Development of Signals Influencing the Growth and Termination of Thalamocortical Axons in...

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Experimental Neurology 156, 363–393 (1999)Article ID exnr.1999.7032, available online at http://www.idealibrary.com on

Development of Signals Influencing the Growth and Termination ofThalamocortical Axons in Organotypic Culture

Zoltan Molnar1 and Colin BlakemoreUniversity Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom

Received November 13, 1998; accepted December 3, 1998

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Explants of embryonic or postnatal rat cortex, orga-otypically cultured in serum-free medium, maintainheir structural integrity and their upper layers con-inue to mature. Coculture of portions of embryonichalamus with cortical slices taken at different ageseveals a temporal cascade of cortical signals. (1)lices of occipital cortex taken at E19 or earlier stimu-

ate axonal outgrowth from explants of embryonicateral geniculate nucleus but do not allow the fibers tonvade. (2) In cortical slices taken after E19 but before2, thalamic axons enter the slice, from any direction,nd extend radially across the entire depth of theortical plate without branching or terminating. (3) Inlices taken after P2, fibers slow down, arborize, anderminate in the maturing layer 4 of the cortex. If thehalamic explant is placed against the pial surface ofhe cortical slice, axons still enter and branch in theame layer. These findings imply that the developingortex expresses a diffusible growth-promoting factornd then itself becomes growth permissive, and finallyhe maturing layer 4 expresses a ‘‘stop signal.’’ In tripleocultures of one thalamic explant with a ‘‘choice’’ ofwo neighboring slices, thalamic axons will not invadelices of cerebellum but behave indistinguishably inesponse to slices from any region of the hemisphere.hus the initial tangential distribution of the thalamicrojection in vivo (which is achieved by about E16) isnlikely to be controlled by regional variation inignals produced by the cortex. When cortical slicesere precultured alone for 7–14 days before the addi-

ion of an explant of embryonic thalamus for 4 furtherays of coculture, the pattern of innervation was moreppropriate to the chronological age of the slice thanhe age at which it was first taken. Thus the timing ofhe cascade of cortical properties is at least partlyntrinsically determined. This sequence of expressionf these signals suggests that they play a part in vivo inontrolling the outgrowth of thalamic fibers, theirccumulation under the cortical plate, their invasion

1Present address: Institut de Biologie Cellulaire et de Morphologie,ue du Bugnon 9, 1005 Lausanne, Switzerland. E-mail:

[email protected].

363

f the plate, and their arborization in layer 4. r 1999

cademic Press

Key Words: organotypic culture; rat; thalamus; cor-ex; hippocampus; cerebellum; axon guidance; che-ospecificity; ‘‘stop signal’’; trophic factors; growth

ermissiveness

INTRODUCTION

Thalamic axons, which later convey afferent informa-ion to the cerebral cortex, reach the developing cortext a very early stage, before the majority of corticaleurons have even been born. Thalamocortical develop-ent follows a similar pattern in a diverse range ofammals (e.g., 52, 60, 61, 67), even marsupials (e.g.,

4, 70, 71). Extensive studies in rodents have shownhat axons grow down from the dorsal thalamus beforet differentiates into distinct nuclei, funnel through therimordial internal capsule as a topographically or-ered array, and then fan out within the intermediateone to find their way with remarkable precision to theppropriate region of the cerebral wall (see 4, 15, 21,2). Within this pathway lie a number of cell groupsnd early axonal projections that might provide growth-ermissive surfaces and even guidance cues for tha-amic fibers (4, 42–44, 49, 68). In particular, as thalamicxons enter the intermediate zone, they confront theescending axons of cortical preplate cells and growithin this corticofugal scaffold on their way to theppropriate region of the cortex (52, 53).By the time thalamic fibers reach their target area,

he first true cortical plate neurons (which will consti-ute the deepest part of the mature cortex) haveigrated up from the ventricular and subventricular

ones and through the lower part of the preplate layero split it into the marginal zone, above, and theubplate, below. In carnivores and primates, thalamicxons accumulate for a long time (the ‘‘waiting period’’;ee 78) within the subplate layer, forming side branchesnd even temporary synaptic connections on subplateeurons, many of which are destined to die (see 60, 61,
9). In rodents too, thalamic fibers congregate in the
0014-4886/99 $30.00Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

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364 MOLNAR AND BLAKEMORE

ubplate layer for 2–3 days (41, 52), although somendividual axons and side branches do penetrate theeepest part of the cortical plate shortly after theyrrive (14, 15, 36, 48, 51, 52, 56). In marsupials, inhich the lower layers of the cortical plate mature

elatively quickly, there is no obvious waiting period:halamic fibers immediately start to grow slowly intohe cortical plate (54, 70).

After any waiting period there is sudden, massivengrowth by thalamic axons, taking a somewhat erraticut roughly radial course through the lower layers (2,6, 52). This invasion of the cortical plate, whenever itccurs, might be linked to a change in the properties ofhe cortex, dependent on its state of maturation. Ashey approach layer 4, most axons slow down, branch,nd spread laterally. Their growth cones collapse andhey terminate on target neurons (36, 41, 52). A smallraction grow onward, mostly ending in the marginalone (layer 1).The orchestration of thalamocortical developmentust depend on rather precise mechanisms. In particu-

ar, the timing of axon outgrowth, waiting, invasion,nd termination appears to be exquisitely regulated.hese actions might be controlled by an intrinsic ‘‘clock’’

n thalamic neurons, but it might also be influenced byolecular signals in the environment through which

heir fibers grow.The timetable has been extensively studied in the

at. Fibers begin to descend from the dorsal thalamusbout the 13th embryonic day (E13), and they passhrough the primitive internal capsule between E14nd E15 (4, 21, 25, 52). Fibers from the posteriororsolateral thalamus (the primordial lateral genicu-ate nucleus; LGN) advance through the intermediateone beneath the lateral neocortex around E15.5, theyeach the occipital cortex at E16, and they massivelynvade the cortical plate about E19–20, just a couple ofays before birth (15, 21, 52).In eutherian mammals, the major steps in thalamo-

ortical development happen mainly before birth, mak-ng direct observation and experimental interventionery difficult. In any case, the key questions aboutechanisms of axonal behavior demand manipulation

f the relative locations and states of maturation of thehalamus and the cortex, which is virtually inconceiv-ble in vivo. However, modern techniques for observingevelopment in vitro offer an opportunity to examinehe interactions of thalamus and cortex directly inimplified model systems. In vitro studies have alreadyast light on questions of axonal guidance, targetpecificity, neuron–neuron recognition, and the pos-ible role of chemoattractants and trophic factors dur-ng the formation of the thalamocortical connections (7,, 23, 24, 30, 38, 39, 45, 50, 51, 62, 76, 79–81). However,hese studies used five different culturing techniques:
ollagen gel cocultures (40), roller tube cultures (26), a
nterphase-type cultures (65, 81), membrane prepara-ions (30), and a cell-attachment assay on culturedlices (23). Some employed serum-free culturing, butthers used medium containing serum, which itself hasomplex, mainly growth-enhancing effects (see 64).hese various approaches may each be ideal for specificuestions, but they prevent simple comparison be-ween different studies.

In this paper we describe an extensive study ofhalamocortical interactions in the rat, all performed inrganotypic culture under serum-free conditions. Ourope was to generate an overall picture of thalamocorti-al development. In particular we were interested inistinguishing between the inherent properties of thexons of thalamic neurons and the influence on them ofolecular signals from and within the cortex itself. We

ave already published a brief report of part of thisork (50).

METHODS

Our results are based on the culturing and cocultur-ng of explants of rat neocortex and dorsal thalamusand, for control experiments, hippocampus or cerebel-um), taken at ages ranging from E15 to postnatal day1 (P11), as listed in Table 1. The tissue explants wererom Hooded Lister rats, bred in the animal house ofhe University Laboratory of Physiology, Oxford. The‘plug date’’ was taken as E0 for the dating of fetuses,nd the day of birth (E21 or 22) was designated P0.Explants were cultured alone or in combination with

thers. Some experiments involved the coculture of aingle thalamic explant with an adjacent slice of neocor-ex (see Fig. 1); in others, two tissue slices were placedext to the thalamic block. The total period of time initro ranged from 1 to 22 days. In some experiments, alice of cortex was precultured alone for 7 or 14 daysefore a fetal thalamic explant was taken and placedext to it for a subsequent period of 4 days of coculture

see Table 1).

reparation of Explants

Identification of embryonic thalamic subdivisions.t E16, the stage at which most of the thalamicxplants were taken (see below), the entire dorsalhalamus extends approximately 1.3 mm anteroposte-ior, 1.25 mm mediolateral, and 0.8 mm dorsoventral,nd the major subdivisions of the thalamus are onlyust beginning to appear (18). Since the LGN, the targetf the optic nerve, is one of the first nuclei to beecognizable, lying at the surface of the posterolateralhalamus, and since the development of its projectionsas been extensively studied in vivo, we decided tooncentrate on culturing explants of the LGN, usuallyith slices of its normal target region, the presumptive
rea 17 in the occipital cortex.

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365THALAMOCORTICAL DEVELOPMENT IN VITRO

FIG. 1. The illustrations on the left show the procedure for takinglices of neocortex from the neonatal rat brain (in this case from theutative area 17 in the occipital cortex). A tissue chopper was used tout 350-µm-thick, roughly coronal slabs. Radial cuts were then maden order to remove a slice of cortex (stippled), extending from the piao the ventricular surface, which was placed on the membrane of aulture chamber. The diagrams on the right show the techniquessed to take explants from the dorsal thalamus of the fetal brain

usually at E16). The diencephalon was exposed, and roughly coronalnd parasagittal cuts were made in order to remove a tiny block ofissue, most often containing the putative LGN (filled area). Thisxplant was usually briefly exposed to an alcoholic solution of thearbocyanine dye, DiI (see Methods) and then placed in the culturehamber. The chamber was kept stationary with serum-free mediumust covering the surface of the explants (bottom diagram). Thisgure illustrates the most common procedure, in which a fetalhalamic explant and one neonatal cortical slice were cocultured for aew days before fixation and examination of axon growth. Thalamicxplants were taken from fetuses between E15 and P0; cortical slicesere taken at ages ranging from E16 to P11. Procedures used in otherxperiments (see Table 1) included the culture of: (1) a single explantof cortex or thalamus) alone; (2) a thalamic explant flanked by twother tissue slices (choice experiments), one neocortex and the othereocortex, hippocampus, or cerebellum; (3) a thalamic explant sur-ounded by six slices of occipital cortex; or (4) a slice of cortex alone foror 14 days, followed by the introduction of an embryonic thalamic

xplant for a further 4 days in vitro (see Methods).

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In order to learn how to recognize the relativeositions of the LGN and other thalamic nuclei fromurface features of the fetal diencephalon, before wetarted the culture experiments, we used axonal trac-ng methods to reveal the projection of the optic nervento the thalamus (see 19). We applied minute crystalsf the carbocyanine dye, DiI (1,18-dioctadecyl-3,3,3838-etramethylindocarbocyanine perchlorate; Molecularrobes, Inc., Eugene, OR) directly to the exposed retinar optic nerve in paraformaldehyde-fixed E15–16 fetalrains (34). The brains were incubated for 2–4 weeks atoom temperature (22°C) or 37°C, in fixative or phos-hate-buffered saline containing 0.1% sodium azide torevent contamination, to allow diffusion of the dyelong the axons of the optic nerve (28). After incubation,he brains were embedded in 5% agar and coronalections were cut at 70–100 µm on a Vibratome (Gen-ral Scientific, Redhill Surrey, UK). They were viewedn a fluorescence microscope and the terminal distribu-ion of the retinothalamic axons within the primordialGN was reconstructed in relation to the surface

eatures of the posterior diencephalon. This informa-ion enabled us to learn how to dissect tiny explants, asmall as 0.5 3 0.5 3 0.3 mm, from the fetal diencepha-on, containing mainly if not exclusively the developingGN. Once the LGN had been clearly identified atmbryonic stages, it was possible to relate the otherajor divisions of the thalamus to surface landmarks

isible during dissection.Collection of fetal explants. Fetal rats were ob-

ained from time-mated females by cesarean sectionnder pentobarbital anesthesia (100 mg/kg, intraperito-eal). Under sterile conditions, the anesthetized fe-uses were removed, chilled, and then decapitated. Thehole brain was removed and immersed in chillededium: dissection was performed with the aid of an

perating microscope under a sterile hood. One hemi-phere was removed and a tiny explant was quicklyxcised from the region of the LGN (see Fig. 1). Duringarly experiments, we demonstrated the reliability ofur localization of the LGN from surface features bytaining and examining sections of the remaining partf the diencephalon, after removal of the explant, to beure that we had taken the desired region for cultureusing Cogeshall’s atlas of the developing diencephalono identify the remaining structures, see 18).

In experiments in which axon growth was comparedor different regions of the thalamus, the two (or more)xplants were always dissected from the same side ofn individual brain, to ensure that they consisted ofifferent thalamic nuclei.To obtain neocortical explants from E16–18 fetal

rains a ‘‘tissue-ribbon’’ (3–4 3 1–2 mm at the pialurface 3 the entire depth of the embryonic cortex) was
xcised in the coronal plane. Then smaller slices (1–2 3

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.5 mm 3 the entire depth) were cut from the end of theibbon, i.e., in a parasagittal plane. In older animals,rom early E19, slices approximately 350 µm thick wereut directly from the whole brain with a hand-heldissue chopper, as for the postnatal animals.

The tissue blocks were gently transferred (with aodified Pasteur pipette) into a petri dish containing a

ew drops of Hanks’ balanced salt solution supple-ented with extra glucose to a final concentration of

.5 mg/ml, before being labeled (see below) and placedn the culture chambers. Preincubation at room tem-erature (for 1 h) reduced necrosis in the center ofxplants (65).Collection of postnatal explants. Surgery on postna-

al animals was performed under hypothermia or (after5) under ip pentobarbital anesthesia. After decapita-ion, the head was immersed in two baths of chilled0% ethanol in quick succession, for 1–2 s each time (toeduce the risk of contamination during further dissec-ion), and dried with sterile gauze. Under a sterile hoodhe whole brain was quickly removed and placed into aetri dish with a little chilled, glucose-supplementedfinal concentration 6.5 mg/ml) Hanks’ balanced saltolution. After careful removal of the pia, the brain waseld stationary in the dish with a pair of forceps in oneand, while the other hand was employed to push aissue chopper down quickly, perpendicular to theonvexity of the hemisphere, roughly in the coronallane. The tissue chopper consisted of five razor blades,eparated by 350-µm-thick stainless steel metal spac-rs, mounted in a metal holder.The four resulting coronal brain slices (containing

oth hemispheres, corpus striatum, and/or hippocam-us, together with diencephalon and mesencephalon)ere transferred into another petri dish containing a

ew drops of chilled Hanks’ balanced salt solution.eocortical slices were prepared from the dorsolateralspect of these whole-brain slices by making two radialuts with a microsurgical knife (Weck Ophthalmics,ristol-Myers Squibb, Jacksonville, U.S.A.) through

he full depth of the cortex, from the pia down to theentricular surface. The cortical slice so formed wasypically 2–5 mm wide (along the pial surface), 1–3 mmeep from pia to ventricular surface (depending on thege), and 300–350 µm thick (see Fig. 1). For somexperiments, explants of hippocampus were also dis-ected from the coronal whole-brain slices, and in someases roughly parasagittal slices were cut directly fromhe cerebellum with the tissue chopper.After preincuba-ion (65) for 1 h in glucose-supplemented Hanks’ bal-nced salt solution (at room temperature), the slicesere transferred to the tissue culture chamber. In onexperiment (six cultures), explants of the LGN were
aken on the day of birth. H
ulture Techniques

Organotypic culture (46) is aimed at preserving theasic structural organization of tissue. In organotypicultures of immature nervous tissue, cells not onlyontinue to migrate and differentiate but also formharacteristic neural circuits (see 26, 27). Of the vari-us protocols that have been described, we chose annterphase-type, stationary culture system, with serum-ree medium, for the following reasons:

(1) Neuronal explants do not flatten to monolayersven after several weeks in vitro. We felt that this wasmportant because neural growth and the formation ofocal circuitry are likely to be more natural in three-imensional substrates than in flattened preparations.(2) We were interested in demonstrating remote

rophic or tropic influences and therefore wished tovoid the use of serum, which itself stimulates neuriteutgrowth.(3) The specimen can be observed directly during

ulturing.Culture chambers. For most experiments, explantsere placed on the surface of a microporous membrane

pore size 0.4 µm) in Transwell-COL culture chambersCostar, Cambridge, MA), with 24.5-mm diameter in-erts in each six-well cluster plate. In most cases wesed Transwell-COL inserts whose membranes arelready treated with an equimolar mixture of Types Ind III collagen (derived from bovine placenta). Norecoating or other treatment was required (only arief preincubation with the medium for 1–2 h, tooisturize the collagen).Transwell membranes have microscopic parallel

rooves on their surface, which can influence theirection of axonal outgrowth, especially when theocultured explants are relatively far from each other.or ‘‘choice’’ experiments (see below), in which one

issue block was flanked by two different neighboringlices, the row of three explants was lined up parallel tohe grooves, so that any influence of the orientation ofhe grooves on axon growth would apply equally foroth targets. To control for possible effects of therooves on the Transwell membranes, we repeated allhe basic experimental paradigms with other cultureishes without grooved membranes (Petriperm, GmbH,urchased from Bachofer). In these dishes the culturesere placed on a 25-µm-thick, gas-permeable foil mem-rane, which formed the bottom of the dish. All theeneral findings described here were confirmed withhis alternative form of culturing.

In a few experiments we used inserts coated withaminin, which provides a more growth-permissiveurface: the membranes of Transwell or Petripermishes were treated overnight with poly-lysine solution1 mg/ml in Hanks’ balanced salt solution) in order toacilitate the adhesion of laminin (1–2 h, 20 µg/ml in
anks’ balanced salt solution).

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N-2 medium. For rat neocortical explants, N-2 and-16 media have proved to be the best for serum-freeulture (9, 10, 65, 80). We used N-2 medium, made ups follows: 1:1 mixture of Dulbecco’s modified Eagle’sedium and Ham’s F-12, supplemented with insulin (5g/ml), transferrin (100 µg/ml), progesterone (20 nM),utrescine (100 µM), and selenium (30 nM, as Na2SeO4).hese constituents were made up into stock solutions,lter-sterilized (pore size 0.2 µm), and then added tohe medium individually. In some cases we used aommercial N-2 stock solution (GIBCO BRL, Grandsland, NY). The medium was stored at 4°C underterile conditions. If the prepared medium was not usedithin a month, it was supplemented with additional

-glutamine (12.5 ml of commercially available stockolution per liter every 4 weeks).In all cases, medium was immediately added care-

ully, so as just to cover the tissue as soon as it had beenut in the chamber. Cultures were kept stationary at7°C in small incubator chambers (Modular Incubatorhamber; Flow Laboratories) with a continuous flow ofumidified carbogen (5% CO2 and 95% air, at 100%umidity). The level of the medium was checked once orwice each day and adjusted when necessary to keephe surface of the explants just covered: this arrange-ent seemed to optimize the efficiency of oxygenation

nd nutrition. The medium was changed every 3–5ays, depending on the amount of tissue in the cham-er.Preculturing of cortical slices. Some experiments

equired the combination of embryonic thalamic ex-lants with slices of cortex that had already been keptn culture for a period of time (see Table 1). Corticallices from early E19 and newborn (P0) animals wererepared and maintained in culture for 7 or 14 days.fter this period of preculturing, explants of LGN from16 fetuses were labeled with DiI (see below) andlaced next to the ventricular or pial surface of therecultured cortical slice. The cocultures were fixedfter a further 4–5 days in vitro and fiber ingrowth wasxamined with fluorescence microscopy.

taining Techniques

Prelabeling with carbocyanine dye. When being co-ultured for fewer than 6 days, the thalamic explantas prelabeled with DiI, before being placed in culture,

n order subsequently to reveal axonal connections.mmediately after dissection, it was moistened with arop of Hanks’ balanced salt solution followed by a dropf an alcoholic solution of DiI (25 mg DiI dissolved in 10l of 5% dimethyl sulfoxide and 95% ethyl alcohol).fter about 1 s, it was briefly rinsed in Hanks’ balancedalt solution. This caused minute crystals of DiI to formn the surface, which extensively labeled cell bodies
ithin the whole explant. Axons growing out were i
ntensely labeled with the fluorescent dye for a period ofbout 5 days, after which the label became concen-rated back in the cell bodies, presumably because ofembrane recycling.Fixation, staining, and sectioning. Culturing was

erminated by fixation, usually by immersion for 30in to 2 h in 4% paraformaldehyde in 0.1 M phosphate

uffer (pH 7.4, 4°C) followed by a phosphate-bufferedaline rinse. For DiI labeling of thalamic projectionsfter 6 days or more in vitro we placed small crystals ofiI on the thalamic explant after fixation and stored

he entire preparation in fixative at room temperature22°C) or 37°C for 2–4 weeks, to allow diffusion of dyelong the axons.Most cultured explants containing DiI-labeled struc-

ures were counterstained with the chromatin stainisbenzimide (10 min in 2.5 µg/ml solution in 0.1 Mhosphate buffer; Riedel-De Haen AG, Seelze-Hanno-er, Germany) and a few with acridine orange (10 µg/mln buffer; Molecular Probes). This enabled us to visual-ze the outline of the explants and some of the laminaroundaries under appropriate illumination. The area ofhe chamber membrane bearing the cultured tissueas cut out, mounted on a slide in phosphate-buffered

aline or Hydromount (National Diagnostics), andealed under a coverslip for examination in a fluores-ence microscope or a laser-scanning confocal micro-cope.For conventional Nissl staining, some cultured ex-

lants were fixed and rinsed in the same way andmbedded in paraffin (ascending alcohol line, 30 minach; methyl salicylate overnight, then paraffin 2 3 2). Ten-micrometer-thick sections were cut, either par-llel or perpendicular to the surface of the explant inontact with the culture chamber membrane. Otherultures were cryoprotected in 30% sucrose (in phos-hate buffer) overnight and sections were cut at 30 µmn a cryostat. Mounted sections were stained with anqueous solution of cresyl violet (1%) for 5 min, differen-iated, dehydrated, cleared, and mounted in DPX.

Some cultures were prepared for semithin sectioningy fixation in 5% glutaraldehyde in sodium cacodylateuffer (300 mOsm, pH 7.4) for 1.5 h and postfixation in% OsO4 in buffer (for 1.5 h). The explants were thenehydrated and embedded in Epon. Sections (1 µm)ere stained with toluidine blue.

icroscopy

Living cultures were occasionally viewed by phase-ontrast microscopy, which could be done without stain-ng or removal of the explants from the chamber.hase-contrast microscopy was also used to view un-tained explants and axons after fixation with 4%araformaldehyde. Nissl-stained sections were exam-
ned by conventional microscopy. Living or fixed cul-

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ures containing cells and axons stained with DiI, withisbenzimide or acridine orange counterstain, wereiewed in a conventional fluorescence microscope or aaser-scanning confocal microscope (Leica CLSM-luovert). Digitized fine optical sections (up to 64ections) could be captured in the confocal microscopend stacked to generate high-resolution extended-focusmages, from which stereo pairs (67° disparity) coulde constructed.

RESULTS

eneral Characteristics of Cultured ExplantsMacroscopic appearance and maturation of cortical

aminae. Both cortical and thalamic explants stayedompact and tightly organized, with quite sharp bound-ries, throughout the culturing period of up to 22 days.oreover, even to casual observation, cortical slices

emained organotypic. Distinct cell and fiber layeringould be seen, from pial to ventricular surface, even innstained cultured slices. After a few days in culture,ortical slices, especially those taken before P3, becamearger in area, particularly in the radial dimension, androgressively more fan-shaped, developing a prominentonvex pial surface and a less regular, narrower ven-ricular aspect (Figs. 2 and 10–14). This enlargementight be caused by continuing migration of immatureeurons into the more superficial layers, as well as therowth of cells and an increase in the amount of corticaleuropil.Evidence for continued cell migration and differentia-

ion in culture comes from a comparison of corticallices immediately after removal at P0 with similarlices cultured for up to 2 weeks, with or without anccompanying thalamic explant (Figs. 2C and 2D). Onhe day of birth, only the relatively mature layers 6 andcould be distinguished, below a band of newly arrived,

mmature neurons (the dense cortical plate) and thearginal zone (Fig. 2A). However, after several days in

ulture, lamination, at least in the central part of thelice, appeared more appropriate to its chronologicalge than to the age at which it was taken, even thoughot completely normal. Putative layer 4, recognizabley the concentration of small, relatively closely packedomata, first emerged from the bottom of the dense

FIG. 2. Maturation of cortical lamination in vitro. (A) 20-µm-thicrea 17) of a newborn rat, after perfusion and paraffin embedding. Luperficially there is only the dense cortical plate (DCP) of tightly pacWM, white matter). (B) 20-µm-thick, Nissl-stained section of an occultured slice was resectioned in a cryostat in the original coronal planhe upper layers have matured in vitro. (C) Low-power micrograph ofter 7 days in vitro. The LGN (left) has become closely fused to theortical layers (1–6) are labeled. Note the collapse of lamination closedges, with the marginal zone wrapping around to make contact withoculture in C. Lamination is similar to that for the slice in B, culture
lice. Scale bars: A, 200 µm; B, 100 µm; C, 300 µm; D, 100 µm.
ortical plate, about 200 µm below the pial surface,fter 2–3 days in culture. It was then progressivelyisplaced downward, to about 500 µm below the surfacefter about a week in vitro.Maturation was much less normal at the radially cut

dges of the slice, especially for explants taken beforebout P1, in which the edges tended to curl over, toreate double thickness, which then collapsed to form aurved boundary and distorted lamination. The low-ower view of a coculture in Fig. 2C shows clearly theompressed lamination at the radial sides of the slice,ith the marginal zone wrapping around to contact the

unction of the thalamic explant and the ventricularurface of the cortical slice. All this suggests thataminar organization continued to mature fairly nor-

ally in the central portion of cultured slices, butecame abnormal at the edges of slices taken beforebout P1. For that reason, we concentrated our analy-is on the central portions of slices.Thinning of cultured explants. Explants, especially

hose taken at younger ages, became somewhat thinnern culture, especially over the first day in vitro, but noto the same extent as with the roller tube technique26), in which cultures become ‘‘less than 4 cell layershick and often monolayers are obtained’’ (11). Shrink-ge was unequal for different cortical layers, the pialide tending to flatten less than the ventricular. How-ver, the thinnest regions of the explant remained over50–200 µm thick, even after several weeks in culture.ypically, 15–20 cell layers or more could be distin-uished through the whole thickness of a culturedortical slice, from top to bottom surface (see Fig. 3A).mbryonic thalamic blocks flattened somewhat more

han the older cortical slices.The upper face of the slice (which occupied theedium–gas interface) became covered with a thin

ayer of tangled cell processes and other debris, 10–15m thick. Some pyknotic profiles, characteristic ofying cells, were seen, almost exclusively at the lowerurface of the slice, which had been in contact with theicroporous membrane (Fig. 3A). This suggests that

ome degeneration and cell death were still takinglace at the lower surface of the slice even after a weekr more in culture, despite the considerable preserva-ion of overall thickness and cell number. However,

issl-stained coronal section taken from the occipital cortex (putativers 6 and 5 are relatively mature and easily distinguished, but more, recently arrived immature neurons, below the marginal zone (MZ)

tal cortical slice taken at birth and cultured alone for 2 weeks. Thetangential to the culture membrane). Comparison with A reveals that

issl-stained 30-µm section of a coculture of P0 cortex and E16 LGNntricular surface of the cortical slice (right). The white matter andthe radial margins of the cortical slice, because of curling over of thethalamic explant. (D) Higher-power view of the cortical slice from thelone, except that layer 4 appears relatively thicker in the cocultured

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ells throughout the whole of the rest of the depth of thelice, between these thin boundary regions, appeared toe healthy. In surface-parallel semithin sections fromhe middle of the thickness of cultured cortical slices,ells appeared remarkably normal, with cytoplasm,uclei, nucleoli, and even major dendrites clearly vis-

ble (Fig. 3B).Migration of cells out of the explants. Phase-

ontrast microscopy of living, unstained cultures re-ealed relatively small numbers of cells migrating outf the edges of explants, across the surface of theulture chamber membrane. The majority of such mi-rating cells were seen around the thalamic blocks andspecially at the ventricular boundary of the corticalxplants. The degree of such cell migration dependedn the nature of the surface of the microporous mem-rane, confirming the results of Nagata and Nakatsuji55), laminin (Figs. 4–6) being much more permissivehan collagen. The maximum distance of cell migrationrom a P0 cortical slice after 7 days in vitro was about 2m on collagen and 5 mm on laminin.

nfluences of Cortex on the Behavior of Thalamic Axonsin Vitro

Most of the coculture experiments involved thalamicxplants from E16 embryos (see Table 1), when thehalamus is in a state of active axonal extension in vivo.reliminary experiments in which the projection of theptic nerve was traced at E16 (see Methods) helped uso judge the position of the LGN from diencephalicurface landmarks. So, we concentrated on coculturingxplants containing mainly LGN with slices of theppropriate target region in the occipital cortex (puta-ive area 17). Our hope was to infer the nature ofolecular signals produced by the developing cortex

rom the behavior of thalamic axons in the presence oflices of cortex of different ages, between E16 and P11,panning the natural period of thalamic fiber arrival,ccumulation, ingrowth, and arborization. In fact, webserved three distinctly different patterns of axonalrowth from thalamic explants, depending on the age ofhe cortical slices with which they were combined initro.Remote influence of cortex (from E16 or before) on

halamic axon outgrowth. Under the serum-free con-itions that we employed, the cortex appeared to have a

FIG. 3. (A) A slice of cortex taken at P0 was cultured alone forerpendicular to the culture chamber membrane and parallel to the oorder region, reveals the histological appearance through the wholes no obvious gliosis. Through most of the thickness of the slice, cellurface, which had occupied the medium–gas interface, is a thin cell-flice, which had been in contact with the microporous membrane, cehanges). (B) The cytoplasm, nuclei, and nucleoli of individual cellsccipital cortex taken at P0 and cultured alone for 7 days. This 1-µm
lane), from the middle of thickness of the cortical culture, was stained w
emote, growth-promoting effect on thalamic axons,ven from the earliest age at which cortical slices wereaken (E16, when the first true cortical plate neuronsave just migrated into the occipital cerebral wall).We used phase-contrast microscopy of living or fixed

ultures, and fluorescence microscopy of fixed explantsrelabeled with DiI, to observe the degree of neuritextension out of thalamic blocks cultured either aloner in the presence of cortical slices in the same culturehamber (Table 1). There was very little axon out-rowth from E16–17 thalamic explants when they wereultured alone in serum-free medium. Even on a lami-in-coated membrane most neurites extended up tonly about 50 µm from the edge of the thalamic blockFigs. 4C and 4D). However, if an LGN explant (E16)as placed within a few millimeters of an occipital

ortical slice of any age from E16 to P11, within a dayxons were seen streaming out of the thalamic explantFigs. 4A and B), extending more than 1 mm after just aouple of days in culture.4If only one slice of cortex was placed in the chamber,

ess than about 4 mm away on laminin, the outgrowthf axons from the thalamic explant was clearly asym-etrical, being more prolific on the side facing the

ortical slice. The example in Fig. 5 shows a denselexus of axons radiating from an E16 LGN explantoward a P0 cortical slice after 6 days in vitro, whilexons on the other side are sparser and extend aaximum of less than 1 mm. Note, however, that the

ast majority of axons, even from the side opposite theortical slice, simply radiate from the thalamic blocklong fairly straight courses. There was no tendency forndividual axons to turn toward the cortical slice, asould be expected if the cortex were exerting a tropic

nfluence on growth cone behavior (40).If the thalamic explant was placed very close to the

entricular or the pial surface of a cortical slice (withinbout 0.5 mm), the gap between them became filledith a dense mass of fibers and a scattering of migrat-

ng cells. With larger separations (up to 2 mm onollagen, 4 mm on laminin), one or more distinct tissueridges developed between the cocultured structuresfter 2–3 days in vitro (Fig. 6). These bridges consistedf large bundles of axons, presumably running in bothirections.Cortical slices of all ages examined (E16–P11) ap-

week and then, after paraffin embedding, was sectioned at 10 µm,inal pial surface. This Nissl-stained section, from about the layer 4/5kness of the slice. The slice is still more than 20 cells thick and there

ppear healthy, embedded in neuropil. Immediately below the upperlayer of tangled processes and debris. And in the lowest 50 µm of theith small dense nuclei are seen (probably undergoing degenerative

e even more apparent in this semithin preparation from a slice ofick section, cut parallel to the culture surface (the original coronal

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eared capable of stimulating axon outgrowth fromhalamic explants. This could be judged by the amountf thalamic neurite extension in the large number oflosely adjacent thalamocortical cocultures (Table 1),ven from the edges of the thalamic explant not in

FIG. 5. This phase-contrast micrograph shows an E16 LGN explasingle P0 slice of occipital cortex (CTX, ventricular surface about 4 m

oughly radially, in all directions, though more densely and much fartr even farther. There are also more migrating cells between LGN andentricular surface of the cortical slice. Scale bar, 500 µm.

FIG. 4. E16/17 LGN blocks were cultured without (N 5 32) or withese micrographs are of E16 LGN explants kept for 6 days onaraformaldehyde and examined unstained by phase-contrast micrortical slices, whose ventricular surfaces formed the circumference oays in vitro a mass of thalamic fibers has grown out in all directiohalamic and cortical blocks. (B) A higher-power view, taken from thehe explant. (C) This E16 explant was cultured under identical conditrom the region indicated by the outline in C, at the immediate edge of
utgrowth beyond about 50 µm from the border. Migrating cells are also l
ontact with the cortical slice. A dense bridge of axons,imilar to that in Fig. 6A, also formed in six coculturesf E16 LGN with P3 cortex in which the explants wereeliberately placed more than 2 mm apart. Moreover,xon outgrowth was just as great from explants of E16

fter 6 days in culture on a laminin-coated Petriperm membrane withaway, out of view to the left). Fibers have grown out of the LGN block,from the side facing the cortical slice: individual axons extend 2 mmtex than on the other side, but some of these probably came out of the

5 12) embryonic cortical slices (E16), for periods from 4 to 20 days.inin-coated Petriperm dishes. The cultures were fixed with 4%

py. (A) This E16 LGN explant was cultured with six E16 occipital-mm-diameter circle, with the thalamic explant in its centre. After 6Note the fairly high density of cells that have migrated out of theion of the inset outline in A, centered about 350 µm from the edge ofs but without a slice of cortex in the chamber. (D) Higher-power viewe explant. Even after 6 days in vitro, there is very little thalamic fiber

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GN cocultured with P3 frontal cortex (N 5 10), androm E16 anterior thalamic blocks cultured with P3ccipital cortex (N 5 4), as it was from cocultures ofopographically corresponding thalamic and corticalegions.Emergence of ingrowth-permissive properties (after19). Even the youngest cortical slices stimulated theutgrowth of thalamic axons, but if the occipital corticallice was taken before late E19, very few of thosehalamic axons penetrated the cortical explant. Theyrew extensively across the membrane toward theortical slice and formed a dense bundle circling aroundts border (Fig. 7). Only occasional axons penetrated ahort distance into the cortical slice itself (Fig. 7B) orrew right through it to emerge from the oppositeorder of the slice and join the band of fibers coursinground the edge. Some ran in sparse fascicles acrosshe slice, but examination in the confocal microscope atne optical resolution showed that these were coursingver its surface rather than through its interior.Even after 4–5 days in culture with E16–19 cortical

lices, many of the circling band of thalamic axons stillad clear growth cones at their tips (Fig. 7D), recog-ized as enlargements with irregular outlines andlopodia visible in the confocal microscope. This alluggests that embryonic cortex taken before late E19oes not provide an attractive environment for tha-amic fibers.

In some of the E19 cortical explants (N 5 8), and inll taken after E19, there was noticeably more axonalngrowth. In these cases many thalamic axons grewtraight in through the ventricular surface of the slicend radially onward through the cortex itself. Thesexons were coursing through the interior of the corticallice, not just over its surface. In Fig. 8 a stereogramnd orthogonal views created from a high-power confo-al microscopic reconstruction show thalamic axonsrowing among cell bodies within a cortical slice afternly 1.5 days in culture.Fibers grew ever more profusely out of E16 thalamic

locks when combined with cortex taken at progres-ively older ages, from late E19 to P3 (see Table 1).any of them entered the ventricular surface of the

ortical slice and ran upward through the intermediateone and on through the subplate layer (the lowest cellone directly below the cortical plate), without anybvious hesitation, branching, or termination. Theyontinued radially into the cortical plate, as a fairlyniform array across the width of the slice. Growing at

FIG. 6. (A) Under phase-contrast microscopy, a tissue bridge, contentricular surface of a slice of P0 occipital cortex (CTX), to the leftonsiderable spread of cells on the membrane (not in focus), below thaminin, the edge of the E16 LGN explant is just visible (on the righortex, more than 2 mm away to the left (out of sight). Quite large num
00 µm; B, 100 µm.
pproximately 1 mm per day, they continued righthrough the layer of immature neurons of the denseortical plate and reached the marginal zone afterbout 3–4 days in culture (Fig. 9).With cortical slices taken at relatively young ages,

etween E19 and birth, a significant proportion ofhalamic axons still circled around the edge of the slice.n these cases, most axons that penetrated and grewithin the slice emerged through the pial surface and

oined this marginal plexus (Fig. 9, P0). For corticallices taken between P0 and P2, a progressively smallerraction of thalamic fibers ran around the edges of thelice. The penetrating axons still grew up through thentire thickness of the cortical plate. Some burst throughhe pial surface, but many turned to run parallel to andust under the surface (Fig. 9, P2). Even after 5 days initro with cortex taken as late as P2, many of thesehalamic axons still had growth cones at their tips.here was no sign of any preference for a particularector of the cortical plate and little or no evidence ofranching or termination in any layer.We cultured eight P1 occipital cortical explants with

16 LGN for 22 days. Then, after fixation, a smallrystal of DiI was placed on the LGN block to label itsxons. Interestingly, even after this very long period ofulturing, the majority of thalamic fibers still appearedo extend to the pial surface. They no longer hadbvious growth cones and many of them had varicosi-ies along their course within the cortical plate, whichight represent en passant synapses.Maturation of a ‘‘stop signal’’ in layer 4 (after P2). In

ome of the late P2 cortical explants and in more thanalf of the 43 P3 slices cocultured for 4 days or more,any thalamic axons behaved rather differently. When

hey reached the bottom of the dense cortical plate,hey branched and spread laterally about 300 µm belowhe pia in what appeared to be the maturing layer 4.oreover, their growth cones had clearly collapsed and

hey showed every appearance, at the light microscopicevel, of having terminated on cortical neurons. Forortex taken at P2 and P3, thalamic axons growinglose to the cut radial edges of the slice still tended toun up through the entire radial extent of the slice.This sudden change of thalamic axonal behavior in

ortex taken after P2–P3 was consistent among theery large numbers of cocultures (see Table 1). How-ver, just to be sure that it did not result from chanceifferences in conditions, we employed the choice para-igm (see Methods), culturing a single E16 LGN ex-

ing cells and fibers, is seen between an E16 LGN block (right) and thecultured for 6 days on a laminin-coated Petriperm dish. There is alane of the axon bridge. (B) In this example of a similar coculture on

dense array of axons is seen fanning out toward a slice of occipitalrs of cells have also migrated out of the thalamic block. Scale bars: A,

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TABLE 1

ArrangementCulturedstructure

Daysin vitro

No. ofcultures

Single explant cultures

Petriperm dishes with laminin; phase-contrast microscopy

E16 LGN alone 6 6E16/17 LGN alone 4 14

Transwell Col chambers with collagen

E16 LGN 4 6E16 LGN 20 6P0 Occ CTX 14 6P6 Occ CTX 4 8P0 Cereb 4 6

halamic explant cultured with cortex to examine remote growth-promoting influences

Petriperm dishes with laminin

urrounded E16 LGN 1 E16 Occ CTX 6 6E16 LGN 1 E16 Occ CTX 6 6E16 LGN 1 P0 Occ CTX 6 12

2 mm E16 LGN 1 P0 Occ CTX 6 6E16 LGN 1 P3 Occ CTX 3.5 12E16 LGN 1 P3 Occ CTX 4 4

Thalamic explant cocultured with a single adjacent slice

ranswell Col chambers with collagen: DiI labeling of thalamicaxons

E16 LGN 1 E16 Occ CTX 4 6E18 LGN 1 E18 Occ CTX 7 6E16 LGN 1 E19 Occ CTX 4 8E16 LGN 1 E21 Occ CTX 4 8E21 LGN 1 E21 Occ CTX 4 4E16 LGN 1 P0 Occ CTX 1 4E16 LGN 1 P0 Occ CTX 2 4E16 LGN 1 P0 Occ CTX 4 14E16 LGN 1 P0 Occ CTX 7 8P0 LGN 1 P0 Occ CTX 21 6E16 LGN 1 P1 Occ CTX 5 4E16 LGN 1 P2 Occ CTX 4 8E16 LGN 1 P3 Occ CTX 1 4E16 LGN 1 P3 Occ CTX 1.5 10E16 LGN 1 P3 Occ CTX 4 26E16 LGN 1 P3 Occ CTX 10 14

2 mm E16 LGN 1 P3 Occ CTX 4 6E16 LGN 1 P5 Occ CTX 4 5E16 LGN 1 P6 Occ CTX 4 10E16 LGN 1 P11 Occ CTX 4 11E16 LGN 1 P3 Fron CTX 4 10E16 Ant Thal 1 P3 Occ CTX 4 4

nverted E18 LGN 1 E18 Occ CTX 7 6nverted E16 LGN 1 E19 Occ CTX 4 6nverted E16 LGN 1 P0 Occ CTX 5 8nverted E16 LGN 1 P1 Occ CTX 22 8nverted E16/17 LGN 1 P3 Occ CTX 4 6nverted E16 LGN 1 P6 Occ CTX 10 4nverted E16/17 LGN 1 P6 Occ CTX 4 6nverted E16 LGN 1 P8 Occ CTX 4 6ateral E16 LGN 1 P3 Occ CTX 4 6ateral E16 LGN 1 P6 Occ CTX 4 8

E16 LGN 1 P3 Hipp 4 8E16 LGN 1 P6 Hipp 4 8E16 LGN 1 P3 Cereb 4 15E16 LGN 1 P8 Cereb 4 4
o
lant flanked by two cortical slices, one taken early on3, the other at P6. In all five of these triple cultures,oth cortical slices were invaded by the LGN explantut with different patterns of innervation, characteris-ic of the ages of the slices. In the early P3 slices, mosthalamic axons grew up to the pial surface and had noterminated after 4 days in vitro, whereas the majority

TABLE 1— Continued

ArrangementCulturedstructure

Daysin vitro

No. ofcultures

Single thalamic explant flanked by two adjacent slices(‘‘choice’’ paradigm)

Transwell Col chambers with collagen: DiI labelingof thalamic axons

E19 Occ CTX 1 E15LGN 1 E19 Fron CTX

4 4


4 4


4 4

P1 Occ CTX 1 E16 LGN 1 P1Fron CTX

2.5 6

P3 Occ CTX 1 E16 LGN 1 P6Occ CTX

4 5


4 5


5 4


5 10


4 5

E19 Occ CTX 1 E15 AntThal 1 E19 Fron CTX

4 4

E20 Occ CTX 1 E16 AntThal 1 E20 Fron CTX

4 4

P1 Occ CTX 1 E16 AntThal 1 P1 Fron CTX

2.5 6

P8 Occ CTX 1 E16 AntThal 1 P8 Fron CTX

4 5

E18 Occ CTX 1 E18LGN 1 E18 Cereb

4 4

E21 Occ CTX 1 E21LGN 1 E21 Cereb

4 4

P1 Occ CTX 1 E16 LGN 1 P1Cereb

5 4


4 6


4 8


5 4

P3 Occ CTX 1 E16 LGN 1 P3Hipp

4 4


5 4


22 4


4 4

f fibers from the same thalamic explant growing into

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he P6 slice branched and terminated in what appearedo be layer 4.

Thus, it appears that about P3 the occipital cortextarts to express a specific molecule (or set of molecules)hat constitutes a stop signal (50), making thalamicbers branch and terminate in layer 4.Is the behavior of thalamic axons determined by the

athway that they take into the cortex? For most of theoculture experiments, we deliberately placed the tha-amic explant adjacent to the ventricular surface of theortical slice, so that axons growing straight out fromhe thalamic block would have the opportunity toontinue up through the intermediate zone and to grownto the cortical plate along the normal, radial trajec-ory. We were concerned, however, that the unusualorm of confrontation with the cortex might account forhe failure of axons to penetrate cortical slices takenefore E19 and their tendency to circle around thedges of the slice. Such behavior might be due primarilyot to an absence of growth-permissive properties in

TABLE 1—Continued

Preculturing of a single slice of occipital cortex followedby introduction of an LGN explant

Transwell Col chambers with collagen:DiI labeling of thalamic axons

rrange-ment

Originalage of

Occ CTX

Durationof precul-

turingAge of addedLGN explain

Durationof cocul-

tureNo. of

cultures

E16 7 days E16 4 days 12E19 7 days E16 4 days 12P0 7 days E16 4 days 8

Inverted P0 7 days E16 4 days 4P0 14 days E16 4 days 12

Note. The columns represent the arrangement and identity of theultured structures, the number of days in vitro, and the number ofultures of that type examined. Some cultures grown on a lamininurface were examined unstained by phase-contrast microscopy, butost were grown on collagen and the thalamic explant was labeledith DiI (see Methods). Cultures of single explants, some grown on

aminin, some on collagen, were used to examine histological changesn the explants and any spontaneous outgrowth of axons fromhalamic blocks. A number of cocultures of thalamus and cortex wererown on laminin with a clear separation between them (sometimes2 mm) to study the influence of cortex on thalamic axon extension.

n one batch of six such cultures, the LGN block was surrounded byix slices of E16 cortex whose ventricular surfaces formed a ringbout 2 mm from the LGN (‘‘Surrounded’’ in the Arrangementolumn). The two or three explants in cocultures on collagen weresually placed directly in contact with each other, except for those

ndicated by ‘‘.2 mm’’ in the first column. In cocultures, the thalamicxplant was always placed next to the ventricular surface of theortical slice, except for those cases marked ‘‘Inverted’’ and ‘‘Lateral,’’n which the thalamus was adjacent to the pial surface or the radiallyut edge, respectively, of the cortical slice. LGN, dorsolateral poste-ior thalamus containing the lateral geniculate nucleus; CTX, neocor-ex; Occ, occipital; Fron, frontal; Ant thal, anterior dorsal thalamus;ipp, hippocampus; Cereb, cerebellum.

he cortex itself, but to a specific repelling property of

he ventricular layer, deflecting the growing axons andorcing them into their circling pattern.

We also wondered whether the termination of axonsn presumptive layer 4, when growing into corticallices older than P2, might be due to growth-repulsiveroperties in the supragranular layers rather than to apecific signal expressed in layer 4.We therefore prepared thalamocortical cocultures inhich the thalamic explant was placed next to the

leaned pial surface of the cortical slice, taken at agesetween E18 and P8 (Table 1). The behavior of axons inhese ‘‘inverted’’ cocultures was just as predicted fromhe results of the main series. With cortical slicesounger than E19, thalamic fibers did not enter thelice through the pial surface, but simply grew aroundts margins in the usual fashion for slices of this age.nd with slices between E19 and P3, the axons freelyenetrated the pial surface and grew down through thentire cortical plate and on into the intermediate andubventricular/ventricular zones, as shown in the ex-mple in Fig. 10A. Again, very few axons branched orppeared to terminate.Thalamic axons also entered the pial surface, indeedith ever-increasing density, in slices taken at andfter P3. There was no tendency for them to avoid theupragranular layers, even in slices taken as late as P8,n which lamination was already very mature. Thexample in Fig. 9C shows dense invasion, by E16halamic axons, through the pial surface and the upperayers of a P6 slice. After 5 days in culture, most ofhese axons had arborized and terminated some 400–00 µm below the surface, at about the depth at whichhey typically branch and terminate when enteringrom below (compare Fig. 9, P4).

We also made a number of cocultures with an E16GN explant placed adjacent to the cut lateral edge of3 or P6 cortical slices, i.e., against the portion of the

ntermediate zone through which thalamic axons nor-ally grow up toward the occipital cortex. Again, axons

ntered, through both white and gray matter, most ofhem running tangentially for some distance beforeurning up, radially through the cortical plate, toranch and terminate around layer 4.

o Thalamic Axons Show Target Specificity?

Preference for neocortex. The simple coculture ex-eriments show that the occipital cortex develops somettribute that supports the ingrowth of geniculatexons from about E19. To test whether this property ispecific to the neocortex, we cultured E16 LGN withlices from other sites (hippocampus, N 5 32; cerebel-um, N 5 49) taken between E18 and P8 (see Table 1).n the majority of cases we used the choice paradigm toompare axonal growth in response to two differentargets, in the hope of revealing preferences.

Figure 11 illustrates typical cases in which E16 LGN

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locks were confronted, on one side, with a slice ofccipital cortex and, on the other, with a slice ofippocampus or cerebellum from the same donor brain.n each case, the pattern of innervation of the neocorti-al slice was similar to that seen in simple cocultures—

FIG. 7. Most fibers from E16 LGN explants fail to penetrate E16 (n vitro. The diagrams (top) illustrate the coculturing procedure, anark-field micrograph of bisbenzimide (nuclear) counterstaining reveaith an E16 LGN explant. (B) Same view as A but showing DiI-labelhite arrows indicate a single fiber running parallel to the main band

ell bodies. (C) Bisbenzimide counterstaining of an early E19 cortical sial surface visible in the upper right corner. (D) Same view as C buttreaming around the edge of this early E19 slice. The white arrow inortical plate, from the other side of the slice, and grew all the way thr

ither growth through the entire slice at P1 (Fig. 11A) a

r termination in presumptive layer 4 at P6 (Figs.1B–11D). The effectiveness of the choice techniqueas shown by the very different behavior of axons from

he side confronting a slice of cerebellum. At both P1nd P6, only thin fasciculated strands of thalamic

nd B) and early E19 (C and D) occipital cortical explants after 4 daysoxes show the approximate positions of the micrographs. (A) This

the pial edge of an E16 cortical slice that had been cultured for 4 daysthalamic axons forming a dense plexus around the edge of the slice.axons and somewhat closer to the cortex, but still not among corticalwhich had been cultured with an E16 LGN block for 4 days, with the

wing DiI-labeled thalamic axons. The vast majority of them are seentes the growth cone of one of the few axons that actually entered theh it. Scale bars: B (also for A), 100 µm; D (also for C), 200 µm.

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urface of cerebellar slices (Figs. 11A–11C) but did notnter them. In all 49 cultures we have seen only tinyumbers of thalamic axons actually penetrating P1–P8erebellar tissue. Axons did grow into the interior ofippocampal slices (all P3 or older) but, even in olderlices, they ran virtually to the other edge of the slicend did not terminate in an organized manner (Fig.1D).Lack of regional specificity for thalamic innervation

f neocortical slices. Given the demonstration that thehoice paradigm can reveal clear differences in theehavior of thalamic axons when confronted with vari-us alternative targets, we used this approach to testhether any of the signals or properties revealed by the

imple cocultures of LGN and occipital cortex might bepecific for topographically corresponding regions ofortex and thalamus. We cultured single explants ofifferent parts of the embryonic dorsal thalamus (ei-her the LGN or the most anterior part) flanked by

FIG. 8. DiI-labeled axons from an E16 LGN explant have invaderown radially through the ventricular, subventricular, and intermedmages are derived from high-power confocal microscopy of the intewenty 2-µm-thick optical sections were collected and stacked to creatith red–green glasses) shows thalamic axons distributed within th

ollapsed orthogonal view, generated from the same 3-D dataset, shoortical slice and not just on its surface. (C) Same view as B showing tcale bar, 10 µm.

lices of two different regions of cortex (frontal, corre- i

ponding to anterior thalamus, and occipital, appropri-te for the LGN). For each triple culture, the pair ofortical slices was taken from a single donor animal, atges ranging from E19 to P8 (Table 1).In none of these choice cocultures (N 5 66) was there

ny distinguishable difference in the pattern of growthrom the thalamic explant into the two flanking slices,he one positionally corresponding, the other entirelynappropriate. Figure 12 shows three representativexamples. In Figs. 12A and 12B, fibers from E16 LGNnd frontal thalamus, respectively, are seen growing inery similar patterns into P1 occipital cortex (on theeft) and frontal cortex (on the right). In both cases,ome axons grow around the margin of the slice butost, after just 2.5 days in vitro, are growing up

hrough the cortical plate with growth cones at theirips. Some have reached the pial surface and turned toun tangentially, typical of behavior with this age ofortex. Figure 12C shows very similar patterns of

cortical slice (taken at P3). After only 1.5 days in culture they havezones and have reached the upper part of layer 5 of the cortex. Theseof the cortical slice in the region of the tips of the growing fibers.

3-D dataset. (A) This red–green anaglyphic stereogram (to be viewedepth of the slice, with expanded growth cones at their tips. (B) Thisthat the fibers (bright profiles) are running within the depth of the

nuclei of cortical cells revealed with acridine orange counterstaining.

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nd frontal cortex, with obvious branching and termina-ion in layer 4.

oes the Temporal Cascade of Cortical SignalsContinue to Mature in Vitro?

In vivo, thalamic axons invade the occipital corticallate massively from about E19 and they start torborize and terminate in layer 4 a couple of days afterirth. Superficially this correlates well with behavior inoculture. Cortical slices taken after E19 are permis-ive to ingrowth, and after about P2 they promote theermination of axons in layer 4. However, these proper-ies are revealed after a period in culture, when the

FIG. 9. Low-power confocal micrographs showing typical behav-or of axons from an E16 LGN block after 4 days in culture with P0,ate P2, and P4 occipital cortical slices. Each image, from the middlef the width of the slice, shows the pial surface, near the top, and theentricular surface, near the bottom. (Left) With P0 occipital cortex,any axons have run around the edge of the slice, as for virtually all

xons in embryonic slices taken younger than E19: part of the band ofbers has formed above the pial surface. However, a substantial

raction of axons have run up radially or obliquely, through theortical plate to the marginal zone and the pial surface, where theyave turned laterally to run beneath the pial surface or to join theircling band. (Middle) Under the same culturing conditions, noeripheral band of fibers is seen around the late P2 slice and a higherensity of fibers is seen within the cortical slice. Some reach thearginal zone but many have arborized and terminated lower in the

ortical plate. (Right) In P4 cortex, almost all thalamic fibers haveranched and terminated about 300–400 µm below the pial surface,n the presumptive layer 4. Scale bar, 200 µm.

xplants are, of course chronologically that much older. a

his could be taken to indicate that, if there is anntrinsic ‘‘clock’’ in the developing cortex that deter-

ines the timing of expression of molecular signals, itight be ‘‘frozen’’ at the time of removal of the explant.owever, thalamic axons grew so quickly in vitro

about 1 mm per day) that they usually adopted theirharacteristic pattern of innervation within a couple ofays of the start of culturing. Therefore, the crucialnteractions, depending on the growth-permissive prop-rties and the stop signal, were probably happeningnly a day or so after the removal of the corticalxplant, not giving enough time to see whether suchroperties might switch on during the culture of aortical slice, at times appropriate to its chronologicalge.We decided to examine this question by preculturing

ccipital cortex alone for 1 or 2 weeks, before adding andjacent explant of E16 LGN for a further 4 days ofoculture. In these experiments, thalamic axons gener-lly behaved as if the slice of cortex were more maturehan the age at which it was originally removed,lthough not quite equivalent to its actual chronologi-al age (i.e., age at removal 1 preculture period).Figure 13 shows an example of a slice that had been

aken early on E19 and kept alone in culture for 7 dayschronological age approximately P4), before the addi-ion of an E16 LGN explant for a further 4 days. Theuorescence micrographs (B and D) clearly show axonstreaming densely into the slice, with none circlinground its boundary. Contrast this with the densexonal growth around the edge of the early E19 slicewithout preculture) in Fig. 7D, and even the P0 slice inig. 9. However, unlike in a true P4 slice (see P4 in Fig.), many fibers in this precultured slice extended to thearginal zone and only a few arborized about 150 µm

elow the surface.Figures 14 and 15 illustrate three experiments inhich a cortical slice was taken at P0 but kept alone in

ulture for 14 days before the addition of an E16halamic explant. The camera lucida drawing in Fig. 14hows the quite mature pattern of branching and arrestf growth in presumptive layer 4 for most axonsntering the central portion of the slice (chronologicalge about P14). Very few fibers extended up to thearginal zone. This is reminiscent of the behavior of

halamic axons in late postnatal cortical slices (com-are Figs. 11B–11D and 12C). However, axons enteringhe sides of the slice (where the lamination has col-apsed and the marginal zone has wrapped aroundirtually to contact the thalamic block; see Fig. 2)amified tangentially over long distances and were not‘captured’’ by layer 4. Note that in this slice, after soong in culture, the radial dimension of the supragranu-ar layers had increased substantially and layer 4 laybout 750 µm below the pial surface.The micrographs in Fig. 15 show examples of the

rborization of individual axons in such slices, taken at

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FIG. 10. Examples of thalamic axon growth in cocultures in whiortical slice and cultured for 5 days. (A) For this coculture the cortihalamic explant is seen, closely adherent to the pial surface, at the tohe entire depth of the cortex to reach the ventricular zone. The intehite arrow a group of axons close to the ventricular surface of the sial surface (PS), the dense cortical plate (dcp), the more mature layernterrupted line again marks the lower margin of the cortical plate. (Cenetrated the PS and have grown down through the upper layers of telow the surface (white arrow). The outline of the cortical slice is indn C, showing branching thalamic fibers without growth cones, in thince the culture is more than 150 µm thick. Scale bars: A and B, 100 µ

xplant of E16 LGN next to the cleaned pial surface. In g

hese cocultures, many thalamic axons behaved in theame way as when entering the pial surface of cortexaken after the appearance of the stop signal (as in Fig.0C). In the center of the slice, the majority of axons

the LGN explant was placed against the cleaned pial surface of theslice was taken at P0. Under epifluorescent illumination, the brighthalamic fibers have penetrated the slice and have run down throughpted line indicates the lower boundary of the cortical plate and the

. (B) Bisbenzimide counterstaining of the same field as A reveals theand 6, the white matter (WM), and the ventricular surface (VS). Thea cortical slice taken at P6, DiI-labeled thalamic axons have densely

cortex. However, they have virtually all arborized about 300–400 µmed with the interrupted line. (D) High-power view of the outlined boxgion of termination. Many of the DiI-labeled fibers are out of focus,C, 500 µm; D, 100 µm.

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erminated less than 1 mm below the surface. But oncegain, these experiments provided evidence that theadially cut edges of the slice, where curling over hadaused the lamination to collapse and the marginalone to curve around, were abnormal and much lessature in their properties. As shown in Fig. 16, axons

ntering these edges ran quite long distances parallelo the pial surface, along the marginal zone, and someven still had growth cones at their tips (Fig. 16C).

DISCUSSION

Yamamoto et al. (80) first reported that explants ofGN and occipital cortex form layer-specific interconnec-ions after 2 weeks in vitro, geniculate axons terminat-

FIG. 12. Camera lucida drawings show thalamic axons in tripleocultures, in which a thalamic explant confronted a slice from theorresponding region of the neocortex on one side and from anntirely inappropriate area on the other. (A and B) E16 thalamicxplants, LGN in A and anterior dorsal thalamus (ANT) in B wereocultured for 2.5 days with slices of occipital (OCC) and frontalFRO) cortex taken at P1. In each case, fibers have ramified withoughly equal density through both slices, and some of them haveeached the pial surface, without branching, after this short time initro. (C) A single block of E16 LGN is indiscriminate in its innerva-ion of P6 occipital and frontal cortex after 5 days in culture: axonensity is indistinguishable and so is the pattern of termination inresumptive layer 4. Scale bar, 1 mm.

FIG. 11. Results of choice experiments in which explants of E16GN were each cultured for 4 days next to two slices from differenttructures, both taken from the same donor. Camera lucida drawingshow DiI-labeled thalamic axons. (A) Axons grow profusely out of theide of an LGN explant in contact with a slice of P1 occipital cortexOCC; on the left), extending all the way through the slice andamifying tangentially below and over the pial surface. But axonsrom the same thalamic block do not penetrate the slice of P1erebellum (CER; on the right): small numbers grow in fasciclesround the edge of the slice or over its exposed surface. (B and C)gain, faced with P6 slices, thalamic axons grow densely into thelice of occipital cortex (on the left), arborizing in layer 4, but formnly a few sparse fascicles running around and over the surface of theerebellar explant. (D) A coronal slice of hippocampus (HIP; right),aken at P6, was quite densely innervated by thalamic fibers, buthey terminated in a more haphazard pattern than in the occipitalortical slice (left) and some of them continued right up to the surface.cale bar, 1 mm.

ng in layer 4 of relatively mature cortical slices. Our

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tudy has extended this approach to virtually the entirege range of growth of thalamic axons and theirnvasion of the cortex (E16–P11).

Immature slices of neocortex kept in culture, evenithout an accompanying thalamic explant, undergo

FIG. 13. In order to test for the maturation of ingrowth-permissiaken early on E19 and precultured on its own for 7 days, making its chen placed against the ventricular surface and they were left in coculocultures of E16 LGN with cortical slices immediately on removal at eays of coculture, showing bisbenzimide counterstaining (A) andrecultured cortical slice in large numbers, unlike in simple cocultudges (Fig. 7D). (C and D) High-power photomicrographs of the upperiI labeling (D). Many axons are seen running radially up to the marranched and terminated within the plate, but not such a high proportand B, 1 mm; C and D, 100 µm.

hanges that strongly suggest that some cell prolifera- fi

ion, migration, growth, and differentiation continue initro. In particular, the cortical plate expands radially,nd newly arrived immature cells of the dense corticallate grow and separate from each other to createpper cortical layers of fairly normal appearance, con-

properties of the cortex in vitro, a slice of occipital cortex (CTX) wasnological age approximately P4. An explant of E16 LGN (THAL) wasfor a further 4 days. Compare the fiber ingrowth with that in normal

y E19 (Fig. 7D) and P4 (Fig. 9). (A and B) Low-power views after the 4labeling of thalamic fibers (B). The majority of axons enter the

with early E19 cortex, in which the axons circle mainly around thert of the cortical plate showing bisbenzimide counterstaining (C) andal zone, some of them still bearing growth cones (arrows). Some haveas in simple cocultures of E16 LGN with P4 slices (Fig. 9). Scale bars:

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his is not surprising in view of the fact that cells stillivide and migrate in cultured cortical slices (e.g., 1, 57,8). However, the laminar pattern that develops aboveayer 5 is not exactly the same as that in a normalnimal of the same chronological age.Although development appears reasonably normal in

FIG. 14. An experiment demonstrating maturation of the stopignal in vitro. A slice of occipital cortex, taken at P0, was preculturedlone for 2 weeks (making it chronologically equivalent to P14). Alock of E16 LGN was then placed next to the ventricular surface ofhe cortex and they were left for 4 days of coculture. The cameraucida drawing (bottom) shows the invasion of the cortical slice byiI-labeled axons. The pattern is much more mature than in true P0

lices (Fig. 9) but not identical to that in late postnatal slices (e.g.,ig. 12C). Unlike in a true P0 slice, large numbers of axons havelearly branched and terminated in presumptive layer 4. However, areater fraction extends to the marginal zone than in coculture withate postnatal slices. The behavior of axons growing into the dis-orted, collapsed margins of the slice is particular aberrant, many ofhem running long distances tangentially through the marginal zone.igure 15A shows a low-power micrograph of this coculture. Scalear, 1 mm.

he center of cultured slices, abnormal changes occur at R

he radially cut edges of slices taken before about P2,resumably because of folding of the edges (see Fig.C). We presume that this accounts for the fact that, inany coculture experiments with such slices, the pat-

ern of thalamic innervation was rather different (andbnormal) at the edges of the slice compared with thatn the middle (e.g., Fig. 14).

Temporal Cascade of Signals Producedby the Developing Cortex

In vivo in the rat, fibers from the region of the LGNrow toward the cortex between about E14 and E16;hey tend to accumulate below the cortex and thennvade the cortical plate profusely from about E19, andhey arborize and terminate in layer 4 a couple of daysfter birth (see 4, 15, 52). Our main hope was to exploithe coculture technique to reveal properties of theeveloping cerebral cortex that might play a part intimulating, guiding, and halting the growth of tha-amic axons. The results do indeed imply that theortex has three classes of influence on thalamic axons,xpressed in a temporal cascade appropriate in timingo the sequence of events in vivo.

ong-Range Influences of Cortex on Thalamus

Chemotropism as a mechanism of axon guidance wasriginally suggested by Cajal (12, 13). The theory waseformulated by Sperry (73) as his hypothesis of spe-ific chemoaffinity. Recently, interest in chemotropismas reemerged with convincing examples of this mecha-ism operating in various parts of the peripheral andentral nervous system (see 40, 45, 59, 63, 75).Now, Bolz et al. (6) saw no obvious difference in axon

utgrowth from thalamic explants whether they wereocultured with cortex or not. However, they employederum-enriched medium, which stimulates florid axonutgrowth even from isolated thalamic explants, andhis presumably masked the effect of any added corticallice.Under the conditions that we used, little or no

halamic outgrowth occurred without a nearby corticalxplant (Fig. 4), providing firm evidence that the cortexxerts a remote influence on axon extension. Extensivetudies by Rennie et al. (62) and Lotto and Price (38),sing culturing methods very similar to ours, alsoemonstrated a trophic effect of cortex on thalamicxon growth in the mouse. In our experiments, out-rowth was clearly more profuse and extensive fromhe side close to a cortical slice than on the opposite sideFig. 5), although there was no evidence of a specificropic effect on the trajectory of axon growth (40).resumably this asymmetry was caused by the estab-

ishment of a concentration gradient of some growth-romoting factor within the static culture medium.
ennie et al. (62) did not see such anisotropy of neurite

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A) Low-power view of bisbenzimide staining in the coculture reconstructed in Fig. 14 (precultured P0 cortex to the right, LGN explant on theeft). (B) This view from the middle of the slice in A reveals some DiI-labeled axons extending up to the marginal zone, but very few of them stillave growth cones. Many fibers have arborized about 200–300 µm below the pial surface (arrows). The axons running roughly tangentiallycross the upper 200 µm of the slice entered the slice via its collapsed lateral margins (see Fig. 14). (C) Higher-power view from the region ofutative layer 4 in the same slice. The arrows point to the same elaborate arbors as in B. (D and E) Low-power and high-power views,espectively, of another slice taken at P0, precultured for 14 days, and then cocultured for 4 days with E16 LGN. Apart from the tangentiallyunning axons that entered the edges of the slice, most fibers have arborized in layer 4. An axon that branches extensively is indicated by anrrow in D and E. (F and G) Bisbenzimide counterstaining and DiI labeling in matched micrographs from an identical preculturingxperiment. Branching axons (arrow) are seen well below the pial surface and no growth cones are visible. Scale bars: A, 1 mm; B (also applieso D), 100 µm; C, 100 µm; G (also applies to E and F), 100 µm.

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xtension in their mouse cocultures but they employedarger separations between cortex and thalamus. Pre-umably the asymmetrical outgrowth that we saw wasaused by the high concentration of the diffusible factorery close to the cortical explant.It is conceivable that this cortical factor, diffusing

FIG. 16. A P0 cortical slice (top left) was precultured for 1 week (uts pial surface and they were cocultured for 4 days. (A) Low-powerright). Note the convexity of cortical slice, caused by curling of thextended right through into the ventricular zone, as for most axons inhe center of the slice, many axons arborized within the cortical platefter P3 (Figs. 10C and 10D). (B) Dark-field micrograph of the outlinedrowth is more aberrant. Some axons (e.g., left arrow) have brancheddge of the slice with growth cones at their tips (right arrow). (C andxon, respectively. Scale bar: A, 1 mm; B, 100 µm; C and D, 50 µm.

hrough the primordial internal capsule into the dien- s

ephalon, plays a part in promoting the outgrowth ofhalamic axons and perhaps even encouraging growthownward through the ventral diencephalon and to-ard the internal capsule. However, it must be noted

hat Rennie et al. (62) showed that explants of cerebel-um are almost as effective as neonatal cortex in

a chronological age of P7): an E16 LGN block was then placed next tow of bisbenzimide counterstaining shows the LGN (left) and cortext edges. Axons entered the slice through its pial surface and a fewerted coculture with a true P0 slice (Figs. 10A and 10B). However, inwould have been the case for immediate coculture with a slice takenx in A, at the distorted margin of the slice. Here the pattern of axonal

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any parts of the brain produce diffusible factors thatromote axon extension rather indiscriminately.Cunningham et al. (20) showed that the degeneration

f the LGN that normally follows removal of theccipital lobe in newborn rats could be partially pre-ented by replacing the ablated cortical tissue with ael impregnated with medium conditioned with cul-ured cortex. The soluble fraction responsible was heatabile and they believed it to be proteinaceous. Itemains to be seen whether this death-preventingactor is the same as the agent that stimulates axonrowth.

rowth-Permissive Properties of Cortical Slices OlderThan E19

When confronted with cortical slices younger thanbout E19, thalamic axons, stimulated to grow out byhe diffusible factor, grow extensively around the edgesf the cortical slice rather than penetrating it. Thenuddenly, at about E19, the cortex changes its proper-ies and starts to permit axon invasion.

Bolz and his colleagues (5, 6, 30) have argued thatostnatal but not embryonic rat cortex expresses aubstrate-bound factor that encourages thalamic axonngrowth. They deposited membrane suspensions ex-racted from cortical explants onto the surface of cul-ure chambers on which they placed thalamic explants.f the cortex came from E16 embryos, there was little oro axon extension, but if it came from P7 cortex thereas florid growth over the membrane-treated surface.Interestingly, Godfraind et al. (29) and Chung et al.

17) have described the expression of a growth-upporting factor, L1, in the developing rodent fore-rain, but only in the subplate layer and on the axons ofts neurons. Chun and Shatz (16) also described tran-ient fibronectin expression in the subplate of the catortex. The nature of the membrane-bound growth-ermissive factor expressed in the rat cortical platefter about E19 obviously deserves investigation.

he Stop Signal

Another sudden change in the properties of theortex occurs at about P3. In slices taken before thatge, thalamic axons invade but proceed right up intohe marginal zone, even out through the pial surface,nd still have growth cones after several days inulture. But with slices taken after P2, the majorityrborize remarkably normally in what appears to behe future layer 4 (4, 50, 80). Recently Yamamoto et al.79) used time-lapse analysis in living cultures to showhat the growth of thalamic axons slows, they tend toranch, and their growth cones collapse within theeveloping layer 4.The expression of the stop signal, as we have called it

50), appears to be quite critically timed. In vivo, c

halamic axons are growing up through the lowerayers of the rat occipital cortex on the day of birth andhey terminate just a couple of days later, as recentlyigrated cells differentiate from the bottom of the

ense cortical plate to form layer 4. This is just thetage at which cortical slices appear to turn on theirtop signal in vitro. The arrival of thalamic axons atayer 4 in vivo seems quite precisely synchronized withhe onset of the stop signal. Indeed, the small propor-ion of thalamic fibers that extend up into the marginalone in vivo may have arrived slightly too early, beforehe expression of the stop signal. Curiously, the sharpnset of the stop signal contrasts with the wide ageange (E14.5 to E18) over which cells destined for layerare born in the neuroepithelium (41, 47).The slowing, branching, and terminating of thalamic

xons in layer 4 implies that they are influenced by aariety of molecules produced by the cells of that layer.owever, other signals may play a part in confining

halamic axons to layer 4 and preventing their whole-ale invasion of the upper layers. For instance, differen-ial expression of repulsive molecules of the sema-horin family (72) in the maturing supragranularayers might help to limit the growth of thalamic fibersbove layer 4. However, fibers from a thalamic explantlaced against the pial surface readily grow into aortical slice older than E19, and, if it is older than P2,hey terminate in layer 4, just as if they had grown inrom below (Fig. 10). Moreover, in vitro experimentssing isolated membrane preparations have shownhat both the optic tectum (see 74) and the granule cellsf the cerebellum (3) express signals that actuallynitiate arborization and growth cone collapse. It seemsery likely that a similar intrinsic message is switchedn in layer 4 of the cortex.

aturation of Cortical Signals in Vitro

The simplest hypothesis to explain the temporalequence of cortical signals is that they are determinedy an intrinsic timing mechanism in cortical neurons.enes controlling the production of the diffusible tro-hic factor(s) might be turned on as soon as neurons areorn or migrate or even in progenitor cells in theeuroepithelium. Then, the expression of the mem-rane-bound permissive factors (perhaps adhesion mol-cules or other surface markers attractive to thalamicrowth cones) might suddenly switch on in the middlef corticogenesis (E19 for the occipital cortex), when theower part of the infragranular layers has maturedonsiderably. And cells of layer 4 may delay expressionf genes controlling the stop signal until they haveigrated into place and started to differentiate from

he lower part of the dense cortical plate.Alternatively, the production of at least some cortical

actors might be initiated by extrinsic signals, perhaps
hanges in substances circulating in the blood or chemi-

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al signals or neural activity in nearby thalamic axons.halamic fibers accumulating below the cortical plateuring the waiting period (from E16 to E19 for theccipital cortex) are certainly in an ideal position toransmit signals to the more mature lower layers of theortex, which might somehow trigger the onset ofermissiveness. Equally, as those axons approach themerging layer 4 (about P2 for the occipital cortex) theyight switch on the stop signal.The fact that a thalamic explant left in contact with a

lice of cortex taken as late as early E19 still does notnter the cortex extensively after 4 days in vitro (Fig.D) argues against a simple contact-induction of growthermissiveness. However, it also militates against theossibility that the expression of permissiveness isntirely intrinsically determined in cortical tissue,ince such a slice has a chronological age of about P1 athe end of coculture, past the time when permissive-ess would normally appear.Thalamic axons also penetrated via the pial surface

f P0 cortical slices (Fig. 10A), but not at E18 or E19.his could be taken as evidence for induction of growthermissiveness by the presence of thalamic axons. Butt could also mean that this property simply switches onoughly synchronously (within a day or two) across thehole pial–ventricular axis of the cortex, by some

ntrinsic mechanism of timing, despite the fact thatells in the upper layers are generally born several daysarlier than those in the lower layers.We tried to clarify this issue by experiments in which

ortical slices were taken at a certain age and thenrecultured alone for 7 or 14 days before the introduc-ion of a thalamic explant (usually next to the ventricu-ar surface). The obvious question was whether theemporal cascade of cortical signals would emerge atoughly the correct chronological age, despite isolationrom thalamic axons and any circulating factors. Thesexperiments support the hypothesis that expression ofrowth permissiveness is at least partly intrinsicallyetermined, although the underlying clock may runore slowly in culture than in vivo.The results of the preculturing experiments are

llustrated schematically in Fig. 17. When placed nexto a cortical slice that had been taken at E16 or early19 but kept in culture for 7 days, a thalamic explant

nnervated the slice readily, as if the slice were consid-rably older. At E16, thalamic axons are only justpproaching the occipital segment of the cerebral wallnd hence explants taken at that age had not beenxposed to thalamic fibers before their first confronta-ion after preculturing. Early E19 slices placed inreculture for 7 days had a chronological age of about4 at the start of the subsequent coculture. The facthat some axons actually branched and lost theirrowth cones within the cortical plate of such slices
uggests that the stop signal had even started to a
ppear, although certainly not to the extent seen inormal P4 slices (e.g., Fig. 9).Preculturing of neonatal cortex, from P0 to a chrono-

ogical age of P7 or P14, provided further evidence thatayer 4 can, to a large extent, mature and develop itstop signal in culture (Fig. 14). However, these proper-ies do not appear completely normal: many thalamicxons ‘‘escape’’ the constraints of layer 4 and run up tohe marginal zone in such a preparation and theaminar pattern of termination is much less precisehan in coculture with a normal late postnatal slice.his result is, in any case, not so definitive as themergence of permissive properties in preculture, sincet could be interpreted simply to mean that layer 4

atures sufficiently in vitro to be able to respond toignals from thalamic fibers that then switch on thetop signal. Certainly blockade of impulses in theeveloping cortex, even in vitro, does alter the arboriza-ion of thalamic axons (31, 77). In the absence of anndicator of the stop signal independent of the behaviorf thalamic axons, it is impossible to discriminateetween complete control by an inherent clock anderely the maturation of competence to respond to

nducing signals from arriving fibers.

pecificity of Thalamic Axons for Cortical Targets butthe Lack of Regional Preference

We were interested in the possibility that the tangen-ial distribution of thalamic fibers to the entire hemi-phere might be influenced by regional variation of oner other of the factors produced by the developingortex. The use of ‘‘stripe assays’’ and ‘‘collapse assays,’’n which axons are grown on membrane extracts inulture, has convincingly demonstrated specific signalsxpressed across the optic tectum that affect the distri-ution and arrest of axons from different parts of theetina (see 74). Our choice paradigm, in which onexplant is grown adjacent to two potential targets, mayot be so sensitive in revealing such specific compo-ents. However, it does provide the opportunity toompare the growth of axons from a single source,nder identical conditions, when confronting the differ-ntly organized, more naturalistic environments of tworganotypic cultures.The choice experiments provided clear evidence that

halamic axons have a particular affinity for the cere-ral cortex. When thalamic explants were combinedith slices of cerebellum taken at ages from P1 to P8,nly small numbers of axons ventured around or overhe surface of the cerebellar slice, in sparse, fascicu-ated bundles (Figs. 11A–11C). The mouse cerebellumoes have some remote growth-promoting influence onhe thalamic axons in vitro (62). However, it seems thaterebellar tissue is not ingrowth permissive for tha-amic fibers (at least over the age range we studied),
lthough it will support the growth of mossy fiber

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fferents from the pontine nuclei and their arrest andermination on cerebellar granule cells (3). Interest-ngly, slices of P3–P6 hippocampus (archicortex), whicheceives a modest projection from the anterior thalamicucleus in vivo, do permit the ingrowth of thalamicxons in vitro, although they do not appear to contain aiscretely organized stop signal (Fig. 11D).In 80 cocultures, we combined a fragment of E15–16

halamus, taken from either the posterior or the ante-ior portion of the dorsal thalamus, with a slice from anntirely inappropriate part of the cortex (E19–P8),aken from the opposite end of the cerebral hemisphereo the correct target region. The majority of these werehoice experiments in which the thalamic explantimultaneously confronted a slice from the appropriate

FIG. 17. These diagrams summarize the results of normal coculthe growth permissiveness of cortical slices and the stop signal canependent on extrinsic cues for their expression.

ortical target, allowing direct comparison of the pat- s

ern of innervation of corresponding and noncorrespond-ng cortical areas. Since each pair of cortical slices wasaken from a single donor animal, all other obviousariables were minimized, providing an opportunity tobserve regional differences in the potency, type, oriming of expression of any of the presumed corticalignals.Among this large number of cocultures we saw no

vidence that the behavior of thalamic axons varied inny obvious way in response to target slices fromifferent cortical areas. First, in simple cocultures, thextent of initial axon outgrowth was indistinguishablehatever the position of origin of the cortical slice,

onfirming the results of Rennie et al. (62) in the mouse.n young cortical slices, from late E19 to P2, axons

and preculturing followed by coculture, which provide evidence thatture to a considerable extent in vitro and are therefore not entirely

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ional origin, but did not terminate. After about P2,hey arborized some distance below the pial surface,hether in a slice from the corresponding region of

ortex or from a part of the hemisphere that suchhalamic axons would never approach in vivo (see Fig.2). This implies that none of the signals produced byhe cortex is highly specific for the correspondinghalamic nucleus. This result is entirely compatibleith the fact that explants of embryonic cortex trans-lanted elsewhere in the hemisphere in vivo accept thengrowth of thalamic fibers and even take on theytoarchitectonic characteristics of the host region (66).It could be that the cortex expresses regionally

pecific properties that are too subtle to be revealed byeterotopic transplantation or our simple choice cul-ure technique. Indeed, Bolz and Gotz (5) and Hubenert al. (35) provided evidence that, at very late stages,he substrate-bound permissive property of the cortexoes varies slightly across the hemisphere. They foundhat outgrowth from an LGN explant is somewhat moreigorous on a surface treated with a membrane fractionrom P7 occipital cortex than on one from P7 frontalortex. At first sight, this result seems to contradict ourhoice results and suggests that the distribution ofhalamic axons to their cortical target areas might benfluenced by gradients of target properties, as clearlyeems to be the case for the optic tectum (see 74).However, there seems to be no possibility that either

he diffusible trophic influence or the substrate-boundrowth permissiveness in the cortical plate could guidehalamic axons, which arrive under their correspond-ng areas of the whole hemisphere between E14 and16 (4, 15, 21, 52). Like Rennie et al. (62), we showed

hat occipital and frontal cortex have indistinguishableemote growth-promoting effects on LGN explants. Inny case, the mixing that would occur during diffusionown through the telencephalon, through the tinyperture of the putative internal capsule and up intohe diencephalon, would surely mask any selectivity.nd the substrate-bound, growth-permissive property

s not expressed until after E18 in rat occipital cortex,s Bolz and Gotz (5) have confirmed. Therefore, even ifhere is regional variation in permissiveness at latertages, it cannot play a part in the basic process ofrdered guidance of thalamic axons.The slight difference in the influence of membrane

ractions from P7 occipital and frontal cortex on out-rowth from LGN seen by Bolz and Gotz (5) couldonceivably be due to fact that the cortex from whichhese membrane extracts were taken had already beennnervated in vivo and therefore the extracts included

embranes of thalamic axons. There might be a ten-ency for axons from any one thalamic nucleus to growreferentially on axons from the same nucleus rather
han others. Indeed such selective adhesion might y
ontribute to the maintenance of fiber order in thedvancing front of thalamocortical afferents in vivo.We believe that the weight of present evidence ar-

ues against chemoaffinity as a major factor in deter-ining the initial topographic distribution of the thala-ocortical projection, en route to the cortex. It may,

owever, play some part in the important process ofocal reorganization of thalamic fibers within the cor-ex, which is known to be influenced by activity in thosexons (see 37) and presumably involves interactionith cortical cells.This leaves, then, the puzzle of how axons actually

re guided over the considerable and quite tortuousathway from thalamus to cortex, establishing topo-raphic order even as they start their journey, beforeny cells have migrated into the cortical plate! We haveresented evidence and argued elsewhere (51–53) thatuidance may depend in part on simple thalamicber–fiber ordering, facilitated by an ordered chronologi-al sequence of outgrowth of axons. However, it seemsikely that interactions with a number of pioneeringescending axons systems and ‘‘guidepost’’ cell groupslso play a part. In particular, there is now a wide rangef evidence (see 52, 53) that, as they emerge into thentermediate zone (at about E14–15 in the rat), theave front of thalamic fibers meets and interminglesith a ‘‘scaffold’’ of descending axons that have grownown, in topographic order, from cells of the corticalreplate (42, 43). The thalamic axons then grow inssociation with fibers from the corresponding region ofhe subplate, enter it, and apparently form synapses onubplate cells (32, 33). Indeed, the association withubplate axons in the intermediate zone may encour-ge thalamic axons to accumulate and ‘‘wait’’ in theubplate layer.It is, of course, conceivable that a chemospecific

radient of growth-permissive properties is expressedt the tips of preplate axons earlier than permissive-ess appears in the cortical plate itself and that this is

mportant in establishing the correct relationship be-ween thalamic fibers and the preplate scaffold.

It is slightly puzzling that we saw no hint of behaviorndicative of a waiting period in our coculture experi-

ents. For instance, thalamic axons did not enter16–19 cortical slices and terminate specifically over

he subplate, but this might have been due to theirnability to grow through the ventricular zone to reacht. They also showed no distinctive reaction to theubplate layer as they grew over it in slices older than19, but this is less surprising since thalamic axons arebandoning the subplate and entering the cortical platen vivo at that stage. It would be interesting to look forints of waiting behavior and formation of synapses inhe subplate in cocultures in which thalamic explantsre placed adjacent to the cut intermediate zone of
oung (E15) cortical slices, the age at which they would

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e growing through the intermediate zone within thecaffold of subplate axons.The studies reported here have further validated the

echnique of serum-free organotypic culture and havehown that patterns of axon growth in coculture caneveal the expression of probable molecular cues. Iteems very likely that the cascade of cortical signals—rowth promoting, growth permissive, and the stopignal—plays an important role in the choreography ofhalamocortical development.

ACKNOWLEDGMENTS

This research has been supported by grants from the Medicalesearch Council, the Wellcome Trust, the Human Frontier Sciencerogram, the Oxford Centre for Cognitive Neuroscience, the Nuffieldoundation, and the Hildegard Doerenkamp–Gerhard Zbinden Foun-ation. Z.M. held a Junior Research Fellowship at Merton College,xford, and an MRC Training Fellowship. We are very grateful toichard Adams, for help with confocal microscopy and valuableiscussion, and to Pat Cordery, William Hinkes, Laurence Waters,orraine Chappell, Eric Bernardi, and Mark Berney for technicalssistance.

REFERENCES

1. Adams, R. 1998. Metaphase spindles rotate in the neuroepithe-lium of rat cerebral cortex. J. Neurosci. 16: 7610–7618.

2. Agmon, A., L. T. Yang, D. K. O’Dowd, and E. G. Jones. 1993.Organized growth of thalamocortical axons from the deep tier ofterminations into layer IV of mouse barrel cortex. J. Neurosci.13: 5365–5382.

3. Baird, D. H., M. E. Hatten, and C. A. Mason. 1992. Cerebellartarget neurons provide a stop signal for afferent neurite exten-sion in vitro. J. Neurosci. 12: 619–634.

4. Blakemore, C., and Z. Molnar. 1990. Factors involved in theestablishment of specific interconnections between thalamusand cerebral cortex. Cold Spring Harbor Symp. Quant. Biol. 55:491–504.

5. Bolz, J., and M. Gotz. 1992. Mechanisms to establish specificthalamocortical connections in the developing brain. Pages179–192 in S. C. Sharma and A. M. Goffinet, Eds., Developmentof the Central Nervous System in Vertebrates. Plenum, NewYork/London.

6. Bolz, J., N. Novak, and V. Staiger. 1992. Formation of specificafferent connections in organotypic slice cultures from rat visualcortex cocultured with lateral geniculate nucleus. J. Neurosci.12: 3054–3070.

7. Bolz, J., M. Gotz, M. Hubener, and N. Novak. 1993. Reconstruct-ing cortical connections in a dish. Trends Neurosci. 16: 310–316.

8. Bolz, J., N. Novak, M. Gotz, and T. Bonhoeffer. 1990. Formationof target-specific neuronal projections in organotypic slice cul-tures from rat visual cortex. Nature 346: 359–362.

9. Bottenstein, J. E., and G. H. Sato. 1979. Growth of a ratneuroblastoma cell line in serum-free supplemented medium.Proc. Natl. Acad. Sci. USA 76: 514–517.

0. Brunner, G., K. Lang, R. A. Wolfe, D. B. McClure, and G. H.Sato. 1982. Selective cell culture of brain cells by serum-free,hormone-supplemented media: A comparative morphologicalstudy. Dev. Brain Res. 2: 563–575.

1. Caeser, M., T. Bonhoeffer, and J. Bolz. 1989. Cellular organiza-tion and development of slice cultures from rat visual cortex.
Exp. Brain. Res. 77: 234–244.
2. Cajal, S. R. 1893. La retine des vertebres. Cellule 9: 119–258.3. Cajal, S. R. 1909–1911. Pages 1952–1955 in Histologie du

Systeme Nerveux de l’Homme et des Vertebres. Instituto Ramon yCajal del C. S. I. C., Madrid. [Translated by L. Azoulay]

4. Catalano, S., R. T. Robertson, and H. P. Killackey. 1991. Earlyingrowth of thalamocortical afferents to the neocortex of theprenatal rat. Proc. Nat. Acad. Sci. USA 88: 2999–3003.

5. Catalano, S. M., R. T. Robertson, and H. P. Killackey. 1996.Individual axon morphology and thalamocortical topography indeveloping rat somatosensory cortex. J. Comp. Neurol. 367:36–53.

6. Chun, J. J. M., and C. J. Shatz. 1988. A fibronectin-like moleculeis present within the developing cat cerebral cortex and iscorrelated with subplate neurons. J. Cell Biol. 106: 857–872.

7. Chung, W. W., C. F. Lagenaur, Y. M. Yan, and J. S. Lund. 1991.Developmental expression of neuronal cell adhesion moleculesin the mouse neocortex and olfactory bulb. J. Comp. Neurol.314: 290–305.

8. Coggeshall, R. E. 1964. A study of diencephalic development inthe albino rat. J. Comp. Neurol. 122: 241–269.

9. Colello, R. J., and R. W. Guillery. 1990. The early development ofretinal ganglion cells with uncrossed axons in the mouse:Retinal position and axonal course. Development 108: 514–523.

0. Cunningham, T. J., F. Haum, and P. D. Chantler. 1987. Diffus-ible proteins prolong survival of dorsal lateral geniculate neu-rons following occipital cortex lesions in newborn rats. Dev.Brain Res. 37: 133–141.

1. De Carlos, J. A., and D. D. M. O’Leary. 1992. Growth andtargeting of subplate axons and establishment of major corticalpathways. J. Neurosci. 12: 1194–1211.

2. De Jong, B. M., J. M. Ruiter, and H. J. Romijn. 1988. Cytoarchi-tecture in cultured rat neocortex explants. Int. J. Dev. Neurosci.6: 327–339.

3. Emerling, D. E., and A. D. Lander. 1994. Laminar specificattachment and neurite outgrowth of thalamic neurons oncultured slices of developing cerebral neocortex. Development120: 2811–2822.

4. Emerling, D. E., and A. D. Lander. 1996. Inhibitors and promot-ers of thalamic neuron adhesion and outgrowth in embryonicneocortex: Functional association with chondroitin sulfate. Neu-ron 17: 1089–1100.

5. Erzurumlu, R. S., and S. Jhaveri. 1992. Emergence of connectiv-ity in the embryonic rat parietal cortex. Cereb. Cortex 2:336–352.

6. Gahwiler, B. H. 1988. Organotypic cultures of neural tissue.Trends Neurosci. 11: 484–489.

7. Gahwiler, B. H., M. Capogna, R. A. Debanne, R. A. McKinney,and S. M. Thompson. 1997. Organotypic cultures: A techniquehas come of age. Trends Neurosci. 20: 471–477.

8. Godement, P., J. Vanselow, S. Thanos, and F. Bonhoeffer. 1987. Astudy in developing visual systems with a new method ofstaining neurones and their processes in fixed tissue. Develop-ment 101: 697–713.

9. Godfraind, C., M. Schachner, and A. M. Goffinet. 1988. Immuno-histochemical localisation of cell adhesion molecules L1, J1,N-CAM and their common carbohydrate L2 in the embryoniccortex of normal and reeler mice. Dev. Brain Res. 42: 99–111.

0. Gotz, M., N. Novak, M. Bastmayer, and J. Bolz. 1992. Membrane-bound molecules in rat cerebral cortex regulate thalamic inner-vation. Development 116: 507–519.

1. Herrmann, K., and C. J. Shatz. 1995. Blockade of actionpotential activity alters initial arborization of thalamic axonswithin cortical layer 4. Proc. Natl. Acad. Sci. USA 92: 11244–
11248.

3

3

3

3

3

3

3

3

4

4

4

4

4

4

4

4

4

4

5

5

5

5

5

5

5

5

5

5

6

6

6

6

6

6

6

6

6

6

7

7


2. Herrmann, K., A. Antonini, and C. J. Shatz. 1994. Ultrastruc-tural evidence for synaptic interactions between thalamocorti-cal axons and subplate neurones. Eur. J. Neurosci. 6: 1729–1742.

3. Higashi, S., Z. Molnar, T. Kurotani, H. Inokawa, and K. Toyama1996. Functional thalamocortical connections develop duringembryonic period in the rat: An optical recording study. Soc.Neurosci. Abstr. 22: 1976.

4. Honig, M. G., and R. I. Hume 1989. DiI and DiO: Versatilefluorescent dyes for neuronal labelling and pathway tracing.Trends Neurosci. 12: 333–335.

5. Hubener, M., M. Gotz, S. Klostermann, and J. Bolz. 1995.Guidance of thalamocortical axons by growth-promoting mol-ecules in developing rat cerebral cortex. Eur. J. Neurosci. 7:1963–1972.

6. Kageyama, G. H., and R. T. Robertson. 1993. Development ofgeniculocortical projections to visual cortex in rat: Evidence forearly ingrowth and synaptogenesis. J. Comp. Neurol. 335:123–148.

7. Katz, L. C., and C. J. Shatz. 1996. Synaptic activity and theconstruction of cortical circuits. Science 274: 1133–1138.

8. Lotto, R. B., and D. J. Price. 1995. The stimulation of thalamicneurite outgrowth by cortex-derived growth factors in vitro: Theinfluence of cortical age and activity. Eur. J. Neurosci. 7:318–328.

9. Lotto, R. B., and D. J. Price. 1996. Effects of subcorticalstructures on the growth of cortical neurites in vitro. Neurore-port 7: 1185–1188.

0. Lumsden, A. G. S. 1991. Chemotaxis in the developing nervoussystem. Pages 167–180 in P. Letourneau, S. B. Kater, and E. R.Macagno, Eds., The Nerve Growth Cone. Raven, New York.

1. Lund, R. D., and M. J. Mustari. 1977. Development of thegeniculocortical pathway in rats. J. Comp. Neurol. 173: 289–305.

2. McConnell, S. K., A. Ghosh, and C. J. Shatz. 1989. Subplateneurons pioneer the first axon pathway from the cerebral cortex.Science 245: 978–982.

3. McConnell, S. K., A. Ghosh, and C. J. Shatz. 1994. Subplatepioneers and the formation of descending connections fromcerebral cortex. J. Neurosci. 14: 1892–1907.

4. Metin, C., and P. Godement. 1996. The ganglionic eminence maybe an intermediate target for corticofugal and thalamocorticalaxons. J. Neurosci. 16: 3219–3235.

5. Metin, C, D. Deleglise, T. Serafini, T. E. Kennedy, and M.Tessier-Lavigne. 1997. A role for netrin-1 in the guidance ofcortical efferents. Development 124: 5063–5074.

6. Maximov, A. 1925. Tissue cultures of young mammalian em-bryos. Contrib. Embryol. Carnegie Inst. 16: 47–114.

7. Miller, M. W. 1988. Development of projection and local circuitneurons in neocortex. Pages 133–175 in A. Peters and E. G.Jones, Eds., Cerebral Cortex, Vol. 7, Development and Matura-tion of Cerebral Cortex. Plenum, New York/London.

8. Miller, B., L. Chou, and B. L. Finlay. 1993. The early develop-ment of thalamocortical and corticothalamic projections. J.Comp. Neurol. 335: 16–41.

9. Mitrofanis, J., and R. W. Guillery. 1993. New views of thethalamic reticular nucleus in the adult and developing brain.Trends Neurosci. 16: 240–245.

0. Molnar, Z., and C. Blakemore. 1991. Lack of regional specificityfor connections formed between thalamus and cortex in co-culture. Nature 351: 475–477.

1. Molnar, Z., and C. Blakemore. 1995. How do thalamic axons findtheir way to the cortex? Trends Neurosci. 18: 389–397.

2. Molnar, Z., R. Adams, and C. Blakemore. 1998. Mechanisms

underlying the early establishment of thalamocortical connec-tions in the rat. J. Neurosci. 18: 5723–5745.

3. Molnar, Z., R. Adams, A. M. Goffinet, and C. Blakemore. 1998.The role of the first postmitotic cortical cells in the developmentof thalamocortical innervation in the reeler mouse. J. Neurosci.18: 5746–5765.

4. Molnar, Z., G. W. Knott, C. Blakemore, and N. R. Saunders.1998. Development of thalamocortical projections in the southAmerican grey short-tailed opossum (Monodelphis domestica).J. Comp. Neurol. 398: 491–514.

5. Nagata, I., and N. Nakatsuji. 1990. Granule cell behaviour onlaminin in cerebellar microexplant cultures. Dev. Brain Res. 52:63–73.

6. Naegele, J. R., S. Jhaveri, and G. E. Schneider. 1988. Sharpen-ing of topographical projections and maturation of geniculocorti-cal axon arbors in the hamster. J. Comp. Neurol. 277: 593–607.

7. O’Rourke, N. A., M. E. Dailey, S. J. Smith, and S. K. McConnell.1992. Diverse migratory pathways in the developing cerebralcortex. Science 258: 299–302.

8. O’Rourke, N. A., D. P. Sullivan, C. E. Kazanowski, A. A. Jacobs,and S. K. McConnell. 1995. Tangential migration of neurons inthe developing cerebral cortex. Development 121: 2165–2176.

9. Placzek, M., M. Tessier-Lavigne, T. Yamada, J. Dodd, and T. M.Jessell. 1990. Guidance of developing axons by diffusible che-moattractants. Cold Spring Harbor Symp. Quant. Biol. 55:279–289.

0. Rakic, P. 1976. Prenatal genesis of connections subservingocular dominance in the rhesus monkey. Nature 261: 467–471.

1. Rakic, P. 1977. Prenatal development of the visual system in therhesus monkey. Philos. Trans. R. Soc. London B Biol. Sci. 278:245–260.

2. Rennie, S., R. B. Lotto, and D. J. Price. 1994. Growth-promotinginteractions between the murine neocortex and thalamus inorganotypic co-cultures. Neuroscience 61: 547–564.

3. Richards, L. J., S. E. Koester, R. Tuttle, and D. D. M. O’Leary.1997. Directed growth of early cortical axons is influenced by achemoattractant released from an intermediate target. J. Neu-rosci. 17: 2445–2458.

4. Romijn, H. J. 1988. Development and advantages of serum-free,chemically defined nutrient media for culturing of nerve tissue.Biol. Cell 63: 263–268.

5. Romijn, H. J., B. M. De Jong, and J. M. Ruijter. 1988. Aprocedure for culturing rat neocortex explants in a serum-freenutrient medium. J. Neurosci. Methods 23: 75–83.

6. Schlaggar, B. L. and D. D. M. O’Leary. 1991. Potential of visualcortex to develop an array of functional units unique to somato-sensory cortex. Science 252: 1556–1560.

7. Shatz, C. J. 1992. How are specific connections formed betweenthalamus and cortex? Curr. Opin. Neurobiol. 2: 78–82.

8. Shatz, C. J., A. Ghosh, S. K. McConnell, K. L. Allendoerfer, E.Friauf, and A. Antonini. 1990. Pioneer neurons and targetselection in cerebral cortical development. Cold Spring HarborSymp. Quant. Biol. 55: 469–480.

9. Shatz, C. J., and M. B. Luskin. 1986. Relationship between thegeniculocortical afferents and their cortical target cells duringdevelopment of the cat’s primary visual cortex. J. Neurosci. 6:3655–3668.

0. Sheng, X.-M., L. R. Marotte, and R. F. Mark. 1990. Developmentof connections to and from the visual cortex in the wallaby(Macropus eugenii). J. Comp. Neurol. 300: 196–210.

1. Sheng, X.-M., L. R. Marotte, and R. F. Mark. 1991. Developmentof the laminar distribution of thalamocortical axons and cortico-thalamic cell bodies in the visual cortex of the wallaby. J. Comp.
Neurol. 307: 17–38.

7

7

7

7

7

7

7

7

8

8


2. Skaliora, I, W. Singer, H. Betz, and A. W. Puschel. 1998.Differential patterns of semaphorin expression in the develop-ing rat brain. Eur. J. Neurosci. 10: 1215–1229.

3. Sperry, R. W. 1963. Chemoaffinity in the orderly growth of nervefibre patterns and connections. Proc. Natl. Acad. Sci. USA 50:703–709.

4. Stahl, B., Y. von Boxberg, B. Muller, J. Walter, U. Schwatz, andF. Bonhoeffer. 1990 Directional cues for retinal axons. ColdSpring Harbor Symp. Quant. Biol. 55: 351–357.

5. Tessier-Lavigne, M., and M. Placzek. 1991. Target attraction:Are developing axons guided by chemotropism? Trends Neuro-sci. 14: 303–310.

6. Tuttle, R., B. L. Schlaggar, J. E. Braisted, and D. D. M. O’Leary.1995. Maturation-dependent upregulation of growth-promotingmolecules in developing cortical plate controls thalamic andcortical neurite growth. J. Neurosci. 15: 3039–3052.

7. Wilkemeyer, M. F. and K. J. Angelides. 1996. Addition oftetrodotoxin alters the morphology of thalamocortical axons inorganotypic cocultures. J. Neurosci. Res. 43: 707–718.

8. Wise, S. P., and E. G. Jones. 1976. The organization andpostnatal development of the commissural projection of thesomatic sensory cortex of the rat. J. Comp. Neurol. 168: 313–344

9. Yamamoto, N., S. Higashi, and K. Toyama. 1997. Stop andbranch behaviors of geniculocortical axons: A time-lapse studyin organotypic cocultures. J. Neurosci. 17: 3653–3663.

0. Yamamoto, N., T. Kurotani, and K. Toyama. 1989. Neuralconnections between the lateral geniculate nucleus and visualcortex in vitro. Science 245: 192–194.

1. Yamamoto, N., K. Yamada, T. Kurotani, and K. Toyama. 1992.Laminar specificity of extrinsic cortical connections studied inculture preparations. Neuron 9: 217–228.

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