oxy-genated artificial cerebrospinal fluid (ACSF), pH 7.4, at 300 µm using aVibroslice. Coronal slices obtained from the anterior half of the cerebralhemispheres were mounted onto porous nitrocellulose filters (0.45 µm;Millipore, London, UK) and transferred to 12-well culture plates. Sliceswere allowed to recover for 1 h in defined medium with 5% CO2 at 37°Cand then either were incubated with Oregon Green BAPTA 488 AM orreceived focal applications of fluorescent dyes. The culture medium con-tained DMEM (Sigma), 5% heat-inactivated fetal bovine serum (Gibco,UK), 1× N-2 (Gibco), 100 µM L-glutamine, 2.4 g/liter D-glucose andpenicillin/streptomycin (1:1,000, Sigma). To follow the migration oflabeled cells from the GE, slices were cultured for 2 DIV and, for prolif-eration assays, pulsed with BrdU (10 µg/ml) for varying lengths of time.

Time-lapse confocal imaging. Time-lapse imaging of migrating cells wasdone as described previously22. Briefly, acute brain slices were incubatedwith Oregon Green BAPTA 488 AM (10 µg/ml in DMSO; MolecularProbes, Eugene, Oregon) and pluronic acid (0.0025%) at 37°C for 2 h.They were then transferred to a temperature-controlled (35–37°C) glasschamber fitted onto a Bio-Rad confocal microscope stage and perfusedthroughout the recording period with oxygenated medium (40–50 ml/h).Images of labeled cells from the dorsomedial neocortex were collectedusing 488-nm excitation and 522/535-nm emission filters. Cells wereselected for imaging only if their somata and processes were clearlylabeled. To follow the migratory movements of cells over substantiallylonger periods (>3 h), stacks of images were collected in the z-plane every15 min through regions encompassing several labeled cells of interest.The speed of migration and the trajectory of migrating cells were sub-sequently analyzed using Metamorph software (Universal Imaging, WestChester, Pennsylvania).

Application of fluorescent dyes. To label the population of corticalinterneurons arising in the ventral telencephalon11, tungsten particlescoated with fluorescent markers 4,4-chloromethylbenzoylaminotetram-ethylrhodamine (CMTMR) or 5-chloromethylfluorescein diacetate(CMFDA; Molecular Probes) were applied to the subventricular zone ofthe LGE of slice cultures using micropipettes. To coat tungsten particles,stock solution of CMTMR or CMFDA (10 mM) in dimethyl sulfoxidewas diluted in ethylene dichloride to yield a final concentration of 1 mM;50 µg of tungsten particles were then spread evenly on a glass slide towhich 100 µl of the fluorescent dye was added.

Immunohistochemistry. Cultured brain slices were fixed with 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), embedded in 3%agar, and sectioned at 50–70 µm with a vibrotome before processing forimmunohistochemistry as described previously22. Brains of embryonicmice or rats, fixed in 4% paraformaldehyde, were cut with a cryostat at15 µm and processed for immunohistochemistry. To characterize the phe-notype of migrating cells, sections were incubated overnight with prima-ry antibodies against Tuj1 (mouse monoclonal, 1:1,000, DevelopmentalHybridoma Bank, Iowa City, Iowa; rabbit polyclonal, 1:1,000, RDIResearch Diagnostics, Flanders, New Jersey), GABA (rabbit polyclonal,1:750, Sigma, St. Louis, Missouri) or calbindin (rabbit polyclonal, 1:750,Swant, Bellinzona, Switzerland). After washing, sections were incubatedwith FITC-conjugated secondary antibodies (against mouse or rabbit,1:500, Molecular Probes) at room temperature for 2 h. For BrdU label-ing, sections were treated with 2 M HCl at room temperature for 1 h,rinsed in 0.1 M sodium borate buffer and processed for immunohisto-chemistry (using mouse monoclonal antibody, 1:500, Sigma). Labeled

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10. Mione, M. C., Cavanagh, J. F., Harris, B. & Parnavelas, J. G. Cell fatespecification and symmetrical/asymmetrical divisions in the developingcerebral cortex. J. Neurosci. 17, 2018–2029 (1997).

11. Parnavelas, J. G. The origin and migration of cortical neurones: new vistas.Trends Neurosci. 23, 126–131 (2000).

12. Tan, S. S. et al. Separate progenitors for radial and tangential cell dispersionduring development of the cerebral neocortex. Neuron 21, 295–304 (1998).

13. De Carlos, J. A., Lopez-Mascaraque, L. & Valverde, F. Dynamics of cellmigration from the lateral ganglionic eminence in the rat. J. Neurosci. 16,6146–6156 (1996).

14. Anderson, S. A., Eisenstat, D. D., Shi, L. & Rubenstein, J. L. Interneuronmigration from basal forebrain to neocortex: dependence on Dlx genes.Science 278, 474–476 (1997).

15. Tamamaki, N., Fujimori, K. E. & Takauji, R. Origin and route of tangentiallymigrating neurons in the developing neocortical intermediate zone.J. Neurosci. 17, 8313–8323 (1997).

16. Lavdas, A. A., Grigoriou, M., Pachnis, V. & Parnavelas, J. G. The medialganglionic eminence gives rise to a population of early neurons in thedeveloping cerebral cortex. J. Neurosci. 19, 7881–7888 (1999).

17. Wichterle, H., Turnbull, D. H., Nery, S., Fishell, G., & Alvarez-Buylla, A. Inutero fate mapping reveals distinct migratory pathways and fates of neuronsborn in the mammalian basal forebrain. Development 128, 3759–3771 (2001).

18. Anderson, S., Mione, M., Yun, K., & Rubenstein, J. L. Differential origins ofneocortical projection and local circuit neurons: role of Dlx genes inneocortical interneuronogenesis. Cereb. Cortex 9, 646–654 (1999).

19. Denaxa, M., Chan, C.-H., Schachner, M., Parnavelas, J. G. & Karagogeos, D.The adhesion molecule TAG-1 mediates the migration of corticalinterneurons from the ganglionic eminence along the corticofugal fibersystem. Development 128, 4635–4644 (2001).

20. Rakic, P. Specification of cerebral cortical areas. Science 241, 170–176 (1988).21. McConnell, S. K. & Kaznowski, C. E. Cell cycle dependence of laminar

determination in developing neocortex. Science 254, 282–285 (1991).22. Nadarajah, B., Brunstrom, J. E., Grutzendler, J., Wong, R. O. L. & Pearlman,

A. L. Two modes of radial migration in early development of the cerebralcortex. Nature Neurosci. 4, 143–150 (2001).

23. Edmondson, J. C. & Hatten, M. E. Glial-guided granule neuron migration invitro: a high-resolution time-lapse video microscopic study. J. Neurosci. 7,1928–1934 (1987).

24. Komuro, H. & Rakic, P. Dynamics of granule cell migration: a confocalmicroscopic study in acute cerebellar slice preparations. J. Neurosci. 15,1110–1120 (1995).

25. Anderson, S. A., Marin, O., Horn, C., Jennings, K. & Rubenstein, J. L. Distinctcortical migrations from the medial and lateral ganglionic eminences.Development 128, 353–363 (2001).

26. Luskin, M. B. Restricted proliferation and migration of postnatally generatedneurons derived from the forebrain subventricular zone. Neuron 11, 173–189(1993).

27. O’Rourke, N. A., Chenn, A. & McConnell, S. K. Postmitotic neurons migratetangentially in the cortical ventricular zone. Development 124, 997–1005(1997).

28. Neyt, C., Welch, M., Langston, A., Kohtz, J. & Fishell, G. A short-range signalrestricts cell movement between telencephalic proliferative zones. J. Neurosci.17, 9194–9203 (1997).

29. Rakic, P. Principles of neural cell migration. Experientia 46, 882–891 (1990).30. Gray, G. E., Leber, S. M. & Sanes, J. R. Migratory patterns of clonally related

cells in the developing central nervous system. Experientia 46, 929–940(1990).

31. Miller, M. W. Cogeneration of retrogradely labeled corticocortical projectionand GABA-immunoreactive local circuit neurons in cerebral cortex. Dev.Brain Res. 23, 187–192 (1985).

32. Cavanagh, M. E. & Parnavelas, J. G. Development of somatostatinimmunoreactive neurons in the rat occipital cortex: a combinedimmunocytochemical–autoradiographic study. J. Comp. Neurol. 268, 1–12(1988).

articles

sections were examined using a confocal microscope, and the images weresubsequently reconstructed using Metamorph imaging software.

Quantitative analysis of CMTMR-labeled cells. For quantitative analysis,slices were fixed after 2 DIV and imaged using the confocal microscope. Toascertain the position of labeled cells within the cortical anlage, the offsetsettings of the confocal microscope were adjusted to enhance tissue back-ground. Stacks of images were collected in sequence from the corticostri-atal boundary to the dorso-medial cortex, thus covering the largest aspect ofneocortex. Each stack of images, consisting a number of optical sectionscollected in the z-plane through a depth of 100 µm tissue thickness, werethen collapsed into single images using Metamorph imaging software. Amontage of the neocortex was subsequently assembled from the series ofcollapsed images and the labeled cells located in the VZ, IZ and CP werecounted. Labeled cells, positioned at various levels of the cortical anlage,with leading processes oriented towards the ventricle, were considered asthose showing ventricle-directed migration. The fraction of labeled cellsthat showed ventricle-directed features was subsequently normalized to thethickness of the zones appropriate to the stage of development.

Note: Supplementary Web Movies can be found on the Nature Neuroscience

website (http://neurosci.nature.com/web_specials).

AcknowledgementsThe work was supported by grants by the Wellcome Trust to B.N. and J.G.P.

(grant number 050325) and by the US National Eye Institute to R.O.L.W.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 3 DECEMBER 2001; ACCEPTED 16 JANUARY 2002

1. Rakic, P. Mode of cell migration to the superficial layers of fetal monkeyneocortex. J. Comp. Neurol. 145, 61–83 (1972).

2. Rakic, P. Neuronal migration and contact guidance in the primatetelencephalon. Postgrad. Med. J. 54 Suppl 1, 25–40 (1978).

3. Angevine, J. B., Jr. & Sidman, R. L. Autoradiographic study of the cellmigration during histogenesis of cerebral cortex in the mouse. Nature 192,766–768 (1961).

4. Berry, M. & Rogers, A. W. The migration of neuroblasts in the developingcerebral cortex. J. Anat. 99, 691–709 (1965).

5. Rakic, P., Stensas, L. J., Sayre, E. & Sidman, R. L. Computer-aided three-dimensional reconstruction and quantitative analysis of cells from serialelectron microscopic montages of foetal monkey brain. Nature 250, 31–34(1974).

6. Hatten, M. E. Central nervous system neuronal migration. Annu. Rev.Neurosci. 22, 511–539 (1999).

7. O’Rourke, N. A., Dailey, M. E., Smith, S. J. & McConnell, S. K. Diversemigratory pathways in the developing cerebral cortex. Science 258, 299–302(1992).

8. Tan, S. S. & Breen, S. Radial mosaicism and tangential cell dispersion bothcontribute to mouse neocortical development. Nature 362, 638–640 (1993).

9. Reid, C. B., Liang, I. & Walsh, C. Systematic widespread clonal organizationin cerebral cortex. Neuron 15, 299–310 (1995).

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