postnatal development and adult organisation of the olivocerebellar projection map in the...

Postnatal Development and AdultOrganisation of the OlivocerebellarProjection Map in the Hypogranular

Cerebellum of the Rat

MARTA ZAGREBELSKY AND FERDINANDO ROSSI*Department of Neuroscience, University of Turin, I-10125 Turin, Italy

ABSTRACTThe olivocerebellar system is characterised by a precise topographical organisation, in

which distinct subsets of inferior olivary axons project to neurochemically heterogeneousPurkinje cell subpopulations, arranged into parasagittally oriented compartments in thecerebellar cortex. Adult climbing fibres and Purkinje cells are linked by a one-to-onerelationship, which is established during postnatal development after a transitory phase ofmultiple climbing fibre innervation. The elimination of redundant climbing fibre synapses isthought to be regulated by granule cell-mediated activity-dependent processes. In order toassess whether this developmental remodelling is also important for the construction of themature olivocerebellar projection map, we examined the hypogranular cerebella of ratstreated by means of methylazoxymethanol acetate (MAM) during early postnatal life, inwhich multiple climbing fibre innervation persists in the adult. In these animals weinvestigated the distribution of calcitonin gene-related peptide (CGRP)-immunoreactiveolivocerebellar axons and arbours during early postnatal development, and the correspon-dence between climbing fibre strips and zebrin II-defined Purkinje cell bands in the adult. Ourresults show that: (1) the pattern of CGRP-immunoreactive climbing fibres observed duringthe first three postnatal weeks is not disrupted after granule cell degeneration; and (2) thealignment between olivocerebellar axon subsets and zebrin II1/2 Purkinje cell compartmentsis normally achieved in adult rats. In contrast, the climbing fibre-Purkinje cell relationship isabnormal, and single arbours innervate restricted dendritic regions of several neighbouringtarget neurons. These results indicate that the normal distribution of olivocerebellar axonsubsets to distinct cerebellar cortical compartments can be established independently fromgranule cell-mediated remodelling processes. Thus, the postnatal climbing fibre plasticity,which is needed to achieve the normal climbing fibre-Purkinje cell relationship, appears to beconfined within the framework of a projection map established during earlier developmentalphases. J. Comp. Neurol. 407:527–542, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: multiple climbing fibre innervation; zebrin II Purkinje cell bands;

methylazoxymethanol acetate; calcitonin gene-related peptide; synapse

elimination; olivocerebellar topography

Point-to-point projection systems of the adult brain arecharacterised by highly specific patterns of connectivity,which are thought to be established during developmentby means of two different mechanisms. Specific molecularrecognition, matching compatible pre- and postsynapticcues, first creates a coarse projection map, which is thenrefined by activity-dependent processes (Changeux andDanchin, 1976; Purves and Lichtman, 1985; Mariani andDelhaye-Bouchaud, 1987; Goodman and Shatz, 1993).Among such systems, the mature olivocerebellar projec-tion shows a precise topographical organisation. Distinct

subsets of inferior olivary axons project to parasagittallyoriented cortical compartments corresponding to the distribu-

Grant sponsor: Ministero dell’Universita e della Ricerca Scientifica eTecnologica; Grant sponsor: Consiglio Nazionale delle Ricerche; Grantsponsor: European Community Biotechnology Programme; Grant number:ERBBIO4-CT96-0774.

*Correspondence to: Ferdinando Rossi, Department of Neuroscience,University of Turin, Corso Raffaello 30, I-10125 Turin, Italy.E-mail: [email protected]

Received 22 September 1998; Revised 7 January 1999; Accepted 8January 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 407:527–542 (1999)

r 1999 WILEY-LISS, INC.

tion of neurochemically heterogeneous Purkinje cell sub-populations (Gravel et al., 1987; Hawkes et al., 1992;Wassef et al., 1992a). In the adult cerebellum, a one-to-onerelationship exists between climbing fibres and Purkinjecells (Ramon y Cajal, 1911; Eccles et al., 1967), which isachieved, during postnatal development, after a transitoryphase of multiple climbing fibre innervation (Crepel et al.,1976; Crepel, 1982). The remodelling processes, whicheliminate the redundant climbing fibre-Purkinje cell con-nections, are partly dependent on the presence of granulecells and on the development of functional parallel fibre-Purkinje cell synapses. Multiple innervation of Purkinjecells persists in the adult hypogranular cerebella of sev-eral mutant mouse strains (weaver, Crepel and Mariani,1976; reeler, Mariani et al., 1977; staggerer, Crepel et al.,1980), or in rats in which granule cell progenitors havebeen killed during early postnatal development by X-irradiation (Woodward et al., 1974; Puro and Woodward,1978; Crepel and Delhaye-Bouchaud, 1979; Mariani et al.,1990; Bravin et al., 1995), viral infections (Benoit et al.,1987), or methylazoxymethanol acetate (MAM) administra-tion (Bravin et al., 1995). Furthermore, the retraction ofsupernumerary climbing fibre synapses is impaired follow-ing pharmacological block of N-methyl-D-aspartic acid(NMDA) receptors (Rabacchi et al., 1992a) or tetrodotoxinapplication in vivo (Kakizawa et al., 1998), and also inseveral mice, in which different genes involved in thegranule-Purkinje cell signalling pathways are mutated(Lurcher, Rabacchi et al., 1992b) or have been knocked out(d2 glutamate receptor, Kashiwabuchi et al., 1995; phospho-kinase C g, Kano et al., 1995; metabotropic glutamatereceptor, Kano et al., 1997, Levenes et al., 1997).

The role played by this granule cell-mediated climbingfibre remodelling in the acquisition of the mature olivocer-ebellar topography is still unclear. Recent in vitro experi-ments indicate that the early formation of the olivocerebel-lar projection map during embryonic development isregulated by chemospecific axon-target recognition mecha-nisms (Chedotal et al., 1997). In addition, neuroanatomi-cal studies show that the basic organisation of the olivocer-ebellar pathway is established before postnatal remodellingtakes place (Crepel, 1982; Sotelo et al., 1984; Chedotal andSotelo, 1992; Paradies and Eisenman, 1993), and it is notgrossly altered in the hypogranular cerebella of mutantmice (weaver, Blatt and Eisenman, 1993). Nevertheless,the recent observation that the climbing fibre vibrissalprojection to the cerebellar cortex is disrupted in thehypogranular cerebella of X-irradiated rats (Furham et al.,1994) indicates that granule cell-dependent mechanismsmay also be required to construct the adult olivocerebellarprojection map.

To address this issue and to ask whether granulecell-dependent climbing fibre remodelling is required toestablish the precise alignment between olivary axonsubsets and Purkinje cell compartments, we examined thehypogranular cerebella of rats treated with MAM duringearly postnatal life. In these animals we assessed whether:(1) the typical pattern of calcitonin gene-related peptide(CGRP)-immunoreactive climbing fibres (Morara et al.,1989; Chedotal and Sotelo, 1992) is maintained during thefirst postnatal weeks; and (2) the strict alignment betweenclimbing fibre strips and zebrin II-defined Purkinje cellbands (Brochu et al., 1990) is present in the adult. Inaddition, we studied the evolution of the climbing fibre-Purkinje cell relationship to assess how its maturation is

related to the formation of the olivocerebellar projectionmap. A preliminary report of this work has been publishedelsewhere (Zagrebelsky et al., 1997a).

MATERIALS AND METHODS

Animals and procedures to induce granulecell degeneration

Granule cell degeneration was induced in the cerebel-lum of Wistar rats (Charles River, Calco, Italy) by means ofpostnatal administration of MAM (Sigma Co., St. Louis,MO), according to a previously described protocol (Bravinet al., 1995). Briefly, MAM was diluted to 23 mg/ml in 0.9%sterile saline and two subcutaneous injections (20 mg/kgeach) were made on postnatal days 1 and 2 (P1 and P2) to42 rat pups, belonging to four different littermates. An-other group of 18 intact rats was used as age-matchedcontrols.

A first set of treated and control animals was killedwithin the third postnatal week (see Table 1) and pro-cessed for anti-CGRP and anti-calbindin immunohisto-chemistry to examine the development of the olivocerebel-lar projection and Purkinje cells. The remaining animals(Table 1) were allowed to survive until adulthood (2–3months) and used for anterograde axonal tracing of theolivocerebellar system and anti-zebrin II or anti-calbindinimmunocytochemistry. All surgical procedures (see below)were carried out under deep general anaesthesia obtainedby intraperitoneal injections of a mixture of ketamine(Ketalar, Parke Davis, Barcelona, Spain, 100 mg/kg) andxylazine (Rompun, Bayer, Leverkusen, Germany, 5 mg/kg). The experimental plan was designed according to theNIH guidelines and the Italian law for care and use ofexperimental animals (DL116/92), and approved by theItalian Ministry of Health.

Immunocytochemical labelling of developingclimbing fibres and Purkinje cells

Under deep general anaesthesia, the rat pups weretranscardially perfused with 200–500 ml of 4% paraformal-dehyde in 0.12 M phosphate buffer (pH 7.2–7.4). Thebrains were dissected, stored overnight in the same fixa-tive at 4°C, and finally transferred to 30% sucrose inphosphate buffer and kept at 4°C until they sank. Thecerebellum and brainstem were cut on a cryostat inalternate series of 20-µm-thick frontal sections, collected

TABLE 1. Number of MAM-Treated and Intact Animals Examinedat Each Age1

Age(postnatal day)

MAM-treatedrats

Intactrats

P4 5 2P6 4 2P8 4 2P10 4 2P12 3 2P14 3 2P16 3P21 4 2Adult (2–3 months) 12 4

1For each age the number of methylazoxymethanol acetate (MAM)-treated and intactanimals considered in this study is reported. Rats killed within P21 were used for theanalysis of the distribution of calcitonin gene-related peptide (CGRP)-immunoreactiveclimbing fibres. Adult rats were used for biotinylated dextran amine (BDA) tracing andzebrin-II immunocytochemistry. Among the latter experimental set, 10 MAM-treatedand 2 control rats received tracer injections in the inferior olive to label olivocerebellaraxons, whereas in the remaining animals the BDA was applied to the cerebellar cortexto stain parallel fibres.

528 M. ZAGREBELSKY AND F. ROSSI

on gelatine-coated slides. The section series were incu-bated with either rabbit anti-CGRP antibody (Amersham,Amersham, United Kingdom, 1:600 in phosphate-bufferedsaline, PBS, pH 7.4 with 0.25% Triton X-100 and 1.5% goatserum) or mouse anti-calbindin monoclonal antibody(Swant, Bellinzona, Switzerland, 1:3,000 in PBS, pH 7.4with 0.25% Triton X-100 and 1.5% horse serum) overnightat 4°C. Immunohistochemical labelling was performed accord-ing to the avidin-biotin-peroxidase method (VectastainABC elite kit, Vector, Burlingame, CA) and revealed byincubation in 0.03% 3-38-diaminobenzidine (DAB) in Tris-HCl (pH 7.6) with 0.01% H2O2. The treated slides wereincubated for 30 minutes at 37°C in a propidium iodidesolution (0.1% in Tris-HCl, pH 7.6) to obtain a fluorescentcounterstain of the different cerebellar cell populations.Then they were washed in the same buffer, air-dried,dehydrated and coverslipped.

Tracer applications andimmunocytochemistry of adult cerebella

In the adult animals, the olivocerebellar system wasvisualised by means of the anterograde axonal tracerbiotinylated dextran amine (BDA; 10,000 MW, MolecularProbes, Eugene, OR) applied according to a previouslydescribed protocol (Zagrebelsky et al., 1996, 1997b). Six toten days before the end of the survival period, the ratswere deeply anaesthetised and placed in a stereotaxicframe. The tracer was injected into the right inferior olive,approached from the ventral side. As a rule, four iontopho-retic injections were made by means of glass micropipettes(inner tip diameter 20–30 µm) filled with a few microlitersof a 10% BDA solution in 0.12 M phosphate buffer (pH 7.2),placed 0.5–0.8 mm deep in the medulla oblongata near theconfluence of the vertebral arteries and just lateral to thefirst segment of the basal artery. Each injection consistedof 7 µA positive current pulses (five seconds on and fiveseconds off over 15 minutes) delivered by a Midgard CS4iontophoretic device (Stoelting, Wood Dale, IL). Finally, inorder to visualise parallel fibres, in another set of animals(two treated and two intact rats) similar tracer injectionswere made into the posterior vermis, exposed by drilling asmall hole in the posterior aspect of the occipital bone.

Six to ten days later, after renewed general anaesthesia,the rats were transcardially perfused with 1,000 ml of 4%paraformaldehyde. The brains were immediately dis-sected and cryoprotected as described above. The cerebellawere cut in 30-µm-thick frontal sections (a few cases werecut in the sagittal plane), collected free-floating in PBS,incubated for two hours in the avidin-biotin-peroxidasecomplex (Vectastain ABC elite kit, Vector; 1:100 in PBSwith 0.25% Triton X-100), and finally incubated with theDAB solution to which 0.04% nickel ammonium sulphatewas added in order to yield a black reaction product. Themajority of these treated sections were subsequently incu-bated with the monoclonal anti-zebrin II antibody (thekind gift of Dr. Richard Hawkes; 1:150 in PBS with 0.25%Triton X-100, 1.5% normal horse serum) to visualise thecerebellar cortical compartments, whereas the others wereincubated in the monoclonal anti-calbindin antibody(Swant, Bellinzona, Switzerland, 1:3,000 in PBS, pH 7.4with 0.25% Triton X-100 and 1.5% horse serum) to label allPurkinje cells. Incubations with these primary antibodieslasted 30 minutes at room temperature and the sectionswere then processed according to the ABC method. TheDAB reaction was performed without nickel ammonium

sulphate to give a brown reaction product. By this well-established double-labelling procedure (see Rossi andStrata, 1995; Strata and Rossi, 1998 for refs.) the black-stained traced axons can be easily distinguished fromother brown-immunolabelled structures. The reacted sec-tions were finally mounted on gelatine-coated slides, air-dried, dehydrated and coverslipped.

Histological analysis

The histological preparations were examined using aZeiss Axiophot light microscope. To map the distribution ofCGRP-immunolabelled olivocerebellar axons in develop-ing rats, as well as that of BDA traced fibres and zebrinII-defined bands in adult animals, several frontal cerebel-lar sections at different rostrocaudal levels were chosenfrom selected cases. Such sections were carefully repro-duced by means of the Neurolucida software (Microbright-field, Colchester, VT) based on a Pentium PC connected toan E-800 Nikon microscope at 103 magnification. Thedrawings obtained were assembled with Micrografx De-signer 3.1 software to produce the final figures.

In order to study the relationship between climbingfibres and Purkinje cells in adult hypogranular cerebella, anumber of traced arbours together with their target neu-rons were reproduced by Neurolucida from single frontalor sagittal cerebellar sections. The climbing fibres andPurkinje cells to be reconstructed were selected accordingto the following criteria: (1) the labelled terminal arbourshad to be strongly stained and contained for a large extentwithin the examined section; (2) they had to be sufficientlyisolated from other traced axons, so that their reconstruc-tion was not hindered by the presence of other climbingfibre branches; (3) the somata of their target Purkinje cellshad to be located close to the granular-molecular layerinterface and the dendritic trees well developed into themolecular layer; and (4) the cell bodies and substantialportions of the proximal dendritic domain of these Pur-kinje cells had to be fully contained within the section. Anumber of climbing fibres and Purkinje cells matchingthese criteria were reproduced by three-dimensional recon-structions at 1003 magnification by means of the Neurolu-cida system as above (see Bravin et al., 1995 for furtherdetails about such reconstructions and technical dataabout the system).

RESULTS

Effects of MAM treatmenton cerebellar development

The severe alterations of cerebellar development in-duced by MAM administration to neonatal rodents havebeen thoroughly described by several previous studies(Woodward et al., 1975; Lovell et al., 1980; Bejar et al.,1985; Chen and Hilmann, 1988; Hillman et al., 1988;Bravin et al., 1995; Ji and Hawkes, 1996). Our presentobservations are fully consistent with these reports. Twodays after MAM injection made at P1–P2, a massivedegeneration of the external granular layer (EGL) wasevident over large areas of the cerebellar cortex (Fig. 1a,b).Two days later (P6), however, distinct clusters of tightlypacked cells appeared scattered along the cortical surface(Fig. 1c). Such clusters gradually spread and at P10 theEGL was almost completely restored, with only a fewagranular gaps in the depth of the fissures (Fig. 1d).

OLIVARY PROJECTION MAP IN HYPOGRANULAR CEREBELLA 529

Fig. 1. Effects of methylazoxymethanol acetate (MAM) administra-tion on postnatal cerebellar development. Micrograph a shows anintact cerebellum at postnatal day (P)4; the external granular layer(egl) is constituted by several cell rows intensely stained by propidiumiodide. By contrast, at the same age in an animal treated by MAM atP1–P2 (b), this germinal layer (pointed by arrowheads) is severelyatrophic and contains loosely packed cells with numerous picnoticnuclei. Two days later (c), clusters of granule cell progenitors (arrow-heads) appear scattered along the cortical surface. Such clustersgradually spread to cover most of the cerebellar surface (d, taken atP10); arrowheads point to small agranular gaps that remain in the

depth of the fissures. The survey micrograph e shows an anti-calbindin-immunostained frontal cerebellar section passing through the poste-rior vermis of an adult MAM-treated cerebellum. Note the numerousexuberant folia symmetrically arranged around midline (indicated bythe dotted line). Also note the defective alignment of Purkinje cells.The micrograph f shows a sagittal section from a MAM-treatedcerebellum into which biotinylated dextran amine (BDA) was injected.Arrowheads point to labelled parallel fibres which are abnormallyoriented along the parasagittal axis of the folium. Scale bars 5 60 µmin a–d; 500 µm in e; 50 µm in f.


During the same period, numerous parasagittally orientedfissures were formed, which divided the lobules into manydefined folia arranged in a highly reproducible symmetri-cal pattern (Fig. 1e). In addition, other typical MAM-induced abnormalities of the cortical cytoarchitectonics,including defects in Purkinje cell alignment (Figs. 1e, 4a)and an altered orientation of parallel fibres (Fig. 1f),gradually became evident (see also Woodward et al., 1975;Chen and Hilmann, 1988; Hillman et al., 1988; Bravin etal., 1995).

Distribution of CGRP-immunoreactiveclimbing fibres during the development

of MAM-treated cerebella

To investigate the terminal distribution pattern of olivo-cerebellar axons during postnatal ontogenesis in the hypo-

granular cerebella, we examined the CGRP-immunoreac-tive inferior olivary axons. This peptide is transientlyexpressed during embryonic and postnatal developmentby a subpopulation of olivocerebellar axons which projectto the cerebellar cortex forming a well-defined set ofparasagittally oriented strips (Kubota et al., 1987; Moraraet al., 1989; Chedotal and Sotelo, 1992).

Despite the anatomical alterations of MAM-treated cer-ebella, the pattern of CGRP-immunoreactive climbingfibre bands was essentially similar to that seen in controlanimals (Fig. 2). During the first postnatal week, frontalcerebellar sections displayed two prominent paramedianbands, symmetrically arranged along the whole rostrocau-dal extent of the cerebellum. In addition, two narrowerstrips were located more laterally in the vermis andanother one in the intermediate cortex. The most intense

Fig. 2. Neurolucida reconstructions of several cerebellar sections(top–bottom is caudal–rostral) from an intact and a methylazoxymetha-nol acetate (MAM)-treated animal aged postnatal day 8, showingthe distribution of calcitonin gene-related peptide (CGRP)-immunore-active climbing fibre strips. The symmetrical pattern of CGRP-

immunoreactive climbing fibres, characterised by two prominentparamedian strips and several thinner bands located more laterally inthe vermis and in the intermediate cortex, is essentially maintained inthe hypogranular cerebellum. Note that abnormal foliation is not fullydeveloped at this age.


CGRP immunostaining was observed between P6 and P10,when an additional climbing fibre band appeared in theflocculus. Labelling intensity gradually decreased thereaf-ter, and at P21 only a few dimly stained climbing fibresremained in the flocculo-nodular lobe (Chedotal and So-telo, 1992).

At all ages, the CGRP-immunoreactive olivocerebellaraxon and climbing fibre bands were characterised by verysharp boundaries with only a minimal intermingling offine terminal branches between the neighbouring parame-dian strips (Fig. 3a; see also Zagrebelsky et al., 1997b). Inaddition, the analysis of adjacent sections stained foranti-calbindin antibodies to label Purkinje cells revealedthat the strips of CGRP-immunoreactive climbing fibreswere precisely aligned to distinct Purkinje cell clusters(Fig. 3a,b). Such clusters, which disappeared in intactanimals by P4–P6, when the final Purkinje cell monolayerwas achieved, remained evident up to P10 in MAM-treatedrats. Altogether, these observations show that the typicalorganisation of CGRP-immunoreactive olivary axon andclimbing fibre bands, as well as their alignment to Pur-kinje cell clusters (Chedotal et al., 1996), are not disruptedafter MAM-induced granule cell degeneration.

Climbing fibre maturation in thehypogranular cerebellum

The typical time course and three-stage evolution of thenormal climbing fibre development (Ramon y Cajal, 1911;see also Mason et al., 1990; Chedotal and Sotelo, 1992)could be also recognised in MAM-treated rats. The pericel-lular nest phase was observed between P4 and P10, whena dense meshwork of fine CGRP-immunoreactive axonscovered the entire surface of Purkinje cell bodies (Fig. 3c).Then, between P10 and P14 the terminal olivary axonbranches gradually moved to the apical pole of the soma toform the typical capuchon (Fig. 3d). From P16 onwards, allthe labelled fibres had completed their peridendritic trans-location and attained the phase of young climbing fibrearborisations (Fig. 3e), made of a few thick and smoothstem branches and numerous fine varicose tendrils, cover-ing the proximal Purkinje cell dendrites.

Despite this essentially normal course of maturation,the developing climbing fibres in hypogranular cerebelladid show some unusual morphological features associatedwith concomitant alterations of Purkinje cell dendritogen-esis. As previously observed in Golgi preparations fromMAM-induced hypogranular cerebella (Woodward et al.,1975), starting from P6 anti-calbindin-immunolabelledPurkinje cells displayed apical dendrites which emanatedfrom the nascent dendritic tree and elongated straight intothe EGL up to the pial surface (Fig. 3f). In alternatesections from the same animals, several CGRP-immunola-belled climbing fibres also showed thick branches, whichpenetrated into the EGL and ran towards the cerebellarsurface (Fig. 3c,g,h). Although we could not label bothclimbing fibres and Purkinje cells in the same sections, themorphological features and orientation of these climbingfibre processes strongly suggest that they were growingalong the apical Purkinje cell dendrites protruding into theEGL. Such Purkinje cell and climbing fibre features, whichwere never encountered in intact animals, were presentduring the same developmental period and graduallydisappeared together with the outgrowth of the Purkinjecell dendritic tree, the translocation of the climbing fibrearbour, and the concomitant resorption of the EGL.

Alignment between climbing fibre stripsand zebrin II-identified Purkinje cell

compartments in the adult hypogranularcerebellum

In the normal adult rat cerebellum, anti-zebrin II anti-bodies reveal a characteristic pattern of alternating immu-nopositive and negative Purkinje cell bands (Brochu et al.,1990; Hawkes et al., 1992), to which distinct subsets ofolivocerebellar axons are aligned (Fig. 4; Gravel et al.,1987; Wassef et al., 1992a; Hawkes et al., 1992). Despitethe severe foliation abnormalities, the typical pattern ofzebrin II1/2 Purkinje cell bands is established in MAM-treated rats (Ji and Hawkes, 1996). In these animals,however, the correspondence between zebrin II-definedcortical compartments and the terminal distribution of thespinocerebellar mossy fibre projection is disrupted (Ji andHawkes, 1996). Thus, we asked whether the normalalignment between climbing fibre strips and zebrin II1/2

Purkinje cell bands is achieved in the adult hypogranularcerebellum.

In the MAM-treated cerebella, zebrin II1/2 Purkinje cellbands were easily identifiable by their sharp boundaries(Fig. 5a). However, due to the abnormal orientation ofPurkinje cell dendritic trees, the dendritic branches ofPurkinje cells located at the edge of a compartmentfrequently radiated into the adjacent one (Fig. 5b). Inaddition, we occasionally encountered single zebrin II-positive Purkinje cells displaced within a negative band(Fig. 5f).

The distribution of olivocerebellar fibres into the hypo-granular cerebellar cortex was examined by double label-ling with anti-zebrin II immunocytochemistry and BDAtracing to stain inferior olivary axons (Figs. 4, 5c–f). Tracerinjections were placed into a restricted portion of the rightinferior olive and, hence, only labelled a subset of theolivocerebellar fibres. The traced axons exclusively pro-jected to the contralateral hemicerebellum and were typi-cally arranged into several distinct bundles terminating inwell-defined parasagittal strips in the cerebellar cortex.The climbing fibre strips were consistently aligned to the

Fig. 3. Development of calcitonin gene-related peptide (CGRP)-immunoreactive climbing fibres in the hypogranular cerebella. Micro-graphs a and b show two adjacent frontal sections from a methylazoxy-methanol acetate (MAM)-treated animal (aged postnatal day [P]6),which were immunolabelled for CGRP and calbindin, respectively.Note that climbing fibre strips (arrowheads in a) are precisely alignedto discrete Purkinje cell clusters (arrowheads in b). Micrographs c, d,and e show the maturation of climbing fibres in the MAM-treatedcerebella. At P6 (c), the labelled axons form typical pericellular nestsaround Purkinje cell somata (some are pointed by asterisks). Thearrow points to a climbing fibre branch elongating into the externalgranular layer (egl). After P10 (d, taken at P12) the climbing fibrebranches (arrowheads) gradually move to the apical pole of theirtarget neurons forming the typical capuchon. A few days later (e, takenat P14), climbing fibres complete their translocation and attain theirfinal peridendritic position (asterisk points to the position of thePurkinje cell body, arrowheads indicate the climbing fibre arbour). Themicrograph f shows anti-calbindin-immunolabelled Purkinje cells in aMAM-treated cerebellum aged P8. Note the thick apical dendrites (arrow-heads) which elongate unbranched up to the pial surface. At the sameage (g and h) CGRP immunolabelling reveals climbing fibre branches(arrowheads) which also elongate straight through the externalgranular layer (egl) towards the cortical surface. The course of theseprocesses suggests that they are growing along the apical Purkinje celldendrites. Scale bars 5 40 µm in a,b; 20 µm in c–e; 30 µm in f–h.


Figure 3


Fig. 4. Neurolucida reconstructions of several cerebellar sections(top–bottom is caudal–rostral) from an adult intact and a methylazoxy-methanol acetate (MAM)-treated animal showing the correspondencebetween the distribution of biotinylated dextran amine (BDA)-tracedolivocerebellar axons and zebrin II1/2 Purkinje cell compartments.

Note that in both the intact and the hypogranular cerebellum, labelledclimbing fibres (indicated by the black hatching) are arranged intoseveral parasagittal strips, which are aligned to the zebrin II1/2

Purkinje cell bands (shaded areas represent zebrin II positive corticalcompartments).

Fig. 5. Correspondence between the distribution of biotinylateddextran amine (BDA)-traced olivocerebellar axons and zebrin II1/2

Purkinje cell bands in the hypogranular cerebellum. Micrograph ashows a frontal view of the vermis of an adult methylazoxymethanolacetate (MAM)-treated cerebellum stained by anti-zebrin II antibod-ies. Note that despite the severe foliation defects and the defectivePurkinje cell alignment, the zebrin II1/2 Purkinje cell bands can bereadily identified. However, due to the abnormal orientation of Pur-kinje cell dendritic trees, the dendritic branches of Purkinje cellslocated at the edge of the compartments (pointed by arrowheads in b,taken from a frontal section of the vermis) radiate into the adjacentband. The micrograph c shows a zebrin II-positive Purkinje cell bandwhich is almost completely covered by BDA-labelled climbing fibrearbours (some are pointed by arrowheads). The higher magnificationpicture d illustrates the edge of another zebrin II-positive Purkinje

cell compartment; note that labelled climbing fibre arbours (some arepointed by arrowheads) abruptly stop in coincidence with the compart-ment edge. Two climbing fibre strips that terminate into two adjacentzebrin-positive and -negative bands (arrowheads point to the bound-aries of these bands) are shown in e. Note that the zebrin II-positiveband is only partially covered by the labelled climbing fibre arbours.However, both olivocerebellar axon strips are precisely aligned to therelevant compartment edge. A zebrin II-positive Purkinje cell dis-placed within the adjacent negative band is indicated by the arrow in f.Note that this Purkinje cell is innervated by a labelled climbing fibreas also other zebrin II-positive Purkinje cells in the two adjacentcompartments (arrowheads in f). By contrast, the neighbouring zebrinII-negative Purkinje cells are not contacted by labelled climbing fibres.Scale bars 5 200 µm in a; 50 µm in b, c, e, f; 20 µm in d.


Figure 5


zebrin II1/2 Purkinje cell bands (Figs. 4, 5c–f). Usually, thelabelled climbing fibre strips did not cover the wholeextent of the wide Purkinje cell bands (Fig. 5e), but analmost perfect match between afferent axon subsets andcortical modules was often observed in the case of nar-rower Purkinje cell compartments (Fig. 5c). In all in-stances, however, the labelled climbing fibre strips neverextended beyond the edge of their cortical compartment(Fig. 5d,e). In addition, when a single zebrin II-positivePurkinje cell displaced within the adjacent compartmentwas innervated by a labelled climbing fibre, the nearbyzebrin II-negative neurons were not contacted by anytraced axons (Fig. 5f). Altogether, these observations showthat the alignment between climbing fibre strips andPurkinje cell compartments is properly established in thehypogranular cerebella.

Climbing fibre-Purkinje cell relationship inthe adult hypogranular cerebellum

Although the correct alignment between climbing fibrestrips and Purkinje cell bands is achieved in MAM-treatedcerebella, multiple climbing fibre innervation persists inthe adult (Bravin et al., 1995). Taken together, theseobservations indicate that granule cell-dependent climb-ing fibre remodelling occurs in the boundaries of the zebrinII-defined cortical modules and, hence, that the climbingfibre-Purkinje cell relationship established within suchcompartments is abnormal in the hypogranular cerebel-lum. Indeed, a few years ago we showed that singlePurkinje cells can be innervated by at least two climbingfibres, which are strictly segregated onto distinct targetdendritic domains (Bravin et al., 1995). In the presentstudy, we further analysed the morphology of climbingfibres and their distribution on Purkinje cell dendrites inorder to obtain additional information about this axon-target interaction in the hypogranular cerebellum.

The basic structure of the adult climbing fibre, e.g., a fewthick stem branches with profuse varicose tendrils imping-ing upon the proximal Purkinje cell dendrites, was consis-tently observed in MAM-treated cerebella. However, theanalysis of double-labelled sections for zebrin II or calbin-din and BDA tracing clearly showed that numerous climb-ing fibre arbours only covered a restricted portion of theirtarget Purkinje cell dendritic tree (Figs. 6a–d, 7a,b; seealso Bravin et al., 1995). In addition, such climbing fibresfrequently displayed collateral branches which left thearbour to form another terminal plexus on the dendrites ofa nearby Purkinje cell (Figs. 6a–d, 7a,b). Usually, suchbranches extended from one Purkinje cell dendritic tree toanother neighbouring one (Figs. 6a,c,d, 7a), or lengthenedfarther away in the molecular layer to reach a moredistant target (Figs. 6c, 7b). In some cases, however, thickramifications ran straight up to the pial border, elongatedfurther along the cortical surface (Fig. 6e,f), and eventu-ally re-entered the molecular layer to break into otherterminal arbours (Fig. 6e). In all these instances, thelabelled climbing fibres appeared strictly segregated ondistinct target dendrites, and we never found any evidencethat different arbours could share the same dendriticbranches. Thus, in contrast to the normal adult cerebel-lum, in which each climbing fibre arbour covers the wholeextent of the proximal dendritic domain of a single Pur-kinje cell, following MAM treatment olivocerebellar axonarbours are distributed over restricted dendritic regions ofseveral neighbouring target neurons. The observation of

sections labelled by anti-zebrin II antibodies revealed thateach climbing fibre exclusively innervated Purkinje cells ofthe same zebrin II1/2 phenotype, thus confirming thatsuch an abnormal climbing fibre Purkinje cell relationshiponly occurred within the boundaries of the cortical compart-ments.

DISCUSSION

To assess the role played by granule cell-dependentclimbing fibre remodelling in the establishment of theolivocerebellar projection map, we examined MAM-in-duced hypogranular rat cerebella, in which multiple climb-ing fibre innervation of Purkinje cells is maintained in theadult (Bravin et al., 1995). Our results show that: (1) thepattern of CGRP-immunoreactive climbing fibre stripsremains fundamentally unmodified during the first threepostnatal weeks; and (2) the alignment between climbingfibre and zebrin II1/2 Purkinje cell bands is normallyestablished in adult rats. Nevertheless, within such bandsthe climbing fibre-Purkinje cell relationship is disrupted.Thus, the normal distribution of olivocerebellar axonsubsets to distinct cerebellar cortical compartments can beachieved independently from granule cell-mediated remod-elling processes, which appear to be confined within theboundaries of such cortical modules.

Climbing fibre synaptic remodelling and theacquisition of the olivocerebellar

projection map

A salient feature of the cerebellum is the precise organ-isation of the afferent and efferent cortical projectionsystems into a set of parasagittal modules identifiable bytheir anatomical characteristics (Voogd et al., 1985; Voogdand Ruigrok, 1997) or specific patterns of gene expression(Herrup and Kuemerle, 1997; Hawkes, 1997; Oberdick et

Fig. 6. Abnormal climbing fibre-Purkinje cell relationship in theadult hypogranular cerebellum. Arrowheads in a point to a climbingfibre arbour which covers the dendrites of two nearby Purkinje cells(the cell bodies are indicated by asterisks). Note that the dendrites ofthe Purkinje cell on the left side of the picture are only partiallycovered by the climbing fibre branches (arrows point to the Purkinjecell dendrites which are not innervated by the labelled climbing fibre).The arrow in b points to a collateral climbing fibre branch whichleaves the dendrites of the Purkinje cell on the left (cell body isindicated by the asterisk on the left) and innervates (large arrow-heads) a restricted portion of another Purkinje cell dendritic tree (thecell body is indicated by the asterisk on the right; small arrowheadspoint to the uncovered dendritic branches of this Purkinje cell). Theclimbing fibres and Purkinje cells illustrated in micrographs a and bare reproduced in the Neurolucida reconstructions displayed in Figure 7.The climbing fibre in c (arrowheads) also emits a short branch (largearrow) which forms a terminal plexus on a restricted portion of aneighbouring Purkinje cell dendritic tree (small arrow). The largecrossed arrow points to another climbing fibre branch, which elongatesthrough the molecular layer to break into a small terminal plexus(small crossed arrow) impinging upon the dendritic branches of adifferent Purkinje cell. The micrograph d shows another example of aclimbing fibre (arrowheads) which is also extended over the dendritesof a neighbouring Purkinje cell. The arrows in e indicate a thick climbingfibre branch which elongates towards the cortical border, runs alongthe pial surface and, then, re-enters the molecular layer to formanother climbing fibre plexus (arrowheads). Several of such thick climb-ing fibre branches running towards and growing along the pial surfaceare indicated by the arrowheads in f. Biotinylated dextran amine(BDA) tracing and anti-zebrin II-immunolabelling in a,c,d,f, BDA tracingand anti-calbindin immunolabelling in b, e. Scale bars 5 20 µm.


Figure 6


Fig. 7. Neurolucida reconstructions of climbing fibres and theirtarget Purkinje cells in the adult hypogranular cerebellum. Theclimbing fibre in a completely covers the proximal dendrites of thePurkinje cell on the right (indicated by the dark shading). This arbouris also extended to cover some of the dendritic branches (arrowheads)of another Purkinje cell (light shading). The arrowheads in b point to aclimbing fibre arbour that partially covers the proximal dendrites oftwo neighbouring Purkinje cells (large arrow points to the collateralbranch jumping form one dendritic tree to the other). The dendrites ofthe Purkinje cell on the left (light shading) are also covered by some

other labelled climbing fibre branches (small arrows). Although itcannot be excluded that all labelled branches impinging upon thisneuron belong to the same climbing fibre, the presence of uncoveredramifications in the middle of the dendritic tree suggests that theyderive from two distinct olivocerebellar axon arbours. To increase thereadability of the figure, the thinnest climbing fibre tendrils and allthe varicosities have been omitted in these reconstructions. Micro-graphs showing these climbing fibres and Purkinje cells are displayedin Figure 6a and b.


al., 1998). Superimposed on this anatomical map is thefunctional representation of sensory information which, bycontrast, is organised into discontinuous patches (Shambeset al., 1978; Robertson, 1987; Welker, 1987), leading to theconcept of ‘‘fractured somatotopy’’ (Shambes et al., 1978).How this organisation is achieved during development andhow these anatomical and functional maps are related toeach other is still unclear.

It has been proposed that the olivocerebellar projectionmap is initially formed by chemospecific recognition be-tween phenotypically heterogeneous subpopulations ofinferior olivary neurons and Purkinje cells (Wassef andSotelo, 1984; Wassef et al., 1992a). Indeed, subsets ofPurkinje cells (Wassef and Sotelo 1984; Wassef et al., 1985,1992a,b,c; Oberdick et al., 1993, 1998; Millen et al., 1995;Chedotal et al, 1996; Herrup and Kuemerle, 1997) andinferior olivary neurons (Morara et al., 1989; Wassef et al.,1992b; Chedotal et al., 1996) can be recognised duringdevelopment by specific patterns of gene expression, andtheir reciprocal distribution often matches the terminalorganisation of the olivocerebellar pathway (Wassef et al.,1992c; Chedotal et al., 1996; Paradies et al., 1996). Inaddition, direct experimental evidence indicating thatPurkinje cells provide positional cues for ingrowing olivo-cerebellar axons has been obtained by recent in vitroexperiments (Chedotal et al., 1997). Finally, a different setof biochemical markers defines topographic compartmentsin the adult cerebellar cortex (see Herrup and Kuemerle,1997; Hawkes, 1997 for reviews), which are also aligned tothe olivocerebellar projection map (Gravel et al., 1987;Hawkes et al., 1992; Wassef et al., 1992a).

Altogether, these data emphasise the role of chemoaffin-ity recognition mechanisms in the formation of the olivocer-ebellar topography and, indeed, its basic organisation canbe already recognised during embryonic life (Chedotal andSotelo, 1992; Paradies and Eisenman, 1993) or at birth(Sotelo et al., 1984). Nevertheless, during postnatal devel-opment climbing fibres undergo a substantial remodelling,which is thought to be regulated by granule cell-mediatedactivity-dependent processes (Crepel et al., 1976; Crepel,1982; Rabacchi et al., 1992a; Kakizawa et al., 1998). Arethe latter processes important to achieve the matureorganisation of the olivocerebellar projection? Our resultsconsistently indicate that they are not required to estab-lish the correct distribution pattern of olivocerebellar axonsubsets to specific cortical compartments. Indeed, refer-ring to both CGRP immunolabelling and BDA tracingexperiments, we cannot completely rule out the possibilitythat unstained axons could be misplaced within the stripsof labelled climbing fibres. However, this possibility isunlikely because we did not observe aberrant CGRP-immunolabelled or BDA traced axons terminating outsidethe labelled strips. Rather, the selective interaction be-tween discrete climbing fibre and Purkinje cell subsetswas maintained even in the case of single Purkinje cellsectopically located within the adjacent compartment (seeFig. 5f).

A normal expression pattern of the zebrin II1/2 Purkinjecell phenotype is developed in the absence of all majorcortical afferent inputs (Leclerc et al., 1988; Wassef et al.,1990; Zagrebelsky et al., 1997b), in hypogranular cerebella(Ji and Hawkes, 1996), and following tetrodotoxin-inducedblock of electrical activity in vitro (Seil et al., 1995). Thisindicates that the phenotypically heterogeneous subpopu-lations of Purkinje cells and inferior olivary neurons are

generated by intrinsic cell-autonomous mechanisms, inde-pendent from their reciprocal interaction or from granulecell-mediated processes (Wassef et al., 1992a; Oberdick etal., 1998). Specific molecular recognition mechanismsmatching such heterogeneous axon and target subsets(Chedotal et al., 1997) lead to the formation of the basicolivocerebellar topography. Then, the ensuing climbingfibre remodelling, regulated by granule cell-mediated pro-cesses, occurs within the framework of this early projec-tion map. This conclusion is also supported by the analysisof reinnervation phenomena during postnatal develop-ment (Zagrebelsky et al., 1997b) or in the adult (Zagrebel-sky et al., 1996): climbing fibres are able to interact withand innervate additional Purkinje cells, but only withintheir proper cortical compartment (Strata and Rossi, 1998).

Despite the normal appearance of the anatomical map,the climbing fibre vibrissal representation is altered in theX-irradiated hypogranular cerebellar cortex (Furham etal., 1994). The precise relationships linking the anatomicalorganisation of the olivocerebellar pathway and the distri-bution of sensory information relayed by this input to thecerebellar cortex is still unclear (Robertson, 1987; Hawkes1997; Voogd and Ruigrok; 1997). Thus, it is difficult toreconcile these apparently discrepant results. However,the overall organisation of climbing fibre-mediated somato-sensory inputs to the cerebellar cortex is not overtlydisrupted in the X-irradiated hypogranular cerebellum(Mariani et al., 1987). Hence, the more diffuse distributionof the climbing fibre vibrissal representation (Furham etal., 1994) can be attributed to abnormal synaptic remodel-ling within discrete cortical compartments plus the defec-tive pruning of collateral branches from single olivocerebel-lar axons projecting to distinct parasagittal strips(Armstrong, 1974; Ekerot and Larson, 1982). Taken to-gether, these data suggest that the functional representa-tion of sensory inputs to the cerebellar cortex is con-structed by activity/experience-dependent mechanisms onthe framework provided by the anatomical map estab-lished through chemoaffinity axon-target recognition.

The climbing fibre-Purkinje cell relationshipin the hypogranular cerebellum

The adult climbing fibre phenotype and the final relation-ship with the Purkinje cell are achieved during postnataldevelopment through two temporally coincident thoughdistinct processes: (1) the translocation of the terminalarbour branches from the perisomatic to the peridendriticposition, and (2) the pruning of supernumerary climbingfibres leading to the final one-to-one relationship with thetarget neuron. Our present observations, together withthose published in a previous report (Bravin et al., 1995),clearly show that the climbing fibre translocation processis not affected in hypogranular cerebella. The only clearmorphological alteration displayed by some developingclimbing fibres in MAM-treated cerebella is the outgrowthof processes into the EGL, observed starting from the firstpostnatal week. This phenomenon is most likely a conse-quence of the abnormal elongation of apical Purkinje celldendrites, which provide a substrate for the outgrowingclimbing fibre branches. It is thus likely that both thephysiological translocation and this aberrant growth ofclimbing fibre processes into the EGL result from a com-mon mechanism. The position of climbing fibre ramifica-tions in the different Purkinje cell compartments might bedetermined by the distribution of target-derived cues,


whose dynamic rearrangement regulates the structuralplasticity of climbing fibres during development (Ramon yCajal, 1911) and in the adult (Rossi and Strata, 1995;Strata and Rossi 1998).

Despite the fact that climbing fibres translocate to theperidendritic position and acquire most of their typicalstructural features, their relationship with Purkinje cellsin the adult is profoundly altered. In a previous study(Bravin et al., 1995), we showed that single Purkinje cellscan be innervated by multiple climbing fibres which arestrictly segregated on distinct dendritic domains. Thepresent results further extend these observations by show-ing that single climbing fibres branch in the molecularlayer to innervate restricted dendritic regions of severalPurkinje cells. Taken together, these results indicate thatone fundamental property of the climbing fibre, i.e., thecapability of maintaining a private target domain, is preservedin the hypogranular cerebellum. Nevertheless, whereas innormal conditions the terminal branches issued by a singleolivocerebellar axon stem are confined to cover the wholeproximal dendritic domain of a single target neuron, in thehypogranular cerebellum they partially innervate thedendrites of several Purkinje cells. The mechanisms lead-ing to this condition have still to be elucidated. However, ithas been recently shown that competitive synaptic remod-elling at the developing neuromuscular junction is medi-ated by signals that decrease in potency over short dis-tances (Gan and Lichtman, 1998). In line with thisobservation, competition processes and synapse elimina-tion at the neuromuscular junction (Franck et al., 1975;Kuffler et al., 1977; Grinnell et al., 1979) or in the ciliaryganglion (Hume and Purves, 1981; Forehand and Purves,1984) are effective only if the terminal branches of differ-ent axons are confined onto a restricted target surface(Purves and Lichtman, 1985). Thus, it is tempting to specu-late that also for the pruning of multiple climbing fibreinnervation the competition process should be completed,or at least resolved, before the translocation takes place,when several olivocerebellar arbours are intermingled onthe cell body of a single target neuron. In other words, theestablishment of the normal one-to-one climbing fibre-Purkinje cell relationship would require that granulecell-dependent competition processes and arbour transloca-tion are precisely timed. In the MAM-induced hypogranu-lar cerebellum, the development of the parallel fibre inputneeded to complete a full competition process is neverattained, or it is delayed in time due to the degenerationand regeneration of granule cell precursors. As a conse-quence, multiple climbing fibres succeed in translocatingto the dendrites of a single Purkinje cell, where they segre-gate into distinct target domains. The abnormal climbingfibre branches, which likely grow along the apical Purkinjecell dendrites protruding into the EGL, may also contrib-ute to this phenomenon by facilitating and anticipatingthe segregation of different terminal arbours on distincttarget territories.

In conclusion, our results show that the olivocerebellarprojection map is normally established in the hypogranu-lar cerebellum. In contrast, the climbing fibre-Purkinjecell relationship is altered. This indicates that granulecell-mediated processes are required to refine olivocerebel-lar connections within previously established topographicmodules in order to achieve the proper cortical connectiv-ity and, probably, to construct the functional sensoryrepresentation maps.

ACKNOWLEDGMENTS

We are grateful to Dr. Richard Hawkes and Prof. Piergior-gio Strata for critical comments on the manuscript. We arealso indebted to Dr. Richard Hawkes for the generous giftof anti-zebrin II antibodies. We thank Mrs. Luisella Mi-lano for valuable technical help and to Miss GraziellaMilano for secretarial assistance. This work was supportedby grants from Ministero dell’Universita e della RicercaScientifica e Tecnologica, Consiglio Nazionale delle Ricer-che, and European Community Biotechnology Programme(ERBBIO4-CT96-0774 to Prof. P. Strata).

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postnatal development and adult organisation of the olivocerebellar projection map in the...

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