Development 99, 577-587 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

577

Pathfinding by dorsal column axons in the spinal cord of the frog tadpole

NIGEL HOLDER1, J. D. W. CLARKE12 and DAVID TONGE2

Anatomy & Human Biology1 and Physiology2 Departments, King's College London, Strand, London WC2R 2LS, UK

Summary

Sensory fibres from dorsal root ganglia (DRG) enterthe spinal cord and run within a clearly definedipsilateral pathway, the dorsal column, which lies inthe dorsal funiculus. We have examined the charac-teristics of this pathway as a defined substrate fordorsal column axons in Rana temporaria tadpoles byrotating the thoracic spinal cord through 180° fromdorsal to ventral. Using HRP as a neuronal tracer weestablish that many dorsal column axons from thehindlimb locate the ipsilateral or contralateral dorsalcolumn pathway in the rotated cord. Other axonslocate and grow caudally down the contralateral

dorsal column returning to the lumbar region. Axonsof the dorsal column never take an inappropriatepathway except at the transection sites where theynegotiate abnormal routes to reach the contralateralor ipsilateral dorsal columns in normally positionedor rotated cord. The results demonstrate that thedorsal columns act as highly specific pathways foraxons from DRG neurones but the axons' interactionswith the pathway do not control the craniocaudal orleft-right options for growth.

Key words: neurone, specificity, growth, guidance,substrate, Rana temporaria.

Introduction

The generation of neuronal specificity during devel-opment and regeneration can be examined broadly intwo stages; the growth of an axon to the near vicinityof its target cell and the creation of a functionalsynapse with that cell. In the first phase the axon mustnegotiate a terrain made up of other cell types, otheraxons and extracellular materials that may somehowmark out a course that governs directed axon growth.In any attempt to understand the basis of neuronalspecificity, therefore, it is crucial to establish thecharacteristics that define axon pathways in the cen-tral and peripheral nervous systems (Katz, Lasek &Nauta, 1980).

In this paper we describe the results of an exper-iment designed to examine the pathfinding ability ofaxons in the spinal cord of the developing frogtadpole. The purpose was to confront growing axonswith a region of spinal cord that they would notnormally encounter in order to assess whether axonscould seek and find preferred pathways. Results ofsimilar experiments in which developing eye primor-dia from embryonic frogs were grafted to variouspositions along the forming neural tube show thataxons from retinal ganglion cells grow along defined

pathways within the spinal cord (Constantine-Paton& Capranica, 1976; Katz & Lasek, 1979). In thepresent study, however, rather than transplanting theneurones whose axons' trajectories are being assessedwe have altered the spatial relationship of the tissuethrough which the axons will grow; in this case byrotating the thoracic region of the spinal cord through180° from dorsal to ventral. The operation is per-formed at a stage at which axons are still being addedto dorsal columns and are known to be able to growacross a completely transected cord (Clarke, Tonge &Holder, 1986); although it is not known in this or thepresent study whether these axons are regeneratingor developing. The results demonstrate that axonsfrom lumbar dorsal root ganglia neurones, which aregrowing cranially along the dorsal column, selectpredetermined pathways which exist within the devel-oping spinal cord.

Materials and methods

All experiments were performed on tadpoles of Ranatemporaria obtained from commercial suppliers or fromeggs stripped from gravid females and fertilized in vitro.Tadpoles were raised in standing tap water at 17-20 °C andwere fed daily on a mix of rabbit chow, gelatin and agar.

578 N. Holder, J. D. W. Clarke and D. Tonge

OperationsTadpoles at stage V or VI (stages of Taylor & Kollros (1946)for Rana pipiens) were anaesthetized in a solution of MS222(Sigma) and removed from the water. The thoracic spinalcord was exposed by a dorsal laminectomy and the dorsalroots were located and identified (Fig. 1A). After severingthe dorsal and ventral roots at segments 4, 5 and 6 with afine needle, the intact spinal cord was lifted with a hook-shaped needle and cut with fine iridectomy scissors betweenroots 3 and 4. The cord was then cut more caudally betweenroots 5 and 6 and the thoracic piece was removed, rotatedthrough 180° dorsal to ventral and replaced in situ. Thewound was then flushed with fresh Ringer solution, coveredwith a flap of skin and the animal was placed in a cooledincubator for l h to facilitate healing. The tadpoles werethen returned to a shallow tank of aerated tap water.

Control operations involved the same procedure de-scribed above except that the isolated piece of spinal cordwas replaced in situ in a normal orientation. In addition,several normal tadpoles at relevant stages were selected forfurther analysis of dorsal column development.

AnalysisAnimals were taken for analysis either two weeks or onemonth after surgery. Lumbar dorsal roots were labelled byapplying horseradish peroxidase (HRP, Boehringer Grade1) to the end of the right limb which was amputated at theupper thigh. After 24 to 48 h the animals were reanaesthe-tized and the central nervous system (CNS) removed andfixed by immersion in 2-5% glutaraldehyde in 0-1 M-phosphate buffer (pH7-4) for 2-3 h. The entire CNS wasthen embedded in a gelatin/albumin mixture and seriallysectioned in either transverse or longitudinal planes at50fim on a vibratome. The sections were then reacted with3,3'-diaminobenzidine (DAB) (0-08% by mass in phos-phate buffer pH7-4) and a 0-01% solution of H2O2.Sections were counterstained with cresyl violet beforecover-slipping.

One control animal was analysed in the same way as theexperimentals; the remainder were labelled by crushingHRP onto severed dorsal roots of isolated CNS's fromwhich the brain had been removed rostral to the cerebel-lum. The preparations were then left for 12-24 h in frogRinger at 10 °C after which time they were fixed and treatedwith DAB and H2O2 as wholemounts before being waxembedded and sectioned transversely at 1011m. This pro-cedure reveals dorsal column axons as effectively as the invivo methods described above.

Because of the small size of the tadpoles we found itimpossible to reliably perfuse the animals and remove redblood cells (RBC's) from the capillaries. Therefore, theperoxidase staining is contaminated with stained RBC's andalso the capillary endothelial cells show some positiveperoxidase activity. Nonetheless, under the microscopethis background labelling was clearly distinguishable fromHRP-labelled axons which, therefore, could be easilyfollowed from section to section. In the black and whitephotomicrographs included here this difference betweenaxonal and nonaxonal labelling is difficult to discern and isfurther hindered by the presence of dark melanin granulesin some of the cells within the spinal cord and in the

meninges surrounding it (Fig. IB). Therefore, most of theresults are presented as camera-lucida drawings.

Results

(A) SurvivalA total of over 60 tadpoles was operated on and thegreat majority of these recovered from the initialsurgery. However, during the subsequent days overhalf of the tadpoles died, mainly, it seemed, from aninability to digest food that had been swallowed.Those animals that survived began to move their tailsin response to gentle stimulation and often swam incircles and upside down. Over the next weeks thetadpoles began to feed and to carry on developing,although at a slower rate than normal. One monthafter surgery 13 experimental and 6 control tadpoleswere analysed with HRP and an additional 3 exper-imental cases were analysed two weeks after surgery.These 3 cases showed no obvious differences fromthose analysed after longer survival times and are notdiscussed separately.

Of the 16 experimental animals all but one showedclear HRP in the dorsal and ventral roots, the lumbardorsal columns and motor neurones of the lumbarlateral motor columns (Fig. IB). These 15 cases formthe basis of the experimental results described belowand summarized in Table 1.

(B) Normal anatomy of the dorsal columns inR. temporariaFrom control and normal animals it is evident that theaxons from the dorsal root ganglion (DRG) neuronesin R. temporaria take basically similar paths and giveoff collaterals in similar positions to those in otherfrogs, such as R. esculenta (Antal, Tornai & Szekely,1980), R. catesbeiana (Joseph & Whitlock, 1968;Forehand & Farel, 1982; Jhaveri & Frank, 1983;Liuzzi, Beattie & Bresnahan, 1984), and Xenopuslaevis (Nikundiwe, De Boer-Van Huizen &Donkelaar, 1982). After entering the cord, axonsrun cranially or caudally in the ipsilateral dorsalcolumns which are situated in the dorsal funiculus(Fig. 1). In the lumbar region two collateral subfieldsare seen; the dorsal neuropil (terminology afterJhaveri & Frank, 1983) in the region of the dorsalhorn and the less-dense ventral neuropil lying in theintermediate grey matter dorsal to the lateral motorcolumn. In many cases clear overlap was seen be-tween collaterals from dorsal column axons anddendrites from the motor neurones (Fig. IB). In thethoracic region the dorsal column was narrower andlay more medially and few collaterals were seen ineither dorsal or ventral neuropil positions (Fig. 2A).In the brachial and cervical cord and hindbrain thedistribution of the lumbar DRG axons in the dorsal

Pathfinding by spinal cord axons 579

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Fig. 1. (A) Drawing of the spinal cord and medulla of a Rana temporaria tadpole at approximately stage XIII showingthe position of the graft and the dorsal roots. The levels of the sections shown in the remainder of this figure areindicated by the arrows. (B) The appearance of the dorsal column in the lumbar region in a normal animal followingHRP application to the cut limb stump. Endogenous peroxidase activity of red blood cells (large solid arrow) andendothelial cells lining capillaries (small solid arrows) is shown following the DAB reaction. Pigment within segmentalmotoneurones (open arrow) is also evident; in black and white pictures melanocytes appear to obscure axon staining.dr, dorsal root; dc, dorsal column; dn, dorsal neuropil; vn, ventral neuropil; mn, motor neurones; D, dorsal; V, ventral;R, right; L, left. Bar, 200/an. (C-F) Representative sections from levels of spinal cord (caudal to rostral) from a controltadpole in which the cord segment shown in A had been removed and replaced in normal orientation. The levels of thesections are shown in A. Note that the dorsal column axons remain on the host dorsal side through the transection andinto the centre of the graft, sc, spinal canal; e, ependymal region; gm, grey matter; wm, white matter. Bar in C is200 /an and is the same magnification as D, E and F.

580 N. Holder, J. D. W. Clarke and D. Tonge

B

Fig. 2. Camera-lucida drawings to show the positions ofdorsal column axons labelled with HRP in the dorsalmidthoracic region of (A) unoperated spinal cord;(B) control, unrotated gTaft segments of spinal cord;(C) dorsoventrally rotated graft segments of spinal cord.In A and B dorsal is up, but in C the drawing has beenrotated through 180° for easy comparison, thus the hostdorsal is down. In each case the axons are present in thesame area of dorsal white matter, just lateral to themidline. In the dorsoventrally rotated cord (C), someaxons occupy more lateral positions than in the controls;these positions would normally contain axons fromthoracic and brachial dorsal roots. In C labelled axons arepresent on both sides of the midline and a few axons canbe seen invading the dorsal neuropil. g, boundary of thegrey matter; nc, neural canal. Scale bar, 100^m.

column is as described previously (Clarke et al.1986).

(C) ControlsIn the six controls examined five were well filled withHRP but in the sixth the axons were too faintlystained to analyse. The overall result in each of thecontrols was the same with axons crossing the first cutand remaining on the host dorsal side of the cord inthe normal dorsal column position for lumbar DRGaxons in the thoracic region. Some disorganization ofthe dorsal column was seen at the first transectionsite, although this was not as extensive as in theexperimental group; it involved axons moving onlyinto the dorsal grey matter immediately ventral to thenormal dorsal column and axons were never seen

moving either contralaterally or towards the hostventral position of the cord (Fig. 1C-F). As axonsgrew through the thoracic piece of cord they becamefewer and moved medially, to the normal midlineposition (Fig. 2B), reaching the second cut in all fivecases. However, as with the experimentals describedbelow, only one tadpole showed axons which success-fully crossed the second transection site, after whichthey grew onto the dorsal column nucleus regionlateral to the obex. In each case the disruption to thegrey matter at the transection site was minimal, theneural canal was patent at both ends of the initiallyisolated cord and the diameter of the healed regionswas close to that of the cord on either side.

(D) Overall anatomy of the cord in experimentalanimals

All of the experimental animals showed normaloverall anatomy in the regions of the cord caudal androstral to the rotated piece. Within the body of thegraft the normal organization of the white and greymatter was well preserved but was clearly reversed byapproximately 180° dorsal to ventral relative to thesurrounding tissues (Fig. 3E). Normally the whitematter is much thinner dorsally than laterally andventrally, but in the rotated grafts the white matterwas thinnest ventrally. Also, the motoneurones thatinnervate the segmental muscles, which are easilyidentified as they contain dark melanin granules(Fig. IB), were present in the dorsal grey matter ofthe grafts in contrast to their normal ventral pos-itions. At the cut ends of the graft the basic cordstructure was lost (Fig. 3B-D): Generally the greymatter remained central although it was often in-vaded by large bundles of white matter (Fig. 3C,D)and sometimes the white matter did not completelysurround the grey matter. The diameter of the cord inthe transection sites was invariably smaller than thaton either side and, in a few cases, narrowed to nomore than a quarter of the normal cord diameter. Inthese instances no spinal canal was present. Onaverage the disorganization of the cord at the transec-tion site lasted for 250 to 350/.an. In some caseshealing at one end of the graft was clearly better thanthe average and alterations in overall anatomy in-volved only a slight reduction in diameter of the cordand minor alterations in outline of the grey matter.These alterations were evident for only 100 nm andthe spinal canal was clearly continuous. In thesedisrupted regions it was not possible to say whichtissues originated from normally ventral or normallydorsal positions.

No well-defined ventral or dorsal roots could beseen attached to the body of the graft at the time offixation. However, the sectioned material revealedthat in some cases a few poorly developed roots were

Pathfinding by spinal cord axons 581

present, but as the sections were only counterstainedwith cresyl violet it was not possible to tell if theseroots contained axons. It is possible that these smallroots may have contributed a few sensory axons(which would not be labelled by HRP) to the whitematter in the graft. Local propriospinal axons andmotoneurone axons would also probably be present,but the major component is likely to be from long-projecting axons that entered the graft either fromthe nongrafted segments of the spinal cord or fromthe brain.

(E) Pathways of DRG neurone axons in the cordBefore describing the patterns of axon pathways indetail it would be useful to make some general points.Except through the transection sites axons of DRGneurones were never located in pathways other thanthe dorsal columns. At the caudal border of therotated region of cord HRP-nlled axons from thelumbar cord were found to have grown along one ofthree routes; (1) they were found to have crossed intothe graft and to have grown along the graft dorsalcolumns which now run ventrally relative to the hosttissue; (2) some were found to have made a U-turncaudal to the graft tissue, crossing to the contralateralside of the cord and then projecting caudally in thecontralateral dorsal column; (3) in some the axonsended in or at the border of the transection site. Oneor a combination of these outcomes was seen in eachexperimental animal (Table 1). Most axons thatentered the graft dorsal column were not seen to crossthe cranial transection site; however, in one remark-able case the axons returned to the dorsal side of the

brachial spinal cord and projected, in a normal dorsalcolumn position, to the dorsal column nucleus, thenormal target site for such axons (Fig. 6).

(F) Detailed analysis of axon pathwaysThe DRG axons of the fifteen experimental animalsall showed normal dorsal column projections andcollateral fields in the dorsal and ventral neuropil inthe ipsilateral lumbar cord. As the axons approachedthe rotated piece of cord in the midthoracic region thedorsal and ventral collateral fields faded out and thedorsal column formed a clear and sickle-shaped tract,somewhat wider medially than laterally.

A number of cords had axons in the contralateraldorsal column caudal to the graft (Figs 3, 4). Suchaxons were present in increasing numbers as thegrafted piece was approached from the lumbar re-gions and occasional collaterals were seen from thesecontralaterally projecting axons in both dorsal andventral neuropil regions in the lumbar area. It isevident from the sections that these axons in thecontralateral dorsal column caudal to the graft werecontinuous with those in the ispilateral dorsal columnin this region; that is they crossed the midline at thefirst transection site and then projected caudally. Inone case this was the only route taken by axons thatreached the transection site whereas in the othercases axons also entered the graft (Table 1, Fig. 3).

In all cases where axons projected into the graft orcaudally in the contralateral dorsal column there wasno obvious alteration in axon trajectory further than100/on from the first transection, and in several casesthe change in trajectory occurred within one 50 fjm

Table 1. Summary of results

Case TS/LS*

Axons descendcontralaterallyfrom first cut

Axons crossfirst cut

Axons moveto graft dorsal

Axons runlength of graft

Axons crosssecond cut

1234t5t6789

10l i t12131415

Positivecases

TSTSTSTSTSTSTSTSTSLSTSTSTSLSLS

*TS, transverse section analysis; LS, longitudinal section analysis,t Fixed two weeks after surgery.

582 N. Holder, J. D. W. Clarke and D. Tonge

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Fig. 3. Photomicrographs and camera-lucida drawings of HRP-filled axons from sections of spinal cord from a tadpoleone month after rotation of the thoracic cord region. HRP was applied to the cut right hind limb. Levels of sectionsshown in A through E are indicated on the inset diagrammatic view of the cord. Numbers indicate distance in ^m fromthe nearest transection. (A) Just caudal to the first transection, axons from the ipsilateral dorsal column can be seenspreading to the contralateral dorsal column where they run caudally. Filled axons also run to the host ventral positionas the transection is crossed through 200/zm of cord (B-D). Axons locate the dorsal column of the graft on bothipsilateral and contralateral sides in the rotated thoracic region (E). This sequence is case 2 (Table 1). c, contralateraldorsal column caudal to the transection; D, dorsal side of cord; V, ventral side of cord; R, right side of cord; L, left sideof cord. Scale bar, 200 fim.

Pathfinding by spinal cord axons 583

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section. This was particularly clearly seen in the singleexample sectioned longitudinally which showed axongrowth to the dorsal side of the graft (Fig. 5). In thisinstance, axons were not seen to move from thedorsal column until the first transection was reached.Indeed, they appeared to follow the line where thecord narrows at the transection.

Axons within the healed transection appeared torelocate in fascicles. Although instances of individualaxons projecting in abnormal directions were seen,in general the axons traversed the cord as a group(Figs 3, 5, 6). The trajectory of growth was direct insome cases and tortuous in others. Commonly axonsleft their normal dorsal position and grew amongthe cells of the dorsal-medial grey. Some axons,although clearly the minority, moved through thelateral white matter to the graft dorsal column. Whencontralateral axon growth occurred in the cord caudalto the transection, some axons grew through thecontralateral grey to enter the contralateral dorsalcolumn of the graft.

When axons were present within the graft theywere never seen in any region other than the dorsalcolumn; indeed, most were found on the ipsilateral

E-D-C >•--<

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side which corresponds to the contralateral dorsalcolumn following rotation. As they progressed ros-trally the number of axons decreased although, in allbut one case, some axons reached the second transec-tion (Table 1). On the whole the rostral transectionhealed better than the caudal one and the centralcanal was patent through the wound site in themajority of cases. Nonetheless, of the eight examplesin which axons reached the rostral cut in only one didthey successfully negotiate a path back to the hostdorsal side (Fig. 6), although in several cases thelimited number of axons began to enter the dorsalgrey.

Discussion

The results presented in this paper demonstrate theability of a particular group of axons to select adefined pathway within the spinal cord. This abilityhas also been demonstrated for misplaced retinalganglion cell axons (Constantine-Paton & Capranica,1976; Katz & Lasek, 1979) and Mauthner cell axons(Katz & Lasek, 1981), and implied from experiments

584 N. Holder, J. D. W. Clarke and D. Tonge

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Fig. 4. A sequence of sections from case 3 (Table 1) inwhich the majority of filled axons run contralaterally andcaudally at the first transection with the result that axonsare evident in the left dorsal column in the lumbar region(arrow). A few axons grew towards the dorsal side of therotated thoracic cord but none entered the body of thegraft. Inset indicates the level of sections as in Fig. 3.Scale bar, 200 /zm.

examining the regeneration of spinal axons in thegoldfish (Bunt & Fill-Moebs, 1984). In the presentstudy, frog tadpole DRG axons, when faced with anenvironment with reversed dorsal-ventral axial po-larity, negotiate an abnormal path through the tissueat the wound and locate the appropriate region ofspinal cord in the rotated piece. Once in the graft,axons originating caudal to the cut, without excep-tion, grew along the dorsal column in the graft; noaxons were seen in any other region of white matter.It is clear, therefore, that some form of matchingmust exist between the axons located in the dorsal

columns and their environment within the spinalcord.

It is of considerable interest to establish whatunderlies this matching. With respect to the dorsalcolumns, insights into this question can be gainedfrom examination of the normal neuroanatomy of theDRG cell axons, the growth of axons from trans-planted retinal ganglion cells in the spinal cord andthe results described here. It is evident from neuro-anatomical studies on various frogs (see Joseph &Whitlock, 1968; Antal et al. 1980; and review byEbbesson, 1976) that DRG axons run caudally androstrally in the dorsal column following entry throughthe dorsal root, and this is true also for R. temporaria(Clarke et al. 1986). This means that axons in thenormal dorsal column have a choice of growingcaudally or rostrally and, whatever the basis of themechanisms outlining dorsal column pathways, it isnot prohibitive in terms of rostrocaudal polarity.Consistent with this property of the dorsal column isthe result of Constantine-Paton & Capranica (1976)who showed that embryonic retinal ganglion cellstransplanted to the ear position in leopard frogs sendaxons caudally in the dorsal column. These authorsargued that this directed growth in a caudal dorso-lateral direction was an intrinsic property of theaxons, but this need not be the case if one considersthe dorsal column to be a general substrate pathwayfor certain classes of axon of which DRG cell andretinal ganglion cells are members. This generalinterpretation is shared by Katz & Lasek (1979) whoalso grafted eye primordia but, in this case, placedthem towards the tail and obtained rostral growth ofretinal ganglion cell axons in the spinal cord ofXenopus. The trajectories of the axons in theseexperiments were not clearly in the dorsal columns,however; rather the axons were located in what theauthors termed the external and internal sensorytracts. One of these may correspond to the tract ofLissauer, which is also seen in R. temporaria (Clarkeet al. 1986). However, it is difficult to tell whetherthese external and internal sensory tracts correspondto the dorsal columns of R. temporaria partly becauseKatz and Lasek analysed their experimental cases atstages earlier than our initial cord rotations wereperformed, although in the later stages the positionsof the dorsal columns in Xenopus are basically thesame as in R. temporaria (see Nikundiwe et al. 1982).Nonetheless, the eye transplant experiments demon-strate that retinal ganglion cell axons can growrostrally or caudally in the spinal cord. Finally, in thepresent results axons of DRG neurones that encoun-ter a cord reversed in polarity can project eitherrostrally into the graft or caudally in the contralateraldorsal column.

Pathfinding by spinal cord axons 585

Not only does the dorsal column allow rostral andcaudal axon growth but it can also provide a substratepathway for axons from DRG neurones on thecontralateral side. With the exception of a few axonsin the hindbrain of R. esculenta (Antal et al. 1980),such decussation does not occur from the dorsalcolumns in frogs. Nonetheless, in the present studysuch contralateral growth occurred regularly at twopoints. At the first of these, axons reaching thetransection site at the caudal end of the graft crossedthe dorsal midline and grew caudally along thecontralateral dorsal column. In the second case manyaxons from the lumbar region remained ipsilateral,grew through the disorganized tissue at the transec-tion site and located in the dorsal column on thegraft's dorsal margin (Figs 2C, 3, 5, 6). Because of the180° rotation this dorsal column is that which wouldnormally lie contralaterally. In addition to this lo-cation a minority of axons was seen in the adjacentgraft dorsal column, having arrived from either ipsi-or contralateral positions in the lumbar cord. Follow-ing simple cord transections many dorsal columnaxons also emerge from the transection site contra-laterally and grow along their contralateral pathway(Clarke et al. 1986). These results demonstrate con-clusively that axons from DRG neurones are notprohibited from growing along the contralateral dor-sal column. Of course, in unoperated spinal cordsdorsal column axons do not cross the midline to growalong their contralateral pathway, so what normallyprevents them from doing so? One of the strikingfeatures of all the operated spinal cords (rotations,controls and simple transections) is the absence ofany dorsal column axons growing along the dorsal

midline between the areas normally occupied by themost medial dorsal column axons. Sections preparedfor transmission electron microscopy (unpublishedobservations) reveal this area to contain a fewunidentified cells (some of which are presumablyRohon-Beard cells (Roberts & Clarke, 1982)), a fewaxons (some myelinated), radial glia processes andlarge extracellular spaces, but no more substantialphysical barrier to stop axon growth across or alongthis region. Some feature of this terrain must presum-ably prevent the invasion of the dorsal column axonsin normal animals.

In addition to these conclusions with respect to thenature of the information carried by the dorsalcolumn as a substrate pathway, a key feature of theresults presented here is that no labelled axons werelocated in the body of the grafted cord in any regionof white matter other than the dorsal column. In mostcases, many of the axons took up the medial dorsalcolumn location typical of axons of the lumbar DRGneurones, although a few axons were found justlateral to this (Fig. 2C) in a position normally occu-pied by axons from thoracic and brachial DRG's(Antal et al. 1980). Thus, there is a strong matchingfor DRG neurone axons to the dorsal column as asubstrate pathway, but the pathways provide noobligatory information for the direction of axongrowth in terms of laterality or the rostrocaudal axis.

The analysis of results using HRP at the two timepoints following surgery revealed basically the sameresult and did not give any clues as to the mechanismsby which axons growing into the graft negotiated apath into the misplaced dorsal columns. One possi-bility is that the initial growth of axons into the graft is

B

Fig. 5. (A) Montage of longitudinal sections through the first transection site (case 10, Table 1) showing HRP-filledaxons (arrows) of the dorsal column crossing to the dorsal side of the rotated thoracic cord. (B) Composite drawingshowing all HRP-filled axons from the complete set of 50/an longitudinal sections of case 10. dn, dorsal neuropilcollateral field; dc, dorsal column; D, dorsal; V, ventral surfaces of the spinal cord. Scale bar, 200 fan.

586 N. Holder, J. D. W. Clarke and D. TongeFig. 6. Sequence of the one case (case 1, Table1) in which axons from the right dorsal columnran ipsilaterally through the graft and reachedthe target dorsal column nucleus in the medulla(J). No axons gTew to the contralateral side ofthe cord but, because of the rotation, they aregrowing through contralateral dorsal column inthe rotated thoracic region. D, dorsal side ofcord; V, ventral side of cord; R, right side ofcord; L, left side of cord. Inset indicates levelsof sections as in Fig. 3. Scale bar, 200 fim.

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Pathfinding by spinal cord axons 587

randomly directed and that this is followed by aretraction of those axons that take an inappropriatepath. If this mechanism operates it must be com-pleted within the first two weeks of axon growth forno axons were inappropriately placed after 14 days. Asecond possibility is that the dorsal columns maysecrete a tropic factor capable of attracting theappropriate axons from long range. Long-range at-traction may not be necessary, however, if the healingprocesses of the graft draw the normal and misplaceddorsal columns into close proximity. This would allowthe axons to follow local pathway cues directly intotheir appropriate locations. A third possibility is thatgrowing axons may be following cues left by dorsalcolumn axons that degenerate immediately aftersurgery. In addition, the current results do not clarifywhether the axons traversing the cuts are regenerat-ing or whether they are derived from neurones bornat or after the time of cord rotation in which case theywould still be developing. At the time of grafting,stages V and VI, some axons have already grownalong the dorsal column pathway to the obex (Clarkeet al. 1986) and, therefore, will have been severed bythe operation. However, the full adult complement ofdorsal column axons probably has not been reachedat this stage.

In conclusion, our results strongly support thenotion that at least some tracts of axons in the spinalcord in particular (and the CNS in general) arecreated by a matching of axons to a preferred pathlocated in the substrate. This conclusion is consistentwith several other studies of axon guidance in theCNS of anurans (see review by Katz et al. 1980).Because of its accessibility and the ability of axons togrow across transected cords at relatively late stagesof morphogenesis, the system holds considerablepromise for the further elucidation of the nature ofsubstrate axon guidance cues in the CNS.

We wish to thank Donna Fekete and Philip Gordon-Weeks for their comments on the manuscript and ActionResearch for the Crippled Child for financial support.

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(Accepted 16 December 1986)