Developmental Modifications ofOlivocerebellar Topography: The

Granuloprival Cerebellum RevealsMultiple Routes from the Inferior Olive

BETTY FOURNIER,1 ANN M. LOHOF,1 ADRIAN J. BOWER,2 JEAN MARIANI,1

AND RACHEL M. SHERRARD2,3*1Laboratoire Developpement et Vieillissement du Systeme Nerveux, Unite Mixte de

Recherche 7102 Neurobiologie des Processus Adaptatifs, Centre National de la RechercheScientifique et Universite Pierre et Marie Curie, Paris 75005, France2School of Medicine, University of Notre Dame Australia, Fremantle,

Western Australia 6959, Australia3Developmental Neuroplasticity Laboratory, School of Anatomy and Human Biology,

University of Western Australia, Crawley, Western Australia 6009, Australia

ABSTRACTCorrect function of neural circuits depends on highly organized neuronal connections, refined

from less precise projections through synaptic elimination, collateral regression, or neuronaldeath. We examined regressive phenomena that define olivocerebellar topography during mat-uration from Purkinje cell polyinnervation to monoinnervation. We used bilateral retrogradetracing to determine the source of olivocerebellar afferents to posterior vermis lobules VII–VIII ina model of retained immature Purkinje cell polyinnervation, the granuloprival cerebellum. Incontrols, labelled neurons were found only in the contralateral inferior olive (ION) clustered in asmall ventromedial locus that is congruent with known olivocerebellar topography. In granulo-prival animals, olivary labelling appeared more dispersed and was present in homologousipsilateral regions. Double-labelled neurons were never seen. Retrograde tracing following uni-lateral olivocerebellar transection in adult granuloprival rats revealed: 1) the origin of the normal(remaining) path projecting through the contralateral inferior peduncle was more localized thanin irradiated nonpedunculotomized rats, 2) a small double-crossed path, and 3) a projection thatascends the peduncle ipsilateral to the ION of origin, part of which crosses the midline within thecerebellum. Electrophysiological and immunohistochemical assessment in the neonatal cerebel-lum revealed that transcommissural paths are not present during development but sprout withinthe irradiated cerebellum. Therefore, the olivocerebellar projection in the granuloprival rat, as amodel of the immature path, shows parasagittal organization similar to that of controls in itsnormally crossed path but possesses additional abnormal projections. Thus, maturation of olivo-cerebellar topography involves removal of whole developmental paths to define laterality plussynapse elimination within largely predefined parasagittal zones. J. Comp. Neurol. 490:85–97,2005. © 2005 Wiley-Liss, Inc.

Indexing terms: inferior olivary complex; climbing fibers; plasticity; X-irradiation; synapse

elimination

Grant sponsor: Centre National de la Recherche Scientifique (to J.M.);Grant sponsor: Fondation Cino et Simone del Duca (to R.M.S.); Grantsponsor: Ville de Paris (to R.M.S.).

*Correspondence to: Rachel M. Sherrard, School of Anatomy and HumanBiology (M309), The University of Western Australia, 35 Stirling Highway,Crawley, Western Australia 6009, Australia.E-mail: rsherrard@anhb.uwa.edu.au

Received 24 September 2004; Revised 26 November 2004; Accepted 18April 2005

DOI 10.1002/cne.20648Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 490:85–97 (2005)

© 2005 WILEY-LISS, INC.

For the nervous system to function normally, neuronalcircuitry has to be organized very accurately. In manypathways, this organization is observed as precise topo-graphical maps. These maps emerge through remodellingof initial broad projections so that the topography is re-fined and the final circuitry determined (Katz and Shatz,1996). Such refinement involves the removal of redundantsynapses, axon collaterals, and even neurons (Oppenheim,1991; Katz and Shatz, 1996). However, the relative con-tribution of each of these processes to the precise topo-graphic organization of any given pathway remains ill-defined.

To address this question, the structural changes thatoccur during the development of each path must be exam-ined. The rodent olivocerebellar projection is one exampleof a topographically organized path (Buisseret-Delmasand Angaut, 1993), which matures from broad longitudi-nal zones (Wassef et al., 1992; Paradies and Eisenman,1993) to narrow microzones (Sugihara et al., 2001). Thismaturation is concurrent with several regressive phenom-ena: elimination of supernumerary synapses (Mariani andChangeux, 1981; Crepel et al., 1981), narrowing of para-sagittal zones (Dupont et al., 1981), removal of an ipsilat-eral projection (Lopez-Roman et al., 1993), and olivary celldeath (Bourrat and Sotelo, 1984; Delhaye-Bouchaud et al.,1985; Cunningham et al., 1999). However, the relativecontribution of these regressive phenomena to refiningolivocerebellar topography has not been clarified becauseneither the topography nor the axonal trajectories havebeen fully examined in the immature system.

Examination of olivocerebellar topography during de-velopment is difficult because the pathway matures in theearly postnatal period (Mason et al., 1990), when thecerebellum changes rapidly and extensively (Altman,1982). Analysis becomes possible when the system’s mat-uration is arrested and the immature structure is main-tained into adulthood. Developmental arrest of this kindoccurs in the agranular cerebellum induced by neonatalX-irradiation. Perinatal X-irradiation prevents the gener-ation of cerebellar granule cells (Altman and Anderson,1972), whose axons (the parallel fibers) form one of twomajor inputs to cerebellar Purkinje cells (PCs), the otherbeing climbing fiber (CF) terminals of olivocerebellar ax-ons. Insofar as the development of the parallel fiber inputto PCs is crucial for the maturation of CF–PC interaction(Delhaye-Bouchaud et al., 1978), the olivocerebellar pro-jection to the adult X-irradiated cerebellum resemblesthat of the immature path (Fig. 1). In the normal adultanimal, olivocerebellar axons cross the midline in the me-dulla to enter the cerebellum via the inferior cerebellarpeduncle and terminate contralaterally in narrow para-sagittal microzones (Sugihara et al., 2001), within whicheach PC receives only a single olivocerebellar axon (Mari-ani and Changeux, 1981). In contrast, both adultX-irradiated and immature olivocerebellar paths are bi-lateral projections (neonate, Bower and Payne, 1987;Lopez-Roman et al., 1993; X-irradiated, Fuhrman et al.,1995; Sugihara et al., 2000). They terminate in broadparasagittal zones (neonate, Sotelo et al., 1984;X-irradiated, Fuhrman et al., 1994; Sugihara et al., 2000),within which each PC receives several CFs (neonate,Mariani and Changeux, 1981; Crepel et al., 1981;X-irradiated, Crepel et al., 1981; Mariani et al., 1990).However, it is not yet clear whether the broader bilateralprojections of the immature and granuloprival olivocer-

Fig. 1. Diagram illustrating the current view of the olivocerebellarprojections arising from the left ION to normal adult (A), immature(B), and granuloprival (C) cerebella. A: In the normal mature path,there is defined topography organized in narrow parasagittal micro-zones of the contralateral hemicerebellum (1, between vertical lines).In these microzones, each PC receives a single climbing fiber (CF).B: During development, olivocerebellar axons terminate in broaderparasagittal zones (5, between vertical dotted lines), in which PCsreceive multiple CFs. There are also ipsilaterally projecting axons,which pass through unilateral (2: (Bower and Sherrard, 1986) ortranscommissural double-crossed (3: Lopez-Roman and Armengol,1994) routes. C: In the granuloprival cerebellum, olivocerebellar ax-ons terminate in broad parasagittal zones (5, between vertical dottedlines), in which PCs receive multiple CFs. Also, there is an ipsilateralprojection that is thought to be transcommissural axons (4: Sugiharaet al., 2000; Fournier et al., 2002).

86 B. FOURNIER ET AL.

ebellar paths are due to extraneous collateral brancheswithin the cerebellar cortex and/or separate transientpaths or, therefore, which regressive phenomena are in-volved in the maturation of this path.

In this study, we examined olivocerebellar topographyin the adult X-irradiated cerebellum as a model of theimmature configuration. To identify whether the bilateralprojections were intracerebellar collaterals or separatepathways, we used double-fluorescent retrograde tracing.Then, we combined this retrograde tracing with unilateralpedunculotomy to transect the projection on one side ofadult granuloprival cerebella thus examining in thismodel system the route by which olivocerebellar axonsreach their target PCs. Such information contributes toour understanding of which regressive phenomena,whether terminal remodelling and/or removal of wholedevelopmental paths, are involved in the refinement of theolivocerebellar topography.

MATERIALS AND METHODS

Animals and X-irradiation

Experiments were performed on Wistar rats under li-cence from the Comite National d’Ethique pour les Sci-ences de la Vie et de la Sante, in accordance with Euro-pean Communities Council Directive 86/609/EEC andNIH guidelines. Pups were irradiated on the fifth postna-tal day (P5) with a single dose of 5,500 mGy delivered tothe posterior cerebellum, following the protocol describedby Fuhrman et al. (1994). This dose is sufficient to retainmultiple CF innervation of PCs (Fuhrman et al., 1994).The rest of the animal was protected by lead shielding.Postirradiation, the pups were returned to the dam forcare and nourishment and allowed to grow to adulthood.

Injection of tracer

Adult irradiated (n � 24) or control (n � 10) rats wereanesthetized with a mixture of ketamine (50 mg/kg), xy-lazine (10 mg/kg), and acepromazine (0.75 mg/kg) andthen placed in a stereotaxic frame, and the entire widthsof lobules VII and VIII of the posterior cerebellar vermiswere exposed. By using a Picopump (PV820 WPI) at 5.5psi for 8 seconds, up to 300 nl tetramethylrhodaminedextran solution [4% in saline; Fluoro-Ruby (FR), D-181710,000 MW; Molecular Probes, Eugene, OR] was injectedthrough a glass micropipette (tip diameter 20 �m) into onehemivermis just medial to the paravermal vein at a depthof 700 �m. A second injection of fluorescein dextran[Fluoro-Emerald (FE), D-1820 10,000 MW; MolecularProbes] was then made into an equivalent location on theopposite side of the midline. The side (left or right) of FRor FE injection was varied between animals. Pipettes wereleft in situ for 3 minutes after the injection before theywere withdrawn. Some animals then underwent left uni-lateral cerebellar pedunculotomy (see below). The woundwas cleaned, the skin was sutured, and the animals wereallowed to recover.

Transection of olivocerebellar axons

To assess the presence of intracerebellar transcommis-sural olivocerebellar axons, the olivocerebellar projectionwas removed from one hemicerebellum by left unilateralinferior cerebellar pedunculotomy. This analysis wasmade 1) in the normal immature cerebellum at P5 when

PC polyinnervation is maximal or 2) in the adult irradi-ated rat, in which the immature pathway is presumed tohave been retained.

Twelve of the twenty-four adult irradiated rats thatwere anesthetized for cerebellar injection (see above) un-derwent unilateral pedunculotomy. Neonatal rats (n � 18)of either sex aged 5 days were anesthetized with diethylether (BDH, Poole, United Kingdom). Animals in eithergroup underwent unilateral transection of the inferiorcerebellar peduncle (ICP) as has been previously de-scribed (Bower and Waddington, 1981). After full recoveryfrom the anesthetic in a warm box, adult animals survivedfor 4 days (see below under Histological preparation), andall neonatal animals were returned to the dam to survivefor 1–3 days when they were taken for electrophysiologicalrecording.

Electrophysiological recording

Cerebellar slices (250 �m) were prepared from pedun-culotomized animals aged 6–8 days by using standardprocedures (Llano et al., 1991). Whole-cell patch-clamprecordings were made from PCs by using a pipette solu-tion containing 13 mM biocytin, and CF currents wereelicited by stimulation in the granular layer, as describedby Sugihara et al. (2003). CF currents were identified bytheir all-or-none character and by the demonstration ofpaired-pulse depression. Multiple CF currents, if present,were recorded by gradually increasing the stimulationintensity and observing discrete response amplitudes. Af-ter recording, the slice was fixed in 4% formaldehyde inphosphate-buffered saline (PBS) for several days prior torevelation of the biocytin-filled PCs using the VectastainElite ABC kit (Vector Laboratories, Burlingame, CA) andcytological verification of recorded PCs.

Calbindin and VGLUT2immunohistochemistry

Cerebellar slices used for electrophysiology were alsotreated for immunohistochemical localization of calbindin,which specifically labels PCs in the cerebellum (de Camilliet al., 1984) and the glutamate transporter VGLUT2,which is localized in CF and mossy fiber terminals (Hiokiet al., 2003). The rabbit polyclonal anticalbindin antibodyis raised against purified 28kD calbindin, has �10% cross-reactivity with other calcium binding proteins (SWant,Bellinzona, Switzerland; CB-38), and has been extensivelycharacterized as a marker for cerebellar PCs. The distri-bution of labelling in our slices was identical to that pre-viously described (Bouslama-Oueghlani et al., 2003; Gi-anola and Rossi, 2004). VGLUT2 was visualized withguinea pig polyclonal anti-VGLUT2 raised against a syn-thetic VGLUT2 peptide that has no overlap with VGLUT1(Chemicon, Hampshire, United Kingdom; AB5907). Thedistribution of VGLUT2-like immunoreactivity was notonly identical to that previous described in the cerebellum(Kaneko et al., 2002; Hioki et al., 2003; Miyazaki et al.,2003) but also was reproducible with a different affinity-purified rabbit polyclonal anti-VGLUT2 (a generous gift ofDr. Salah El Mestikawy; Herzog et al., 2001). Controls forantibody specificity were abolition of staining followingomission of the primary antibody or preabsorption of theantiserum with the immunogen peptide.

After washing in PBS containing 0.25% Triton (T-PBS)and blocking for 1 hour in T-PBS containing 0.2% gelatin(T-PBS-G), slices were incubated overnight in T-PBS-G

87DEVELOPMENT OF OLIVOCEREBELLAR TOPOGRAPHY

containing two primary antibodies, rabbit anticalbindin(SWant; dilution 1:5,000) and guinea pig anti-VGLUT2(Chemicon; dilution 1:3,000). Primary antibodies were re-vealed for 2 hours with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit and Cy3-conjugated donkeyanti-guinea pig secondary antibodies (Jackson Immunore-search, West Grove, PA) diluted 1:200 in T-PBS-G. Sliceswere washed in PBS and mounted in Mowiol.

Histological preparation and analysis

Four days after tracer injections, animals were killedwith an overdose of anesthetic and perfused transcardi-ally with heparinized (5 IU/ml) 0.05 M sodium phosphatebuffer (pH 7.4) and 4% paraformaldehyde fixative. Thebrainstem and cerebellum were removed and cryopro-tected, and serial coronal sections (30 �m) of the inferiorolive (ION) and cerebellum were cut, mounted on glassslides, air dried, and coverslipped with Eukitt. Sectionsfrom two animals were stained with Luxol blue and ana-lyzed for histological changes induced by the irradiationprotocol. Unstained fluorescent sections were analyzed for1) location of the cerebellar injection sites and 2) completetransection of the ICP in pedunculotomized animals(Sherrard et al., 1986). Therefore, 12 (two control, fiveirradiated, and five irradiated-pedunculotomized) animalswere excluded from the study, nine because their injectionsites encroached upon the midline and three because ofincorrect irradiation or pedunculotomy.

In all the remaining animals, both IONs were searchedfor retrogradely labelled cells and their number and posi-tion documented. Neurons of the right ION are present inanimals pedunculotomized as adults, because these neu-rons survive for several weeks following axotomy in adult-hood (Buffo et al., 1998). To ensure that no labelled neu-rons were missed, neuronal counts were made on all(serial) sections of the inferior olives in all animals of eachgroup. Equally, to ensure that no neuron was countedtwice in two adjacent sections, only those cells that hadvisible neurites and contained an “empty” nuclear areadevoid of fluorescent granules were included. Compari-sons of the numbers of retrogradely labelled olivary neu-rons in different groups were made by using the Kruskal-Wallace test and post-hoc analysis with the Mann-Whitney U test when applicable. All photographs weretaken on a Photometrics Coolsnap-fx (Roper Scientific Inc,Tucson, AZ), and, where appropriate, red or green colorwas added and a digital montage of fluorescent labellingmade.

RESULTS

Although the adult irradiated rat cerebellum is knownto maintain Purkinje cell CF polyinnervation (i.e., reflectimmature olivocerebellar connectivity) because of the ab-sence of granule cell parallel fibers, the spatial organiza-tion and trajectory of the olivocerebellar path to thegranuloprival cerebellum have remained poorly defined.Therefore, we mapped olivary afferents to a severelygranuloprival region of the posterior cerebellum. In addi-tion to confirming atrophy of the irradiated cerebellumwith PC misalignment and severe granuloprivation inposterior lobules (Fig. 2; Mariani et al., 1987; Fuhrman etal., 1994) and the presence of afferents from the ipsilateralION (Fuhrman et al., 1995; Fournier et al., 2002), ourstudy adds important new information about the regionaltopography and multiplicity of routes taken by olivocer-ebellar axons to the granuloprival cerebellum.

Injections of fluorescent dextran conjugates were cen-tered in lobules VII and VIII but in some animals spreadinto the adjacent lobules VIc or IX. All retrogradely la-belled inferior olivary cells displayed the characteristicovoid soma of inferior olivary neurons, from which a fewlabelled neurites extended (Fig. 3). The distribution ofretrogradely labelled ION neurons contralateral and/oripsilateral to the injection was identical irrespective of theside into which FR or FE was injected. Therefore, forclarity, the results will be described as though all injec-tions of FR were on the right and FE on the left of themidline.

Fig. 3. Retrogradely labelled olivary neurons had normal morphol-ogy, with ovoid soma and visible neurites (arrows). The cellular struc-ture was identical in both control (A) and irradiated (B) olives, con-firming that the irradiation protocol does not adversely affect olivaryneurons. Scale bars � 10 �m.

Fig. 2. The neonatally irradiated granuloprival cerebellum issmaller than normal. A: The greatest depletion of the granule cells isin the dorsal vermis (lobule VII). In lobules that are more distant, athin granular layer exists (arrowheads). VII, VIII, and IX, vermis

lobules VII–IX. B: A higher magnification of the area outlined in A.The PCs are misaligned into a multilayer two to four cells thick (blackarrows) and the internal granular layer is almost devoid of granulecells (white arrow). Scale bars � 500 �m in A; 50 �m in B.

88 B. FOURNIER ET AL.

Precise topography in the normalolivocerebellar path

To provide a standard with which to compare the olivaryprojections to the granuloprival cerebellum, we mappedthe location of inferior olivary neurons retrogradely la-belled by small injections of fluorescent-dextran into theleft or right hemivermis of normal adult rats. In controlanimals (n � 8), the distribution of labelled olivary neu-rons was in accordance with the known olivocerebellartopography (Buisseret-Delmas and Angaut, 1993); i.e., af-ter injections localized within lobule VII (five rats), FR-labelled neurons were identified in the left ION and FE-labelled neurons in the right, the olivary labelling being inboth cases contralateral to the cerebellar injection. Theseneurons were located symmetrically about the midline,clustered in a narrow diagonal ventromedial-dorsolateralband of the medial accessory olive subnucleus c (MAO “c”;Fig. 4B). When the injections extended into lobules VIc orVIII (three rats), additional labelling was observed in nu-cleus � (n�).

Ipsilateral and contralateral olivocerebellarinputs to the granuloprival cerebellum arise

from separate populations of neurons

It is known that the olivocerebellar projection to thegranuloprival cerebellum is bilateral (Fournier et al.,2002) and terminates in broader parasagittal zones(Fuhrman et al., 1995). To identify whether these broaderprojections arose from a separate population of olivaryneurons or were collaterals of the normal path, we exam-ined olivary labelling after double fluorescent cerebellarinjections in six adult rats rendered granuloprival by ir-radiation on day 5.

Each cerebellar injection retrogradely labelled neuronsin homologous areas of both the ipsilateral and the con-tralateral IONs (Fig. 4D), as previously described(Fournier et al., 2002). However, our double-fluorescenttracing has demonstrated the nature of these projections.First, we never observed double-labelled cells. Therefore,each ION contained two separate populations of retro-gradely labelled neurons, the majority being labelled fromthe contralateral injection and a minority labelled by thedye injected ipsilaterally; i.e., the left ION contained manyFR-labelled cells (crossed path: Fig. 4E, path 1) plus someFE-labelled cells (ipsilateral path: Fig. 4E, paths labeled?2 or ?3) and vice versa for the right ION (Fig. 4D). Second,neurons in the ION contralateral to the injection (i.e.,FR-labelled cells in the left ION and FE-labelled cells inthe right ION) appeared to be more widely dispersedthroughout “c” and n� than in control animals (Fig. 4B vs.4D). Thus, in the irradiated rat, the apparently less well-defined olivocerebellar projection arises from populationsof neurons that project either ipsilaterally or contralater-ally, but not both.

Unilateral pedunculotomy revealsolivocerebellar axons that ascend the

inferior cerebellar peduncle ipsilateral tothe ION of origin

To understand whether the aberrant ipsilateral projec-tion in the granuloprival system is directly ipsilateral ordouble-crossed, we also analyzed olivary labelling in twonormal and eight granuloprival adult rats in which theleft ICP was completely transected at the time of cerebel-

lar injection. This allowed examination of olivocerebellaraxons from either ION, which entered the cerebellumthrough only one (the right) ICP.

In pedunculotomised control rats, only FR-labelled neu-rons were observed. They were located in the left (con-tralateral) ION as expected from normal olivocerebellartopography (Buisseret-Delmas and Angaut, 1993) and in-jection of FR into the right hemivermis (not shown). Theabsence of cells in the right ION labelled by the left vermalFE injection was also expected, in that olivary axons tothis region (the left ICP) had been transected.

In pedunculotomized X-irradiated rats, there were ret-rogradely labelled neurons in both IONs. In the left ION,most labelling was FR-positive neurons in a band-likedistribution between ventromedial “c” and n� similar tothat seen in the control animals (Fig. 4F,G, path 1) andnormal animals pedunculotomized in adulthood. Whenthe cerebellar injections spread into lobule IX, FR-labelledneurons were also found in the dorsomedial cell column;again, this is congruent with established olivocerebellartopography (Buisseret-Delmas and Angaut, 1993). A veryfew FE-filled, i.e., labelled by the ipsilateral injection,neurons were also observed among the FR-labelled cells infour of these eight animals (not shown). Because the leftICP had been transected, these axons must have crossedthe midline from the left ION to ascend the right ICP andthen recrossed as transcommissural axons within the cer-ebellum (i.e., equivalent to Fig. 4E, path ?3).

The presence of retrogradely labelled neurons in theright ION demonstrated olivocerebellar axons that ascendthe inferior peduncle ipsilateral to the ION of origin, apathway not observed in the normal adult animal (Fig.4G, paths 2, 4). In all eight irradiated, pedunculotomizedrats, there were FR-labelled neurons in the right ION, i.e.,ipsilateral to the injection. They were located in the samesubnuclei, symmetrically about the midline to their coun-terparts on the left (Fig. 4F,G, path 2). Furthermore, in sixanimals, FE-labelled neurons were also noted (Fig. 4F)adjacent to the ipsilaterally placed FR-labelled cells. Be-cause the left ICP had been transected in these irradiatedanimals, FE labelling in the right ION contralateral to theinjection indicates that some axons ascend the right (ip-silateral) inferior peduncle and then cross the midline ascerebellar transcommissural axons (Fig. 4G, path 4). Al-though both FR- and FE-labelled cells displayed the samehistological features, each neuron was distinct and neverdoubly labelled.

In summary, data from the unilaterally pedunculoto-mized irradiated cerebella indicate that olivocerebellaraxons that enter the cerebellum via the contralateral ICP(Fig. 4G, path 1) arise from a focal origin similar to thatseen in normal animals. The data also reveal a smalldouble-crossed path (Fig. 4E, path ?3) and a separateolivocerebellar projection that ascends the peduncle ipsi-lateral to the ION of origin (Fig. 4G, path 2), part of whichcrosses the midline within the cerebellum (Fig. 4G,path 4).

Ipsilateral olivocerebellar projection islarger than intracerebellar

transcommissural paths

To examine the relative contribution of each olivocer-ebellar component identified above, we counted the num-ber of neurons retrogradely labelled in each pathway. To

89DEVELOPMENT OF OLIVOCEREBELLAR TOPOGRAPHY

Figure 4

90 B. FOURNIER ET AL.

normalize the data for interanimal variation in injectionsize, the number of labelled neurons from each rat wasexpressed as a percentage of the number of neurons la-belled within the normal crossed pathway (Fig. 4C,E,G,path 1) and the mean obtained for each group (Table 1).

As indicated above, in control animals, labelling wasobserved only contralaterally to each injection, thus thenormal crossed projection (Fig. 4C, path 1) accounted for100% of labelled neurons in each ION. In the irradiatedpedunculotomized group, each aberrant pathway is iso-lated. In these animals, the number of FR-labelled neu-rons in the right ION (i.e., ipsilateral to the injection)indicated that the unilateral ipsilateral projection (Fig.4G, path 2) was �13% the size of the normal path. Fur-thermore, FE-labelled neurons in the right ION revealedthat olivocerebellar axons that ascend the right (ipsilat-eral) ICP to form intracerebellar transcommissural axons(Fig. 4G, path 4) were a smaller proportion, being 8% ofnormal. In contrast there were very few FE-labelled neu-rons in the left ION, indicating that double-crossed ipsi-laterally projecting path (Fig. 4E, path 3) is very small,�3% of normal (Table 2).

In the irradiated nonpedunculotomized cerebella, indi-vidual projections could not be isolated. In each ION,labelling from the contralateral injection arises from

transport via two routes (Fig. 4E, path 1, and Fig. 4G,path 4); i.e., FR-labelled neurons in the left ION resultfrom the normal (path 1) and abnormal (path 4) routesand vice versa for the right ION. In addition, neuronslabelled by the ipsilateral injection, i.e., FE-labelled neu-rons in the left ION and FR-labelled neurons in the rightION, also result from transport along two possible paths,ipsilateral and double-crossed (Fig. 4E, paths 2, 3). Hencequantitative data can only indicate the relative size ofipsilateral vs. contralateral olivocerebellar projections,but not the individual components. Quantitation revealedthat these ipsilaterally projecting paths were large, being7–16% the size of contralaterally projecting paths (Table1).

Thus quantitative data from all groups confirm thequalitative observations that a significant proportion ofolivocerebellar axons ascend the peduncle ipsilateral tothe ION of origin (Fig. 4G, path 2) but that only some ofthese cross the midline within the cerebellum (Fig. 4G,

Fig. 4. Digital montage photomicrographs (A,B,D,F) showing thecerebellar injection sites and inferior olivary labelling and diagrams(C,E,G) showing the olivocerebellar routes in the three experimentalgroups. In each section, the midline is shown by the white dotted line.A: An illustration of typical injection sites (outlined) that are sym-metrical about the midline and located in the lateral part of lobuleVII. B: In control animals, there are a few retrogradely labelledneurons in subnucleus “c” of the ION contralateral to the injection.This labelling is consistent with a small cerebellar injection andnormal olivocerebellar topography (Buisseret-Delmas and Angaut,1993). C: Diagram in which the red and green lines (1) demonstratesymmetrical crossed olivocerebellar topography in control animals, asindicated by neuronal labelling in B. D: Inferior olivary labelling froman irradiated rat in which the injections were located in lobules VIIand VIII. Retrogradely labelled neurons were located in “c” and n� inaccordance with normal topography but appeared more widely dis-tributed than in control animals (compare B). In addition to labellingfrom the contralateral injection, in each inferior olive there were alsoneurons in homologous locations retrogradely labelled from the ipsi-lateral injection (arrowheads). The areas outlined by the boxes areshown in higher power at the lower corners of the image on the sameside. These neurons are only singly labelled. E: Diagram in which the

red and green lines demonstrate the olivocerebellar projection inirradiated rats, as indicated by neuronal labelling in D. In addition tothe normal crossed paths (1, solid lines), there are ipsilateral projec-tions, although whether they are totally unilateral (?2) or double-crossed (?3) cannot be deduced in these animals. F: Inferior olivarylabelling from an irradiated-pedunculotomized rat in which the injec-tions were located in lobules VII and VIII. There were a few FR-labelled neurons in the left (contralateral) “c” and n�, consistent withthe normal olivocerebellar projection. In the homologous location ofthe right olive, there were also neurons labelled by both FE (thecontralateral injection; arrow) and FR (the ipsilateral injection; ar-rowhead). The area outlined by the box is shown at higher power inthe right lower corner and shows that this neuron is singly labelled.G: Diagram in which the red and green lines demonstrate the olivo-cerebellar projection observed in irradiated-pedunculotomized ani-mals, as indicated by neuronal labelling in F. There is the normalcrossed path (1, solid red line), plus a unilateral ipsilateral path (2,red dotted line) as well as a path that ascends the right peduncle andthen crosses the midline in the cerebellum (4, green dotted line). c,Subnucleus c; n�, nucleus �; VII, lobule VII; VIII, lobule VIII. Scalebars � 250 �m in A; 50 �m in B,D; 100 �m in F.

TABLE 1. Percentage (Mean � SEM) of Neurons in Each IONRetrogradely Labelled Following Injection of Fluorescent Dyes into the

Normal or Irradiated Cerebellum1

Group (n)

Left ION Right ION

FR-labelled

FE-labelled

FR-labelled

FE-labelled

Control (8) 100 0 0 100IR � Px (8) 100 2.9 � 2.4 12.9 � 3.7 8.1 � 2.9IR (6) 100 15.8 � 7.9 7.1 � 2.1 100

1The proportions of retrogradely labelled neurons in each ION, expressed as a percent-age of the crossed path, in control, irradiated (IR), and irradiated-pedunculotomised(IR � Px) animals. For each animal, the number of neurons labelled by the normalcrossed path was defined as 100%. In IR � Px animals, each pathway is isolated, so eachpercentage of labelled neurons reflects an individual path. In the IR group, individualprojections could not be isolated, so percentage of retrogradely labelled neurons indi-cates only the ratio of ipsilateral vs. contralateral projections. n, Number of animals pergroup.

TABLE 2. Percentage (Mean � SEM) of Retrogradely Labelled NeuronsContributing to Normal and Aberrant Olivocerebellar Paths in Control and

Irradiated Rats1

Group (n)

Contralateral projection Ipsilateral projection

Path 1(normal)

Path 4(crosses incerebellum)

Path 2(unilateral)

Path 3(double-crossed)

Control (8) 100 0 0 0IR � Px (8) 100 8.1 � 2.9 12.9 � 3.7 2.9 � 2.4IR (6) 108 (paths 1 � 4)† 12.3 � 4 (paths 2 � 3)*

1The relative contribution of abnormal olivocerebellar projections to the X-irradiatedhypogranular cerebellum as a percentage of the normal path. Each path conforms to thenumeric identification shown in Figures 4 and 6. In the IR � Px animals, the percentageof labelled olivary neurons reveals that aberrant olivocerebellar paths (paths 2–4) aresignificant projections. In the IR group, labelling from the contralateral injection(dagger) arises from transport via both normal (path 1) and abnormal (path 4) routes.It is therefore greater than 100% of the normal projection. We have redefined this as108%, being the sum of contralaterally projecting paths identified from the IR � Pxgroup. The percentage of ipsilaterally labelled neurons also represents transport alongtwo possible path, ipsilateral (path 2) and double-crossed (path 3). For each animal inthe IR group, the percentage of ipsilaterally labelled neurons (asterisk) has beenrecalculated for the corrected percentage of contralaterally labelled neurons in thatgroup (i.e., 108%). The result is not significantly different from the combination ofipsilaterally projecting paths (paths 2 and 3) found in the IR � Px group (MannWhitney U, P � 0.57). n, Number of animals per group.

91DEVELOPMENT OF OLIVOCEREBELLAR TOPOGRAPHY

path 4). Furthermore, the double-crossed path (Fig. 4E,path ?3) is very small (Table 2).

Absence of transcommissuralolivocerebellar axons in the normal

neonatal cerebellum

To clarify whether the transcommissural olivocerebellaraxons of the irradiated rat are a retained transient neo-natal path or develop in response to the perturbed de-velopment, the presence of CFs was assessed bothelectrophysiologically and anatomically, by VGLUT2 im-munohistochemistry, in the deafferented hemicerebellumover 3 days following unilateral cerebellar pedunculotomyat P5.

CF-PC synaptic currents. CF-excitatory postsynap-tic currents (CF-EPSCs) were examined in 66 Purkinjecells from the left hemivermis of 15 successfully unilater-ally pedunculotomized rats (as evidenced by gross inspec-tion of the lesion site) and in age-matched normal rats.Twenty-four hours postpedunculotomy (P6), CF-EPSCscould not be elicited from the majority of PCs in the left(denervated) hemivermis (Fig. 5E, left). In only 2 of 29PCs from six rats could one or two small possible CF-EPSCs be recorded (Fig. 5E, left inset). In contrast, in acontrol vermis, CF-EPSCs were readily elicited from allPCs (Fig. 5E, middle). Furthermore, these control PCsshowed evidence of innervation by several CFs; that is, theCF-EPSCs had multiple distinct amplitudes correspond-ing to the activation of more than one CF (n � 14; mean �SEM � 4.2 � 0.32 CFs/PC). However, by 2 days postlesion(P7), CF-EPSCs were recorded from 86% of PCs (19 of 22cells) of the left hemivermis and indeed showed evidenceof innervation by more than 1 CF (mean � SEM � 2 �0.05 CFs/PC; Fig. 5E, right). By 3 days postdenervation,multiple CF-EPSCs were elicited from all PCs in the leftpedunculotomized hemivermis (n � 7; 2.28 � 0.12 CFs/PC).

These results indicate that, after unilateral pedunculo-tomy at the height of PC polyinnervation, transcommis-sural olivocerebellar axons with normal function are notpresent but rapidly sprout to reinnervate denervated PCswithin at the first 24–48 hours, as previously described byZagrebelsky et al. (1997).

Distribution of CF terminals. To clarify whether theapparent absence of functional transcommissural CFs atP5 was due to an inability to locate PCs innervated by asparse projection or to genuine absence of the pathway, wemade a histological examination of labelled CFs in thesame cerebellar slices from which PC currents were re-corded. Twenty-four hours after left pedunculotomy (i.e.,P6), dense VGLUT2-like immunoreactive terminals wereobserved only in the internal granular layer of the dener-vated hemicerebellum. There was no VGLUT2-like label-ling in the Purkinje cell or molecular layers (Fig. 5B). Incontrast, in control P6 cerebella, VGLUT2-like immuno-reactive terminals were observed both in the internalgranular layer and clustered around the PC somata, cor-responding, respectively, to mossy and climbing fibers atthis age (Mason and Gregory, 1984; Fig. 5A).

Two days after pedunculotomy (P7), VGLUT2-like im-munoreactive terminals were seen in both the internalgranular and the PC layers of both the pedunculotomizedand the control hemicerebella (Fig. 5C,D), which is con-sistent with CF reinnervation of the PC layer in the pe-

dunculotomized animals. Qualitatively, the immunoreac-tive terminals appeared to be less dense in the PC layer ofthe left (pedunculotomized) hemicerebellum comparedwith the control (Fig. 5C,D). Thus, transcommissuralolivocerebellar axons are not present at P5 during devel-opment but sprout into a hemicerebellum whose develop-ment is perturbed.

DISCUSSION

To clarify the topography of the immature olivocerebel-lar path, first we examined the olivary projection to thegranuloprival cerebellum as a model of “frozen” develop-mental status, and then we investigated whether multipleCF innervation of PCs resulted from the maintenance oftransient developmental paths or sprouting within theperturbed granuloprival environment. Our data show thatthe olivary projection to the cerebellum in the irradiatedrat contains multiple paths, which arise from separatepopulations of neurons; specifically, these aberrant pathsare 1) retained ipsilateral axons or 2) transcommissuralsprouting in response to the irradiation. We also demon-strate that in the granuloprival cerebellum the origin ofthe olivocerebellar projection traversing the contralateralICP is more localized than previously thought, being sim-ilar to that of controls. Thus, in that this is a model of theimmature path, relatively accurate parasagittal organiza-tion exists within the normally crossed olivocerebellarprojection early in development. Therefore, maturation ofolivocerebellar topography involves synapse eliminationresulting from 1) local refinement within a predefinedzone and 2) olivary neuronal death of a distinct populationof neurons, which removes abnormally projecting paths todefine laterality.

Neonatal X-irradiation retains an ipsilateralolivocerebellar projection

It is well-known that olivocerebellar input to the imma-ture (Bower and Payne, 1987) and granuloprival (Marianiet al., 1990) cerebella are bilateral projections (Fuhrmanet al., 1995; Sugihara et al., 2000; Fournier et al., 2002),although the nature of the ipsilateral path remained un-clear. In this study, the absence of double-labelled IONneurons, despite symmetrical cerebellar injections and bi-lateral olivary labelling, indicates that axons terminatingipsilaterally in the granuloprival hemicerebellum arisefrom separate populations of neurons, i.e., constitute anentirely separate path. In addition, our combination ofretrograde tracing and unilateral pedunculotomy hasdemonstrated the route by which an ION innervates ipsi-laterally placed PCs. After left unilateral pedunculotomyand FR injection into the right hemivermis, FR-labelledneurons in the right ION must result from axons arisingin the right ION, passing through the right ICP to theright hemicerebellum (Figs. 4F, 6D, path 2). The ipsilat-eral olivocerebellar path demonstrated here is consistentwith previous studies in the granuloprival cerebellum.First, a few olivary axons have been traced from the IONthrough the ipsilateral peduncle (Sugihara et al., 2000),although in that study they were thought to representtracer uptake by fibers-of-passage through the injection.Second, physiological and anatomical studies indicate thatfunctionally homologous regions of both IONs innervate asingle region of the cerebellar cortex (Fuhrman et al.,

92 B. FOURNIER ET AL.

1995, 2002). Furthermore, our data suggest that this ip-silateral projection (being �13% of the normal path) con-tributes a significant number of CFs to the PC multiin-nervation observed in the granuloprival cerebellum(Crepel et al., 1981).

An ipsilateral olivocerebellar projection has also beenobserved during development (Bower and Payne, 1987;Lopez-Roman and Armengol, 1994). Thus, its presence inthe granuloprival cerebellum is consistent with retentionof a transient developmental ipsilateral path, a phenom-

Fig. 5. Immunohistochemistry (A–D) and electrophysiology (E) inthe P6 and P7 cerebellum. Digital montage photomicrographs ofdouble-immunostained slices, showing localization of calbindin(green) in PCs and VGLUT2 (red) in mossy and climbing fiber termi-nals, from normal (A,C) or pedunculotomized (B,D) cerebella. A: Ver-mal slice from a control P6 rat. VGLUT2-like staining is found in boththe granule cell layer (GL) and in the PC layer around the PC somata(arrows), which is consistent with both mossy fiber and CF labelling,respectively. B: Slice from the left hemivermis of a P6 rat peduncu-lotomized on P5 (Px5). VGLUT2-like staining is restricted to the GL(arrowheads), consistent with the presence of mossy fibers but notCFs. C: In a control P7 rat, the VGLUT2-like immunoreactive climb-ing fibers surround the PC somata (arrow) and developing apicaldendrites. D: Slice from the left hemivermis of a Px5 rat on P7. The

pattern of VGLUT2-like staining is around the PCs (arrow) in asimilar location to the control although apparently less denselypacked (compare with C). E: Electrophysiological recordings from PCsin pedunculotomized (left and right panels) or control (middle panel)cerebellar slices. The left panel (Px5 P6) shows an example of record-ings from the majority of PCs in the P6 cerebellum the day afterpedunculotomy; CF-EPSCs were rarely found. The inset shows cur-rents recorded from one of the very few apparently innervated PCs.These rare currents are smaller than CF-EPSCs recorded from acontrol slice (middle panel; control P6) at the same day. The rightpanel (Px5 P7) shows the CF-EPSCs from a PC 2 days after pedun-culotomy. The multiple steps in the current amplitude indicate inner-vation by three CFs. Scale bars � 25 �m.

93DEVELOPMENT OF OLIVOCEREBELLAR TOPOGRAPHY

enon that has been demonstrated in other systems (Barthand Stanfield, 1990; Thompson et al., 1993, 1995) in re-sponse to perturbed development. Therefore, our datastrengthen the hypothesis that X-irradiation “freezes” theolivocerebellar projection in its immature state and re-veals that the immature path includes a separate, entirelyunilateral projection, which displays topography consis-tent with the normal path, albeit to the incorrect side ofthe midline (Fig. 6C, path 2).

X-irradiation induces transcommissuralolivocerebellar sprouting

Previous studies on the olivocerebellar projection in theirradiated rat proposed that the broader parasagittalzones and ipsilateral projections are due to retainedtranscommissural axons (Mariani et al., 1990), i.e., thatthey are present in the immature pathway (Sugihara etal., 2000). Our data do not corroborate this view.

Fig. 6. Diagrams of the contralateral (solid lines) and ipsilateral(dotted lines) olivocerebellar projections arising from the left ION(A–C) and a proposed structure of parasagittal microzones in thegranuloprival cerebellum (D). A: In the normal mature path (solidline), there is defined topography organized in narrow parasagittalmicrozones (1; vertical solid lines between arrows), in which each PCreceives a single CF. B: After neonatal X-irradiation, there aretranscommissural projections in the adult granuloprival cerebellum.There are two different transcommissural paths; one ascends theipsilateral peduncle (4; arising from the right ION in this diagram)and crosses within the cerebellum and a second is double-crossed (3;crossing in the medulla and then again in the cerebellum). C: In thegranuloprival cerebellum, each ION (for clarity, only the left is shown)projects multiple pathways to the cerebellum. First, a “normal” con-tralateral projection (thick solid line). Second, an aberrant contralat-

eral path (4) which ascends the peduncle ipsilateral to the ION andcrosses the midline within the cerebellum. Third, the ipsilateral pro-jection (Sugihara et al., 2000; Fournier et al., 2002) is formed byunilateral (2) and double-crossed (3) paths. D: Thus, each area ofcerebellar cortex in the granuloprival cerebellum receives multipleolivocerebellar projections from both inferior olives. The presence ofthese aberrant paths may explain the PC multiinnervation and broadparasagittal microzones observed in physiological studies (Fuhrmanet al., 1994). We propose that the “normal” contralateral projection(thick solid line) terminates in a narrow microzone (see inset; verticalsolid lines between arrows, 1) and that it is the aberrant contralateral(4) plus ipsilateral projections (2, 3) that widen the zone (see inset;vertical dotted lines, 5). Among these paths, 2 is present duringdevelopment (Bower and Sherrard, 1986; Lopez-Roman and Armen-gol, 1994), but 3 and 4 have sprouted in the granuloprival cerebellum.

94 B. FOURNIER ET AL.

First, we assessed the presence of transcommissuralCFs on P5–6, during peak CF-PC polyinnervation, byusing left inferior cerebellar pedunculotomy on P5 to re-move normal CF input. The lack of either normal multipleCF-EPSCs or visible CF terminals after 24 hours suggeststhat transcommissural olivocerebellar axons do not existin the normal neonatal system. The very few small, atyp-ical CF currents recorded 24 hours after unilateral pedun-culotomy are consistent with an extremely small numberof transcommissural olivocerebellar axons, such as havebeen documented in the normal adult animal (Sugihara etal., 1999). However, they are not congruent with the ex-tensive transcommissural innervation of the adult granu-loprival cerebellum, which is �8-fold greater than normal(Sugihara et al., 2000). These few currents are more con-sistent with early transcommissural sprouting, whichcrosses the cerebellar midline 18 hours after denervation(Zagrebelsky et al., 1997). This latter interpretation issupported by the subsequent development of PC CF-EPSCs during the following 2–3 days, which not onlyfollows the time course of transcommissural reinnervationpreviously described (Zagrebelsky et al., 1997) but also isin agreement with the very rapid axonal elongation ob-served in the immature rat brain (Jhaveri et al., 1991)during transcommissural sprouting into denervated areas(Sabel and Schneider, 1988). In either case, our data donot demonstrate an extensive transcommissural compo-nent to the neonatal olivocerebellar projection.

Second, for the adult irradiated cerebellum, our datarevealed transcommissural olivocerebellar axons by thepresence of retrogradely labelled neurons in both IONsfollowing left cerebellar injection and left cerebellar pe-dunculotomy. Such labelling can be explained onlythrough intracerebellar transcommissural axons thathave entered the cerebellum through the right ICP andsubsequently crossed the midline (Fig. 6B). These data areconsistent with increased transcommissural axons in theirradiated system identified by anterograde tracing (Sugi-hara et al., 2000). However, retrogradely labelled neuronsin both IONs in these animals (left cerebellar injection andleft pedunculotomy) demonstrates two separate transcom-missural phenomena: 1) a double-crossed path that ulti-mately projects to the ipsilateral cortex (Figs 4G, 6B,C,paths 3) and 2) a single-crossed path that has ascendedthe peduncle ipsilateral to the cells of origin and thencrossed the midline within the cerebellum to project con-tralaterally (Fig. 6B,C, path 4).

The ipsilaterally projecting olivocerebellar path, albeitvia a double-crossed route (Fig. 6B,C, path 3), is consistentwith the ipsilateral projection seen in the adult irradiatedcerebellum both physiologically (Fuhrman et al., 1995)and anatomically (Sugihara et al., 2000; Fournier et al.,2002). However, the paucity of olivary labelling (�3% ofthe normal path; see Table 1) indicates that this ipsilat-eral (i.e., double-crossed) pathway forms only a minorcomponent of additional olivocerebellar axons to thegranuloprival cerebellum. In contrast, contralaterally pro-jecting axons, which cross the midline within the cerebel-lum (Fig. 6B,C, path 4) rather than the medulla, providean additional crossed pathway that is 8% of the size of thenormal crossed path. Such a projection may account forthe apparently broader olivary origin (i.e., reduced preci-sion) of the contralateral olivocerebellar projection in theirradiated rat. These transcommissural paths were previ-ously believed to be retained collaterals of the normally

projecting path (Fuhrman et al., 1994; Zagrebelsky andRossi, 1999), but our data from the nonirradiated neonatalcerebellum (see above) indicate that they are postlesionsprouting.

In summary, the olivocerebellar projection in the adultirradiated rat contains two transcommissural paths, oneprojecting contralaterally and the second double-crossed.Neither of these transcommissural paths exists to anygreat extent during development. Therefore, we proposethat their presence within the adult granuloprival cere-bellum represents sprouting in response to the perturba-tion of normal development induced by the X-irradiation.

The normally crossed olivocerebellar path isaccurately organized

In the irradiated cerebellum, the parasagittal organiza-tion of the “normal” olivocerebellar projection, whichcrosses the midline in the medulla to traverse the oppositeICP, displays a refinement similar to that in the normalrat. Evidence for this comes from similar focal olivarylabelling, located exactly according to known olivocerebel-lar topography (Buisseret-Delmas and Angaut, 1993), inboth control and irradiated animals, in which pedunculo-tomy removed any abnormal projections passing throughthe ipsilateral peduncle (Figs. 4F,G, 6C,D, path 4). Suchapparent normality of this projection is not surprising,insofar as PCs, which are not destroyed by the irradiation,determine olivocerebellar organization (Blatt and Eisen-man, 1993) and CFs in mildly hypogranular cerebella arerestricted to zebrin-defined compartments (Zagrebelskyand Rossi, 1999). Such topographic accuracy is also com-patible with the broader zones previously identified in thegranuloprival cerebellum (Fuhrman et al., 1994; Sugiharaet al., 2000); in those studies, multiple olivocerebellarroutes had not been identified.

Olivocerebellar topography in the irradiatedrat: relevance to the immature pathway

If the olivocerebellar projection to the granuloprival cer-ebellum truly represents the developmental structure (de-spite the addition of a small amount of transcommissuralsprouting), our data suggest that parasagittal zoning inthe developing pathway is more precise than previouslythought; i.e., immature olivocerebellar axons, forming thenormal path through the contralateral peduncle, are or-ganized into predefined narrow parasagittal zones. Thusour data indicate that subsequent olivocerebellar matura-tion associated with regression of PC polyinnervation in-volves two separate phenomena: 1) elimination of super-numerary synapses at a local cellular level within ratherthan between predefined zones and 2) regression of entirepaths that inappropriately ascend the ipsilateral pedun-cle.

Similar examples of early topographic organization andipsilateral projections during development are found inother parts of the neuraxis. First, early topographic defi-nition, within which there is synaptic regression, occurs inthalamocortical (Shepherd et al., 2003) and barrel cortex(Bureau et al., 2004) circuits. It is also consistent withearly olivocerebellar targeting by cell-surface molecules(Chedotal et al., 1997; Plagge et al., 2001). Second, theregression of an ipsilateral path is widely observed duringmaturation of other projection pathways (O’Leary andStanfield, 1989; Thompson et al., 1993, 1995; Chan andGuillery, 1993). Although the function of transient ipsilat-

95DEVELOPMENT OF OLIVOCEREBELLAR TOPOGRAPHY

erally projecting axons remains unclear, it has been pro-posed that, because of their early outgrowth, they act aspioneers, which direct later-arriving axons (Marcus andMason, 1995). Given the topographic accuracy and num-ber of ipsilateral olivocerebellar axons in this study, wepropose that they have this function; that is, they areinvolved in directing the ingrowing normal (i.e., crossed)olivocerebellar axons in the perinatal period. In addition,our data indicate that the loss of this ipsilateral pathinvolves olivary neuronal death, in that the absence ofdouble-labelled neurons suggests that it arises from aseparate population of olivary neurons. This phenomenon,death of a subpopulation of neurons forming transientipsilateral projections, is not new and has been docu-mented in other systems (Serfaty et al., 1990; Gramsber-gen and Ijkema-Paasen, 1991; Reese and Urich, 1994;Thompson et al., 1995). Indeed, not only are there moreolivary neurons in the irradiated rat, at least until day 20(Geoffroy et al., 1988), but also olivary neuronal death istemporally associated with maturation of the olivocerebel-lar path (Delhaye-Bouchaud et al., 1985; Armengol andLopez-Roman, 1992; Cunningham et al., 1999). Thus ourdata reveal that the immature olivocerebellar projectiondisplays early topographic definition, within which thereis synaptic regression plus a transient ipsilateral pathwhose elimination involves olivary neuronal death.

CONCLUSIONS

We have used the olivocerebellar projection to thegranuloprival cerebellum as a model of the immaturepathway. Our results suggest that broader parasagittalzones and ipsilateral olivary innervation during develop-ment derive primarily from abnormally projecting axonsthat ascend the ICP ipsilateral to the ION of origin. Fur-thermore, the distribution of retrogradely labelled neu-rons and the absence of double-labelled cells indicate that,in the immature rat, the contralaterally projecting olivo-cerebellar path possesses the same parasagittal organiza-tion as is found in adults. Thus, maturation of olivocer-ebellar topography involves synapse elimination relatedto two phenomena: removal of whole developmental pathsto define laterality plus local collateral retraction thatrefines the precise map within predefined narrow para-sagittal zones.

ACKNOWLEDGMENTS

The authors thank Dr. Salah El Mestikawy for provid-ing the rabbit anti-VGLUT2 antibody.

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