Projection of Reconstructed Single Purkinje Cell Axons ...

Projection of Reconstructed Single Purkinje Cell Axons inRelation to the Cortical and Nuclear Aldolase CCompartments of the Rat Cerebellum

IZUMI SUGIHARA,1* HIROFUMI FUJITA,1 JIE NA,1,2 PHAM NGUYEN QUY,1 BING-YANG LI,2

AND DAISUKE IKEDA1

1Department of Systems Neurophysiology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519,Japan2Laboratory of Brain and Cognitive Science, Shenyang Normal University, Shenyang 110034, China

ABSTRACTAlthough the overall topography of the cerebellar cortico-nuclear projection formed by Purkinje cell (PC) axons hasbeen described, only a few studies have dealt with theorganization of this projection at the level of individual PCaxons. Thus, we reconstructed 65 single PC axons that werelabeled with biotinylated dextran amine in the rat. We thenanalyzed the relationship between the projections of thesePCs and the compartmentalization of the cerebellar cortexand nuclei based on the topography of olivocerebellar pro-jection and aldolase C expression in PCs. After giving rise toshort local recurrent collaterals near the soma, a PC axonformed a terminal arbor in a specific small area in the cer-ebellar nuclei (CN). The terminal arbors of vermal PCs werespread more widely than those of hemispheric PCs andsometimes extended to extracerebellar targets. PCs located

in any of the aldolase C-positive (Groups I and II) and-negative (Groups III and IV) stripes consistently projectedto the caudoventral and rostrodorsal parts of the CN, re-spectively, precisely in accordance with the compartmen-talization of the cortex and nuclei. Mediolateral segregationand rostrocaudal convergence were seen between projec-tions of separate PCs in a single aldolase C compartment.The results revealed a tight link between the projectionpatterns of individual PC axons, the topography of the olivo-cerebellar pathway, and the aldolase C expression pattern.Their overall correspondence seems to reflect a basic as-pect of cerebellar organization, although some area-dependent variation in the relationship of these three enti-ties was also present. J. Comp. Neurol. 512:282–304, 2009.© 2008 Wiley-Liss, Inc.

Indexing terms: cerebellar cortex; cerebellar nucleus; vestibular nucleus; biotinylated dextranamine; zebrin; topography

The organization of the cerebellar system is character-ized by the topography and compartmentalization of theconnections of its input and output fibers. Among thesefibers, the Purkinje cell (PC) axons constitute the sole out-put of the cerebellar cortex and the major input to thecerebellar nuclei (CN) to connect these two essential cere-bellar structures, and thus significantly contribute to theorganization of the cerebellar system (Brodal, 1981; Ito,1984; Voogd, 2004).

The cerebellar cortex has been longitudinally subdividedaccording to the arrangements of projecting axons and ace-tylcholinesterase activity (Voogd, 1967; Voogd and Bigare,1980). These subdivisions have been reflected or followed bythe topographic projection patterns of olivocerebellar climb-ing fibers and PC axons. PCs in each subdivision (designatedzones A, B, C1-3, and D0-2) are innervated by neurons indistinct subnuclei of the inferior olive and project to differentareas of the CN (Groenewegen and Voogd, 1977; Azizi andWoodward, 1987; Buisseret-Delmas and Angaut, 1993).

Much finer longitudinal compartmentalization of the cere-bellar cortex has recently been evidenced by longitudinalstripe-shaped expression patterns of specific molecules, suchas aldolase C (�zebrin II), in a population of PCs in the rat(Hawkes and Leclerc, 1987; Brochu et al., 1990; Voogd et al.,2003; Sugihara and Shinoda, 2004, 2007). Aldolase C is a rel-atively brain-specific isozyme of fructose-1,6-(bis)phosphatealdolases, which is involved in glycolysis (Mukai et al., 1991).

Grant sponsor: Japan Society for the Promotion of Science; Grant num-ber: Grant-in-Aid for Scientific Research 20300137.

*Correspondence to: Dr. Izumi Sugihara, Dept. of Systems Neurophysi-ology, Tokyo Medical and Dental University Graduate School of Medicine,1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.E-mail: [email protected]

Received 26 December 2007; Revised 18 June 2008; Accepted 23 Sep-tember 2008

DOI 10.1002/cne.21889Published online in Wiley InterScience (www.interscience.wiley.com).

The Journal of Comparative Neurology 512:282–304 (2009)

© 2008 Wiley-Liss, Inc.

About 20 longitudinal compartments, in which PCs show pos-itive, negative, or lightly positive expression of aldolase C,have been defined in the rat cerebellar cortex. A specific namesuch as 1�, 1� and so on (usually a numeral and/or a letterfollowed by a sign indicating positive or negative) has beengiven to each compartment. Tracing studies have identifiedspecific olivocerebellar projection to each compartment andclarified the correspondence between the aldolase C com-partments and zones A-D (Voogd et al., 2003; Sugihara andShinoda, 2004; Sugihara and Quy, 2007). For example, aldo-lase C compartments 1�, 1�, 2�, 2�, 3�, 3�, and 4� belongto zone A in the caudal cerebellum. Furthermore, it has beensuggested that aldolase C-negative and -positive areasmainly receive somatosensory and other (cerebral, tectal, ves-tibular, and visual) inputs, respectively, through the area-specific inputs to the inferior olive and the topographic olivo-cerebellar projection (Sugihara and Shinoda, 2004). Thus,aldolase C compartments may represent fundamental func-tional organization of the cerebellar cortex.

Recently, fine compartmentalization in the CN has alsobeen demonstrated by tracing nuclear collaterals of olivocer-ebellar axons (Sugihara and Shinoda, 2007). According to thisstudy, the CNs are primarily divided into caudoventral aldo-lase C-positive and rostrodorsal aldolase C-negative parts, incontrast to the conventional mediolateral nuclear subdivi-sions. The aldolase C-positive and -negative areas in the CNare then further subdivided into multiple compartments basedon the topographic olivonuclear projection. This aldolase-Ccompartmentalization within the CN has no trace of the lon-gitudinal stripes that characterized cortical compartmental-ization.

These fine but different compartmentalizations in the cere-bellar cortex and nuclei based on the olivocerebellar projec-tion raise the question of whether PC projection is organizedaccording to the same compartmentalizations. To answer thisquestion, understanding the morphology of the axonal arbor

of single PCs is essential. Although the labeling of single PCaxons has been reported in the paramedian lobule and ante-rior lobe (cat, Bishop et al., 1979), flocculus (rabbit, de Zeeuwet al., 1994; rat, Sugihara et al., 2004), and nodulus (rabbit,Wylie et al., 1994) and in cortical recurrent collaterals (cat,Bishop, 1982; O’Donoghue and Bishop, 1990), projection pat-terns of PC axons have not been systematically studied inrelation to cerebellar compartmentalization. Therefore, wesought to trace the entire trajectories of single PC axons toinvestigate the basic and general structural organization oftheir projection in relation to cortical and nuclear compart-mentalizations by using biotinylated dextran amine as atracer.

MATERIALS AND METHODSTracer injection and histological procedure

Reconstruction of PC axons was performed in 24 Long–Evans adult rats (Kiwa Laboratory Animals, Wakayama, Ja-pan). All of the experimental animals in this study were treatedaccording to the Guiding Principles for the Care and Use ofAnimals in the Field of Physiological Sciences of the Physio-logical Society of Japan (2001 and 2002 editions). The exper-imental protocols were approved by the Institutional AnimalCare and Use Committee of Tokyo Medical and Dental Uni-versity (numbers 0040089, 0050041, 0060121, and 0070133).The anesthesia, surgical, and histological procedures weresimilar to those described previously (Sugihara et al., 1999,2001).

Briefly, the animals were anesthetized with an intraperito-neal injection of ketamine (130 mg/kg body weight) and xyla-zine (8 mg/kg). Atropine (0.4 mg/kg) was also given intraperi-toneally. Supplemental doses of ketamine (13 mg/kg) andxylazine (1 mg/kg) were given every 30 minutes starting 1 hourafter the initial dose, as required. Some animals were anes-thetized with an intraperitoneal injection of pentobarbital so-

Abbreviations

4V Fourth ventricleI-X Lobules I-Xa-d Sublobules a-dAICG Anterior interstitial cell groupAIN Anterior interposed nucleusBDA Biotinylated dextran amineC CaudalcMAO Caudal part of the medial accessory oliveCN Cerebellar nucleiCop Copula pyramidisCP Caudal poleCrI crus I of ansiform lobuleCrII crus II of ansiform lobuleD Dorsald-Y Dorsal Y nucleusdas Dorsal acoustic striaDC Dorsal cap of KooyDCoN Dorsal cochlear nucleusdDAO Dorsal fold of the dorsal accessory oliveDLH Dorsolateral hump (of the AIN)DLP Dorsolateral protuberance (of the FN)DM Dorsomedial group subnucleusDMC Dorsomedial crest (of the AIN)DMCC Dorsomedial cell column subnucleusDN Dentate (lateral) nucleusDPFL Dorsomedial paraflocculusdPO Dorsal lamella of the principal oliveFL Flocculus

FN Fastigial (medial) nucleusICG Interstitial cell groupicp Inferior cerebellar peduncleIVN Inferior vestibular nucleusL LateralLVN Lateral vestibular nucleusM MedialMVN Medial vestibular nucleusn7 Facial nervePar Paramedian lobulePIN Posterior interposed nucleusPBN Parabrachial nucleusPC Purkinje cellpf Primary fissureR RostralrMAO Rostral part of the medial accessory olivescp Superior cerebellar peduncleSim Simple lobulesp5 Spinal trigeminal tractSVN Superior vestibular nucleusV, v- VentralvDAO Dorsal fold of the dorsal accessory oliveVLO Ventrolateral outgrowthVPFL Ventral paraflocculusvPO Ventral lamella of the principal oliveX Nucleus XY Nucleus Y

The Journal of Comparative Neurology

283SINGLE PURKINJE CELL AXONS

dium (90 mg/kg body weight). Atropine (0.4 mg/kg) was alsogiven intraperitoneally. Supplemental doses of pentobarbitalsodium (20 mg/kg) were given every 40 minutes starting 1hour after the initial dose, as required. Biotinylated dextranamine (BDA, D-1956, 10,000 MW or D-7135, 3,000 MW; Mo-lecular Probes, Eugene, OR; 10% solution in saline) waspressure-injected with a Picopump PV820 (WPI, Sarasota, FL)in the molecular layer at various locations in the cerebellarcortex (putative volume, 1–5 nL) for anterograde labeling.Spontaneous complex spike activities were recorded to lo-cate the molecular layer through the injection pipette by usinga Micro 1401 recording system and Spike2 software (CED,Cambridge, UK) with a bandpass frequency range of 150–3,000 Hz. After injection, complex spike activity was tempo-rarily inactivated (Fig. 1A). Multiple injections (up to six) weremade in separate locations throughout the cortex in each ratsince it was not difficult to distinguish axons from differentorigins by following them. Some injections labeled one toseveral axons intensely. Other injections labeled no PC axonsintensely or too many PC axons to trace individual ones. Aftera survival period of 6–8 days the rats were anesthetized as inthe first operation but with a 1.5-times larger dose of anes-thetics. They were perfused intracardially with phosphate-buffered saline (PBS) followed by fixative containing 5% para-formaldehyde, 2% sucrose, and phosphate buffer 50 mM (pH7.4). Eighty-�m-thick serial frozen sections were cut coronallyfrom the cerebellum and medulla.

BDA was visualized in black with an Elite ABC kit (PK6100,Vector Laboratories, Burlingame, CA). In most cases, aldolaseC was then immunostained in brown. The immunohistologicalprocedures that were used to double-label BDA (black reac-tion product) and aldolase C (brown reaction product) withdiaminobenzidine have been described previously (Sugiharaand Shinoda, 2004). The anti-aldolase C antibody used in thisstudy was raised in our laboratory by immunizing a rabbit witha synthetic peptide that represented amino acids 322–344from rat aldolase C (Sugihara and Shinoda, 2004). This anti-body stains a single band on Western blot with rat cerebellartissue and the addition of the immunizing peptide to theprimary antibody solution abolishes immunostaining (Sugi-hara and Shinoda, 2004). Some sections were counterstainedwith thionine after they were mounted on glass slides.

In three Long–Evans adult rats, BDA was injected in thefastigial (medial) nucleus (FN) by a procedure similar to thatdescribed above (diameter 0.2–0.3 mm, volume 4–13 nL) tolabel PCs retrogradely. The brains of these rats were cutparasagittally and treated for BDA visualization. Otherwise,the procedures for these brains were the same as those de-scribed above.

In seven Long–Evans adult rats, fluorescent (red and/orgreen) latex microspheres (Lumafluor, Naples, FL) were in-jected into the CN by a procedure similar to that describedabove (diameter 0.2–0.3 mm, volume 4–13 nL) to label PCsretrogradely. The surgery, survival, and histological proce-dures for these rats were the same as those for other rats. Thebrains of these rats were cut coronally and immunostained foraldolase C with diaminobenzidine. The sections weremounted on glass slides and photographed after being cov-erslipped using PBS.

One Long–Evans adult rat was used for immunostaining ofaldolase C in the cerebellar cortex to label recurrent collater-als of PC axons. This rat was anesthetized, perfused, andfixed in the same way as described above. Serial frozen sec-tions of the cerebellum were cut coronally. The immunohisto-logical procedures used to label aldolase C black with diami-nobenzidine have been described previously (Sugihara andShinoda, 2004).

Reconstruction of individual axons andphotomicroscopic procedure

Axonal trajectories of single-labeled olivocerebellar axonswere reconstructed from serial coronal sections using a three-dimensional imaging microscope (Edge R400; SNT Micro-scopes, Los Angeles, CA) equipped with a camera lucidaapparatus. Cut ends of an axon on one section were con-nected properly to the corresponding cut ends of the sameaxon on the successive section (Shinoda et al., 1981; Sugiharaet al., 1999; Wu et al., 1999). Only axons that were well labeled,isolated from other axons, and could be traced from theinjection site to every end were considered to be completelyreconstructed, whereas axons that could not be traced at anypoint on their pathway due to poor labeling or interminglingwith other axons were regarded as “not fully reconstructed.”Reconstructions in the sectioning plane (coronal) were some-times converted to those in another plane (parasagittal). Indrawings of single-axon images, fibers and swellings weredrawn thicker than scale for clarity, as is conventionally donein drawings of reconstructed fibers. Single-axon images in thepresent figures are depicted on a montage of drawings ofsections for the BDA injection site and the center of theterminal arbor. These drawings contained contours of cere-bellar structures and boundaries between areas with differentaldolase C labeling intensities. The cerebellar lobules weredefined according to Larsell (1952) and Voogd (2004). Aldo-lase C compartments in the cerebellar cortex were definedaccording to Sugihara and Shinoda (2004), who basicallyadopted previous nomenclatures (Hawkes and Leclerc, 1987;Voogd et al., 2003). The name of an aldolase C compartment

Figure 1.Labeling single PC axons. A: Spontaneous complex spike activity, which indicated that the glass microelectrode is located in the molecular layer, andits temporary inactivation after pressure injection of the tracer (arrowhead). One of the complex spikes (asterisk) is shown with a fast sweep speed(right). B,C: Photomicrographs of an injection site in compartment 1� in lobule IXc (B) and labeled PC axons running in the granular layer in the samecompartment (1�) (C). D: Rise of an axon from a PC soma (arrowhead). The PC soma is partially embedded within the darkly labeled injection spot.E: Some swellings that belong to a single PC axon surrounded a nuclear neuron (arrowheads). F: Terminal arbor of a labeled single PC axon. All labeledfibers and swellings in this panel belong to a single PC axon. Montage of five photographs with different focus depths. Arrowhead indicates the cutend of the proximal side of the axon. G: Entire trajectory of a reconstructed single PC axon in a frontal view. In drawings in this panel and in otherfigures, the shaded areas in the molecular layer of the cerebellar cortex indicate the aldolase C-positive compartments and the shaded areas in theCN indicate the aldolase C-positive caudoventral part. The terminal arbor of this axon belonged to the wide type. H: A compact-type terminal arborof a reconstructed PC axon. Arrowhead indicates a collateral that extended rostrally by about 200 �m from the main terminal arbor. I: Anelongated-type terminal arbor of another reconstructed PC axon. Filled circles in H and I indicate the stem axon. Scale bars � 5 �V and 50 ms in A,left; 500 �s in A, right; 200 �m in B; 50 �m in C,F; 10 �m in D,E; 200 �m in G (applies to H,I).


284 I. SUGIHARA ET AL.

Figure 1



was usually indicated by a numeral plus a sign, such as 5�. Toindicate linked compartments located in the rostral and cau-dal cerebellum, their names were connected with a “//,” as in“4�//5�.” While we traced axons in the cerebellar nuclei, wealso traced the contour of the CN and boundaries betweenaldolase C-positive and -negative areas in the CN (Sugiharaand Shinoda, 2007).

Sections were photographed using a digital camera (DP-50,Olympus, Tokyo, Japan) attached to a microscope (BX41,Olympus). Photographs were assembled using Photoshop LEand Illustrator software (Adobe, San Jose, CA). The softwarewas used to adjust contrast and brightness, but no digitalenhancements were applied.

Sections of rats that had been injected with fluorescentlatex microspheres were photographed with PBS and photo-graphed with a fluorescent microscope and a color digitalcamera (BX51WI and DP-70, Olympus) with 4�, 10�, or 20�objectives. Weak brightfield illumination was applied to visu-alize the contour of the brain structures and aldolase C com-partments labeled in brown in addition to the fluorescence.The colors of photographs were converted to gray scale.

In all figures in this article, drawings and photographs on theright side of a coronal section were flipped about the verticalaxis.

RESULTSMorphology of entire PC axons that were labeled

anterogradelyThe injection of BDA created a darkly labeled spot in the

molecular layer, which typically measured 0.04–0.10 mm inthe transverse direction and about 0.08–0.20 mm in the lon-gitudinal direction (Fig. 1B). A few or several PC axons werelabeled by such an injection (Fig. 1C). We picked up one ormore well-labeled axons in the folial white matter and startedtracing toward the proximal and distal directions. We com-pletely reconstructed 65 PC axons that were labeled in 43injections in 24 rats in the present study. A reconstructed axoncould always be traced back to the injection site. The origin ofthe axon from a PC soma (Fig. 1D) was visible unless the PCwas not completely covered by the dark labeling of the tracerinjection spot (23 of 65 axons). An axon usually gave rise toone or two recurrent collaterals near the PC soma (see below).A PC axon then descended through the folial white matterwithout any branching until it was close to the CN.

A stem axon usually branched into two or occasionally threerelatively thick (diameter �0.8 �m) primary branches shortlybefore or after it entered the CN (Fig. 1G–I). These primarybranches ran roughly parallel to each other in the same regionof the CN for about 0.5–1 mm. Several thinner secondarybranches (diameter �0.5 �m) were given off from the primarybranches or from the stem axon. Abundant short tertiarybranches or branchlets (diameter �0.3 �m) were given offfrom secondary branches. Many en-passant and terminalswellings (diameter: �2–3 �m) were located on tertiarybranches (Fig. 1F). The number of branchings ranged from 27to 155 (mean and SD, 45.8 � 20.1, n � 65) per axon. Thenumber of swellings ranged from 63 to 404 (121.9 � 53.4) peraxon. Occasionally a short fine collateral (length, 1–2 �m) witha small satellite swelling (diameter, 1–1.5 �m) at the endextended from en-passant and terminal swellings. Overall, the

terminal arbor of a single axon resembled loose roots of aplant. Swellings of an axon were scattered along the entirelength of the terminal arbor (Fig. 1F–I). However, some of theswellings of a single axon sometimes strategically surroundedthe soma of a specific nuclear neuron (Fig. 1E).

Since PCs were classified into aldolase C-positive and-negative (including lightly positive) populations (see below),we compared the number of swellings between these PCs.The number of swellings in terminal arbors of aldolaseC-positive (133.0 � 61.9, mean and SD, n � 38) was slightlygreater than that of aldolase C-negative PCs (106.8 � 29.5,n � 27), and this difference was statistically significant in asingle-factor analysis of variance (ANOVA) analysis (F � 4.20,d.f.(between) � 1, d.f.(residual) � 63, P � 0.045).

Variation in terminal arbor morphologyA remarkable difference was observed in the shape and size

of PC terminal arbors in the CN. Therefore, in the presentstudy the terminal arbors in the CN were tentatively classifiedinto elongated, wide, and compact types, although a fewterminal arbors showed conformations intermediate betweenthese types. This variation in the terminal arbor conformationwas related to the location of the arbor within the CN. First,terminal arbors in most of the FN, except for those in theventral FN and the dorsolateral protuberance (DLP) of the FN,were usually widely spread in multiple directions by more than0.8 mm (wide type, Fig. 1G). Second, terminal arbors in theDLP, in the anterior interposed nucleus (AIN) and in most ofthe posterior interposed nucleus (PIN) and the dentate (lateral)nucleus (DN), except for those in their ventral regions, wererelatively compact in size (less than 0.8 mm in spread) anddense with regard to swelling disposition (compact type, Fig.1H). The overall conformation of compact-type terminal ar-bors showed some elongation in a direction that seemedequivalent to the direction of the hilus of the nuclei. Occasion-ally, one or a few thin collaterals extended for a long distancein this direction apart from the main compact part of theterminal arbor (Fig. 1H, arrowhead). Finally, terminal arbors inthe ventral parts of the DN, PIN, and FN were often elongatedin a single (often mediolateral) direction by more than 0.8 mm,but did not spread much in other directions (elongated type,Fig. 1I). Terminal arbors of PCs that projected mainly to ex-tracerebellar targets (n � 6 axons, see below; also refer tofloccular PCs in fig. 10 of Sugihara et al., 2004) would beclassified as elongated-type or wide-type.

The number of swellings in wide, compact, and elongatedterminal arbors in the CN, 130.1 � 85.5 (mean and SD, n � 20),117.1 � 30.0 (n � 31), and 114.5 � 26.0 (n � 8), respectively,were similar, and a single-factor ANOVA analysis found nostatistically significant difference (F � 0.429, d.f.(between) �2, d.f.(residual) � 56, P � 0.653).

Aldolase C compartment-specific PC projection(Groups I–IV)

The cerebellar cortex is divided into about 20 longitudinalstripes that are alternately aldolase C-positive (or -lightly pos-itive) and -negative (Hawkes and Leclerc, 1987; Sugihara andShinoda, 2004; Sugihara and Quy, 2007). Previous studieshave identified the pattern of olivocerebellar climbing fiberprojection to each aldolase C stripe in the cortex (Voogd et al.,2003; Sugihara and Shinoda, 2004). On the other hand, the CNis subdivided into rostrodorsal aldolase C-negative and cau-



doventral aldolase C-positive parts (Sugihara and Shinoda,2007). This aldolase C subdivision is partially compatible withthe conventional subdivision of the CN into the FN, AIN, PIN,and DN. However, the topography of the collateral projectionof olivocerebellar axons to the CN has indicated a fine com-partmentalization in each of the aldolase C-positive and-negative parts of the CN (Sugihara and Shinoda, 2007), whichcorresponds nearly completely to the cortical fine compart-mentalization based on aldolase C stripes. To better under-stand the cortical compartmental organization and relate it tofunctional aspects, we have proposed that aldolaseC-positive and -negative compartments can be classified intothree (Groups I, II, and V) and two (Groups III and IV) groups,respectively (Fig. 2A; Sugihara and Shinoda, 2004, 2007). Thelightly positive compartments and several negative compart-ments have been considered to be akin to each other, andhave been included together in Group IV, since they bothreceive olivary projection from the same subnuclei. The five-group scheme can also be applied to the compartmentaliza-tion of the CN (Fig. 2B,C; Sugihara and Shinoda, 2007). Areasin the cortex and CN that belong to the same group areinnervated by the same population of inferior olive neurons.

Aldolase C is expressed in PCs, including their axonal ter-minals, but not in nuclear neurons (Sugihara and Shinoda,2007). Therefore, the subdivision of the CN into rostrodorsalaldolase C-negative and caudoventral aldolase C-positiveparts indicates that Purkinje cells in aldolase C-positive areas(Groups I, II, and V) in the cortex do not project much toaldolase C-negative areas (Groups III and IV) in the CN. How-ever, much detail remains unclear regarding the cortico-nuclear PC projection. Therefore, we examined whether thetopography of PC projection is also in accordance with thefive-group scheme by analyzing axonal projections of PCsthat belonged to Groups I to IV. Group V, flocculus and nod-ulus, was not considered in the present study.

Cortical Group I has been defined as aldolase C-positivecompartments that extend rostrally to the anterior lobe be-yond the primary fissure (green areas in Fig. 2A). They corre-spond to zones C2, D1, and D2 and some areas in zone A, andare innervated by the ventrolateral parts of subdivisions of theinferior olive (Table 1), which mainly receive midbrain inputs(Sugihara and Shinoda, 2004). Axons of seven PCs that belongto Group I are shown in Figure 3. They projected to the

ventromedial FN from 1� in lobule II (Fig. 3A), to the ventro-lateral FN from medial 2� in lobule VIb (Fig. 3B), to the ventralICG from lateral 3� in lobule VIII (Fig. 3C), and to the centraland caudal PIN from 5� in crus IIb and the copula pyramidis(Fig. 3D,E). Two other axons projected to the lateral PIN fromcrus Ib and to the lateral DN from the dorsal paraflocculus(Fig. 3F,G). Since there are no aldolase C-negative stripes in

Figure 2.Five-group compartmentalization of the cerebellar cortex and nucleibased on the topography of the olivocortical and olivonuclear projec-tions and aldolase C immunostaining. A: Unfolded scheme of the leftcerebellar cortex. B,C: Three-dimensional scheme of the most ventralportions of the left CN (B) and the entire left CN (C) in the dorsocaudalview. Compartments that belong to each group are indicated bydifferent colors: Group I (aldolase C-positive), green; Group II (posi-tive), cyan and blue; Group III (negative) yellow and orange; Group IV(negative and lightly positive) pink and red; Group V (positive) gray.Group or subgroup in the same color in the cortex and nuclei areinnervated by the same subareas of the inferior olive (see fig. 10 ofSugihara and Shinoda, 2007). Arrows represent a fine topographiccorrespondence within each group or subgroup, i.e., areas in thecortex and nuclei that are roughly overlaid by the same portion of thearrows (base to tip) receive divergent projection from a subarea of theinferior olive. Each arrow is colored in the same hue as the (sub)groupto which it is related. These schemes are derived from figure 10A,C ofSugihara and Shinoda (2007) with slight modification.



TABLE 1. Major Olivocortical and Olivonuclear Topographic Projection Patterns in Previous Studies in the Rat, to Which Corticonuclear PC Projection inThis Study Was Compared

1 Aldolase C compartment as major termination area (Sugihara and Shinoda, 2004). See Results for details.2 Voogd and Bigare, 1980; Buisseret-Delmas and Angaut, 1993; Voogd et al., 2003; Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2004.3 Including subareas of the vestibular nucleus that are innervated by the inferior olive.4 This column lists the PC axon that originated from the aldolase C compartment listed in the same line in this table. The target of the PC axon usually coincided with the CN subareathat is also listed in the same line in this table.5 Not including subareas of the vestibular nucleus that are innervated by the inferior olive.6 The target of the PC axon depicted in the indicated figure does not fully agree with the CN subarea listed in the same line in the table, which was obtained in the previous studyof olivonuclear projection (Sugihara and Shinoda, 2007).7 Speculated based on the results of the present study (Fig. 8D).8 Sugihara et al., 2004.



Figure 3.Projections of reconstructed axons of PCs that belonged to Group I (green areas in Fig. 2). A–G: Trajectories of axons in the caudal view. In thedrawings of the molecular layer and the cerebellar nuclei, the aldolase C-positive parts are indicated by shading. In the drawings of thecerebellar nuclei, areas that belong to Groups I–V are indicated by (I), (II), and so on by referring to Fig. 2 (and also to fig. 8 of Sugihara andShinoda, 2007). Origin, destination, and type of the terminal arbor of each axon: 1� in lobule II, ventromedial FN, elongated (A); medial 2� inlobule VIa, ventrolateral FN, wide (B); lateral 3� in lobule VIII, ventral ICG to caudal PIN, compact (C); 5� in crus IIa, central PIN, compact (D);5� in copula pyramidis, caudal PIN, compact (E); 5� or 6� in crus Ib, lateral PIN, compact (F); dorsal paraflocculus, lateral DN, elongated (G).H: Mapping of injection sites for the reconstructed axons depicted in this figure (filled circles with a letter indicating panels A–G). Open circlesindicate other Group I injections in which PC axons were reconstructed in this study. Scale bar � 500 �m.



crus Ib or the dorsal paraflocculus, it was not straightforwardto determine the compartments for the injection sites in thesecases. The target areas of these axons generally coincidedwith the Group I areas in the CN (green areas in Fig. 2B,C).Furthermore, the topography was rather straightforward, atleast in the mediolateral directions; PCs in more lateral stripesin the cortex projected more laterally in the CN (in the order ofA to G in Fig. 3). This topography generally agreed with thetopography of olivocortical and olivonuclear projections (Ta-ble 1). For example, olivocerebellar axons originating from therostral part of subnucleus a of the caudal part of the medial

accessory olive innervate lateral compartment 3� in lobuleVIII and the ventral ICG, which were the origin and target of theaxon depicted in Figure 3C (line 6 in Table 1), respectively.

Cortical Group II has been defined as aldolase C-positivecompartments that do not extend rostrally beyond the primaryfissure (cyan and blue areas in Fig. 2A). They correspond tomost of zone A in lobules VI-IX and zone X-CX, and areinnervated by several medial subnuclei and adjacent areas inthe inferior olive (Table 1), which mainly receive vestibular andcollicular inputs (Sugihara and Shinoda, 2004). Axons of sixPCs that belonged to Group II are shown in Figure 4. They

Figure 4.Projections of reconstructed axons of PCs that belonged to Group II (blue and cyan areas in Fig. 2). Trajectories of axons in the caudal view(A–G) and mapping of injection sites (H) for the reconstructed axons in this figure (filled circles with alphabet letters) and for other axons in thisstudy (open circles) were prepared in the same format as in Figure 3. Origin, destination, and type of the terminal arbor of each axon: a� in lobuleVIb, midcaudal FN, compact (A); medial 2� in lobule IXa, ventrocaudal FN, wide (B); lateral 2� in lobule VII, midcaudal FN, wide (C); lateral 2�in lobule IXc, ventrolateral FN, wide (D); 4� in lobule IXc, ventral PIN, elongated (E); 5a� in crus IIb, caudal neck of the DLP, compact (F); c�in crus Ia, central FN, compact (G). The drawing of the section containing the FN was shifted upward in A. Scale bar � 500 �m.



projected to the midcaudal FN from a� in lobule VIb (Fig. 4A),to the ventrocaudal FN from 2� in lobule IXa (Fig. 4B), to themidcaudal FN from 2� in lobule VII (Fig. 4C), to the lateral FNfrom ventrolateral 2� in lobule IXc (Fig. 4D), to the ventral PINfrom 4� in lobule IXc (Fig. 4E), to the caudal neck of the DLPfrom 5a� in crus IIb (Fig. 4F), and to the central FN from c� incrus Ia (Fig. 4G). The target areas of these axons generallycoincided with the Group II areas in the CN (cyan and blueareas in Fig. 2B,C). Although the topography of the projectionof these axons seemed complicated, it generally agreed withthe topography of olivocortical and olivonuclear projections(Table 1). For example, olivocerebellar axons originating fromthe caudomedial part of the ventral lamella of the principal

olive nucleus innervate compartment 4� in lobule IX and themost ventral PIN, which were the origin and the target of theaxon depicted in Fig. 4E (line 15 in Table 1), respectively.

Cortical Group III has been defined as aldolase C-negativecompartments in the vermis and the central pars intermedia(yellow and orange areas in Fig. 2A). They correspond to mostof zone A in lobules I–V, some areas of zone A in other lobulesand zones X and CX, and are innervated by the central portion(subnucleus b) of the caudal part of the medial accessory olive(Table 1), which receives somatosensory, vestibular, and mid-brain inputs (Sugihara and Shinoda, 2004). Axons of five PCsthat belonged to Group III are shown in Figure 5. They pro-jected to the rostrodorsal and medial FN from 1� in lobule VIa

Figure 5.Projections of reconstructed axons of PCs that belonged to Group III (yellow and orange areas in Fig. 2). Trajectories of axons in the caudal view(A–E) and mapping of injection sites (F) for the reconstructed axons in this figure (filled circles with alphabet letters) and for other axons in thisstudy (open circles) were prepared in the same format as in Figure 3. Origin, destination, and type of the terminal arbor of each axon: 1� in lobuleVIa, rostrodorsal and medial FN, wide (A); 1� in lobule IXc, rostrodorsal and medial FN, wide (B); 3� in lobule VIII, dorsal ICG, compact (C); c�in crus Ia, dorsal DLP, compact (D); 4b� in crus IIb, dorsal DLP, compact (E). Scale bar � 500 �m.



(Fig. 5A), to the rostrodorsal and medial FN from 1� in lobuleIXc (Fig. 5B), to the dorsal ICG from 3� in lobule VIII (Fig. 5C),and to the dorsal DLP from c� in crus Ia and 4b� in crus IIb(Fig. 5D,E). The target areas of these axons generally coin-cided with the Group III areas in the CN (yellow and orangeareas in Fig. 2B,C). Furthermore, the topography of the pro-jection of these axons generally agreed with the topography ofolivocortical and olivonuclear projections (Table 1). For exam-ple, olivocerebellar axons originating from the rostral part ofthe lateral subnucleus b of the medial accessory olive inner-vate compartment 3� in lobule VIII and dorsal ICG, whichwere the origin and the target of the axon depicted in Fig. 5C(line 18 in Table 1), respectively.

Cortical Group IV has been defined as the negative compart-ments and neighboring lightly positive compartments in the ros-tral and caudal portions of pars intermedia, and all of the nega-tive compartments in the hemisphere (pink and red areas in Fig.2A). They correspond to zones B, C1, C3, and D0, and areinnervated by the dorsal subnuclei of the inferior olive (Table 1),which mainly receive somatosensory inputs (Sugihara and Shi-noda, 2004). Axons of six PCs that belonged to Group IV areshown in Figure 6. Injection sites were located at various posi-tions within Group IV areas in the cerebellar cortex. PCs in all ofthese areas projected to the AIN, which also belonged to GroupIV (red in Fig. 2C). Terminal arbors of these axons were compactand vertically organized in the dorsomedial-to-ventrolateral di-rection. More lateral PCs often project more laterally in the AIN(Fig. 6G) than more medial PCs (Fig. 6A–F). However, a PC in 4�

in lobule IIIB (Fig. 6D) projected more medially than a PC in 3� inlobule VId (Fig. 6E). Comparison of the olivary and PC projectionsindicated that the projections of most PC axons generally agreedwith the topography of olivocortical and olivonuclear projections(Table 1). However, some discrepancy remained in the presentresults, as indicated by the figure numbers in the parentheses inTable 1 (see footnote 6 for Table 1). For example, compartment4� in the rostral cerebellum and compartment 5� in the caudalcerebellum were speculated to be related to the lateral AINbased on olivary projection (line 28 in Table 1). Although theprojection of a PC in caudal 5� to the lateral AIN (Fig. 7B) wasconsistent with this relationship, the projection of a PC in rostral4� to the relatively medial AIN (Fig. 6D) was not. Therefore, wecould not fully explain the topographic relationship of olivary andPC projections for Group IV in the present study.

The above results indicated that the topography of theprojection of PC axons was generally arranged in accordancewith the compartmental organization of the cerebellar cortexand nuclei, which was originally based on the olivocortical andolivonuclear projections, except for a few cases (Table 1).Thus, the basic topography of the PC projections seemed tobe generally interpreted by the five-group scheme, which wasoriginally proposed based on the olivary projection. The pro-jection patterns of all axons other than those depicted inFigures 3–6 (see open circles in Figs. 3H, 4H, 5F, 6H) sup-ported this conclusion (some of these axons are shown inother figures). Basically, with regard to the aldolase C com-partmentalization, aldolase C-positive PCs (Groups I and II) pro-

Figure 6.Projections of reconstructed axons of PCs that belonged to Group IV (pink and red areas in Fig. 2). Trajectories of axons in the caudal view (A–G)and mapping of injection sites (H) for the reconstructed axons in this figure (filled circles with alphabet letters) and for other axons in this study(open circles) were prepared in the same format as in Figure 3. Origin of each axon: b� in lobule IIIa (A); 3� in lobule IIIb (B); f� in lobule VIII(C); 4� in lobule IIIb (D); 3� in lobule V (E); 5� in lateral lobule III (F); 5� (satellite) in lobule V (G). All of these depicted axons terminated in theAIN and their terminal arbors were classified into the compact type. Scale bar � 500 �m.



jected to the caudoventral parts of the CN and aldolaseC-negative (Groups III and IV) and lightly positive (Group IV) PCsprojected to the rostrodorsal parts of the CN. This distinct pro-jection underlay the aldolase C compartmentalization in the CNthat has been reported previously (Sugihara and Shinoda, 2007).

We counted the number of swellings in aldolase C-positiveand -negative parts of the CN for each reconstructed axon.For 23 of 37 aldolase C-positive and 16 of 22 aldolaseC-negative (including lightly positive) reconstructed PC axonsthat projected mainly to the CN, all swellings were located inthe aldolase C-positive and -negative parts of the CN, respec-tively. For the rest of the axons (14 of 37 aldolase C-positiveand 7 of 22 aldolase C-negative axons), a small fraction of theswellings (1.8–34.7%, 14.3 � 10.4%, n � 21 axons) werelocated in the opposite parts of the CN (aldolase C-negativeparts for aldolase C-positive axons and vice versa). On aver-age for all reconstructed axons that terminated mainly in theCN (n � 59 axons), 4.8% of swellings were located in theopposite parts. These swellings were located near the bound-ary between the aldolase C-positive and -negative parts of theCN, as shown in some axons depicted in the figures (Figs. 1G,1I, 3C, 4G, 5E). Thus, the separation of aldolase C-positive and-negative parts was generally, but not completely, exclusive inthe PC axonal projections. This seemed to be at least partiallywhy the boundary itself is not as clear in the CN as in thecortex (Sugihara and Shinoda, 2007).

Nearby PCs had partially overlappingtermination areas when they belonged to the

same aldolase C compartmentWe then examined how strictly the topography of the PC

projection is organized in relation to cortical aldolase C com-

partmentalization. Specifically, we wanted to test whetheradjacent PCs that are located in a single aldolase C compart-ment would project to termination areas in the CN accordingto the same topographic principle. Therefore, we tried toreconstruct multiple axons for small cortical injections andcompared their projections. In 11 injections we could com-pletely reconstruct multiple (2 to 6) PC axons originating froman aldolase C compartment.

Several examples of multiple reconstructed axons areshown in Figure 8. Three axons were reconstructed with aninjection to 4� in crus Ic (Fig. 8A). They terminated in theventral PIN. One axon also terminated in the caudal FN withpart of the terminal arbor (Fig. 8A, blue); however, this doesnot contradict the scheme of Sugihara and Shinoda (2007),since the two termination areas of these axons, as well as theirorigin, belong to Group II, according to the nuclear compart-mentalization of Sugihara and Shinoda (2007). In fact, theresults suggest that the two separate termination areas couldbe functionally related to some extent.

Three axons were reconstructed with an injection to 1� inlobule IXc (Fig. 8B). Although the termination areas of theseaxons were significantly separated, they were still locatedwithin the aldolase C-negative rostral FN. This part of the FN,as well as the origin of these axons, belonged to Group III. Thedistal part of the black axon seemed to slightly extend intoaldolase C-positive areas (Fig. 8B, arrowhead).

Two axons were reconstructed with an injection to 2� inlobule III (Fig. 8C). They mainly terminated in the lateral ves-tibular nucleus (LVN) and anterior interstitial cell group (AICG).The termination areas of these axons hardly overlapped, butwere adjacent. The AICG (Sugihara and Shinoda, 2007) is the

Figure 7.Projections of PCs located slightly separately within the same or different aldolase C compartment(s). A: Projection of PCs in compartments d�and 4� in the rostral folial wall of crus Ia. They innervated the dorsomedial PIN and dorsal ICG (aldolase C-negative), and centrodorsal PIN(aldolase C-positive), respectively. These PCs were labeled by the same BDA injection that was centered at the boundary between d� and 4�.The location of the somata and axonal paths of these PCs indicated that these axons belonged to d� and 4�. B: Projections of two PCs thatwere separated transversely but located in the same compartment. Two small injections were made in medial and lateral 5� in the apex of crusIIa. PC axons that originated from each of the injections were reconstructed. Blue axons indicate those that were partially reconstructed, exceptfor the fine branches in the terminal arbor. The medial PCs projected to the lateral AIN while the lateral PCs projected to the junction betweenthe DLH and lateral AIN. Scale bar � 500 �m.



area located rostral to the ICG and ventral to the AIN. Thisdivergent projection of PCs to the LVN and AICG is parallel tothe olivary projection, since nuclear collaterals of olivary ax-ons that innervate 2� in the anterior lobe project to both theLVN and AICG (Sugihara and Shinoda, 2007).

Two axons were reconstructed with an injection to b� inlobule IV (Fig. 8D). They terminated in the rostromedial AIN.Termination areas of these axons showed some overlap. Ad-

ditionally, the projection to AIN of these axons indicated thata lightly positive compartment (b�) is akin to an adjacentnegative compartment (b�), in which a PC also projected tothe rostromedial AIN (Fig. 6A), as proposed previously basedon the olivocortical projection (Sugihara and Shinoda, 2004).

In sum, terminal arbors of adjacent PCs did not necessarilyshow tight overlap but were sometimes located within areas inthe CN that could be considered to be the same or closely

Figure 8.Projection patterns of adjacent PCs labeled by a single injection of BDA in an aldolase C compartment. A: Three PCs located in 4� in crus Ic.They mainly innervated the ventral PIN. However, one axon (blue) innervated the aldolase C-positive area in the caudolateral FN additionally.B: Three PCs located in 1� in lobule IXc. They all innervated the aldolase C-negative area in the rostral FN. However, the termination areas didnot fully overlap. The axon colored in blue terminated mainly in more dorsocaudal areas than the other axons. C: Axons of two PCs located in2� in the junction between lobules II and III. They innervated similar areas in the LVN. They also innervated the AICG. D: Two PCs in b� in lobuleIV innervated overlapping areas in the rostromedial AIN. Scale bar � 500 �m.



related functional compartments. In the DN (not shown), AIN,and PIN, however, they seemed to overlap more tightly than inthe FN and LVN. This indicated that corticonuclear topogra-phy might be more precisely organized in the DN, AIN, and PIN(see Discussion).

We next compared the projection of adjacent PCs thatare located across the boundary of aldolase C compart-ments. Axons of two PCs labeled by an injection that wascentered at the boundary between d� and 4� in rostralcrus Ia were reconstructed (Fig. 7A). Locations of the so-mata and path of the proximal part of axons indicated thatone PC was located in compartment 4� (aldolaseC-positive) and the other was located in compartment d�(aldolase C-negative). While the aldolase C-positive PC in-nervated the centrodorsal PIN, which is aldolase C-positive,the other aldolase C-negative PC innervated the dorsome-dial PIN and dorsal ICG, which was mostly aldolaseC-negative (Fig. 7A, left and right axons, respectively). Al-though these results also support the idea that the PCprojection is precisely related to the aldolase C compart-mentalization in the cerebellar cortex (as was concludedfrom the results of the previous section), this example wasthe sole case that we could reconstruct PC axons in aninjection across the compartmental boundary.

Projections from separate narrow zones withinthe same aldolase C compartments

Andersson and Oscarsson (1978) have shown electrophysi-ologically that PCs arranged into separate narrow microzoneswithin zone B in the lateral vermis receive different somato-sensory inputs via olivocerebellar projection. A similar obser-vation has been made in zone C3 in the pars intermedia byEkerot et al. (1991). In the rat, PCs arranged in a narrowlongitudinal band show significantly synchronous activity(Lang et al., 1996). This band often equates to an aldolase Ccompartment, but is sometimes narrower (Sugihara et al.,2007). With regard to anatomical studies, olivocerebellarclimbing fibers originating from adjacent olivary neuronsproject to PCs arranged within a longitudinal band-shapedarea (Sugihara et al., 2001). Such areas are generally muchnarrower than a single aldolase C compartment (Sugihara andShinoda, 2004). These findings suggest that PCs that areseparate in the mediolateral direction within a single aldolaseC compartment may be functionally distinct and project dif-ferently to the CN.

Small injections were made into mediolaterally separateareas in the same aldolase C compartment in the same lobule(medial and central 5� in crus IIb) and PC axons originatingfrom each injection site were reconstructed (Fig. 7B). Theaxon originating from medial 5� formed a compact terminalarbor in the lateral AIN and the other axon from central 5�formed a compact terminal arbor more laterally in the junctionbetween the AIN and DLH. These two terminal arbors werecompletely separate in the mediolateral direction (Fig. 7B,black axons). A few other axons were also labeled by thesetwo injections, which were also reconstructed except for finebranches in the terminal arbor. The terminal arbor of eachpartially reconstructed axon overlapped that of the fully re-constructed axon that originated from the same injection site,and was also separate from that of the axons originating fromthe other injection site (Fig. 7B, blue axons).

These results suggested that fine mediolateral organizationwas present within the CN, especially in the dorsal main partsof the DN, AIN, and PIN, which reflected microzones in thecerebellar cortex by the precise topographic projection ofPCs. Although we did not perform additional studies of similaradjacent microzonal anterograde labeling, the results of ret-rograde labeling (see below) supported this suggestion.

Convergent projections from the same aldolase Ccompartments in separate lobules

Single olivocerebellar axons branch several times andproject as climbing fibers to about seven PCs that are gener-ally located in a longitudinal band-shaped area, often in ad-jacent and separate multiple lobules (Sugihara et al., 1999,2001). There seems to be a rule that climbing fibers thatoriginate from a single axon tend to innervate specific com-binations of lobules (for example, simple lobule sublobule b,crus IIa, and crus IIb; see figs. 2A,B and 5b green of Sugiharaet al., 2001; figs. 4C orange and 8D red and orange of Sugiharaand Shinoda, 2004).

With regard to the output of the cerebellar cortex, it is alsopossible that the projection of PCs located in different areasof the cerebellar cortex can converge in the CN. Indeed,Purkinje cells located in zones C1 and C3 in lobule V project tooverlapping areas in the anterior interposed nucleus in the cat(Apps and Garwicz, 2000), which is an example of mediolat-eral convergence. However, the divergent olivocortical pro-jection along with the general longitudinal organization of thecerebellar cortex (Groenewegen and Voogd, 1977; Buisseret-Delmas and Angaut, 1993; Voogd, 2004) suggests that rostro-caudal convergence might be more common than mediolat-eral convergence in PC projection throughout the cerebellum.Here, we tried to find the correlate for such longitudinal con-vergence in the morphology of single PC projection.

We reconstructed PC axons originating from two sitesaligned on the same longitudinal compartment (medial 6�) inneighboring crus IIa and IIb in a rat (Fig. 9A–C). The axonsprojected with compact terminal arbors to the caudal pole(CP), which is the dorsocaudally protruding area in the junc-tion between the DN and PIN (Voogd, 2004). Terminal arborsof these axons significantly overlapped each other, as seen inthe frontal and lateral trajectories of the reconstructed axons(Fig. 9A,B), which indicated convergence of PC projectionsoriginating from crus IIa and IIb. Compartment 6� in thecaudal cerebellar cortex (including crus IIa and IIb) is inner-vated by the ventral lamella of the principal olive, which alsoprojects to the caudal DN including the CP (part of Group I,Sugihara and Shinoda, 2004, 2007). Therefore, the convergentcorticonuclear topography of these axons paralleled the di-vergent olivocortical and olivonuclear topography.

In other rats we reconstructed PC axons originating fromthe most lateral aldolase C-positive compartments (6�//7�) insimple lobule sublobule b and crus IIa (Fig. 9D–H). Thesecompartments, 6� in the rostral cerebellum including simplelobule and 7� in the caudal cerebellum including crus II, havebeen considered to be linked or equivalent based on thespatial pattern of aldolase C compartments and olivocorticalprojection (Sugihara and Shinoda, 2004). These PC axonsterminated in the lateral DN. The small termination areas ofthese axons nearly overlapped each other in their frontal tra-jectories (Fig. 9D,E). Although these axons were labeled indifferent rats, their lateral trajectories were drawn on the same



panel by carefully referring to the contour of the CN in cere-bellar sections of the two rats (Fig. 9F,G). In their lateraltrajectories, their terminal arbors were located in the rostro-dorsal portion of the DN, with one of the crus IIa PC (Fig. 9G)slightly rostral to the other (Fig. 9F). Therefore, the resultsseem to support the possibility that PC axonal projectionsfrom the rostral and caudal cerebellum can converge to someextent in the CN. The linked compartments 6�//7� are inner-vated by the dorsal lamella of the principal olive, which alsoproject to the lateral DN through nuclear collaterals (part ofGroup I, Sugihara and Shinoda, 2004, 2007). Therefore, theconvergent corticonuclear topography of these axons wouldparallel the divergent olivocortical and olivonuclear topogra-phy.

Similarly, target areas in the CN of PC axons projecting fromthe linked aldolase C compartments in separate lobules were

located close to each other between the cases depicted inFigure 5D,E. However, it was difficult to analyze the conver-gent PC projection by anterograde labeling and axonal recon-struction as shown in this section since aldolase C compart-ments are not visible during injection. Therefore, weperformed retrograde labeling (below).

Convergent PC projections revealed byretrograde labeling

To further examine the possibility of the convergence of PCaxonal projections from multiple lobules, a small amount offluorescent latex microspheres (Apps and Ruigrok, 2007) wasinjected into the CN, and the distribution of retrogradely la-beled PCs was mapped (Fig. 10). It has been proposed thatthe putative transverse line in lobule VIc and crus Ib repre-sents the rostrocaudal boundary of the cerebellar cortex

Figure 9.Convergent projection of PCs from multiple lobules. A,B: PCs located in 6� in crus IIa and IIb project to overlapping areas in the CP, as shownin frontal (A) and lateral (B) trajectories. These cases were labeled in the same rat. C: Mapping of the PCs in A and B on the unfolded schemeof the cerebellar cortex. D–G: Two PCs that were located in 7� of crus IIa and 6� of simple lobule sublobule b projected to partially overlappingareas in the DN, as shown in frontal (D,E) and lateral (F,G) trajectories. These cases were labeled in different rats. Note that compartments 6�in the rostral cerebellum (including simple lobule) and 7� in the caudal cerebellum (including crus IIa) are considered to be linked (Sugihara andShinoda, 2004). H: Mapping of the PCs in D-G on the unfolded scheme of the cerebellar cortex. Scale bars � 500 �m in A (applies to B), in F(applies to D,E,G).



based on the stripe pattern of aldolase C expression and thedivergent olivocerebellar projection (dotted transverse lines inFig. 10D,H; Sugihara and Shinoda, 2004). Therefore, the posi-tional relationship of the distribution to the rostrocaudalboundary was primarily examined.

With a small injection into the rostrodorsal FN (aldolaseC-negative part, Fig. 10A), PCs were labeled mostly in centraland relatively medial areas in 1� in lobule II-VIa (rostral cer-ebellar cortex, Fig. 10B,D) and 1� in lobule VIII and IXa (caudalcerebellar cortex, Fig. 10C,D). These compartments, rostral1� and caudal 1� (1�//1�), have been considered to belinked compartments since they receive the divergent projec-tion of the same olivocerebellar axons (Sugihara and Shinoda,2004). The present results indicated that rostrocaudal conver-gence occurs in the PC projection from these linked compart-ments (1�//1�). In this case, the injection site in the rostro-dorsal FN (Fig. 10A) coincided roughly with the terminationarea of the reconstructed axon of a PC in 1�//1� (Fig. 5A) and

that of nuclear collaterals of olivocerebellar axons projectingto 1�//1� (line 16 in Table 1).

Another injection of fluorescent retrograde tracer wasmade into the dorsolateral DN (aldolase C-positive part,Fig. 10E). PCs were labeled in relatively medial 6� in simplelobule sublobules a and b and rostral crus Ia (rostral cere-bellar cortex, Fig. 10F,H) and in relatively medial 7� in crusIIa-b and paramedian lobule (caudal cerebellar cortex, Fig.10G,H), which indicated convergent projection of these PCsin the rostral and caudal cerebellar cortex. These compart-ments (6�//7�) receive divergent projection of the sameolivocerebellar axons and are thus considered to be linkedcompartments (Sugihara and Shinoda, 2004). The injectionsite of this case in the dorsolateral DN (Fig. 10E) coincidedroughly with the termination area of the reconstructed ax-ons of PCs in 6�//7� (Fig. 9D–G) and that of nuclear col-laterals of olivocerebellar axons projecting to 6�//7� (seeTable 1).

Figure 10.Rostrocaudally convergent projection of PCs revealed by retrograde labeling with small injections of fluorescent latex microspheres into the CN.A–D: Injection into the rostrodorsal FN (A, arrowhead). PCs were labeled in centromedial 1� in lobules I-VIa (B, arrowheads) and in VIII-IXa (C,arrowheads). E–H: Injection into the dorsolateral DN (E, arrowhead). PCs were labeled in medial 6� in the simple lobule (F, arrowheads) androstral crus Ia, and in medial 7� in crus IIa-b and the paramedian lobule (G, arrowheads). Dotted lines in A and E indicate contours of the CN.Plus and minus signs (�, �) in A indicate aldolase C-positive and -negative parts in the FN, respectively. The distributions of PCs were mappedon the scheme of aldolase C compartments in the unfolded cerebellar cortex (D,H). Dotted lines in D and H indicate the rostrocaudal boundaryof the cerebellar cortex. Scale bars � 500 �m in B (applies to C), in F (applies to G).



Therefore, these results not only indicated rostrocaudalconvergence in the PC projection but also supported theconclusion in the earlier section that the topography of corti-conuclear PC projection was closely linked to that of olivo-cerebellar projection. In six other injections of retrograde trac-ers into the CN, similar results were obtained. However,further studies will be needed to obtain a systematic under-standing of the details of the convergence of PC projections.

As an additional finding of the experiments shown in Figure10, retrogradely labeled PCs were generally distributed not inthe entire mediolateral range of a single aldolase C compart-ment, but only in a part of it. This supported the notion ofmicrozonal organization in the topography of PC projection(see above).

Extracerebellar innervation of PC axonsCerebellar nuclei are the general targets of PC projection.

However, PCs in some areas of the cerebellum project tostructures outside of the cerebellum. PCs in the flocculus,nodulus, and parts of the uvula project to the medial vestibularnucleus, SVN, and dorsal and ventral Y nucleus, depending onwhich zones they belong to (Bernard, 1987; De Zeeuw et al.,1994; Wylie et al., 1994; Sugihara et al., 2004). In the anteriorlobe of the cerebellum, PCs in the lateral vermis (zone B anda part of zone A that correspond to the lateral compartment1�) project to the LVN (Buisseret-Delmas and Angaut, 1993;Voogd and Ruigrok, 2004). Some PCs in the cardiovascularresponsive parts of the cerebellum including the median andparamedian anterior vermis and the lateral uvula and nodulusare reported to project to the parabrachial nucleus (PBN)(Sadakane et al., 2000; Nisimaru, 2004). However, the detailsof such extracerebellar projection have not been clearly re-ported. For example, it is not known whether such PC axonsproject solely to extracerebellar targets or whether theyproject to both the CN and extracerebellar targets, except forthose from the flocculus and nodulus that have been recon-structed (De Zeeuw et al., 1994; Wylie et al., 1994; Sugihara etal., 2004). Therefore, we analyzed the trajectories of recon-structed PC axons that projected to structures outside of thecerebellum.

Ten of 65 reconstructed PC axons, which did not includethose from the flocculus or nodulus, had extracerebellar pro-jections in the present study. All of these 10 axons originatedfrom the vermis. In four of them projection to the CN waspredominant and one or a few branches of the axon extendedto the extracerebellar target. In the six other axons, projectionto the extracerebellar area was predominant, but often re-tained some projection to the CN.

The PC axons shown in Figure 8C, which originated fromcompartment 2� in lobule III, projected to the LVN. One ofthese axons also projected to the AICG, which is the junc-tional area between the AIN and LVN (Sugihara and Shinoda,2007). The AICG and LVN are the common targets of nuclearcollaterals of the olivocerebellar climbing fibers that project tocompartment 2� (zone B) (Sugihara and Shinoda, 2007).

The PC axons shown in Figure 11A originated from thelateral part of compartment 1� in lobule III. These axonsprojected to the rostrodorsal and lateral FN, ventral LVN, andthe inferior vestibular nucleus (IVN). One of the two axonsextended down to nucleus X, which is located between theIVN and inferior cerebellar peduncle. The IVN and nucleus Xare innervated by olivary axons that also innervate lateral 1�

in the rostral cerebellum and part of the rostral aldolaseC-negative FN (Sugihara and Shinoda, 2007).

The axon shown in Figure 11B, which originated from themedian aldolase C-positive area in apical lobule VII, termi-nated with a wide terminal arbor in the caudoventral aldolaseC-positive area in the FN. One of the branches in the terminalarbor extended rostroventrally to the PBN. This axon had 10terminals in the mediodorsal part of the PBN. PCs in medianlobule VII respond to cardiovascular vagal signals in rabbits(Okahara and Nisimaru, 1991). The PBN is concerned withautonomic functions including cardiovascular regulation (Nisi-maru, 2004; Saper, 2004). Therefore, this axon may be con-cerned with cardiovascular control. Although PC projectionfrom lobule VII to the PBN has not been reported so far, PCprojections to the PBN from other autonomic areas (medianand paramedian anterior vermis and in the lateral uvula andnodulus) have been reported (Sadakane et al., 2000).

The PC axon shown in Figure 11C, which originated fromcompartment 2� in lobule IXc, mainly projected to the cau-domedial SVN. Additionally, this axon had collaterals thatterminated in the rostral aldolase C-negative part of the FN(arrowhead in Fig. 11C). According to reports about olivaryprojection, the SVN is not innervated by olivary axons, whilethe rostral aldolase C-negative part of the FN is innervated byolivary axons that also innervate caudal 2� (Ruigrok andVoogd, 2000; Sugihara and Shinoda, 2004).

The PC axon shown in Figure 11D, which originated fromcompartment 2� in lobule IXa, made a terminal arbor with 30swellings in the caudal FN, but gave rise to two long branches.One of these projected to the rostrolateral SVN with 131swellings. The other projected to the caudal nucleus Y with 49swellings.

Three other axons that originated from lobule IX (compart-ment 1� and 2�) extended one branch to the SVN or LVN witha few swellings, while projecting predominantly to the FN (twoshown in Fig. 4B,D).

In sum, some vermal axons had predominant extracerebel-lar projection and weak projection to the CN (Figs. 8C,11A,C,D). Some other vermal axons had predominant projec-tion to the CN and weak extracerebellar projection (Figs. 4B,D,11B). Various subareas of the vestibular nucleus and the PBNare the extracerebellar targets of PC axons that have beenreconstructed so far. Some extracerebellar targets receive theprojection of collaterals of olivocerebellar axons and othersdo not (Table 1). No hemispheric PCs had extracerebellartargets in this study. Thus, the extracerebellar projectionseems deeply involved in the functional organization of thecerebellar system in the vermis.

Recurrent cortical collaterals of PC axonsThe morphology of local recurrent collaterals of PC axons

has been described in detail (Ramon y Cajal, 1911; Chan-Palay, 1971; Hawkes and Leclerc, 1989). Indeed, their entiremorphology has been reported based on intracellular horse-radish peroxidase labeling in the cat (Bishop, 1982;O’Donoghue and Bishop, 1990). Here we reexamined the mor-phology of these collaterals as part of single axonal recon-struction in relation to the aldolase C compartmental pattern.

With anterograde labeling the morphological isolation oflocal recurrent collaterals was often difficult because of thelocal spread and local concomitant labeling of cells other thanPCs unless the injection was precisely localized in the molec-



Figure 11.Extracerebellar projection of some PC axons. A: Two PCs located in lateral 1� of lobule III projecting to the rostrodorsal aldolase C-negativeFN, ventral part of the LVN, and the IVN. One of the axons also projected to the nucleus X. B: A PC located in median lobule VII projecting tothe caudal aldolase C-positive FN and the PBN (arrowhead). C: A PC located in compartments 2� in lobule IXc projecting to the rostral FN(arrowhead) and the caudomedial SVN. D: A PC located in medial 2� in lobule IXa projecting to the caudal FN, rostrolateral SVN, and caudalnucleus Y. The drawing of the section containing the caudal nucleus Y was shifted downward to improve the view. Scale bar � 500 �m.



ular layer. The proximal part of the axon was isolated down tothe soma of the PC enough to clearly identify the branchingpoints of recurrent collaterals in 26 reconstructed PC axonsthat were labeled anterogradely. In the other reconstructedaxons, the very proximal part was not clear enough to identifybranching because of spread of the injected tracer into theadjacent granular and PC layers. To further examine recurrentcollaterals, we performed retrograde labeling of PC axons byinjecting BDA into the fastigial (medial cerebellar) nuclei. Instrongly labeled PCs (n � 13 PCs in three rats), the stemaxons were well visualized and recurrent collaterals, ifpresent, could be fully reconstructed (Fig. 12A).

Among these 39 PCs (26 anterogradely labeled and 13retrogradely labeled PCs), 28 (71.8%) had one, 8 (20.5%) hadtwo, and 3 (7.7%) had no recurrent collaterals. The average

number of recurrent collaterals was 1.1 (SD � 0.5). When a PCaxon had two recurrent collaterals, they rose from the samebranching point (4 of 8 PC axons) or from different branchingpoints (4 of 8). We completely reconstructed 30 recurrentcollaterals in 22 PCs (10 anterogradely labeled and 12 retro-gradely labeled PCs). The branching points were located 60–210 �m (139 � 37 �m, mean � SD, n � 30) from the soma,which presumably corresponded to the first or second Ran-vier’s node. These collaterals were mainly distributed in thePC layer and the superficial granular layer. The total number ofswellings seen on a recurrent collateral, including itsbranches, ranged from 6 to 38 (26.7 � 7.9). The swellings thatbelonged to a single collateral were distributed within a rangeof 103 � 47 �m (mean � SD, n � 30) in the rostrocaudaldirection and 82 � 75 �m in the mediolateral direction. The

Figure 12.Local recurrent collaterals of PC axons. A: Two cortical collaterals originating from a PC axon that was intensely labeled retrogradely by aninjection of BDA into the FN. The photomicrograph is a montage with five different focuses in a parasagittal section. Arrowheads indicatecollaterals and their branching points. Inset drawing (top right) shows the reconstruction of this axon from three parasagittal sections. B,C:Photomicrographs of aldolase C-positive PCs in lobules VIa (B) and VIII (C) immunostained with a nickel-intensified diaminobenzidine reactionin coronal sections. Recurrent collaterals of aldolase C-positive PCs innervated mainly aldolase C-positive areas (open arrowheads) andsometimes aldolase C-negative areas (filled arrowheads). D: Photomicrograph of a recurrent collateral of a BDA-labeled aldolase C-negative PCin coronal sections. This collateral innervated both aldolase C-positive (filled arrowheads) and -negative (open arrowheads) areas in lobule IXc.Inset drawing (bottom right) shows reconstruction of this axon from four sections that were double-labeled for BDA and aldolase C. Scale bars �500 �m in A, A inset; 50 �m in B–D, D inset.



center of the distribution of the swellings was separated fromthe soma by 104 � 69 �m in the rostrocaudal direction and by80 � 63 �m in the mediolateral direction. These differences inthe direction were not statistically significant (P > 0.05, two-sided t-test with paired samples). Thus, the recurrent collat-erals did not tend to run in any clear specific direction. Inner-vation formed by individual recurrent collaterals in therat seemed weaker than that in the cat (Bishop, 1982;O’Donoghue and Bishop, 1990).

To study the relationship between aldolase C compart-ments and the projection of local collaterals, we visualizedaldolase C-positive PCs including their axonal collaterals byimmunostaining. A dense distribution of collaterals was seenin the PC layer and upper granular layer in the aldolaseC-positive compartment (Fig. 12B,C). This agreed with theprevious observation by Hawkes and Leclerc (1989). However,some aldolase C-positive collaterals were also seen withinaldolase C-negative compartments (filled arrowheads in Fig.12B,C). Such sparse distribution of aldolase C-positive fiberswas seen in all aldolase C-negative compartments throughoutthe cerebellum. Concerning BDA-filled PC axon collateralsreconstructed in aldolase C-labeled cerebellar sections, inmost cases (n � 9 of 10) they terminated in the same aldolaseC compartment in which the PC soma was located. However,one recurrent collateral of the axon that originated from analdolase C-negative PC that was located close to the bound-ary of an aldolase C-positive compartment innervated bothaldolase C-negative and -positive compartments (Fig. 12D).We encountered similar divergent innervation to aldolaseC-positive and -negative compartments for recurrent collat-erals (n � 2) that could not be fully reconstructed. Theseresults indicated that the projection of local recurrent collat-erals was not exclusively specific to aldolase C compart-ments.

DISCUSSIONThe present study clarified the morphology of entire axons

of single PCs located in identified aldolase C compartments inthe rat cerebellum. Their projection patterns supported a pre-cise corticonuclear topography that was generally consistentwith the topography of the olivocerebellar pathway. Their pro-jection patterns also showed some area-dependent variation.The significance and implications of these characteristics incerebellar organization will be discussed.

Innervation of single PC axonsThe present study confirmed that PC axons have local re-

current collaterals and a nuclear main terminal arbor, and thatthey do not have any other branches that might project toother cortical areas in the middle of the axonal path. Projec-tion of local recurrent collaterals was much weaker than pro-jection to the CN and was not completely discriminative withregard to aldolase C compartments.

The nuclear terminal arbor of a PC with an average 122swellings in the present study in the rat may be comparable to474 swellings per PC as estimated in a Golgi staining study incats (Palkovits et al., 1977) and agrees with the strong syn-aptic input from PCs recorded in CN neurons electrophysi-ologically (Shinoda et al., 1987). An electron microscopicstudy has shown that CN neurons receive dense innervationof PC terminals at the soma as well as at dendrites (Chan-

Palay, 1973), although it is unclear whether these terminalsbelong to the same or different PCs. The finding that severalswellings of an axon surrounded a single CN neuron suggeststhat a CN neuron receives strong synaptic input at the somafrom a relatively small number of PCs. The number of CNneurons innervated by a single PC axon could not be deter-mined directly in the present study.

Corticonuclear topography in terms of aldolase Clabeling in the cortex and nuclei

The cerebellar cortex is compartmentalized into some 20longitudinal stripes which contain aldolase C-positive or-negative PCs (Brochu et al., 1990; Voogd et al., 2003; Sugi-hara and Shinoda, 2004), while the CN, in which only PC axonsexpress aldolase C, are generally divided into caudoventralaldolase C-positive and rostrodorsal aldolase C-negativeparts (Sugihara and Shinoda, 2007). This has raised the hy-pothesis that aldolase C-positive and -negative PCs projectdistinctively to the caudoventral and rostrodorsal parts of theCN, respectively. Furthermore, it has been proposed that, inboth the cerebellar cortex and nuclei, aldolase C-positive and-negative areas can be subdivided into three and two groups(five-group scheme) (Sugihara and Shinoda, 2004, 2007). Theprojections of the reconstructed single PC axons in thepresent study were generally consistent with this five-groupscheme (Table 1). Thus, PC projection and olivonuclear pro-jection seem to be based on a common principle of cerebellarcompartmentalization, which is represented partially in thealdolase C expression pattern. This notion suggests that thefurther systematic and detailed analysis of topography of cor-ticonuclear PC projection will help refine the current under-standing of cerebellar compartmentalization. For example, thecompartmental and topographic organization within the AINor Group IV is still obscure (footnotes 6 and 7 for Table 1) and,furthermore, the relationship between the anatomical organi-zation and functional or somatotopic organization (Ekerot etal., 1995; Dum and Strick, 2003; Dimitrova et al., 2006) is alsoambiguous in the CN.

In the conventional scheme, the cerebellar nuclei are sub-divided into the FN, AIN, PIN, and DN, which are innervated bythe vermis (zone A), parts of the pars intermedia (zones C1and C3), other parts of the pars intermedia (zone C2), and thehemisphere (zone D) (Voogd and Bigare, 1980; Brodal, 1981;Buisseret-Delmas and Angaut, 1993; Voogd, 2004). The corti-conuclear topography obtained in the present study, whichconsidered finer compartmentalization, generally agrees withthe conventional scheme. However, when we look at thepresent results in detail, several exceptions to the conven-tional scheme are obvious. For example, a lateral area invermal lobule IX projects to the PIN (compartment 4�, zoneX-CX, Fig. 4E). In the central cerebellum, some areas in thepars intermedia project to the FN including the DLP (compart-ments c�, Fig. 4G; c�, Fig. 5D; 4b�, Fig. 5E, and 5a�, Fig. 4F;all belonging to lateral zone A), while another area in the lateralvermis located more medially than the former projects to thePIN (compartment 4� in crus Ic, putatively zone X-CX, Fig.8A). All of these unusual projections are actually consistentwith the aldolase C expression-based five-group scheme (Ta-ble 1).



Variation in the spatial conformation of PCterminal arbors may be indicative of different

organizations in the CNThree types of PC terminal arbors were seen in different

areas in the CN in the present study. This suggests anotheraspect of the organization of the CN. In the dorsal main part ofthe DN, PIN, and AIN and probably in the dorsolateral protru-sion (DLP) of the FN, the terminal arbors were dense andcompact and were oriented in the dorsoventral direction (dor-solateral to ventromedial direction in the DN), which wasroughly equivalent to the direction from the surface to thehilus located in the center of the CN. Therefore, in these areasthe CN may be composed of columnar or cuneiform functionalentities (units) arranged radially or parallel. Such radial orga-nization was first reported by Chan-Palay (1973) in the DN. Afine mediolateral topographic segregation of axonal projec-tion seen in these areas (Fig. 7B) supports such organization.Thus, these areas in the CN seem to be organized mostregularly in the CN with regard to PC projection. Interestingly,the olivonuclear projection to these parts of the CN also hascolumnar or cuneiform termination areas and is regularly ar-ranged with a two-dimensional topography (Sugihara and Shi-noda, 2007).

In most of the aldolase C-positive and -negative areas in theFN, terminal arbors were more widely spread than in otherareas. Furthermore, the direction of the elongation of terminalarbors was not consistent among them, although they aremost frequently elongated in the mediolateral and caudoros-tral directions. Therefore, these areas may not be as regularlyorganized as the other areas (see above).

In the ventral parts of the DN, PIN, and FN, terminal arborswere remarkably elongated in the mediolateral direction. Thissuggested that functional compartmentalization is organizedmore or less transversely in these areas. These ventral areaswere innervated by compartments 1� and 2� in the vermis,compartments 4� in lobule IX and paraflocculus. Projectionfrom the flocculus to the dorsal Y nucleus and ventrorostralDN (Sugihara et al., 2004) was similarly elongated. An analysisof olivonuclear projection also suggested that the ventralparts of the DN, PIN, and FN may be considered to be orga-nized distinctly from the dorsal main parts of these subnuclei(Sugihara and Shinoda, 2007).

In sum, based on the different structures of the terminalarbor, some parts of the CN seem to differ from other partswith regard to organization. Subdivisions of the CN defined inthis way do not directly correspond to those based on aldo-lase C compartmentalization. Thus, further studies will beneeded to elucidate the organization of the CN.

Extracerebellar projections of PCsSome axons of vermal PCs extended beyond the CN to

other structures. These extracerebellar projections seemed tobe organized according to the cortical compartments, similarto the PC projections to the CN. The lateral vermal area thatprojects to the dorsal part of the LVN (zone B; Groenewegenand Voogd, 1977; Ito, 1984; Buisseret-Delmas and Angaut,1993) has been identified as compartment 2� (Sugihara et al.,2004; Voogd and Ruigrok, 2004). It has recently been reportedthat PCs in lateral compartment 1� in the anterior lobe alsoproject to the LVN based on the results of retrograde labeling(Voogd and Ruigrok, 2004). This study showed that PCs pro-

jected not only to the LVN but also to the IVN and nucleus Xfrom this area. In relation to this finding, collaterals of olivaryaxons that innervate lateral compartment 1� project to nu-cleus X and the IVN (Sugihara and Shinoda, 2007). Our studyalso showed projection from median lobule VII to the PBN. Aprevious study has shown that a different area (the lateralnodulus and uvula) projects to the PBN (Sadakane et al.,2000). Further analysis of the extracerebellar projections ofPCs in relation to their somatotopy or function will be requiredto understand the output system of the vermis.

Variable conformation of the olivo-cortico-nuclear loop

The results in our present and previous studies (Sugiharaand Shinoda, 2004, 2007) generally agree with the hypothesisthat the whole cerebellar system is formed by the parallelassembly of an olivo-cortico-nuclear loop (microcomplex ormodules) (Ito, 1984; Apps and Garwicz, 2005; Pijpers et al.,2005). However, a close look at the morphology of PC axonsindicates that the formation of such loops is not uniformamong areas in the cerebellum, suggesting some area-dependent differentiation in the organization of the cerebel-lum.

An essential part of the olivo-cortico-nuclear loop consistsof three putative projections which interconnect precisely: apopulation of PCs and nuclear collaterals of the olivary axonsthat innervate these PCs project to the same nuclear neurons.The nucleo-olivary projection may be added to the loop as anadditional component (Ruigrok and Voogd, 1990). A possibleexception is that extracerebellar targets of some PCs may notbe innervated by olivary axons. The PBN that is innervated bysome vermal PCs (present study and Sadakane et al., 2000),the SVN that is innervated by PCs in the flocculus, uvula, andnodulus (Bernard, 1987; de Zeeuw et al., 1994; Sugihara et al.,2004; and present study), and the medial vestibular nucleusthat is innervated by some PCs in the flocculus and nodulus(Bernard, 1987; de Zeeuw et al., 1994) may not receive theinnervation of olivary axons (Sugihara and Shinoda, 2007). TheLVN (and AICG) that is innervated by compartment 2� (Fig.8C; Voogd and Ruigrok, 2004) and the IVN (including nucleusX) that is innervated by compartment 1� (Fig. 11A) receiveinnervation from collaterals of the olivary axons that innervatecompartments 2� and 1�, respectively (Sugihara and Shi-noda, 2007). Additionally, it is unclear whether the entire ter-mination area of a wide terminal arbor in the FN or an elon-gated terminal arbor in the ventral PIN, ventral DN, and dorsalY (this study and Sugihara et al., 2004) is completely inner-vated by the same population of olivary axons, since thetermination of the olivonuclear projection is generally local-ized (Ruigrok and Voogd, 2000; Sugihara and Shinoda, 2007).Thus, the organization of the olivo-cortico-nuclear loop hassome variations in the FN and ventral CN.

On the other hand, a typical olivo-cortico-nuclear loopseems to be formed in the rest of the CN (dorsal main parts ofthe AIN, PIN, and DN), in which a PC has a compact and denseterminal arbor. In these areas the basic topographic confor-mation of the olivo-cortico-nuclear loop seem to have a onearea-to-multiple areas-to-one area relationship, since olivaryaxons show a rostrocaudally divergent (branching) projectionto the cortex (Sugihara et al., 2001; Pijpers et al., 2006) and PCaxons in rostrocaudally separate cortical areas converge to



the specific area in the CN (Figs. 9, 10), where the collateralsof olivary axons also project to (Table 1).

ACKNOWLEDGMENTThe authors thank Dr. E.J. Lang for reading the article.

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