timecourse of development of the wallaby trigeminal pathway: iii. thalamocortical and...

Timecourse of Developmentof the Wallaby Trigeminal Pathway:

III. Thalamocortical andCorticothalamic Projections

L.R. MAROTTE,1* C.A. LEAMEY,1,2 AND P.M.E. WAITE2

1Developmental Neurobiology Group, RSBS, Australian National University,Canberra ACT 2601, Australia

2School of Anatomy, University of New South Wales, Sydney NSW 2052, Australia

ABSTRACTThe development of trigeminal projections between the thalamus and cortex has been

investigated in the marsupial mammal, the wallaby, by using a carbocyanine dye, horseradishperoxidase conjugated to wheat germ agglutinin (WGA-HRP), Neurobiotin, and biocytin aspathway tracers. The appearance of whisker-related patterns in the cortex in relation to theirappearance in the brainstem and thalamus was examined, as was the presence or absence of awaiting period for thalamocortical afferents and the identity of the first cortical cells to projectto the thalamus. Thalamic afferents first reached the cortex at postnatal day (P) 15 and weredistributed up to the deep edge of the compact cell zone in the superficial cortical platethroughout development, in both dye and WGA-HRP labelled material, with no evidence of awaiting period. The initial corticothalamic projection, detected by retrograde transport ofWGA-HRP from the thalamus, occurred at P60 from layer 5 cells. This was confirmed bylabelling of corticothalamic axons after cortical injections of Neurobiotin and biocytin.Scattered, labelled cells seen before P60 after dye labelling from the thalamus presumablyresulted from transcellular labelling via thalamic afferents. Clustering of afferents in layer 4and cell bodies and their dendrites in layers 5 and 6 first occurred simultaneously at P76.There is a sequential onset of pattern formation, first in brainstem, then in thalamus, andfinally in cortex, with a long delay between afferent arrival and pattern formation at eachlevel. Independent regulation at each level, likely depending on target maturation, issuggested. J. Comp. Neurol. 387:194–214, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: marsupials; subplate; somatosensory; barrels; pioneer

The study of the development of periphery-related pat-terns in the central nervous system has made extensiveuse of the rodent trigeminal system (reviewed by Davies,1988; Killackey et al., 1990; Waite and Tracey, 1995),where the mapping of the peripheral mystacial vibrissaeat the level of the brainstem, thalamus, and cortex can bereadily demonstrated with routine histological and histo-chemical techniques (Van der Loos, 1976; Belford andKillackey, 1979, 1980; Ivy and Killackey, 1982; Durhamand Woolsey, 1984; Bates and Killackey, 1985; Land andSimons, 1985). Such studies have lead to the idea thatthere is a sequential development of the mystacial vibris-sal representation at each level of the pathway, which hasbeen termed ‘‘the domino theory’’ (Killackey, 1985). How-ever, the development of the rat trigeminal pathway israpid and partly prenatal, with whisker-related patternsappearing over a short time period of several days with

some overlap reported in their appearance in thalamusand cortex (Belford and Killackey, 1979; Killackey andBelford, 1979; Erzurumlu and Jhaveri, 1990; Chiaia et al.,1992). One recent report, which used acetylcholinesterasestaining, suggested that cortical patterns appeared appre-ciably earlier than previously thought (Schlaggar andO’Leary, 1994). This rapid mode of development makes itdifficult to distinguish between sequential, synchronousor, indeed, independently regulated onset of pattern forma-tion at the various levels of the pathway in the rodent.

Grant sponsor: Australian Research Council; Grant number: AO9530653.*Correspondence to: L.R. Marotte, Developmental Neurobiology Group,

RSBS, Australian National University, GPO Box 475, Canberra ACT 2601,Australia. E-mail: [email protected]

Received 28 January 1997; Revised 21 May 1997; Accepted 27 May 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 387:194–214 (1997)

r 1997 WILEY-LISS, INC.

The marsupial mammal, the wallaby (Macropus euge-nii), provides an alternative model to examine thesequestions. It has a largely postnatal and protracted devel-opment. The gestation period is 26–28 days and the pouchyoung occupies the pouch for 8 months (Murphy andSmith, 1970). Its vibrissal follicles are structurally similarto those of the rat (Marotte et al., 1992) and vibrissae-related patches can be demonstrated histochemically inthe brainstem (Waite et al., 1994), the thalamus (Leameyet al., 1996a), and the cortex (Waite et al., 1991). In theprevious two papers in this series, we showed that therewas a delay between the first arrival of afferents and thefirst appearance of vibrissae-related patches in both thebrainstem and the thalamus. Although trigeminal gan-glion cells had innervated the periphery and the trigemi-nal brainstem nuclei by birth, patches demonstrated histo-chemically did not begin to appear until postnatal day (P)40 in the brainstem (Waite et al., 1994). For the secondlevel, afferents from the brainstem reached the somatosen-sory thalamus between P10 and P15, but patches were notseen until P50 (Leamey et al., 1996a). Vibrissae-relatedpatches in the somatosensory cortex, visualized with suc-cinic dehydrogenase (SDH) histochemistry, were first seeneven later, at P85 (Waite et al., 1991). This raises thequestion as to when thalamic afferents first reach thesomatosensory cortex and when clusters, corresponding tothe SDH patches, form. Furthermore, what is their timingin relation to similar events in the brainstem and thethalamus?

As well as addressing questions of relevance to thedevelopment of the trigeminal system, this study alsoprovides information about an area of current interest: thedevelopment of thalamocortical and corticothalamic connec-tions. In the visual cortex of the cat (Shatz and Luskin,1986; Ghosh and Shatz, 1992) and monkey (Rakic, 1977),thalamic afferents are said to undergo a waiting period inthe subplate (SP), a largely transient layer in these speciesbeneath layer 6, before their invasion of the developinglayers of the visual cortex. In contrast, results in ourlaboratory revealed that, in wallaby visual cortex, ingrow-ing afferents did not undergo a waiting period but werewidely distributed up to the cell dense region in thesuperficial cortical plate throughout development, invad-ing the developing cortical layers as they differentiatedfrom the cortical plate (Sheng et al., 1991; Marotte andSheng, 1995). There is now increasing evidence thatrodents show a similar pattern to the wallaby, withthalamic afferents growing into each cortical layer as itdifferentiates from the cortical plate (Naegle et al., 1988;Catalano et al., 1991; Agmon et al., 1993; Miller et al.,1993; Kageyama and Robertson, 1993). There is also somecontroversy over which cortical cells are first to send theiraxons to the thalamus. Some work has suggested that thecells of the SP are first (McConnell et al., 1989; De Carlosand O’Leary, 1992), whereas other studies indicate thatthe first projections are from cortical layer 5 (Clasca et al.,1995) or from cortical layer 5 in the dorsal cortex andcortical layers 5 and 6 in the lateral cortex (Sheng et al.,1991; Marotte and Sheng, 1995).

In this third paper in the series on the development ofthe wallaby trigeminal system, we describe the timecourse of development of thalamocortical and corticotha-lamic projections of the somatosensory pathway and, inparticular, the timing of the initial projections and of theclustering of afferents and/or cortical cells in relation to

the appearance of vibrissae-related SDH patches in thecortex. This has been analysed by using the anterogradeand retrograde transport of a carbocyanine dye in fixedtissue and of horseradish peroxidase conjugated to wheatgerm agglutinin (WGA-HRP) in vivo. In addition, thearrival of the corticothalamic projection in the thalamuswas determined independently by anterograde labelling ofcortical axons in vivo, by using Neurobiotin and biocytin.Some of the results have been published in abstract form(Leamey et al., 1996b).

MATERIALS AND METHODS

Fifty-five wallabies (Macropus eugenii) obtained from abreeding colony were used in this study. Experiments wereapproved by the Animal Ethics Experimentation Com-mittee of the Australian National University. Animalsaged 30 days or younger were of known birthdate andolder animals were either of known birthdate or theirage was determined from a chart of head lengths ofanimals of known age produced from data available in ourlaboratory. A straight line regression of age on head lengthof animals of known age (n 5 122) resulted in a table ofpredicted age as a function of head length, with a standarderror of 2 days. This table is available on our web pagehttp://biology.anu.edu.au/research-groups/DN/.

Pouch young 30 days and younger were anaesthetisedby hypothermia, whereas older pouch young were anaesthe-tised by intramuscular ketamine (1–3 mg/100 g bodyweight, depending on age) and xylazine (0.2 mg/100 g bodyweight). The adults were anaesthetised with an initialdose of intravenous sodium thiamylal (20 mg/kg bodyweight) followed by 20 mg doses as required.

Cortical deposits of horseradish peroxide

Thirteen pouch young aged 10, 18, 21, 30, 42, 53, 54, 65,72, 75, 89 (2 animals), and 91 days and two adults wereused. One adult was used to provide normal histologicalmaterial of the somatosensory thalamus. This animal wasdeeply anaesthetised and perfused with 10% formol saline.Frozen sections were cut at 60 µm and stained withthionin. In one adult and the three oldest pouch young theposition of the whisker representation in somatosensorycortex was determined by recording the evoked response tostimulation of the whisker pad with paired needle elec-trodes. A craniotomy was performed and evoked potentialswere recorded from the cortical surface with a silver ballelectrode (Waite et al., 1991). Once the site of maximumresponse was located, a small amount of undissolvedWGA-HRP on the tip of a sealed micropipette was insertedinto the cortex. In younger animals deposits were made inthe equivalent region of cortex after cutting a skull flap.The skull flap was replaced and the skin sutured. After asurvival period of 8–24 hours, depending on age, animalswere deeply anaesthetised as described above and per-fused briefly with 0.9% NaCl followed by 1% paraformalde-hyde/1.25% glutaraldehyde in 0.1 M phosphate buffer (PB)pH 7.4 for 30 minutes followed by 0.1 M PB containing 10%sucrose. Brains were dissected out, placed in 0.1 M PBcontaining 20% sucrose overnight at 4°C, and then embed-ded in gelatin/albumin hardened with glutaraldehyde.

Frozen sections were cut at 60 µm in a plane at rightangles to the surface of the cortex at the midline, heretermed coronal for descriptive ease. They were then pro-cessed for HRP histochemistry by the tetramethyl benzi-

WHISKER PATHWAY DEVELOPMENT: THALAMUS AND CORTEX 195

dine method of Mesulam (1982). Alternate sections werelightly stained with thionin. In most cases, camera lucidadrawings of alternate sections through the unstainedseries of sections were made by marking the position of thecortical deposit site and the transported label, and stainedsections were used to draw the borders of thalamic nuclei.

Thalamic deposits of DiI

Experiments were performed on 23 pouch young walla-bies aged P10, P15 (2 animals), P20 (3 animals), P30 (5animals), P40 (3 animals), P50 (3 animals), P60 (3 ani-mals), P70, P76, and P81. Pouch young were deeplyanaesthetised as described above and perfused with 4%paraformaldehyde in 0.1 M PB, pH 7.4. Heads were thenfixed for a minimum of 3 days. The cortex was exposed anda cut was made, in the plane described in the previoussection, through the forebrain and the base of the skull at alevel, calculated from sectioned tissue described in the firstsection to pass through the mid-ventroposteromedialnucleus (VPM), after allowance for the shrinkage of thesectioned tissue had been made. In animals older thanP40, the VPM could then be recognised dorsomedial to theexternal medullary lamina. One or more crystals of thecarbocyanine dye, DiI (1,18-dioctadecyl-3,3,38,38-tetra-methylindocarbocyanine perchlorate, 50–100 µm), wereapplied by using fine dissecting pins. In young animals,the distance of the mid-VPM from the midline and dorsalborder of the thalamus was determined from sectionedtissue, and this was used to determine the application site.

Tissue was stored in fixative in darkness at 37°C be-tween 1 week and 3 months, depending on the age of theanimal. Extended periods at 37°C were found to compro-mise the quality of the tissue so brains were checked atregular intervals. As soon as the tissue showed any hint ofdeterioration it was removed from the oven and stored atroom temperature in fresh fixative for periods varyingbetween 5 weeks and 10 months. Tissue was embedded in4% agar and 100-µm coronal sections were cut on aVibratome. In some cases, tangential sections were cutthrough the somatosensory cortex. Sections were counter-stained in 0.001% bizbenzimide, rinsed and mounted in PBand viewed immediately under epifluorescence optics byusing filters appropriate for DiI and bisbenzimide. Se-lected sections were converted to a photostable productwith 3,38-diaminobenzidine (Maranto, 1982; Sandell andMasland, 1988).

Thalamic injections of WGA-HRP

Ten pouch young aged 24, 59, 73, 74, 75, 76, 77, 81, 94,and 113 days received thalamic deposits of 2% WGA-HRPaimed for VPM. Coordinates for the deposits were calcu-lated from Nissl stained coronal sections. Volumes rangedfrom 0.015 to 0.25 µl, depending on age. In the youngestpouch young the telencephalon had still not grown cau-dally to cover the diencephalon and a micropipette ofapproximately 50 µm in diameter coated with a concen-trated, dried solution of WGA-HRP was inserted directlyinto the thalamus. After 12–24 hours survival, dependingon age, tissue was prepared as described in the first section.

Cortical injections of Neurobiotinand biocytin

Seven pouch young wallabies aged P30 (2 animals), P37,P50, P60, P74, and P75 were used. Experiments describedin the first section established the relationship between

somatosensory cortex and external landmarks such asbregma, the midline, and the eye. A small hole was drilledin the skull at this point and the dura removed. For pouchyoung aged P30 and P37 this was at the intersection of aline passing through the dorsal margin of the eye parallelto the midline and a line passing through bregma at rightangles to the midline. For older pouch young a hole wasdrilled at the intersection of a line passing through thedorsal margin of the pupil of the eye parallel to the midlineand a line passing through bregma at right angles to themidline. Glass microelectrodes were filled with 3 µl of 6%Neurobiotin (Molecular Probes, Eugene, OR) or 3% biocy-tin (Molecular Probes), coloured pale blue or pale green bythe addition of a small amount of methyl green. The tip ofthe microelectrode was broken back until a small drop oftracer could be visualised emerging from the tip underpressure. A pressure injection of biocytin or Neurobiotinwas made bilaterally into the depth of the somatosensorycortex. The volume injected was estimated to range be-tween 300 nl and 500 nl. After survival times of 8–16hours, animals were deeply anaesthetised as above andperfused with 4% paraformaldehyde/1.25% glutaralde-hyde in 0.1 M PB (pH 7.4) for 20 minutes. The brain waspostfixed in the same fixative for 2–3 hours before beingcryoprotected in 30% sucrose in 0.1 M PB at 4°C overnight.Tissue was sectioned as described in the first section. Thetracer was visualized by using a Vectastain ABC kit(Vector Laboratories, Burlingame, CA; Horikawa and Arm-strong, 1988), and the peroxidase component of the avidin-biotin or avidin-neurobiotin complex was visualized byusing a nickel-intensified 3,38-diaminobenzidine reaction(Hancock, 1982). Alternate sections were stained withthionin.

RESULTS

Thalamic labelling from corticaldeposits of WGA-HRP

Adult. The whisker area of the adult somatosensorycortex was identified by recording an evoked potential tostimulation of the whisker pad and a localized deposit ofWGA-HRP was made (Fig. 1a). The VPM of the wallabycontains dorsoventrally aligned bands of cells, which canbe seen in Nissl stained sections (Fig. 1b). The deposit ofWGA-HRP resulted in a discrete band of label in VPMrunning dorsoventrally through the nucleus (Fig. 1c),which resembled the bands seen in Nissl stained sections.Both retrogradely labelled cells and anterogradely labelledterminals were present (Fig. 1d).

P10. The development of somatosensory thalamus hasbeen fully described using Nissl, myelin and histochemicalmethods for mitochondrial enzyme activity (Leamey et al.,1996a). At this age, the ventroposterior thalamic nucleus(VP) cannot be recognized on cytoarchitectural grounds.After a large deposit into an area including the putativesomatosensory cortex and similar in extent to the depositsite at P30 (see below) which did result in thalamic label,no transported label was seen in the thalamus (not illus-trated).

P15–21. At these ages, VP was difficult to recognizecytoarchitecturally and the border between VP and theposterior thalamic nucleus (Po) could not always be reli-ably distinguished. Within VP, VPM could not be distin-guished from VPL (Fig. 2b). The position of VPM wasconfirmed by comparison with material in which the

196 L.R. MAROTTE ET AL.

trigeminothalamic projection had been labelled from theprincipal trigeminal sensory nucleus (Leamey, 1996). Inthe three cases at these ages, large deposits of WGA-HRPincluded putative somatosensory cortex (Fig. 2a) andresulted in extensive label in somatosensory thalamus.Label was present in VP and extended dorsally into theputative Po (Fig. 2b,c). In all cases there was retrogradelabelling of cells but the small size and dense packing of

the cells made it difficult to be certain whether there wasalso label that could be interpreted as anterograde.

P30–42. By P30, VP can be identified cytoarchitectur-ally, the division between VPM and VPL is starting tobecome apparent and the packing density of cells hasdecreased (Fig. 2d). A large injection in somatosensorycortex centered on the putative whisker area resulted inextensive label in VPM that extended dorsally into Po (Fig.

Fig. 1. a: Stained coronal section through the somatosensorycortex of an adult wallaby showing the cortical deposit of wheat germagglutinin conjugated to horseradish peroxidase (WGA-HRP; arrow).Shown on the right hand side of the figure is the cortical evokedresponse to whisker stimulation used to locate the deposit site. Thevertical scale represents 50 mV and the horizontal scale, 10 msec. b:Stained coronal section through the somatosensory thalamus of anadult showing the normal histology of the ventroposteromedial nucleus

(VPM), which is characterised by intensely stained cells. Arrows pointto the dorsoventrally arranged bands of cells in the VPM. eml: externalmedullary lamina. c: Unstained coronal section through the VPMshowing the dorsoventrally aligned band of label (arrow) resultingfrom the cortical deposit of WGA-HRP. d: Darkfield high-power view ofretrogradely labelled cells and anterogradely labelled terminals (ar-row) in the VPM after a cortical deposit of WGA-HRP. Scale bars 5 5mm in a, 2 mm in b, 500 µm in c, 100 µm in d.


Fig. 2. a: Stained coronal section through the cortex of a postnatalday (P) 15 wallaby showing the deposit of wheat germ agglutinin con-jugated to horseradish peroxidase (WGA-HRP) in putative somato-sensory cortex. b,c: Adjacent stained coronal sections through thethalamus in brightfield (b) and darkfield (c) showing the label in theventroposterior nucleus (VP) and the posterior nucleus (Po) resultingfrom the cortical deposit of WGA-HRP shown in a. LGN, lateral genic-ulate nucleus; VPL, ventroposterolateral nucleus; eml, external medul-lary lamina. d: Stained coronal section through the thalamus of a P30wallaby showing the retrogradely labelled cells in the ventropostero-

medial nucleus (VPM) and Po after a deposit of WGA-HRP in somato-sensory cortex. e: High-power view of the retrogradely labelled cells inthe VPM, shown in d. No anterograde label is apparent. f: Stainedcoronal section through the somatosensory thalamus on P62 showinglabel in the VPM and Po resulting from a deposit of WGA-HRP insomatosensory cortex. g: High-power view of the label in the VPMshown in f. Both retrogradely labelled cells and anterogradely labelledterminals, seen as fine dust between the cells, are present. Scalebars 5 500 µm in a–d,f, 50 µm in e,g.

2d). At this age the lower cell density allowed label to beclearly identified as being in cells bodies (Fig. 2e). Noanterograde label was obvious in the neuropil amongstcells, but small amounts would be difficult to distinguishfrom light label in cells. Results at P42 were similar.

P53–54. In one case at this age the deposit site wasprimarily in visual cortex with only a small spread into thecaudal region of somatosensory cortex. This resulted inlabel in a narrow band running dorsoventrally through themost lateral and caudal part of VPM and extending into Po(not illustrated). In the second case, the large deposit sitewas more rostral and centered on somatosensory cortex.This resulted in labelling more medially in VPM thatextended into Po (not illustrated). The label was primarilypresent in cell bodies but axonal label, consisting of stringsof granular reaction product, could be followed from theexternal medullary lamina into VPM where these fibrescoursed dorsomedially. It was unclear whether any of thisrepresented anterograde label or the axons of retrogradelylabelled cells projecting to the cortex.

P62–75. At the youngest age in this group labelling inthe form of fine dust was present between the retrogradelylabelled cells (Fig. 2g). We interpret this as anterogradelabel. No significant dendritic labelling of retrogradelylabelled cells occurred, so this is unlikely to be labelleddendrites running through the plane of section. After alarge deposit both types of label were present in VPM andPo (Fig. 2f). A similar pattern of labelling was seen in theolder animals of this group but with an increased densityof the fine dust-type labelling between cells.

P89–91. Of the three animals in this age group, thesmallest deposit was dorsal in the whisker area andresulted in labelled cells and terminals, which in adjacentsections could be traced as a curved band running dorsoven-trally through VPM and extending into Po, similar to thatseen in the adult. A similarly placed, slightly larger depositproduced a similar band slightly wider in mediolateralextent in VPM. The third deposit was larger in dorsoven-tral extent and a correspondingly larger part of VPM andPo was labelled, primarily in lateral regions.

Cortical labelling from thalamicdeposits of DiI

In this series of experiments the cortical distribution ofthalamic afferents and of cells projecting to the thalamusduring development was investigated using DiI as a tracer.The terminology used to describe developing cortical lami-nation is based on that agreed upon by the BoulderCommittee (1970) and that used by Sheng et al. (1991) intheir description of the developing wallaby visual cortex.

Figure 3 illustrates a typical thalamic injection site asseen under the dissecting microscope (Fig. 3a) and thefluorescent microscope (Fig. 3b) after sectioning. Injectionsites were largely confined to VPM, although some spreadof label to neighbouring thalamic nuclei was unavoidable,particularly in younger animals. There was also labellingof radial glia (Fig. 3b). In no case did the deposit sitespread to include the internal capsule (IC). Labelled axonswere consistently seen to enter the IC slightly rostral tothe injection site, and pass laterally, dorsally and rostrallyin the IC to enter the intermediate zone of the presumptivesomatosensory cortex.

P10–P15. By this stage the cortical plate can be recog-nized in the putative somatosensory cortex, as a condensa-tion of cells. This layer has split the primordial plexiform

layer, which was present at birth, into the marginal zone(MZ) and the deeper intermediate zone. The subventricu-lar zone can also be recognized above the ventricular zone(Fig. 3c). The cortical plate is the region of neuron differen-tiation that will give rise to layers 2–6 of the adult cortex,whereas the intermediate zone will form the white matterof the adult cortex. By P15 a deeper, less densely packedregion, referred to here as the loosely packed zone (LPZ),has begun to appear beneath the more superficial compactcell zone (CCZ) of the cortical plate, and the superficialpart of the intermediate zone, termed the SP by Kostovicand Mollivar (1974) can just be discerned (Fig. 3c). At P10,thalamocortical axons had left VPM and entered the ICwhere they ended in complex growth cones (Leamey, 1996),confirming the results of a cortical injection of WGA-HRPat this age that failed to retrogradely label cells in VPM.

By P15, small numbers of thalamocortical fibres hadarrived in the putative somatosensory cortex (Fig. 3d).Some axons passed superficially in the intermediate zoneand ran dorsally within the LPZ for a short distance beforeending in growth cones immediately deep to the CCZ of thecortical plate (Fig. 3d and left inset). Others stayed deep inthe intermediate zone, and made a fairly abrupt turn toterminate in growth cones just deep to the CCZ. Anexample of such an axon and its growth cone at the base ofthe CCZ is shown in the right hand inset of Figure 3d.Others branched in the intermediate zone and one branchpassed superficially to end in a growth cone just deep to theCCZ. Retrogradely labelled cell bodies and their dendriteswere occasionally seen, sometimes in pairs or small clus-ters, but these had no consistent laminar or areal location.

P20–40. By P30 the LPZ is well developed throughoutthe dorsoventral extent of somatosensory cortex and itscharacteristic feature of cells in parallel arrays is clear(Fig. 4a). By P40, the number of thalamocortical axons hadincreased substantially and as at P15 they were distrib-uted from the intermediate zone up to the base of the CCZof the cortical plate (Figs. 4b,c, 5b). A small amount ofbranching occurred in the LPZ but the majority of axonsremained simple, terminating in growth cones at the lowerborder of the CCZ (Fig. 5a,b). Retrogradely labelled cellswere occasionally seen in both superficial and deep parts ofthe cortex but, as at P15, had no consistent laminar orareal location (Figs. 4c, 5b). Some had prominent apicaldendrites (Fig. 4c). Occasionally, labelled cells were seen inthe CCZ and some were in clusters, strongly suggestingtranscellular labelling (Fig. 4c).

P50. In some cases, at this age and also in olderanimals, where the transport distances were longest, therewas a gradual diminution in the intensity of fluorescentlabel as axons approached the cortex, and they could not betraced further than the deeper cortical layers. Such caseswere excluded and transport times were increased. Label-ling of afferent fibres within the MZ was first seen at thisage. Axons could be followed through the CCZ into the MZwhere they ramified superficially (Fig. 5c,d). As at previousages, afferent fibres filled the region between the interme-diate zone up to the base of the CCZ (Fig. 5c,d). However,the density of the thalamocortical projection was increasedcompared with the previous age, particularly in the LPZ(Fig. 5c), possibly reflecting an increase in the branching ofthe afferent fibres. Growth cones were again visible at thelower border of the CCZ (Fig. 5d). Occasional labelled cellswere again seen in both the superficial (Fig. 5d) and deepparts of the developing cortex.


P60. By P60, layer 5 can be identified on cytoarchitec-tural grounds immediately below the CCZ and the cellsparse lamina that separates layers 5 and 6 is visible deep

to this (Fig. 6b). The most significant event at this age wasthe appearance of a row of retrogradely labelled cells in theLPZ, slightly deep to the CCZ (Fig. 6a) identified as

Fig. 3. a: An example of a 1,18 dioctadecyl-3,3,38,38-tetramethylin-docarbocyanine perchlorate (DiI) application site (arrow) in the ventro-posteromedial nucleus (VPM) from a wallaby at postnatal day (P) 30,as seen under the dissecting microscope. A coronal cut was made toexpose the VPM. Cx: cortex; Thal: thalamus. b: A coronal sectionthrough the DiI (carbocyanine dye) application site (arrow) from theP30 preparation shown in a, as seen under the fluorescence microscopefollowing sectioning. The orientation is the same as in a. The applica-tion site is localised in the VPM although there is some labelling ofradial glia (rg). c,d: Low-power fluorescence views of a coronal sectionthrough the somatosensory cortex at P15 exposed for bizbenzimidecounterstain (c) and DiI labelling (d) after transport of DiI from theVPM. The first thalamocortical axons have reached the cortex by this

stage and pass superficially from the intermediate zone (IZ) throughthe loosely packed zone of cells (LPZ) beneath the compact cell zone(CCZ) of the cortical plate to terminate in growth cones (small arrowsin d) immediately beneath the CCZ. Occasional cell bodies arelabelled, sometimes in pairs (large arrow in d). The right hand edges ofc and d are coincident, and in d the surface of the cortex and the extentof the CCZ are marked. MZ, marginal zone; SP, subplate; SVZ,subventricular zone; VZ, ventricular zone. Insets: High-power viewsof growth cones at the base of the CCZ. The axon tipped with thegrowth cone on the left approaches the CCZ obliquely, whereas the oneon the right makes an abrupt turn in the intermediate zone to passsuperficially and approach the CCZ at a right angle. Scale bars 5 1mm in a, 500 µm in b, 100 µm in c,d, 25 µm in insets.


developing layer 5 cells. These cells were very prominentwith basal dendrites and apical dendrites that extendedinto and branched in the MZ (Fig. 6e). Their presence and

location was consistent between sections. In some sectionsa less prominent row of retrogradely labelled cells was alsoseen in the developing layer 6 but these were not consis-

Fig. 4. a,b: Low-power fluorescence views of a coronal sectionthrough the somatosensory cortex at postnatal day (P) 30, exposed forbizbenzimide counterstain (a) and 1,18 dioctadecyl-3,3,38,38-tetramethy-lindocarbocyanine perchlorate (DiI) labelling (b) after transport of DiIfrom the ventroposteromedial nucleus. The dotted lines in b denote theborders of the marginal zone (MZ) and compact cell zone (CCZ). Thenumber of thalamocortical afferents have increased markedly com-pared with P15 and extend up to the base of the CCZ. LPZ, looselypacked zone. c,d: High-power views of the somatosensory cortex

shown in b (c) and in a similarly aged animal (P24) after a deposit ofwheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) in the somatosensory thalamus (d). The layers are aligned andlabelling in c applies to d also. In both cases afferents reach to the baseof the CCZ of the cortical plate. In the DiI labelled material, occasionalretrogradely labelled cell bodies are present (arrows) including pairs ofcells, seen here in the CCZ. In contrast, no retrogradely labelled cellsare seen in the material labelled with WGA-HRP (d). Scale bars 5 200µm in a,b, 100 µm in c,d.


tently seen from section to section and scattered labelledcells were also present as at previous ages (Fig. 6a). Thedistribution of afferent fibres (Fig. 6a,d) was similar to thatseen at previous ages and fibres tipped with growth conescontinued to be observed at the base of the CCZ (Fig. 6d).

P70–72. At this stage the developing layer 4 is presentimmediately deep to the CCZ (Fig. 7c). Thalamocorticalafferents, frequently seen tipped with growth cones (Fig.7a), extended throughout this layer up to the base of theCCZ. The afferents showed no hints of aggregation ineither coronal (Fig. 7a) or tangential (not shown) sections.The prominent row of retrogradely labelled layer 5 cellswas also present and in the lateral part of the presumptivesomatosensory cortex, labelled cells in layer 6 were alsocommon (Fig. 7a). These were absent in the more dorsome-dial region. Occasional scattered cells were seen in otherlayers of the developing cortex including the CCZ (Fig. 7a).

P76. Clustering of retrogradely labelled cells in layers6 and 5 was first apparent at this age. The number oflabelled cells in layer 5 appeared reduced (Fig. 7d) com-pared with the previous age (see Fig. 7a and the followingsection). The labelled apical dendrites of these cells thatcoursed through layer 4 made it difficult to determine theorganisation of the afferent fibres at this stage. However,unbranched thalamocortical fibres tipped with growthcones were present in layer 4 (Fig. 7e). These could bediscriminated from the growth cones associated with thedendrites of cortical cells by following the fibres from theintermediate zone. Relatively little branching was visible

in layer 4 from either the thalamocortical fibres or from thedendrites of cortical cells (Fig. 7e).

P81. Layer 3 was now visible beneath the CCZ (Fig.8b). Clustering of labelled cells and processes was moreprominent by this stage, and more branching was visiblein layer 4 (Fig. 8a). Again, the prominence of the retro-gradely labelled cells and their processes made it difficultto discriminate what contribution the thalamocorticalafferents made to the clustered organisation (Fig. 8a).Thalamocortical fibres tipped with growth cones wereoccasionally seen in layer 4 but were less common than atP76. The clustered organisation was also obvious in tangen-tial sections through layer 4 (Fig. 8d).

Cortical labelling from thalamicinjections of WGA-HRP

A second tracer, WGA-HRP, was chosen to examineconnections between thalamus and cortex at selected agesfor two main reasons. First, the scattered retrogradelylabelled cells in the deep layers of the cortex, and indeed inthe CCZ of the cortical plate, apparent with DiI tracingearly in development, were not seen in visual cortex at thesame stages of development by using WGA-HRP as atracer (Sheng et al., 1991). This raises the possibility thattranscellular labelling may have been occurring with DiI.Second, the retrograde labelling of the entire dendritic treeof cortical cells projecting to the thalamus with DiI ob-scured the distribution of thalamic afferents, making the

Fig. 5. a,b: Low-power fluorescence views of adjacent coronalsections through the somatosensory cortex at postnatal day (P) 40,exposed for bizbenzimide counterstain (a) and 1,18 dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI) labelling (b) after trans-port of DiI from the ventroposteromedial nucleus (VPM). A thalamocor-tical axon tipped with a growth cone is seen at the base of the compactcell zone (CCZ) (small arrow) as well as one within the CCZ (smallarrow). Occasional retrogradely labelled cells are seen superficially aswell as deep (large arrows). MZ, marginal zone; LPZ, loosely packed

zone. c: Low-power fluorescence view of a coronal section through thesomatosensory cortex at P50, exposed for DiI labelling after a depositin the VPM. Thalamocortical afferents are seen in the MZ for the firsttime and there is a large increase in density of thalamocorticalafferents below the CCZ of the cortical plate. d: High-power view of thesomatosensory cortex shown in c. Afferents, some of which are tippedwith growth cones (arrows) occupy the developing cortex up to the baseof the CCZ. A retrogradely labelled cell is present in the LPZ (arrow-head). Scale bars 5 100 µm in a–d.


Fig. 6. a–c: Low-power views of coronal sections through thesomatosensory cortex after a deposit of 1,18 dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI) in the ventropostero-medial nucleus (VPM) at postnatal day (P) 60 (a) and an injection ofwheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) in the somatosensory thalamus, including the VPM, at P59 (b,c).A fluorescent view of DiI labelling is shown in a, whereas b and c areadjacent sections shown in brightfield after Nissl staining (b) and indarkfield showing WGA-HRP labelling (c). Sections have been alignedso the layers are in register. At this age, a row of retrogradely labelledcells is present in layer 5 just below the compact cell zone (CCZ) of thecortical plate in both DiI (a, arrow) and WGA-HRP labelled material

(b,c, arrow). A high-power view of one of these DiI labelled cells isshown in e. Scattered retrogradely labelled cells are seen in deeperlayers with DiI labelling (a) but not with WGA-HRP (c). Afferentsextend up to the base of the CCZ in both cases. d,e: High-powerfluorescent views of the upper layers of the somatosensory cortexshown in (a). Sections have been aligned so the layers are in register.Thalamocortical afferents tipped with growth cones (arrows) arepresent at the base of the CCZ (d) and afferents are dense in themarginal zone (MZ). Retrogradely labelled cells in layer 5 haveprominent apical dendrites that branch in the MZ (e). Scale bars 5 100µm in a–c, 50 µm in d,e.

Fig. 7. a–c: Low-power views of coronal sections through thelateral region of the somatosensory cortex after a deposit of 1,18dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI) inthe ventroposteromedial nucleus (VPM) at postnatal day (P) 70 (a) andan injection of wheat germ agglutinin conjugated with horseradishperoxidase (WGA-HRP) in the somatosensory thalamus, including theVPM, at P73 (b,c). DiI labelling after photoconversion is shown in (a),while (b) and (c) are adjacent sections reacted for WGA-HRP andunstained (b) and Nissl stained (c). The sections have been aligned sothe layers are in register and the paired lines in a and b mark theborders of the compact cell zone (CCZ). Two rows of retrogradelylabelled cells in layers 5 and 6 are now present (a,b). Occasionalscattered retrogradely labelled cells are still seen in other layers in DiIlabelled material (a) but not in WGA-HRP labelled material (b). Layer

4 can now be recognized beneath the CCZ (c) and thalamocorticalafferents are present within it up to the base of the CCZ (a,b). In DiIlabelled material these axons can be seen tipped with growth cones (a,arrows). d: Low-power fluorescent view of DiI labelling in the somato-sensory cortex at P76 after a deposit of DiI in the VPM. The pair ofwhite bars mark the boundaries of the CCZ. At this age, the first signof clustering of retrogradely labelled cells in layers 6 and 5 and theirprocesses is seen (arrows). Retrogradely labelled cells in layer 5 havedecreased and those in layer 6 increased compared with that seen atthe previous age. e: High-power fluorescent view of DiI labelling inlayer 4 of the somatosensory cortex shown in d. A thalamocorticalafferent, tipped with a growth cone, is present (small arrow). Branch-ing of both axons and dendrites is not frequent (large arrows). Scalebars 5 100 µm in a,b (bar in b applies to c), 200 µm in d, 50 µm in d.

Fig. 8. a–c: Low-power views of coronal sections through thesomatosensory cortex at P81 after a deposit of 1,18 dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI) in the ventropostero-medial nucleus (VPM) (a,b) and after an injection of wheat germagglutinin conjugated with horseradish peroxidase (WGA-HRP) in thesomatosensory thalamus, including the VPM (c). a,b: Adjacent sec-tions exposed for DiI labelling and bizbenzimide staining, respectivelyand these have been aligned with the section in (c) so that the layersare in register. Clustering of cells, primarily located in layer 6, andprocesses are again prominent and there is increased branching inlayer 4 compared with the previous age (a). Labelling with WGA-HRP,which does not fill the dendrites of retrogradely labelled cells, con-

firmed that thalamocortical afferents are also clustered (c).MZ, mar-ginal zone; CCZ, compact cell zone. d: Fluorescent view of a tangentialsection through layer 4 of the somatosensory cortex at postnatal day(P) 81 showing labelling after a deposit of DiI in the VPM. Discretecircular clusters of labelled processes are obvious (diamonds). e:Darkfield unstained coronal section through the somatosensory cortexat P113, showing labelling after a deposit of WGA-HRP in thesomatosensory thalamus, including the VPM. Well-defined clusters ofafferents are present in layer 4. Retrogradely labelled cells areprimarily in layer 6 and are sparse in layer 5. Scale bars 5 200 µm in a(applies to b), 100 µm in c–e.


onset of their segregation into patches indeterminate inDiI labelled material.

In the adult, injections of HRP into VPM (Mayner, 1985)resulted in patches of anterograde label in layer 4 of theregion of somatosensory cortex identified as the whiskerarea in the present study. Lower amounts of anterogradelabel were also present in layers 6 and 3. Retrogradelylabelled cells were seen in high numbers in layer 6 and inlow numbers in layer 5. The latter may reflect spread atthe injection site into Po. These cells were in clusters inregister with the patches of anterograde label in layer 4.

P24. This age point was examined as it is a stage whenscattered retrogradely labelled cells are seen in deepercortical layers in DiI material but it is before the appear-ance of a row of retrogradely labelled cells beneath theCCZ of the cortical plate. A large thalamic injectionincluding somatosensory thalamus produced anterogradelabel in putative somatosensory cortex that extended fromthe intermediate zone up to the base of the CCZ of thecortical plate (Fig. 4d). No segregation of afferents waspresent and no retrogradely labelled cells were apparent.

P59. At this age both anterograde and retrograde labelwas seen in somatosensory cortex (Fig. 6b,c) after a largethalamic injection that included the lateral part of somato-sensory thalamus including VP and Po. The anterogradelabel was distributed up to the base of the CCZ of thecortical plate as at the younger age and was evenlydistributed throughout the labelled region (Fig. 6c). Aprominent row of retrogradely labelled cells in layer 5 wasalso present just deep to the CCZ (Fig. 6c), in the sameposition as the row of retrogradely labelled cells in DiImaterial at this age. In contrast to DiI labelled materialthough, there were no retrogradely labelled cells apparentdeep to this row. The injection extended into visual thala-mus, and in visual cortex retrogradely labelled cells couldbe recognized in layer 6 as well as in layer 5 laterally.Further dorsally, such cells in layer 6 gradually decreasedin number and were absent most dorsally, confirmingprevious results (Sheng et al., 1991).

P73–75. Afferents were evenly distributed in layer 4up to the base of the CCZ and retrogradely labelled cells inlayer 5, and for the first time in layer 6, were presentlaterally (Fig. 7b,c), whereas more dorsally they were onlypresent in layer 5.

P76. By this age the first hints of patchiness in theanterograde label in layer 4 were evident but the patcheswere somewhat irregular and indistinct. Retrogradelylabelled cells were present in both layers 5 and 6.

P81–94. At P81 the patches of anterogradely labelledafferents in layer 4 were more distinct (Fig. 8c) than at theprevious age, ranging in size from around 180 to 215 µm inwidth. The number of retrogradely labelled cells in layer 5had decreased compared with P76 and retrogradely la-belled cells were prominent in layer 6 (Fig. 8c), similar tothe labelling obtained with DiI at the same age (Fig. 8a).There was little change at P94.

P113. By this age, well-developed clusters of thalamicafferents were labelled in layer 4 and these extended intothe deeper part of layer 3 at lower density (Fig. 8e). Axonspassed through layers 3 and 2 to reach layer 1. Theclusters in layer 4 were somewhat variable in size rangingfrom around 140 to 210 µm in width. Retrogradely labelledcell were present in large numbers in layer 6 and in lowernumbers in layer 5. Clustering of the cells was not asprominent as seen in some examples in DiI material, but

there was a hint of this in layer 5. This may have been dueto the plane of section as only a small region of the whiskerarea was labelled and this was close to the parietal sulcusdorsally. The injection site included the lateral part of VPand Po.

Labelling of corticothalamic axons withNeurobiotin and biocytin

Given the conflicting results obtained on the time ofarrival of corticothalamic axons in VPM by using retro-grade tracers and the difficulty in detecting low amounts ofanterograde terminal label in VPM by using WGA-HRP,the aim of these experiments was to obtain an independentassessment of the time of arrival of corticothalamic axons.Neurobiotin and biocytin were chosen as tracers becausethey completely fill axons, permitting both the position ofaxons tipped with growth cones and the onset of axonalarborization to be followed during the development of theprojection.

Injections of either Neurobiotin or biocytin through thedepth of the developing somatosensory cortex yielded bothanterograde labelling of corticofugal projections and retro-grade labelling of thalamocortical projection cells in VPMand Po but the use of relatively small amounts of eithertracer minimised the amount of retrograde label. In addi-tion to the subcortical labelling described below, there wasalso intracortical labelling of both axons and cortical cells(not shown). Results with Neurobiotin and biocytin werecomparable and are not described separately.

P28–31. This was the youngest age investigated here.An example of an injection site is illustrated in Figure 9a.It passes through the thickness of the developing corticalmantle to the intermediate zone. Labelled axons coursedventrally from the injection in the intermediate zone (Fig.9a). Retrogradely labelled cells within VPM confirmed thatthe injection site was within the somatosensory region(Fig. 9b). A slightly more caudal section than that shown inFigure 9a illustrates the position of labelled corticofugalfibres tipped with growth cones in the IC at this stage (Fig.9c). Examples of these are shown in Figure 9d,e. No axonstipped with growth cones were found in labelled axonbundles caudal or ventral to this region or within thethalamus. Other labelled axons present in the IC wereretrogradely labelled thalamocortical axons.

P37–42. Labelled axons took the same course as at theprevious age (Fig. 9f) after injections through the depth ofthe cortex. Again the retrogradely labelled axons of thala-mocortical projection cells composed a large component ofthe axons labelled at this time, but anterogradely labelledaxons tipped with growth cones (Fig. 9g) were also identi-fied in the IC, at more caudal levels than seen at P30 (Fig.9f). Near the lateral border of the thalamus a few labelledfibres continued ventrally past the thalamus to enter thecerebral peduncle. No axons with growth cones arisingfrom descending inputs were seen in the thalamus. Cellswith label largely confined to their somata were scatteredwithin the lateral half of VPM and to a lesser extent in Poconfirming the accuracy of the injection site.

P50–52. By this stage labelled corticofugal axons haddescended in the IC to reach the lateral border of thethalamus. At this level the pathway branched (Fig. 10a,b).Some of the axons turned medially to enter the rostralthalamus (Fig. 10b), often as branches of fibres thatcontinued ventrally to form the cerebral peduncle (Fig.


Fig. 9. a: Coronal section through the somatosensory cortex show-ing an example of an injection of Neurobiotin (arrow) at postnatal day(P) 30. Note that the tracer penetrates into the developing corticallayers to the level of the intermediate zone. Labelled axons extend intothe internal capsule (IC). b: Coronal section through the thalamus atP30, from the case shown in (a). Retrogradely labelled cells are presentin the lateral half of the ventroposteromedial nucleus (arrow) confirm-ing that the injection was located in the somatosensory cortex. eml,external medullary lamina. c: Coronal section through the somatosen-sory cortex slightly caudal to the injection site shown in (a). The mostcaudal corticofugal axons have entered the IC. The location of the

leading edge of the projection is indicated by the arrow. d,e: Examplesof axons tipped with growth cones in the IC. The example shown in d isjust dorsal to the leading edge of corticofugal axons indicated by thearrow in c, whereas the example in e is at the leading edge. f: Coronalsection through the forebrain showing the position of the leading edgeof the corticofugal projection in the IC (arrow) after an injection ofNeurobiotin in the somatosensory cortex at P40. This position isfurther caudal to that seen at P30. g: High-power view of the leadingedge of corticofugal axons tipped with growth cones (arrows). Theirposition is shown in f. Scale bars 5 500 µm in a,b, 1 mm in c,f, 20 µm ind,e, 50 µm in g.


10c). These axons in rostral thalamus ended in growthcones (Fig. 10d) and none could be traced into VPM.

P60–62. At this stage anterogradely labelled axonsfrom cortical cells could first be traced into VPM and Po(Fig. 11a). Retrogradely labelled thalamic cells were againseen in VPM and Po, and by this stage the label appearedas a dorsoventrally aligned finger (Fig. 11b) similar to thatseen after a small cortical injection of WGA-HRP in theadult (see Fig. 1a,c). Cortical axons were seen to terminatein growth cones in both VPM (Fig. 11c) and Po (Fig. 11d),and others appeared to have begun branching (Fig. 11c). Anumber of fibres tipped with growth cones had not yetreached VPM or Po.

P74–75. By this age axons were more highly branchedbut axons tipped with growth cones were still present inVPM and Po.

DISCUSSION

This study has used the transport of DiI in fixed tissueand the transport of WGA-HRP, Neurobiotin, and biocytinin vivo to describe the development of the thalamocorticaland corticothalamic projections in the wallaby. The firstthalamocortical axons arrived beneath the cell dense re-gion in the superficial cortical plate by P15. Over the ensu-ing weeks the projection became more extensive and fibresramified evenly to fill the region between the intermediatezone and the deep border of the CCZ. There was noevidence for a waiting period in the SP. Axons invaded thedeveloping cortical layers as they began to form deep to theCCZ. From the combined results of the experiments using

the three different types of tracers, we conclude that theinitial corticothalamic projection arose from layer 5 cellsbetween P50 and P60. By P76 far more corticothalamicprojections were seen from layer 6, whereas those fromlayer 5 were reduced. Also at this age thalamic-projectingcells in cortical layers 5 and 6 began to form clusters andthalamic afferents showed the first signs of patchinesswithin the cortex, typical of the whisker area of the adult.

Technical considerations

Four main issues need to be addressed here. First, werethe appropriate somatosensory structures correctly tar-geted? For cortical injections, in older animals evokedpotentials to whisker stimulation were used to identifysomatosensory cortex. In all other cases correct placementwas confirmed by the fact that retrogradely labelled cellswere always found localised in VPM and Po. For placementof DiI in the thalamus, the application site was verifiedhistologically in sectioned tissue. In animals older thanP30, VPM can be identified (Leamey et al., 1996a). Inyounger animals, the identification was possible aftercomparison with the results of anterograde tracing of thetrigeminothalamic pathway (Leamey, 1996). The injectionsites of WGA-HRP were similarly verified.

A second issue of concern was the degree of spread oftracer at the injection site. Because DiI is transportedtranscellularly to radial glial cells, it was often difficult todiscern how much spread of label at the application site inthe thalamus was actually present. However the use ofsmall amounts of tracer in younger animals, combined withthe limitation of the region of analysis to the area that had

Fig. 10. a: Coronal section through the brain at postnatal day 50showing axons labelled after an injection of Neurobiotin in thesomatosensory cortex. By this age, a medially directed bundle of axonsleaves the descending corticofugal pathway (arrow) and some axonsare entering the thalamus. b: Higher power view of the regionindicated by the arrow in a showing axons entering the thalamus. D,

dorsal; M, medial. c,d: High-power views of axons from the regionshown in b. A collateral branch (arrow) from a corticofugal axonextends medially toward the thalamus (c). Axons entering the thala-mus are tipped with growth cones (arrows). Scale bars 5 1 mm in a,100 µm in b, 50 µm in c,d.


been identified as somatosensory cortex, from corticalinjections of WGA-HRP, minimised these problems,whereas the larger size of the nucleus in older animalsmade the spread of tracer less of a problem. Because axonsof Po pass through VPM the possibility that some of thelabelling from DiI reflected that associated with Po cannotbe ruled out, and thalamic injections of WGA-HRP gener-ally included Po. These issues are considered below in theinterpretation of the results. The very limited diffusion ofNeurobiotin and biocytin at cortical injection sites meansthat the spread of label was not a problem in theseexperiments.

Third, successful transport of label to the end of theaxons was confirmed in both the Neurobiotin/biocytin andDiI labelling experiments by the consistent observation of

labelled growth cones on the tips of axons at all agesincluded in this study. Also, if transport times for DiI werenot sufficient in older animals, inadequate labelling re-sulted in thalamic afferents apparently being confined todeeper cortical layers. These cases were not included.

Lastly, there was no evidence that transcellular trans-port of WGA-HRP, Neurobiotin or biocytin occurred. Thattransneuronal transport occurred in the DiI labelled mate-rial is a significant issue in the interpretation of the resultsand is discussed in detail below. Although such labelling isbelieved to have occurred, resulting in the spurious label-ling of cortical cells, it constituted a relatively smallproportion of the total label. It is believed to occur underconditions of close contact between apposing cell mem-branes (Godement et al., 1987).

Fig. 11. a,b:Adjacent stained (a) and unstained (b) sections throughthe ventroposteromedial nucleus (VPM) showing the label resultingfrom an injection of Neurobiotin in the somatosensory cortex atpostnatal day (P) 60. Label is in the form of a dorsoventrally orientedfinger running through the VPM and extending into the posterior

nucleus (Po) (arrow in b). The label is shown at high power in c and d.c,d: Labelled axons and cells in the VPM (c) and the Po (d) at P60.Retrogradely labelled cells (large arrows) and corticothalamic axonstipped by growth cones (small arrows) are present. Some have begunto branch (arrowhead in c). Scale bars 5 500 µm in a,b, 25 µm in c,d.


Distribution of developing thalamocorticalafferents: No waiting period

Thalamic afferents labelled from thalamic injectionsfirst reached the somatosensory cortex by 15 days afterbirth and this coincided with the time that thalamic cellscould first be retrogradely labelled from somatosensorycortex. From the time of their arrival in the cortex,thalamic afferents were found throughout the LPZ rightup to the deep border of the CCZ of the cortical plate,where they frequently terminated in growth cones. Noevidence was found for a waiting period in the superficialpart of the intermediate zone, identified in the presentstudy as the SP. However, Luskin and Shatz (1985a) on thebasis of cell birthday and cell fate determination andKostovic and Rakic (1990) on the basis of histologicalanalysis have defined an upper SP, which extends furtherdorsally into the zone of loosely packed cells at the base ofthe cortical plate. This raises the question of the identity ofcells in the LPZ in the wallaby.

The presence of a loosely packed layer of cells, oftenarranged in parallel arrays, beneath a relatively thin CCZappears to be a characteristic of cortical development inmarsupial mammals, and has been previously reported(Reynolds et al., 1985; Saunders et al., 1989; Sheng et al.,1991; Harman et al., 1995). Reynolds et al. (1985) identi-fied this region as the SP in animals up to P75, but clearlyby P60 it must be considered as part of the cortical plate insomatosensory cortex as layers 5 and 6 could be recognizedwithin it. Similarly in visual cortex cells in layers 5 and 6within the LPZ could be retrogradely labelled from tha-lamic injections (Sheng et al., 1991), even earlier, at P40.At earlier times, cells within the LPZ could not be identi-fied on morphological grounds. The results of thymidinebirthdating studies have shown that some of the cells thatwill form layer 6 of the visual cortex had already left theCCZ and were in the LPZ by P23 (Marotte and Sheng,1995). Layer 6 cells in somatosensory cortex are bornseveral days earlier than those in visual cortex (Leameyand Marotte, unpublished data). By extrapolation from thedata for visual cortex, layer 6 cells can be expected to be inthe LPZ in somatosensory cortex even earlier than P23.Thus, by P23, at least some if not all of the LPZ must beconsidered as part of the cortical plate.

At all times during development the afferents were foundimmediately deep to the CCZ, meaning that they invadedthe cortical layers from the time that each layer began toform deep to the CCZ, even before the layers could beidentified histologically. The same mode of developmenthas also been described in wallaby visual cortex (Sheng etal., 1991) and is consistent with results reported in boththe visual and somatosensory systems of rodent species(Naegle et al., 1988; Catalano et al., 1991, 1996; Erzurumluand Jhaveri, 1992; Kageyama and Robertson, 1993; Milleret al., 1993), but contrary to that reported in the visualsystem of the cat (Ghosh and Shatz, 1992) and monkey(Rakic, 1977).

One reason advanced to explain the apparent absence ofa waiting period of thalamic axons in the SP duringthalamocortical development in rodents, in contrast to itsreported presence in the cat and monkey, is the much morerapid developmental timecourse found in rodent species(Catalano et al., 1991). However, the lack of a waitingperiod in the wallaby, with its protracted development,longer than that of the cat and similar to that of the

monkey, suggests this is not an explanation of the observeddifference. In rodents, the period between conception andthe ingrowth of afferents to layer 4 occupied just 24–25days (22 days gestation with ingrowth to layer 4 at P1–2;Kageyama and Robertson, 1993). In the cat the periodseparating these events was 55–60 days (ingrowth to layer4 occurred between E55–60; Ghosh and Shatz, 1992),whereas in the wallaby it occupied almost 100 days (28days gestation with ingrowth to layer 4 occurring at P70;current study). In the cat, thalamocortical fibres werereported to wait in the SP for a period of 2 weeks (Ghoshand Shatz, 1992). Therefore, if a more protracted develop-mental pattern was related to the presence and length of awaiting period in the SP we would expect to see a period of3–4 weeks in the wallaby where thalamocortical axonswere confined to the SP. This was not observed.

It has been suggested that the necessity for a waitingperiod in the SP in species with a more protracted develop-ment may be related to the timing of arrival of thalamocor-tical fibres in the cortex in relation to the genesis of theirprimary target, layer 4 (Reinoso and O’Leary, 1990; Ghoshand Shatz, 1992; Erzurumlu and Jhaveri, 1992). In thevisual system of the cat, the geniculocortical fibres arrivedbelow the developing cortex at E36 (Ghosh and Shatz,1992), whereas layer 4 cells did not begin to be born untilafter this at E37 (Luskin and Shatz, 1985b) and were inthe cortical plate by around E55 (Shatz and Luskin, 1986).

In the cat, geniculocortical axons are reported not tobegin their invasion of the deeper cortical layers until E50,and it was suggested that the presence of layer 4 cells maytrigger the end of the waiting period (Ghosh and Shatz,1992). It was unclear whether the authors of this studybelieved that it was the arrival of layer 4 at the corticalplate that provided the trigger, or whether it was related tointeractions between the thalamocortical fibres and thelayer 4 cells because the cells migrated through the affer-ent fibres. In the wallaby, thalamocortical fibres arrived inthe developing somatosensory and visual cortex by P15(Sheng et al., 1991). There was a delay of around 2 weeksbetween their arrival and the commencement of the gene-sis of their primary target, layer 4 at P28 in somatosensorycortex (unpublished observations) and P32 in visual cortex(Marotte and Sheng, 1995). Despite this, the thalamocorti-cal axons were found to be present in the developing deepercortical layers before this time, as soon as the layers beganto appear deep to the CCZ. Thus, in this species, althoughthere was a time lag between the arrival of thalamocorticalfibres and the genesis and subsequent migration of layer 4cells, as in the cat, thalamocortical axons did not experi-ence a period of confinement in the SP. It is also difficult tounderstand why the thalamocortical fibres should wait forthe presence of layer 4 cells before they invade the corticalplate, when the earlier generated and differentiated layer6 is also a target of the thalamocortical fibres in all thesespecies (LeVay and Gilbert, 1976; Mayner, 1985; Jensenand Killackey, 1987; Sheng et al., 1991).

Interestingly, the ingrowth of thalamocortical axons intothe most superficial layer of the cortex, layer I, occurredseveral weeks ahead of the innervation of layer 4. At thistime layer 4 could still not be recognized beneath the CCZ.A similar delay between the innervation of layers 1 and 4has been reported in the cortex of the ferret (Clasca andSur, 1996).


The initial corticothalamic projection

The first cortical axons to reach the thalamus came fromthe cells of layer 5, at P60. We conclude that cells labelledby DiI before this time were not the result of retrogradelabelling from the thalamus, but rather were a result oftranscellular labelling, a common problem associated withthis method (Godement et al., 1987; Catalano et al., 1991;Miller et al., 1993; Clasca et al., 1995). There are severalarguments to support transcellular labelling. The pres-ence and location of these cells was variable both betweenand within animals. The occasional small groups of closelyapposed, labelled somata provided further support thattranscellular labelling did occur. Furthermore, HRP label-ling from VPM produced no retrogradely labelled corticalcells up to and including P50. At P60, as in the DiI labelledmaterial, a prominent row of labelled cells was consis-tently seen slightly deep to the CCZ in layer 5. Thiscoincided with the time that anterogradely labelled corti-cofugal projections were first traced into VPM. Someretrogradely labelled cells were also seen in layer 6 at thistime in DiI material, but the presence of these cells was notas consistent as those seen in layer 5. Because these werenot seen after retrograde transport of HRP from VPM tothe cortex, we conclude that they were the result oftranscellular labelling, as found at younger ages. By P70,two rows of labelled cells in layers 5 and 6 were consis-tently seen in the more lateral part of the somatosensorycortex. This is interpreted as indicating the formation of agenuine layer 6 projection to the thalamus by this age,because both of these layers were also labelled by HRPfrom the thalamus at a similar age. After this age theprojection from layer 6 predominated (see below).

The arrival of the cortical fibres in VPM at P60 wasconsiderably delayed in comparison to the arrival of thethalamic fibres in the somatosensory cortex. A similardelay between the arrival of thalamic axons in the cortexand the invasion of the thalamus by cortical axons hasbeen reported in the visual system in the wallaby (Shenget al., 1991) and in rodents (Miller et al., 1993) and in thesomatosensory and visual systems of the ferret (Clasca etal., 1995). The identification of the initial corticothalamicprojection in the somatosensory system as originatingfrom layer 5 is in agreement with findings for dorsalregions of the visual cortex in the wallaby (Sheng et al.,1991) and for somatosensory and visual cortex in the ferret(Clasca et al., 1995). Retrograde transport of DiI from theLGN to the visual cortex in the rat also revealed that thefirst substantial projection arose from layer 5 (Miller et al.,1993). It is possible that the smaller numbers of cellslabelled before this in the SP and layer 6 in the rodent werein fact the result of transneuronal labelling as found in thecurrent study and reported by Clasca et al. (1995). Theearlier arrival of the layer 5 neurons in VPM is somewhatsurprising considering their later neurogenesis comparedwith layer 6 cells. However, Clasca et al. (1995) demon-strated that the delayed arrival of layer 6 projections to thethalamus was a result of their slower axonal growth.

The conclusion that the initial corticothalamic projec-tion arose from layer 5 is in disagreement with studies inthe cat (McConnell et al., 1989) and rodent (De Carlos andO’Leary, 1992), which have reported that the first cortico-thalamic projection originated from the SP. Part of thereason for this disparity may be methodological, as both ofthese studies utilised DiI deposits in the thalamus like the

study of Miller et al. (1993), where transcellular labellingis likely to be a problem. This is supported by the results ofClasca et al. (1995) who, when they used tracers otherthan DiI in the ferret, a carnivore like the cat, found noevidence for an early projection to the thalamus fromthe SP.

Many of the corticothalamic axons were noted to invadethe thalamus as branches of axons that had already grownpast the thalamus and into the cerebral peduncle. This hasalso been reported by Clasca et al. (1995) in the ferret, andCabana and Martin (1986) in the opossum. The ingrowthof cortical fibres into the thalamus after they have ex-tended to more caudal regions has also been reported inthe visual system of the rabbit (Distel and Hollander,1980). It has been suggested that locally acting signalsmay trigger the formation of the collateral branches oncaudally projecting corticofugal axons to invade the thala-mus (Clasca et al., 1995). Because the collateral branchesthat will innervate the thalamus emerged in the vicinity ofthe perireticular nucleus (which itself sends a topographicprojection to the thalamus; Mitrofanis and Baker, 1993), itis possible that this nucleus plays a role in this.

In the wallaby, as in the rodent (Bourassa et al., 1995),the main corticothalamic projection to VPM in the adultarose from layer 6 cells (Mayner, 1985). This is consistentwith the finding here that much of the initial layer 5projection to VPM had disappeared within 3 weeks afterits inception, whereas the layer 6 projection was promi-nent by this time. The initial layer 5 projection to VPM inferrets was also greatly reduced in the adult (Clasca et al.,1995).

Appearance of whisker-related patterns

Patches of thalamic afferents in cortical layer 4 andcorresponding clusters of thalamic-projecting cell bodies incortical layer 6 reflecting the mystacial vibrissae havebeen demonstrated in the adult wallaby somatosensorycortex (Mayner, 1985). The first hint of patches of thalamo-cortical afferents in the emerging layer 4 in the presentstudy was coincident with the clustering of cell bodies andtheir dendrites in layers 5 and 6, at P76. This was slightlyearlier than what could be demonstrated by using SDH, atP85 (Waite et al., 1991). In the somatosensory thalamus ofthe wallaby there is also a well-defined whisker-relatedpattern (Leamey et al., 1996a). There, the onset of forma-tion of the branching arbors of trigeminothalamic affer-ents in dorsoventrally aligned fingers (Leamey et al., 1995)was not only coincident with the formation of similarlyarranged fingers of cells seen with Nissl stain, but alsowith the same pattern demonstrated with CO staining(Leamey et al., 1996a). This may indicate that CO stain-ing, which is more sensitive than SDH for demonstratingbarrels (Leamey, personal observation), would detect theonset of cortical barrel formation earlier than SDH. Alter-natively unlike in the thalamus, there is no cellularspecialization within cortical layer 4 corresponding to theSDH pattern (Waite et al., 1991). Thus, oxidative enzymehistochemistry, reflecting mitochondrial activity, may bedependent on further maturation of axon terminals and/orpost-synaptic components before the pattern can be demon-strated.

In the rat somatosensory cortex it has been reportedthat labelling of afferents with DiI revealed the appear-ance of the pattern in the first few postnatal days (Erzu-rumlu and Jhaveri, 1990) just prior to the emergence of the


pattern revealed by metabolic markers (Killackey andBelford, 1979). In our material labelled by DiI the clus-tered dendrites of retrogradely labelled cells did not allowus to decide whether the afferents, also labelled by DiI,were clustered. Although Erzurumlu and Jhaveri (1990)commented on the fact that the labelled dendrites ofcorticothalamic cells contributed to the labelling observedin tangential sections, they believed that the whisker-related patterns forming on P2 accurately reflected theorganisation of the thalamocortical fibres since the cortico-thalamic cells did not begin clustering until P4. Agmon etal. (1993) circumvented the complication of the presence ofretrogradely labelled cortical cells and their dendritic treesin the mouse by preparing oblique thalamocortical slicesbefore the insertion of the DiI crystal and clearly showedthe radial ingrowth of the thalamocortical fibres into layer4 and subsequent branching to form patches of termina-tions corresponding to the whiskers on P4. This was a dayafter the cellular pattern could be detected (Rice et al.,1985; Agmon et al., 1993) and suggested that the patternsobserved by Erzurumlu and Jhaveri (1990) in animals atP2 may actually have been the result of labelling in thedeep layers. More recently, Catalano et al. (1996) havereported in the rat that periodicities could be detected inthe density of the thalamocortical afferents by P1, and byP3 clusters of afferents could be seen within layer 4.

Despite these small differences in reported timings inpattern formation, presumably exacerbated by the shortdevelopmental period in rodents and perhaps by speciesdifferences, it is clear that the vibrissae-related pattern isreflected early in the development of thalamocortical con-nections and arises as layer 4 begins to differentiate fromthe cortical plate (Catalano et al., 1996) or even earlier (seeAgmon et al., 1993).

Agmon et al. (1993) commented on the tangential pas-sage of thalamic axons within the deep tier of terminations(in lower layer 5 and upper layer 6) which contrasted withthe punctate radial ingrowth to layer 4 from the deep tierof terminations in mouse cortex. This led to the suggestionthat a precise topographic map may be set up firstly in thedeep layers, before fibres grow into layer 4. This sugges-tion is supported by the observation made here that thecells labelled retrogradely in the thalamus following corti-cal injections of similar size, became progressively moreclosely packed with increasing age, and by P60 were in aformation which resembled the fingers which were re-ported as beginning to emerge in CO and Nissl stainedmaterial at this stage (Leamey et al., 1996a). Since, in thewallaby, the corticothalamic projection first arose at P60,and the cells from the infragranular layers formed clustersbetween P70 and P76, it is possible that interactionsbetween the corticothalamic axons and the thalamic cellsmay contribute to the emergence of clustering in thecortical projections cells.

Another possibility is that such interactions could occurat the level of the cortex. If the discrete topographyemerging in the thalamic cells projecting to the cortex atthis time was reflected in their axons in the cortex, thenthese axons could interact with the cells of layer 5 and 6 toimpose clustering. Filling of small numbers of thalamocor-tical axons, uncontaminated by retrogradely labelled den-drites of cells would help answer this question. Further tothis is the observation that the clustering of the corticotha-lamic cells was coincident with the appearance of a com-plete whisker-related pattern in the thalamic cells (Leamey

et al., 1996a). Since evoked potentials could first berecorded in vivo from the somatosensory cortex followingperipheral stimulation at P62 (Mark and Waite, 1995), it ispossible that activity may play a role in this process.

Timing of pattern formationin the wallaby trigeminal pathway:

Comparison with rodents

This is the third in a series of studies which haveinvestigated the development of the wallaby trigeminalpathway. This series was undertaken with the aim of usingto advantage the protracted developmental timecourse ofthe wallaby, to address the question of the factors control-ling the onset of whisker-related pattern formation. Specifi-cally we were interested to know whether the initiation ofwhisker-related clusters in the brainstem, thalamus andcortex was controlled by the afferent fibres arriving at eachlevel or by the maturation of the target cells. Studies inrodents have shown that there is a closely timed sequenceof pattern development in the brainstem, thalamus andcortex (Belford and Killackey, 1979). This has led to thesuggestion that a peripherally derived signal, carried byafferent fibres, may spread rapidly along the pathway,initiating clustering at each level (reviewed by Killackey,1985). However, the short duration of neural developmentin the rat, where less than a week separates afferentarrival and pattern formation at all central levels, hasmade it difficult to separate the contribution of afferentfibres and target tissues. In vitro cocultures of rat trigemi-nal ganglion and brainstem have indicated that the arbori-zation of afferents in the trigeminal nucleus is dependenton the maturity of the target cells not the afferent fibres(Erzurumlu et al., 1993).

Our results in the wallaby, like those in rodents, haveshown that clusters in the brainstem, thalamus and cortexdeveloped in sequence. However, the onset of clusteringalong the wallaby pathway was separated by weeks (brain-stem P40, thalamus P50, cortex P76) rather than the fewdays seen in the rat. This suggests that, rather than asingle peripheral signal sweeping through the pathway,initiation of pattern formation is likely to be independentlycontrolled at each level. Several factors indicate that themost probable candidates for such control are the targetcells rather than the afferent fibres. Firstly, each levelshowed long delays between the arrival of the afferentinput and the start of pattern formation. In the brainstemthis delay was around 40 days (Waite et al., 1994), in thethalamus 35 days (Leamey et al., 1996a) and the presentstudy has shown a delay of about 60 days from arrival ofthe first afferent fibres (P15) to the initiation of patterns(P76). Secondly, even though afferents first reached thethalamus (Leamey et al., 1996a) and the cortex at thesame time, there was a delay in the onset of patternformation between these two regions. Finally, in thecortex, the close coincidence in the appearance of a recog-nizable layer 4 (about P70–72) and the onset of clustering(P76) suggests maturation of the target cells in layer 4provides an essential signal to the afferent terminals.

ACKNOWLEDGMENTS

We thank Dr. A.C. James for statistical analysis of thehead lengths of animals of known age, Professor R.F. Markfor criticism of the manuscript, Ms. A. Devlin and Mr. K.Williams for care of the wallaby colony, Dr. R. Meischke for


veterinary advice, Mr. J. Wilson for photography, and Mrs.M. Donohue for word processing.

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