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CHAPTER 6

Regenerating slow motor axons navigate toward their ownregions within adult skeletal muscles of the rat

L.C. Wang, J.C.V.M. Copray, N. Brouwer, M.F. Meek and D. KernellDepartment of Medical Physiology, University of Groningen,

P.O. Box 196, 9700 AD Groningen, The Netherlands.

Submitted for publication, 2001

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Summary

1. In limb muscles of adult rats, measurements were made of how well thetopographic patterns of fibre type distribution (“regionalization”) wererestored after denervation and reinnervation..

2. In an initial operation, the sciatic nerve was unilaterally sectioned andre-ited above the knee. Following a survival time of 21 weeks, muscleswere removed from both lower hindlimbs after determining their intra-limb position (extensor digitorum longus, flexor digitorum and hallucislongus, gastrocnemius medialis, peroneus longus, and tibialis anterior).

3. For each muscle, cryostat sections from seven equidistant proximo-distallevels were stained for myofibrillar ATPase. A distinction was madebeween type I (“slow”) and II (“fast”) fibres. The muscle outline and theposition of each type I fibre was determined.

4. In comparison to contralateral controls, reinnervated muscles consistentlyshowed a very high number of “close type I pairs”with inter-fibre dis-tances< 50 µm (data normalized in relation to total number of type Ifibres).

5. Within each cross-section, the direction of type I fibre accumulation wasquantified; this “type I fibre vector angle” was, on average, almost iden-tical in reinnervated muscles and contralateral controls.

6. On average, a significant proximo-distal decline of type I fibre density(fibres / mm2) was found for each muscle species, in reinnervated casesas well as in controls.

7. In conclusion, the mean directions of type I fibre regionalization reco-vered after reinnervation, and there were also signs of widespread re-specification of muscle fibre types by the ingrowing nerve fibres (increaseof “close type I pairs”). The results imply that “slow” motor axons pre-ferentially grew back toward their own “slow” muscle regions,indicatingthe presence of (molecular) mechanisms for appropriate axonal guid-ance in the adult mammal.

Key words: Skeletal muscle; Reinnervation; Axon guidance; Muscle fibre type; Fibretype grouping; Rat

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Introduction

In mammals (including man), cut periph-eral nerve fibres can regenerate and re-establish functional connections. A ma-jor problem is, however, that adult motoraxons lack the capacity for finding theircorrect target muscle (Bodine-Fowler etal., 1997; Brushart & Mesulam, 1980;Gillespie et al., 1986; Thomas et al., 1987;Weiss and Hoag, 1946). In the presentstudy we will describe results indicatingthat, within the more limited spatial con-text of single muscles, regenerating adultslow motor axons are still capable of find-ing their “correct” intramuscular regions.

Most skeletal muscles contain mix-tures of fast and slow muscle fibre types(identifiable in histochemistry), each be-ing innervated by its own type of moto-neurone (Burke, 1981; Kernell, 1992).During early embryological development,the two main muscle fibre types may dif-ferentiate even in the absence of innerva-tion (Condon et al., 1990). In adult life,muscle fibre types may change due to theinfluence of patterns of activation (re-views: Gordon, 1995; Kernell, 1992),stretch and force load (review: Goldspink,1999), hormones (e.g. Izumo et al., 1986;Larsson et al., 1995) and altered innerva-tion (Romanul and Van der Meulen,1966). The fibre type changes seen afterreinnervation may partly depend on theactivation patterns produced by varioustypes of motoneurones. In muscles thathave become reinnervated following par-tial or complete denervation, a frequentlyoccurring sign of fibre type re-specifica-tion is the appearance of “fibre typegrouping”, i.e. that fibres of one his-tochemical type tend to lie clumped to-gether more often than what is normally

the case (Kugelberg et al., 1970). How-ever, the degree to which this phenom-enon is manifested varies considerablywith the circumstances under which rein-nervation takes place (e.g. Rafuse andGordon, 1996; Unguez et al., 1996) and(some of the) fast or slow muscle fibresmay be resistant to the potential type-changing influences of the innervatingaxon (Unguez et al., 1993; Unguez et al.,1995).

Muscle fibre types are often unevenlydistributed across and along the muscle(“fibre type regionalization”; review:Kernell, 1998). In a few published reportsit was noted that relatively normal pat-terns of fibre type regionalization seemedto appear also after denervation and rein-nervation (fast fibres, cat gastrocnemiuslateralis, Foehring et al., 1987; fast fi-bres, mouse tibialis anterior, Parry andWilkinson, 1990; slow fibres, cat gastroc-nemius medialis, Rafuse and Gordon,1996). These interesting observationswere, however, of limited extent and openfor alternative interpretations. Few musclespecies were studied, measurements werelimited to single-level cross-sections andlengthwise aspects of fibre type regio-nalization were not investigated. Further-more, the authors themselves felt some-what uncertain whether pre- and post-reinnervation patterns of regionalizationmight have resembled each other simplybecause the original fibre types had failedto change, i.e. the fibres might largelyhave been resistant to the potential type-changing influences of the reinnervatingaxons.

We have recently completed an ex-tensive survey of the normal patterns offibre type regionalization within musclesof the rat’s lower hindlimb (Wang &

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Kernell, 2000; Wang & Kernell, 2001).In the present investigation, we use thisbackground knowledge and the associatedquantitative methods (Kernell & Wang,2000) for an experimental study of theextent to which regionalization of the“slow” type I fibres re-emerges after den-ervation and reinnervation in five differ-ent muscles of the rat hindlimb. The ex-tent to which reinnervation was associ-ated with a re-specification of fibre typeswas evaluated using quantitative assess-ments of fibre type grouping. The presentresults have been briefly described in acongress abstract (Wang & Kernell,1999).

Methods

Five male Wistar rats were used (weightsat initial operation about 225 g; finalweights 520 ± 50 g (SD)). Initial surgicalprocedures were performed under generalanaesthesia (1 % isofluorane and O2/N2O).Between the hip and knee a 12 mm seg-ment of the sciatic nerve was resected, re-versed and re-implanted as an autologousnerve graft (fixed with fine sutures). Theinversion was done to decrease the chanceof axonal re-growth into original fascicles.Post-operative survival time was 21weeks.

In a terminal experiment under gen-eral pentobarbitone anaesthesia (50 mg/kg i.p., additional doses as required), tar-get muscles of both lower hindlimbs wereexposed. The target muscles included ex-tensor digitorum longus (ED), flexordigitorum and hallucis longus (FD), gas-trocnemius medialis (GM), peroneus lon-gus (PE) and tibialis anterior (TA). In ourcases, the two components of FD (flexordigitorum longus and flexor hallucis lon-

gus) seemed fused and could not be sepa-rated without causing tissue damage (cf.Wang & Kernell, 2001). Posterior and/orlateral muscle sides were labelled usingwater-insoluble stains. After removal,muscles were weighed and fixed by freez-ing in isopentane cooled by liquidnitrogen.The animal was killed with anoverdosis of pentobarbitone. Cryostatcross-sections of 10 µm were stained formyofibrillar ATPase and fibres were clas-sified into types I (“slow”) and II (“fast”),using standard methods and criteria(Brooke & Kaiser, 1970; Lind & Kernell,1991). The normally innervated right-sidemuscles served as controls, with regardto the topography of type I fibre distribu-tion their properties were very similar tothose obtained in our corresponding mea-surements from untreated rats (Wang &Kernell, 2000; Wang & Kernell, 2001).

For each muscle, cross-sections werecut at 7 evenly spaced proximo-distal lev-els. For one cross-section from each level,enlarged high-contrast photo-copies wereused for tracing muscle profiles and fibrepositions, using a PC with a graphic tab-let and custom-made software. The posi-tion of each type I fibre was indicated(Fig.1) and the following measures werecalculated:

(1) Muscle cross-section area (mm2)and equivalent diameter (mm; diameterof circle with muscle cross-section area);

(2) Type I fibre density (fibres / mm2),as determined for the whole muscle cross-section;

(3) Mutual distances between all typeI fibres;

(4) Direction and degree of type I fi-bre eccentricity, as given by the directionand length of a “type I fibre vector” con-necting the centre of mass for the muscle-

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section as a whole to that for only the typeI fibres (arrows in Fig.1). The centre ofmass was in each case calculated accord-ing to standard rules for mechanics, withthe cross-section area of the muscle rep-resented by a similarly shaped sheet ofuniform thickness and density and withthe type I fibre positions represented bypoints, each with an equal amount ofmass. The direction of the type I fibre vec-tor (vector angle, VA) was given in de-grees with muscle sections oriented suchthat posterior was up and lateral to the left(0° and 360° medial, 90° posterior, 180°lateral, 270° anterior). Different meanvector angles were found for different spe-cies of normally innervated muscles(Wang & Kernell, 2001). The length ofthe type I fibre vector (vector length, VL)was given in percent of the equivalentdiameter of the corresponding musclecross-section.

(5) Degree of restriction of type I fi-bres to part of the muscle cross-section,as indicated by the relative area of the re-gion containing the type I fibres (“type Ifibre region”, FRh). FRh was expressedas a percentage of the total area of thesame cross-section, and its extent was au-tomatically delineated using the “convexhull” method (e.g. Cormen et al., 1990;Wang & Kernell, 2000; Wang & Kernell,2001).

Whenever applicable, mean values inthe text are given ± SD. Pearson correla-tion coefficients were calculated for ana-lyzing the degree of co-variation betweendifferent variables. Differences in prop-erties between different groups of muscleswere analyzed using standard t test pro-cedures. Calculations were made usingExcel (Microsoft) or the software pack-age SYSTAT. Cases with P < 0.05 were

considered statistically significant.

Results

General remarks

With the exception of ED, all the rein-nervated muscles showed some degree ofdecrease in weight and muscle cross-sec-tion area (Table 1). Furthermore, afterreinnervation the midlevel number of typeI fibres per muscle was significantly largerthan normal in ED and TA, close to nor-mal in FD and PE, and significantly lowerthan normal in GM (Table 1). Qualita-tively similar results were obtained alsowhen comparing total type I fibre countsfor all analysis levels together.

Type I fibre grouping: relative number ofclose type I fibre pairs

Figure 1 shows the traced muscle outlineand the positions of all type I fibres withincross-sections from a reinnervated muscle(B) and its contralateral control (A; bothextensor digitorum longus). When look-ing at the fine-grain pattern of the fibredistribution it is obvious that the two pan-els differ: the type I fibres of panel 1Bare much more clumped together in smallgroups than is the case in panel 1A. Wequantified the degree of this “fibre typegrouping” by calculating, for each cross-section, the relative number of “short”type I inter-fibre distances (“Close type Ipairs”) i.e. the number of distances of <50 µm, normalized in relation to the totalnumber of type I fibres in the same cross-section.

The cross-section of Fig.1A does notshow all the slow fibres of this particularmuscle because, for ED as for most other

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Table 1. General properties of reinnervated and control muscles

Muscle weight Muscle midlevel Muscle midlevel(mg) cross- section area (mm2) number of type I fibres

Controls

ED 241.0±21.9 8.7±1.0 91.4±25.4FD 667.8±57.7 22.6±2.0 250.6±150.8GM 1295.4±95.7 44.9±4.1 783.2±128.3PE 224.2±10.1 6.2±0.9 192.0±60.0TA 1005.0±52.4 46.7±3.6 290.0±77.0

ReinnervatedED 243.6±48.8 8.6±2.1 333.2±95.9FD 449.0±38.8 16.8±3.3 282.4±117.5GM 863.2±125.0 33.7±4.2 530.0±152.3PE 163.6±35.6 4.2±1.0 159.8±90.6TA 797.6±139.8 37.1±5.3 930.8±425.3

Ratio R/C (%)ED 101.1 98.6 364.6*FD 67.2* 74.1* 112.7GM 66.6* 75.0* 67.7*PE 73.0* 68.3* 83.2TA 79.4* 79.4* 321.0*

Means ± SD (n = 5 rats) of muscle weight (mg), muscle cross-section area at midlevel (mm2), andnumber of type I fibres at midlevel. Separate means for rightside control muscles and leftside reinnervatedmuscles. “Ratio R/C (%)” gives the ration between the respective averages. The presence of a signifi-cant difference between corresponding experimental and control data indicated by bold print and “*” (ttest, P < 0.05 or better). Muscle abbreviations: ED, extensor digitorum longus; FD, flexor digitorum andhallucis longus; GM, gastrocnemius medialis; PE, peroneus longus; TA, tibialis anterior.lower hindlimb muscles (Wang &Kernell, 2000), the length of individualmuscle fibres is less than half of that forthe whole muscle (pinnate fibre arrange-ments). Thus, in such muscles differentcombinations of individual fibres will beseen in cross-sections taken at differentproximo-distal levels. Therefore, for eachmuscle species in the present study, cross-

sections were investigated at seven dif-ferent proximo-distal “analysis levels”.

The five panels of Fig.2 demonstratethat, in each one of the present 5 speciesof muscles, the relative number of closetype I fibre pairs was markedly and sig-nificantly higher after reinnervation thanin contralateral controls (Fig.2; see Leg-end for statistics). In each muscle, this was

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true for all proximo-distal analysis lev-els. Furthermore, in reinnervated muscles,“clumps” of type I fibres were presentacross the whole of the respective type Ifibre regions (cf. Fig.1B).

Most of the present experimentalmuscles showed a significant degree ofatrophy following the denervation andreinnervation (Table 1). A general de-crease of fibre diameter would, of course,in itself have caused a decrease of inter-fibre distances. We estimated the approxi-mate magnitude of this effect by calcu-lating, for the control muscles, how therelative number of “close type I pairs”would become influenced by appropriatechanges in the criterion-distance for“closeness”. For instance, in the rein-nervated GM muscles, the midlevel cross-sectional area had become decreased to75% of that found on the control side(Table 1). This implies that, at the proxi-mo-distal midlevel, the linear dimensions

Fig.1. Regionalization of slow type I fibres within transverse sections from a normally innervated muscle(A) and a reinnervated one (B). Digitized data from sections through the proximo-distal middle of exten-sor digitorum longus (ED), both muscles from the same rat. Arrow drawn from “centre of mass” forwhole muscle section to that for only the type I fibres (“type I fibre vector”). Interrupted antero-posteriorline drawn through muscle centre. Note uneven regional distribution of type I fibres in both cases (A, B)and marked “clumping” of type I fibres after reinnervation (B, “fibre type grouping”).

had changed by a factor of 0.750.5=0.866,i.e. inter-fibre distances might generallyhave been about 1/0.866=1.155 timesgreater on the control side than on the ex-perimental side. Hence, in the extra set ofatrophy-adjusted calculations, type I fi-bres of GM control muscles were consid-ered “close” if situated within 50/0.866=57.7 µm from each other. Whenaveraged for all muscle species together,such atrophy-adjusted values for the rela-tive number of “close type I pairs” were1.49 times greater than those obtained forthe same control muscles using the stan-dard “closeness” criterion of 50 µm. Incontrast, for the reinnervated muscles therelative number of “close type I pairs”was, on average, 19.70 times that for thestandard controls (cf Fig.2), and for eachmuscle species this valuee was also mark-edly and significantly higher than that ofthe atrophy-adjusted control values(P<0.001). Hence, the atrophy could not

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Fig.2. All reinnervated muscles showed an extensive type I fibre grouping. Type I fibre grouping quan-tified by determining, within each cross-section, the number of type I inter-fibre distances < 50 µm,expressed as a percentage of the total number of type I fibres (”Close type I pairs (%)”). In each panel,means ± SE (n = 5 muscles) plotted vs. proximo-distal level for reinnervated muscles (filled triangles)and contralateral controls (open circles). Each panel shows the data for a separate muscle species. Abbre-viations as in Table 1. Note large and consistent difference between experimental and control values (ttest, P < 0.001 for each panel).

in itself have caused the huge increase of“close type I pairs” in the reinnervatedmuscles.

Direction of type I fibre regionalizationwithin cross-sections

Figure1A demonstrates a case of markedfibre type regionalization in a normallyinnervated hindlimb muscle of the rat (ex-tensor digitorum longus). In this muscle,the type I fibres tended to be more com-mon medially than laterally. The sectionof panel 1B is taken from the same rat

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Fig.3. Post-reinnervation re-appearance of normal direction of type I fibre regionalisation: transverseanalysis. A - E Transverse direction of type I fibre regionalisation quantified by determining, in cross-sections, the angle of the “type I fibre vector” (VA; cf. arrows in Fig.1). In each panel, means ± SE (n= 5 muscles) for VA plotted vs. proximo-distal level for reinnervated muscles (filled triangles) andcontralateral controls (open circles). Each panel A - E shows the data for a separate muscle species. FSummary of overall average results. For each of the five muscle species, means ± SE were calculatedfor VA values of all muscle cross-sections together (including all proximo-distal levels from all 5 rats; n= 30 or more for each mean value). Average data for reinnervated muscles plotted versus those forcontra-lateral controls. Note great similarity between experimental and control values (r = 0.998, P <0.001). The regression line had the equation: y = 0.99 x - 5.

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and muscle species (ED) as panel 1A, butfrom the contralateral reinnervated side.The pattern of regional fibre distributionin Fig.1B resembles that of 1A: the type Ifibres tend to be more common mediallythan laterally. In both cases about 82 %of the type I fibres are to the right of theinterrupted vertical line through themuscle centre.

The direction of regionalizationwithin cross-sections was investigated bycalculating the angle of the type I fibrevector (VA; arrow, Fig.1). The panels inFig.3 A-E show, for each proximo-distalanalysis level and muscle species, that themean values of VA were similar inreinnervated muscles and in their con-tralateral controls. In none of the five spe-cies of muscle was there a significant dif-ference between the two sets of values (P> 0.4 or worse).

In Fig.3F these observations havebeen summarized. Here mean values ofVA were, for each muscle species, calcu-lated for all analysis levels together. Re-sults for reinnervated muscles are seen tobe practically identical to those for thecontrol muscles: the slope and correlationcoefficient for the regression line inFig.3F are both very close to 1 (see Leg-end).

Degree of type I fibre regionalisationwithin cross-sections

We have recently made a distinction be-tween two potentially independent aspectsof “transverse” type I fibreregionalization: the extent to which thetarget fibres are restricted to part of thetotal cross-section area (“area regio-nalization”) and the degree to which the

Fig.4. Post-reinnervation degree of type I fibre regionalisation. A Plot of reinnervated vs. control valuesfor “type I fibre region” (FRh), expressed in percent of total cross-section area. B Plot of reinnervated vs.control values for relative length of the “type I fibre vector” (VL), i.e. a measure of type I fibre eccentric-ity. In both panels, plotted values are the means of the respective parameters, as determined at all proximo-distal analysis levels for each muscle species (n = 30 or more). Interrupted line in A and B: unity line forx = y. Plot symbols: extensor digitorum longus (E), flexor hallucis and digitorum longus (F), gastrocne-mius medialis (G), peroneus longus (P) and tibialis anterior (T). Rings around plot symbols indicate thepresence of a statistically significant difference between reinnervated and control values (t test, P < 0.05or better).

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Fig.5. Post-reinnervation re-appearance of normal direction of type I fibre regionalization: proximo-distal analysis, absolute data. A - E. Means ± SE for the density of type I fibres (fibres mm-2)plottedversus proximo-distal level for reinnervated muscles (filled triangles) and their contra-lateral controls(open circles). Separate panel for each muscle species (see labels). Note negative slope for both experi-mental and control values.

target fibres are eccentrically accumu-lated toward one direction within themuscle cross-section (“vector regionali-zation”; Wang & Kernell, 2001). The de-gree of area regionalization was measuredas the relative size of the automatically

delineated “type I fibre region” (FRh, seeMethods), and the degree of vectorregionalization was given by the relativelength of the type I fibre vector (VL; ar-row, Fig.1).

The effects of reinnervation on the de-

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gree of transverse type I fibre regionali-zation were different for different musclespecies (Fig.4). One of the muscle spe-cies (FD) showed an increased regio-nalization after reinnervation (valid onlyfor area regionalization, F in Fig.4 A). Ingastrocnemius medialis and peroneus lon-gus, the area and vector regionalizationwere both about the same in reinnervatedand control muscles (G and P in Fig.4).In extensor digitorum longus and tibialisanterior, both aspects of regionalizationbecame significantly less distinct afterreinnervation (i.e. larger FRh and smallerVL; E and T in Fig.4). The latter twomuscles were also the only ones showinga significant increase in type I fibre num-bers after reinnervation (Table 1).

Proximo-distal type I fibre regionalization

We have recently shown that, also in thelengthwise direction, muscles of the rat’slower hindlimb show a marked degree oftype I fibre regionalization: the densityof the type I fibres normally becomes pro-

gressively lower at progressively moredistal levels (Wang & Kernell, 2000). Thisbehaviour is demonstrated by the controldata of Fig.5 (circles) and Fig.6A. Afterreinnervation, essentially the sameproximo-distal behaviour was also, on av-erage, seen in all the five species ofmuscle in spite of the fact that the abso-lute figures for type I fibre density wereoften different (Fig.5, filled triangles).Thus, a statistically significant negativecorrelation between mean type I fibredensity and proximo-distal level wasfound for all control muscles and all thereinnervated ones (P < 0.05 or better) ex-cept PE. However, even in reinnervatedPE, a significant negative correlation wasfound when restricting the calculations tolevels 1 - 6 (P < 0.05).

The similarity in lengthwise type Ifibre regionalization between the musclespecies, and between control and rein-nervated cases, became particularly ap-parent when normalizing the data for eachmuscle separately (% of maximum value;Fig.6).

Fig.6. Post-reinnervation re-appearance of normal direction of type I fibre regionalisation: proximo-distal analysis, normalized data. Mean normalized values for the relative density of type I fibres plottedversus proximo-distal level for the five species of reinnervated muscles (B) and their contra-lateralcontrols (A). Type I fibre densities normalized (%) vs. the largest mean value for each muscle group.Note similarity between experimental and control values. Symbols: ED, filled diamonds; FD, opensquares; GM, filled squares; PE, filled triangles; TA, open triangles.

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Effects of reinnervation on the variabil-ity of regionalization parameters

Although the average directions ofregionalization agreed well betweenreinnervated and control cases (Figs. 3,5, 6), the scatter of individual values wasgreater in reinnervated muscles than in thecontrols (cf. fibre distributions of Fig.1 Avs. B). Thus, the type I fibre vector anglehad almost identical mean values in bothgroups of muscles (Fig.3F), but its stan-dard deviation was significantly larger forthe reinnervated muscles (73 ± 40°, n=35)than in the controls (23 ± 22°, n=33; t test,P < 0.001; cf. Fig.3). Similarly, althoughboth groups of muscles showed, on aver-age, a negative correlation between typeI fibre density and proximo-distal level(Figs.5-6), such a correlation more oftenreached a significant level in individualcontrol muscles (P < 0.05 for 24 of 25cases) than in individual reinnervatedmuscles (P < 0.05 for 12 of 25 cases; 0.1> P > 0.05 for 4 further, borderline cases).

Discussion

For adult mammalian muscles, our resultsprovide the so far most comprehensivequantitative analysis of fibre type distri-bution following reinnervation, incorpo-rating transverse (Fig.3-4) as well as lon-gitudinal (Fig.5-6) aspects. Our findingsare consistent with a few preceding ob-servations of a more limited nature (cross-sections only, less quantification) con-cerning the intra-muscular distribution ofslow (Rafuse & Gordon, 1996) or fast(Foehring et al., 1987; Parry &Wilkinson, 1990) fibres following rein-nervation. Thus, although there was some

increase in the general scatter of type Ifibres after reinnervation (cf. Fig.1 A vs.B), the mean direction of type I fibreregionalization was practically the sameafter reinnervation as in the normally in-nervated muscles (Figs.3, 5, 6). Further-more, this re-appearance of a normal di-rection of type I fibre regionalization wasassociated with a very marked increaseof type I fibre grouping (Fig.2), i.e. theingrowing nerve fibres had evidently re-specified the histochemical properties inmany of the post-reinnervation type I fi-bres (see also Rafuse & Gordon, 1996).Hence, after reinnervation the regional-ized distribution of type I fibres could in-deed be taken to reflect the similarly re-gionalized distribution of nerve endingsof slow motor axons.

It is interesting to compare our re-sults to those of Laskowski & Sanes(1988), who used axial muscles of adultrats (diaphragm, serratus anterior) fordemonstrating a partial recovery afterreinnervation of another aspect of intra-muscular organization: the topographicalcorrespondence between motoneuronalpositions in the spinal cord and the sitesof their terminals within the muscle. How-ever, it is important to note that, at leastfor hindlimb muscles, there is no stronggeneral link between such topographicalaspects of muscle innervation and fibretype regionalization; also for cases withan evident spinal cord vs. muscle topog-raphy (Donselaar et al., 1985), fast andslow (or larger and smaller) hindlimbmotoneurones may be widely inter-mingled within the spinal cord (Kernellet al., 1985; see also Clamann & Kukulka,1977; Ishihara et al., 1995).

During embryological development,powerful guidance mechanisms exist for

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directing the neurites of each functionalgroup of motoneurones to their correcttarget muscle or fast vs. slow muscle com-partment (e.g. Milner et al., 1998; Rafuseet al., 1996). Furthermore, during devel-opment a correct matching also takesplace between the functional propertiesof the slow and fast motoneurones andthose of their muscle fibres (Jansen &Fladby, 1990). For this latter aspect,(re-)matching mechanisms are stillpresent in adults, although to varying de-grees in different fibres and situations (e.g.Burke, 1981; Kernell, 1992; Mendell etal., 1994; Rafuse & Gordon, 1998;Unguez et al., 1995). However, with re-gard to the guidance mechanisms for re-generating motor axons, inter-muscularpath-selections apparently become dys-functional soon after birth (ratsAldskogius & Thomander, 1986;Hardman & Brown, 1987) whereas in-tra-muscular path-selections remain(partly) available also in adults (Figs.3,5, 6; see also Foehring et al., 1987;Laskowski & Sanes, 1988; Parry &Wilkinson, 1990; Rafuse & Gordon,1996). The possible reasons for these dif-ferences are intriguing: are, for instance,the greater distances involved in adult vs.embryonic inter-muscular path-selectionsa limiting factor with regard to the effi-ciency of the guidance mechanisms? Out-growing axons are guided by a combina-tion of attracting and repellent actions,arising due to the interactions between re-ceptor molecules in the growth cone anddiffusible or tissue-attached guidance fac-tors in its immediate surrounding. Al-though many such guidance moleculeshave been found in other studies of cen-tral and peripheral axons (Goodman,1996; Stoeckli & Landmesser, 1998),

little is still known about the molecularnature of the axon guidance between andwithin mammalian muscles. In this con-text, the present results provide an inter-esting experimental paradigm for the fur-ther search for such (molecular) mecha-nisms.

The increased variability in regio-nalization parameters after reinnervationsuggests that, although some kind ofaxon-guidance mechanism must havebeen active to lead “slow” and “fast”axons toward their appropriate target re-gions, this took place in competition withother mechanisms influencing the courseof the ingrowing fibres. For a closer un-derstanding of these various competingmechanisms it would be of interest tocompare the efficiency of restoration offibre type regionalization after variousmanipulations of the experimental con-ditions (e.g. varying path of ingrowth,original identity of the motoneurones,position of the muscle etc.; L.C. Wang &D. Kernell, in preparation).

Following reinnervation by (presum-ably) a random mixture of sciaticmotoneurones, an increased number ofcounted type I fibres was noted in ED andTA. This might simply have been a con-sequence of the fact that these musclesnormally tend to have rather few type Ifibres (cf. Ariano et al., 1973; Armstrong& Phelps, 1984; Wang & Kernell, 2001);hence, a random selection of sciatic moto-neurones would be likely to include agreater proportion of slow motoneuronesthan that normally valid to these twomuscles. It seems intuitively understand-able (but not self-evident) that, in thesetwo muscles, the increased number of typeI fibres might have led to a decreased de-gree of type I fibre regionalization (Fig.4).

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However, such relationships were notconsistently present: for reinnervated FDmuscles there was a decreased area

regionalization and no change in type Ifibre number and for reinnervated GMmuscles the opposite was true (cf. Fig. 4,Table 1).

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cial nerve transection and regeneration in the neonatal rat. Brain Res. 1986; 375:126-134.Ariano MA, Armstrong RB, Edgerton VR. Hindlimb muscle fibre populations of five mammals. J.

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