olivo-cortico-nuclear localizations within crus i of the cerebellum

Olivo-Cortico-Nuclear Localizationswithin Crus I of the Cerebellum

LUIS HERRERO,1,2 MIN YU,1 FRASER WALKER,1 DAVID M. ARMSTRONG,1AND

RICHARD APPS1*1Department of Physiology, School of Medical Sciences, University of Bristol,

Bristol BS8 1TD, UK2Departamento de Fisiologia y Zoologia, Facultad de Biologia, Universidad de Sevilla,

41012 Seville, Spain

ABSTRACTRetrograde and anterograde tracers were microinjected into the folia of crus I of the cat

cerebellum to investigate spatial localization in olivo-cerebellar and cortico-nuclear projec-tions. The folia were shown to be mainly occupied in rostrocaudal succession by three zonesreceiving their olivo-cerebellar climbing fiber afferents from parts of, respectively, the dorsallamella of the principal olive, the ventral lamella of the principal olive, and the rostral halfof the medial accessory olive. These zones are presumably parts of the D2, D1, and C2cerebellar cortical zones, as earlier proposed by Rosina and Provini ([1982] Neuroscience7:2657–2676). Their respective nuclear target territories were found to be in the rostroventralquadrant of nucleus lateralis, the caudoventral quadrant of nucleus lateralis, and the ventralhalf of nucleus interpositus posterior. The medial-to-lateral width of each zone was shown tobe innervated by different groups of olive cells and to project respectively to medial andlateral parts of the nuclear territory for that zone, consistent with the existence in crus I ofolivo-cortico-nuclear microcomplexes (cf. Ito [1984] New York: Raven Press). Parts of thelength of each zone located within different folia were also shown to relate to different groupsof olive cells and to different regions of the zone’s overall nuclear territory. Interfoliallocalizations, which were heavily overlapping in nature, intersected orthogonally with thosefor zone width. The fine-grain topography implies that individual microzones exist withineach of the zones present within crus I. The results also have implications for the possibilitythat lateral cerebellar pathways are involved in cognition. J. Comp. Neurol. 497:287–308,2006. © 2006 Wiley-Liss, Inc.

Indexing terms: inferior olive; cerebellar cortex; cerebellar nuclei

It is widely accepted that the cerebellar cortex of the catand other mammals includes from medial to lateral a D1

and a D2 zone (initially recognized by Voogd, 1964, 1967,1969). Their climbing fibers originate from the principalsubnucleus of the inferior olive (PO) and their Purkinjecells project to the lateral (dentate) cerebellar nucleus(NL). These zones are present in the ansiform lobule (crusI and crus II), the part of the anterior lobe lateral to the C3

zone, the lateral part of the paramedian lobule, and also inthe paraflocculus (for reviews, see Brodal and Kawamura,1980; Voogd and Bigare, 1980).

However, the precise topographical relationship be-tween the two lamellae of PO and the two D zones has longbeen debated. Voogd and Bigare (1980) concluded thatanterograde tracing studies (Groenewegen and Voogd,1977: Groenewegen et al., 1979) offered little help becauseof the difficulty of making sufficiently restricted lesions or

tracer injections into PO. However, they cited work sub-sequently published by Rosina and Provini (1982), inwhich olive cells were retrogradely labeled after horserad-ish peroxidase (HRP) injections into different parts of crusI and crus II, as indicating that the D1 zone received itsclimbing fibers from the ventral lamella (vlPO) and the D2

zone from the dorsal lamella (dlPO). They also pointed out

Grant sponsor: Wellcome Trust; Grant number 059814.*Correspondence to: Dr. Richard Apps, Department of Physiology,

School of Medical Sciences, University of Bristol, University Walk, BristolBS8 1TD, UK. E-mail: [email protected]

Received 6 October 2005; Revised 22 December 2005; Accepted 30 Jan-uary 2006

DOI 10.1002/cne.20976Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 497:287–308 (2006)

© 2006 WILEY-LISS, INC.

that studies by others (Beitz, 1976; Martin et al., 1976;Chan-Palay, 1977) and their own unpublished observa-tions demonstrated that the rostromedial and caudola-teral parts of NL provide nucleo-olivary projections torespectively dlPO and vlPO. The nucleo-olivary and olivo-cerebellar projections are thought to be reciprocally orga-nized (Ruigrok, 1997), implying an additional link be-tween the D1 compartment and vlPO and between the D2compartment and dlPO. This arrangement is further sup-ported by the findings of a recent electrophysiologicalmapping and bidirectional tracing study in which theolivo-cortical and cortico-nuclear connections of the medi-almost folium of crus I was charted in cats (Edge et al.,2003). Similar conclusions to those reached by Rosina andProvini (1982) in the cat have also been recently proposedin the rat (Voogd et al., 2003; Sugihara and Yoshida,2004).

By contrast, Brodal and Kawamura (1980) reached theopposite conclusion, namely, that the D1 and D2 corticalzones receive their climbing fibers from, respectively,dlPO and vlPO. This was primarily on the basis of aretrograde tracing study in which HRP was injected intothe most lateral parts of the cat paramedian lobule (Wal-berg and Brodal, 1979). Findings in monkeys (Brodal andBrodal, 1981, 1982; Whitworth and Haines, 1986) and inthe rat (Azizi and Woodward, 1987; Buisseret-Delmas andAngaut, 1989, 1993) have been similarly interpreted.

Uncertainty also exists concerning the cortico-nuclearprojections. This is particularly important to resolve be-cause Purkinje cells are the sole output neurons of thecerebellar cortex, and thus the cortico-nuclear projectionis central to cerebellar function. According to Voogd andBigare (1980), the D2 and D1 zones in the cat projectrespectively to the rostromedial (magnocellular) and thecaudolateral (parvocellular) parts of NL. However, on thebasis of HRP studies in the same species, Dietrichs andWalberg (1979, 1980) and Dietrichs (1981a,b) reached dif-ferent conclusions. Moreover, in the rat Buisseret-Delmasand Angaut (1993) indicated that the D2 and D1 zonesrespectively target parvocellular and magnocellular NL.

In view of the conflicting evidence and because of ourinterest in the involvement of the cerebellar hemispheresin the control of volitional movements and the possibilitythat it may also be involved in nonmotor processes, includ-ing cognition (see, e.g., Leiner et al., 1986; Schmahmann,1991; Middleton and Strick, 2000), we reinvestigated theolivo-cortico-nuclear connections of crus I of the ansiformlobule. The olivo-cerebellar and cortico-nuclear projectionswere traced using microinjections into the four differentfolia of crus I, using cholera toxin subunit B (CTB) or amix of fluorescent-labeled beads and fluorescent dextranamines, employed, respectively, as bidirectional, retro-grade, and anterograde tracers. Most injection sites wereconfined to one folium to help us test the prediction that ifindividual zones are located in similar parts of successivefolia, then in crus I, where the folial long axes run rostro-caudally (rather than mediolaterally as, for example, inthe anterior lobe), the boundaries between zones should beorientated mediolaterally (rather than rostrocaudally, asin the anterior lobe).

Finally, since our initial findings suggested the exis-tence of intrazonal patterns of topographical localization,additional experiments were included to investigate theirnature. This is because of their relevance to the importantconcepts that the zones comprise assemblages of smaller

microzones, and that microzones are parts of olivo-cortico-nuclear “microcomplexes” (e.g., Oscarsson, 1969; Ito, 1984,2000; Garwicz and Ekerot, 1994; Garwicz et al., 1996; seeApps and Garwicz, 2005, for a review).

MATERIALS AND METHODS

Microinjections of axonal transport tracer agents weremade into the cerebellar cortex in 17 purpose-bred youngadult cats (2.3–4.5 kg). All surgical procedures were car-ried out aseptically and with surgical levels of generalanesthesia. The animals were initially deeply sedated viasubcutaneous injection of medetomidine hydrochloride(0.2 mg/kg; Domitor; SmithKlein Beecham, UK) and sub-sequently anesthetized via continuous intravenous infu-sion of propofol (Rapinovet; Schering Plough AnimalHealth, UK) at a rate sufficient to abolish flexion reflexes(�0.1–0.25 mg/kg/min). Airway mucous secretions weresuppressed via subcutaneous injection of 0.3 mg atropinesulfate (Atrocare Animal Care, UK) and antibiotic coverwas provided via subcutaneous injection of the broad-spectrum antibiotic ampicillin (Amfipen, Intervet, UK).Analgesia during and for 24 hours after operation wasobtained via a single intravenous dose of the nonsteroidalantiinflammatory analgesic and antipyretic carprofen (4mg/kg Rimadyl; Pfizer Animal Health, UK). The experi-ments were carried out in accordance with the UnitedKingdom Animals (Scientific Procedures) Act of 1986.

Tracer injections

In each animal a craniotomy was made to expose thefolia (typically n � 4) of crus I of the ansiform lobule of theleft cerebellar hemisphere. The most medial folium (fo-lium 1) was �10 mm long; more lateral folia were progres-sively shorter with the most lateral (folium 4) �7 mm inlength. In each animal a scale drawing and/or photographof the exposed surface was used to chart the position of oneor more tracer injection sites, produced in selected folia bypressure-injecting small volumes of tracer via the tip of aglass micropipette (tip diameter �50 �m) inserted to adepth of �0.5 mm below the pial surface. The pipette wasattached to a 1-�l syringe (Hamilton, Reno, NV) attachedto a 3D micromanipulator.

Details of all 32 injection sites are provided in Table 1.At most sites a total of 200 nl of tracer was injected in foursuccessive 50-nl aliquots injected at the same locus, but inseven instances tracer was injected at multiple loci toproduce a larger composite site. In five of these cases asingle injection was made into each of two (or in one casethree) adjacent crus I folia, the line joining the loci beingapproximately at right angles to the folial long axis. At theother two composite sites (both in one animal) injectionswere spaced �1 mm apart along the whole length of achosen folium.

At 28 sites a fluorescent tracer was employed. At 14sites, referred to below as red (R) sites, this was a 20%solution of the fluorescent dextran amine Fluoro-Ruby(FR; Molecular Probes, Eugene, OR) in a saline suspen-sion of red beads (Lumafluor, Naples, FL). At 14 other(green; G) sites the fluorescent tracer was a solution of20% of the fluorescent dextran amine Fluoro-Emerald(FE; Molecular Probes) in a saline suspension of greenbeads (Lumafluor). Beads are highly efficient retrogradetracers (see, e.g., Katz et al., 1984; Cornwall and Phillip-son, 1988; Katz and Iarovici, 1990; King et al., 1998) while

The Journal of Comparative Neurology. DOI 10.1002/cne

288 L. HERRERO ET AL.

the dextran amines were employed here as anterogradetracers (see Results). Previous studies of cerebellar corti-cal connections have shown that the two types of tracer,when mixed together, produce very similar effective re-gions of uptake and axonal transport for retrograde andanterograde labeling of olivo-cerebellar and cortico-nuclear projections (Apps and Garwicz, 2000; Pardoe andApps, 2002; Edge et al., 2003). At four sites the tracer wasCholera Toxin Subunit B (CTB), which is a bidirectionaltracer (although in our hands it functioned better in theretrograde than the anterograde direction; see Results).

After the injections the pial surface was protected withan overlay of gelfoam, the craniotomy was sealed withcold-curing dental acrylic cement, and the wound wasclosed in layers. All postoperative recoveries were rapidand uneventful. After a survival period of 7–9 days, theanimal was deeply anesthetized via intraperitoneal injec-tion of sodium pentobarbitone (60 mg/kg; Sagatal; RhoneMerieux, UK) and perfused transcardially. In most cases2 l of a heparinized 0.9% saline rinse was followed by 2 l of4% paraformaldehyde fixative followed by 2 l of 0.2 Mphosphate buffer (pH 7.4) containing 10% sucrose. In CTBcases, 2 l of 0.05 M phosphate buffered saline (pH 7.4) wasfollowed by 2 l of 0.05 M phosphate buffer (pH 7.4) con-taining 4% paraformaldehyde, 0.1% glutaraldehyde, and4% sucrose.

The cerebellum and brainstem were removed and thepositions of the fluorescent injection sites (visible as red oryellow-green dots on the cerebellar surface) were verifiedrelative to the folial pattern. The tissue was allowed to

sink overnight in 30% sucrose at 4°C (in CTB cases, after1 hour of postfixing in the perfusion fixative).

Histological processing

The cerebellum and brainstem were separated, a brain-stem block containing the inferior olive was sectionedtransversely and the cerebellum was cut into right andleft halves, embedded in aqueous 10% gelatin, 10% su-crose, and sectioned sagittally. Blocks were frozen-sectioned serially at 50 �m and except in the cases involv-ing a CTB site were mounted as two series of alternatesections: one for scrutiny, the other as a reserve. To min-imize fading of fluorescence the sections were not clearedor coverslipped and were stored at 4°C in the dark.

In cases that included a CTB injection site the sectionswere collected as three series each including every thirdsection. Two series were treated as above, while to visu-alize CTB distribution the third was processed immuno-cytochemically according to the protocol described in fullby Voogd et al. (2003).

Microscopy

The distribution of the fluorescent tracers was deter-mined using a Leica DMRB microscope fitted with a 50-Wmercury UV light source (Ploemopak) and a range of highnumerical aperture objectives (PL Fluotar). Red fluores-cence was viewed with an N2.1 filter block (Dichroic mir-ror 580 nm, BP 515–560 nm, LP 580 nm, BP 420–490 nm,LP 520 nm). Green fluorescence was viewed with an H3filter block (Dichroic mirror 510 nm, BP 420–490 nm, LP

TABLE 1. Injection Site Details

Case/tracer1

(n � 32)

Identityof

folium2

Tracervolume3

(nl)

Site center tofolium end4

(mm)Site length5

(mm)Site size6

(mm2)

7G (1) 2 200 0.3 1.0 0.9212R (2) 1 150 0.7 ? 0.9910CTB (3) 2,3 2 � 100 0.7 ? 1.419G (4) 3 200? 0.8 0.3 0.1311R (5) 2,3 200,150 0.8 0.9 0.9110G (6) 2,3,4 3 � 100 0.9 1.0 1.351R (7) 4 200 1.1 ? 1.178G (8) 3 2 � 200 1.3 0.5 0.4811CTB (9) 2 135 1.5 ? 1.937R (10) 2 200 2.2 1.3 0.7715G (11) 2 200 2.3 0.7 1.0026R (12) 3 200 2.4 1.0 2.203R (13) 1 200 2.9 1.1 0.87L16R (14) 3 200 3.6 0.7 1.0026G (15) 3 200 3.8 0.9 0.9415R (16) 2 200 3.9 ? 1.632G (17) 1,2 400?,200 4.4 ? 3.87L16G (18) 3 200 4.7 1.1 0.9315CTB (19) 2 200 4.7 ? 1.6217G (20) 2 300? 5.1 0.5 0.393G (21) 1 250 5.6 0.5 0.162R (22) 1,2 2 � 100 6.3 ? 0.16L16CTB (23) 3 200 6.1 ? 1.9419G (24) 4 225 6.0 ? 0.9620R (25) 3 150 6.5 ? 1.0518R (26) 2 100 6.5 ? 0.5417R (27) 2 250 6.6 ? 2.2419R(28) 2 175 7.0 ? 1.3120G (29) 1 250 7.5 ? 0.3918G (30) 2 100 8.5 ? 0.2421G 2 8 � 150 N/A ? 3.4621R 4 8 � 100 N/A ? 2.40

1The prefix L indicates left side in animal 16, other letter(s) indicate the tracer used. Number in parentheses relates to location of injection site in Figure 4.2Column shows which folia were injected with 1 being the most medial and 4 the most lateral folium in crus I: Note that in cases 21R and 21G multiple injections were spaced�1 mm apart along the entire rostrocaudal length of the folium.3Queries indicate cases with tracer escape onto folial surface; such escapes were removed by blotting/flushing.4Distance to rostral end of folium. Where there were separate subsites distance is for the largest. N/A, not applicable (see 2 above).5Distance represents maximum extent of site (or largest subsite) measured along the rostrocaudal length of folium. Queries indicate information not available.6Area of injection site deduced from the numbers of retrogradely labeled olive cells; see Material and Methods.


289CEREBELLAR CRUS I LOCALIZATIONS

520 nm). The distribution of CTB was determined usingtransmitted light, initially employing an M 400 Leitz-Wildphotomacroscope and for higher resolution a Leica DMRBmicroscope with a range of high-power objectives.

Mapping of transported tracers

The distributions of retrogradely labeled neurons in thecontralateral inferior olive were charted on a rostrocaudalseries of standard transverse maps �0.25 mm apart asdescribed by Trott and Apps (1991). Although FR and FEare bidirectional tracers and seemed similar to fluorescentbeads in their efficiency as retrograde tracers, there wasno doubt that lightly labeled cells and unlabeled cells weremost readily differentiated on the basis of whether or notbeads were present in the cytoplasm (e.g., Fig. 1).

Because most cases involved hundreds of retrogradelylabeled cells, these are not represented individually in thefigures except where they were sparsely present. Instead,the population distribution is indicated by hatching. Thenumbers of cells were, however, counted and totals sub-jected to an Abercrombie (1946) fragmentation correction,as described by King et al. (1998).

Olivary labeling was also represented on 2D scale maps(plan views) of the rostral part of the medial accessoryolive (rMAO), vlPO, and dlPO (see, e.g., Figs. 2, 7, and 9,respectively). In each of the three maps the dimensionswere based on measurements from the standard trans-verse sections and the rostrocaudal and mediolateral di-mensions are shown to the same scale. In each case anappropriate relationship was maintained at each rostro-caudal level between the medial edge of the olivary sub-nucleus and the brainstem mid-line (cf. Trott and Apps,1991). As a result, our maps for dlPO and vlPO differsomewhat from those of other authors (e.g., Brodal andKawamura, 1980; Voogd and Bigare, 1980; Rosina andProvini, 1982), who pivoted dlPO and vlPO out flat abouttheir point of junction (at the lateral bend of PO), in amanner akin to opening a book. That procedure has theadvantage of emphasizing (except caudal to AP 11.25,where vlPO is absent) that the two lamellae are in realityfused at the lateral bend, but the disadvantage of mark-edly distorting their shapes because it ignores the factthat at some different rostrocaudal levels the bend lies atsubstantially different distances from the midline. Notealso that rMAO has in parts a substantial thickness andthis is not evident in a plan view.

Anterograde labeling of Purkinje cell stem axons andtheir terminal arborizations with CTB, FE or FR (see forexample Fig. 1) was also charted on a series of standardsagittal diagrams of the cerebellar nuclei between themedial pole of the nucleus fastigius and the lateral pole ofNL, as described by Trott et al. (1990). Hatching is used torepresent terminal labeling (cf. Trott et al., 1998 a and b)and wavy lines to indicate stem axons (e.g., Fig. 2).

Injection sites

Although the position of each injection locus relative tothe crus I folial pattern was noted at operation, histolog-ical confirmation was also obtained of the position and sizeof each injection site (for the typical appearance of aninjection site in sagittal section see Fig. 1). Site positionwas expressed in terms of the distance (in mm) betweenthe center of the site (in the section where the site waslargest) and a chosen landmark which was the rostral endof the folium where it abutted on the dorsal parafloccular

folial chain. Because the folial long axes were parallel tothe (parasagittal) plane of section the distance could bemeasured directly.

Site size was expressed as the area (in mm2) of thecerebellar cortical sheet that was involved in the site. Inall cases an estimate was obtained via a method fullydescribed by King et al. (1998). This exploits the fact that1) each Purkinje cell is innervated by one climbing fiberand therefore receives input from only one olive cell, and2) local branching of climbing fibers is very limited, sotypically each olive cell provides only one climbing fiberper mm2 of cortex. Thus, dividing the number of retro-gradely labeled olive cells by a literature estimate of thenumber of Purkinje cells present per mm2 of cortical sheet(373; see King et al., 1998 for refs), provides an estimate ofthe area of cortical sheet from which retrograde olivarylabeling occurred.

Photomicrograph production

Images of sections were captured with a digital camera(Axiocam HRc Color; Zeiss) mounted on a Zeiss epifluo-rescence microscope (Axioskop 2) with software Axiovision3.1 (Zeiss, Welwyn Garden City, Herts, UK). The imageswere subsequently processed in Adobe Photoshop 7.0(Adobe Systems Inc., San Jose, CA) to adjust contrast andbrightness.

RESULTS

Injection sites

In 17 animals a total of 32 crus I injection sites wereproduced as described in Materials and Methods, of which28 involved fluorescent tracers and 4 involved choleratoxin subunit B (CTB). The present report is focused,however, on consideration of results obtained primarilyfrom a total of 22 cases in which the olive cell labeling wasrestricted to only one subdivision of the contralateral in-ferior olive (rMAO, dlPO or vlPO, see below). For theremaining additional cases not considered in detail (be-cause the olive cell labeling indicated spread of injectate toneighboring zones, see Table 2), the overall distribution oflabeled olivary cells and Purkinje cell axon terminals wasentirely consistent with the pattern that would arise if thelabeling from injections confined to individual zones wascombined. Note that individual animals with more thanone tracer injection site enabled the analysis of overlaps(or their absence) between populations of olivary neuronsretrogradely labeled with different tracers and betweencortico-nuclear termination fields anterogradely labeledwith different tracers.

Injection site locations and sizes

Table 1 indicates for each case (numbers in bracketsrelate to cortical location in Fig. 4) the folium or foliainjected, with folium 1 the most medial and folium 4 themost lateral. It also shows the distance (estimated histo-logically; see Materials and Methods) from the center ofthe injection site to the rostral end of the injected folium.With a few exceptions (see below), the sites were centeredin the cortical layers, extending down to, or a little beyond,the junction with the subcortical white matter (Fig. 1A,B).As described in Materials and Methods and by King et al.(1998), an estimate of injection site size was obtained forall cases. Table 1 shows that most injection sites were



small, with only five involving more than 2 mm2 of corticalsheet.

Cases with retrograde cell labeling in rMAO

All 32 cases are represented in Table 2 (in the sameorder as in Table 1), which shows the number of olive cellsretrogradely labeled and their percentage distribution be-

tween the different subnuclei of the inferior olive, and alsoindicates the distribution of anterograde terminal labelingwithin the cerebellar nuclei. Inspection shows that therewere 12 cases (in seven animals) in which a crus I injectionled to olivary labeling only in rMAO. The numbers of cellsinvolved were always substantial, ranging from a mini-mum of 60 (case 3G) to a maximum of 837 (case 17R).

Fig. 1. Photomicrographs to show the appearance of an injectionsite, retrogradely labeled cells, and anterogradely labeled terminals.A: Camera lucida drawing of a sagittal section of the cerebellum toshow the location and extent of a typical injection site (case 15G).B: Photomicrograph of the boxed area in A to show the center of thegreen injection site. C: Camera lucida drawing of a sagittal section ofthe cerebellar nucleus (level 17) showing the location of anterogradelylabeled Purkinje cell axons (wavy lines) and labeled preterminals/terminals (stippling, case 17R). D: Photomicrograph of the boxed areain C showing fibers anterogradely labeled with Fluoro-Ruby. A plexus

of labeled preterminals/terminals is demarcated by a dashed line.E: Standard transverse outline of the inferior olive at AP level 10.0.F: Photomicrograph of the boxed area in E, showing olive cells retro-gradely labeled with beads (case 11R). In this example red labeledcells (arrowheads) were located in the dorsal lamella of the principalolive (dlPO). G: Photomicrograph of the boxed area in F, to showlabeled cells at higher power. NIA, nucleus interpositus anterior; NIP,nucleus interpositus posterior; Ant, anterior; Post, posterior; Pt/t,preterminals/terminals.



Figures 2 and 3 illustrate the findings in six cases (inthree animals) from this group. By analogy with otherregions (paravermal part of the anterior lobe, paramedianlobule, paraflocculus), we conclude that all 12 injectionsites were confined to the C2 cortical zone.

In nine of these cases (see Table 2) the tracer succeededin producing anterograde terminal labeling of Purkinjecell axons and this was always confined to NIP, which isthe sole cerebellar nuclear target for the Purkinje cells inthe parts of the C2 zone that lie elsewhere in the cerebellarcortex.

The locations on the cerebellar surface of the 12 cases inwhich all of the labeled olivary neurons were in rMAO areshown by filled circles in Figure 4. These sites constitute12 of the 16 most caudal sites in crus I; three of theadditional cases are indicated in Figure 4 by half-filledcircles (cases 15 (26G), 17 (2G), and 18 (L16G)) becausethey were centered rostrocaudally in the middle of crus Iand involved labeling in rMAO and also vlPO. This sug-gests that in each of these “mixed” cases the injection siteinvolved two neighboring zones. The one additional case(case 22 (2R), shown by two linked crosses) involved twoinjection sites that were centered in more caudal parts offolium 1 and 2 and resulted in some labeling in rMAO, butmost labeled cells were located in rDAO. A possible reasonfor this discrepancy is given in the Discussion. Note thatno attempt has been made in Figure 4 to represent sitesizes, but when the information available in Table 1 iscombined with the distribution of the sites it is clear thatcollectively cases with cell labeling confined to rMAO in-volved much of the caudal halves of all four folial crests.

In summary, no fewer than 12 cases provided unequiv-ocal evidence for the existence, in the caudal halves of thefolia of crus I, of a substantial region of cortex identifiableas part of the C2 zone because it receives its climbingfibers exclusively from rMAO and its Purkinje cells projectonly to NIP.

“Total” olivary and nuclear territories ofthe crus I part of the C2 zone

Among the 12 cases confined to the C2 zone, there wereappreciable variations in the distribution of the labeledneurons within rMAO and, likewise, variations in thelocation of the anterogradely labeled terminals in NIP(e.g., Fig. 3). These cases (plus case L16G in which 93% ofthe labeled cells were in rMAO) were pooled to reveal thetotal extent of the territories in rMAO and NIP that werelabeled in at least one case and the results are shown inFigure 5. In rMAO the territory was substantial; at caudallevels it was confined to the ventrolateral part but morerostrally it expanded medially so that between levels 9.5and 10.0 it occupied most of the width of rMAO. Veryrostrally, at levels 9.25 and 9.0, it reduced again, beingconfined to the medial part of the cross-section. For thesmall areas of vertical hatching, see the legend of Fig-ure 5.

In NIP Figure 5 shows that the pooled cortico-nuclearterritory (for the n � 9 “pure” rMAO cases with anyterminal labeling plus case L16G) included most medio-lateral levels of the nucleus except those furthest lateraland medial. It mainly involved the ventral half of the

TABLE 2. Label Distributions in Inferior Olive and Cerebellar Nuclei

Case/ FoliaOlive cell

count1

Percentage distribution in olivary subnuclei Corticonuclear terminal labeling2

Tracer Injected rDAO rMAO vlPO dlPO NIA NIP NLc NLr

7G (1) 2 345 0 0 0 100 — — — ��12R (2) 1 370 0 0 0 100 — — — ��10CTB (3) 2,3 526 0 0 0 100 — — — ��9G (4) 3 47 0 0 0 100 — — — ��11R (5) 2,3 337 0 5 4 91 — — — ��10G (6) 2,3,4 505 0 0 4 96 — — � ��1R (7) 4 436 0 4 76 20 — � �� —8G (8) 3 178 0 0 70 30 — — �� —11CTB (9) 2 720 0 0 0 100 — — — ��7R (10) 2 285 0 0 100 0 — � �� —15G (11) 2 374 0 0 100 0 — — — —26R (12) 3 399 0 0 100 0 — — �� —3R (13) 1 325 0 0 100 0 — — �� —L16R (14) 3 374 0 0 100 0 — — �� —26G (15) 3 219 0 11 69 20 — � � —15R (16) 2 608 0 100 0 0 — �� — —2G (17) 1,2 1445 0 31 69 0 — �� —L16G (18) 3 347 0 93 7 0 — �� — —15CTB (19) 2 605 0 100 0 0 — — — —17G (20) 2 145 0 100 0 0 — �� — —3G (21) 1 60 0 100 0 0 — �� — —2R (22) 1,2 60 93 7 0 0 �� — —L16CTB (23) 3 722 0 100 0 0 — — — —19G (24) 4 358 0 100 0 0 — �� — —20R (25) 3 391 0 100 0 0 — �� — —18R (26) 2 200 0 100 0 0 — �� — —17R (27) 2 837 0 100 0 0 — �� — —19R (28) 2 489 0 100 0 0 — �� — —20G (29) 1 146 0 100 0 0 — �� — —18G (30) 2 90 0 100 0 0 — — — —21G 2 1294 0.5 50 28.5 21 (�) �� 21R 4 896 0 19 26 55 (�) ��

1Column shows total number of retrogradely labeled neurons in the contralateral inferior olive corrected as described in Material and Methods.2Numbers of plus signs indicate the approximate relative abundance within a case of terminal labeling in the different cerebellar nuclei; — indicates no terminal labeling. Incompiling the table NL was divided at each mediolateral level by a dorsoventral line drawn so as to divide the nucleus into caudal (c) and rostral (r) portions approximately equalin size (see text). Bracketed signs (�) indicate labeling in the extreme ventrocaudal tip of NIA at mediolateral levels 9 and 10.See Table 1 for further details.



nucleus and at levels 12–17 it formed a rough crescentwith its concave surface facing dorsally.

Width of the C2 zone in crus I

Among the cases in which the olivary labeling was inrMAO there were four “pairs” in which both injection sites

were in the same folium in the same animal. These werecases 15R and 15 CTB, 17G and 17R (see Fig. 3), 18G and18R (see Fig. 2), and L16G and L16CTB (not shown) inwhich the injection site centers were, respectively, 0.8, 1.5,2.0, and 1.4 mm apart along the length of the folium.Clearly, the C2 zone cannot have been narrower and it

Fig. 2. Distributions of retrograde olivary and anterograde cere-bellar nuclear labeling in cases 18R and 18G. Circles in dorsal view ofthe left crus I show the red and green injection sites centered 2 mmapart in the caudal part of folium 2 in the same animal; note circle sizedoes not represent site size (see Table 1). Hatched areas on scalediagrams to left show the territories containing red- and green-labeled neurons as found in transverse sections through rostral levelsof the right inferior olive; numbering indicates stereotaxic AP levels;levels between 12.25 and the caudal pole of the olive at 14.75 areomitted. Lower middle diagram shows the territories in MAO re-represented on a scaled horizontal reconstruction of the whole of thatolivary subnucleus. Scale diagrams to right show sagittal sections

through the left cerebellar nuclear complex; out of 32 levels �0.25 mmapart levels 1–19 are shown but the more medial levels that includenucleus fastigius are omitted; wavy lines and hatched outlines, re-spectively, represent labeled cortico-nuclear stem axons and termina-tion fields; only red nuclear labeling is shown because in this exper-iment the green tracer labeled stem axons approaching NIP but failedto yield terminal labeling. m, med, medial; l, lat, lateral; r, rostral; c,caudal, d, dorsal; v, ventral; dmcc, dorsomedial cell column; �, nucleus�; DAO, dorsal accessory olive; MAO, medial accessory olive; dlPOand vlPO, respectively, dorsal and ventral lamella of principal olive;vlo, ventrolateral outgrowth; NL, nucleus lateralis; NIP, nucleus in-terpositus posterior; NIA, nucleus interpositus anterior.



must, indeed, have been significantly wider. The length ofeach site along the folium could be estimated assumingthe injection site occupied an approximately circular patchof cortex and for each pair of sites an estimate couldtherefore be obtained of the distance from the rostral edgeof the rostral site to the caudal edge of the caudal site.These estimates were 2.2, 2.6, 2.5, and 2.8 mm, respec-tively. Because all the labeled olivary neurons were con-fined to rMAO, these values must be regarded as conser-vative estimates of zone width and the C2 zone is likely tobe appreciably wider. Interestingly, the rostrocaudal dis-

tance between the centers of the most caudal and the mostrostral of the sites restricted to the C2 zone (i.e., 18G and15R; both in folium 2) was 4.6 mm; i.e., 8.5 minus 3.9 mm;see Table 1). Overall, therefore, it seems reasonable toconclude that the C2 zone is at least 3 mm wide and veryprobably 4.5 mm wide in crus I of the cat cerebellum.

Fine-grain topographical organizationwithin the C2 zone in crus I

Differences between cases with regard to the preciselocations of the labeling in rMAO and NIP suggested that

Fig. 3. Distributions of olivary and cerebellar nuclear labeling infour cases (in two animals) with olivary labeling restricted to rMAO.Hatched areas in A represent the findings in cases 15R and 15CTB.Likewise in B for cases 17R and 17G. Circles in the dorsal view of thecrus I folia indicate the positions of the injection site centers but notthe extent of the sites. In fact, in both A and B there was partial

overlap between the two sites. Note that in A the CTB tracer failed toproduce detectable terminal labeling in the cerebellar nuclei; in Beach tracer produced both terminal labeling and labeling of cortico-nuclear stem axons but green stem axons are omitted for clarity;although segregated from the red axons they reached NIP via aparallel course. Conventions and abbreviations as in Figure 2.



intrazonal patterns of localization might exist in both theolivo-cerebellar and the cortico-nuclear projections of theC2 zone, and these possibilities were therefore explored. Itwas of particular interest (see Discussion) to determinewhether any topographical localizations existed that re-lated to the width of the zone, i.e., in the sagittally orien-tated folia of crus I, to its parts lying rostrally and cau-dally within the folium. Also, to determine whether anylocalizations existed within the mediolateral sequence ofthe four different folia making up crus I.

Olivo-cerebellar localization in relation to the width

of the zone. As previously noted, there were four “pairs”of cases in which the two sites were both in the samefolium in one animal. In cases 18R and 18G the red sitewas rostral to the green and as may be seen in Figure 2 thered-labeled olive cells were likewise rostral to the green(and extended slightly further medially in rMAO). Neitherthe injection sites nor the olivary territories overlapped,nor were any olive cells double-labeled. In cases 15 CTBand 15R (Fig. 3A), there was partial overlap between thetwo sites (centered only 0.8 mm apart) and likewise par-tial overlap between the two rMAO cell populations. How-ever, as Figure 3A shows, the population labeled from themore rostral site (15R) was centered more rostrally andmedially than the (caudolateral) population related to theother site. In cases 17G and 17R (see Fig. 3B), the formerinjection site was centered more rostrally but was partlyoverlapped by the latter, which was much larger. Corre-spondingly, the 17G population of labeled olive cells waspartly overlapped by the much larger 17R population, butthe latter extended substantially further caudally andlaterally in rMAO.

In summary, these cases show unequivocally for the C2zone in crus I folia that there is an olivo-cerebellar topog-raphy such that rostral in the folium (and presumably,therefore, lateral in the zone) corresponds to rostromedialin rMAO, while caudal in the folium (and medial in thezone) corresponds to caudolateral in rMAO.

Cortico-nuclear localization in relation to the width

of the zone. Unfortunately, in three of the above pairs ofcases one tracer failed to yield any terminal labeling in thecerebellar nuclei (see Table 2). As a result, the only “same-animal” comparison possible was between cases 17R and17G (see Fig. 3B), where the larger (red) site producedsubstantially more labeling in NIP. The two terminalfields showed only limited overlap and the red field ex-tended further medially, laterally, and dorsally so that noconclusions could be drawn regarding any systematicmapping of zone width onto the nucleus.

However, when between-animal comparisons weremade among the 15 cases with appreciable terminal label-ing in NIP (other than 21G and 21R, which involvedmultiple injections along the entire length of individualfolia), the five sites centered most caudally (between 6.5and 8.5 mm caudal to the rostral end of the folium; seeTable 2) all had terminal labeling that extended furthermedially in NIP than in any of the five centered mostrostrally (between 1.1 and 4.4 mm from the rostral end ofthe folium). On average, labeling in the first group ex-tended to between NIP levels 17 and 18 (range 15–19),whereas in the second it reached level 12 (range 8–14),corresponding to an average difference of over 1 mm (com-pare, e.g., case 17R in Fig. 3B with 15R in Fig. 3A). Suchdifferences were evident even for comparisons restricted

Fig. 4. Location of the injection site centers pooled for 30 cases in16 cats in which tracer was injected into the tips of the folia makingup crus I. To the left is shown a view of the anterior aspect of the catcerebellum. Boxed area is shown to the right in which small numbersrelate to individual cases listed in Tables 1 and 2. Filled circles (n �12) represent cases with olivary labeling restricted to rMAO. Opencircles (n � 5) represent cases with olivary labeling restricted to vlPO.Filled squares (n � 5 sites, one shown by two linked squares) repre-sent cases with labeling restricted to dlPO. Other symbols represent

cases with cell labeling in two or more olivary subnuclei; links indicatecomposite sites involving injections in more than one folium; forfurther details, see text. Note that cases 21G and 21R are not shownbecause multiple injection sites were made along the entire length ofindividual folia. Note also that injection site sizes are not represented(see Table 1 for details). AL, anterior lobe; FP, fissura prima; LS,lobulus simplex. Large numbers to the side of the expanded viewidentify the different folia of crus I: 1, medial; 4, lateral.



to cases involving corresponding folia; it is thereforehighly probable that medial to lateral in the C2 zone ismapped medial to lateral in NIP (cf. Edge et al., 2003,their fig. 5).

Olivo-cerebellar localization in relation to foliation.

In two animals (19 and 20) one fluorescent tracer wasinjected into a lateral folium (respectively, folium 4 and 3)and the other into a more medial one (respectively, 2 and1), the red and green sites being centered, so far as possi-ble, at similar positions along the folial long axis. Alllabeled cells were in rMAO and, as seen in Figure 6, inboth experiments the olivary territory for the lateral fo-lium was centered lateral and marginally rostral to thatfor the medial one so that a line joining the center of thetwo territories would be approximately at right angles toone joining the centers when two injections were made ina single folium (compare Fig. 6 with Figs. 2 and 3). Thesefindings support localization such that medial and lateralcrus I folia are represented respectively caudomediallyand rostrolaterally within the overall C2 territory inrMAO, i.e., in a pattern that intersects orthogonally withthat for zone width.

Cortico-nuclear localization in relation to foliation.

In experiments 19 and 20 (see Fig. 6), each with a red sitein one folium and a green site similarly placed along thelength of a second, both termination fields were centeredin the ventral part of the nucleus, but the field labeledfrom the more medial folium was, at each mediolaterallevel where both were present, centered further dorsally,and there was virtually no overlap between the fields. Inexperiment 21, with multiple injections in two folia, thefindings in NIP were similar but in this instance there wasmore (but still slight) overlap between the two fields.These results suggest that medial to lateral in the folialchain is mapped dorsal to ventral within NIP. However,given that in all three experiments an uninjected foliumwas interposed between the injected ones, it is highlyprobable that there is substantial overlap between theterritories for contiguous folia. The findings in other caseswere generally in good accord with such a pattern oflocalization, although it should be noted that in case 3G,where the injection site was in folium 1, the (small) ter-minal field was near the ventral border of NIP (i.e., moreventral than expected). However, this site was very small

Fig. 5. Total extent of the olivary and cerebellar nuclear territoriesdefined by pooling cases in which there was olivary labeling in rMAO.The filled and vertically hatched areas together include all those partsof rMAO and NIP in which labeling was present in at least one case.The filled olivary territory was derived by pooling the 12 “pure” rMAOcases (see Table 2 and filled circles in Fig. 4) plus case L16G in which93% of the labeled olive cells were in rMAO. The filled nuclear terri-

tory was derived from a total of 10 cases in which the tracer producedterminal labeling confined to NIP. Vertical hatching in MAO showsthe very small additions to the labeled territory that resulted fromincluding in the pooling all cases with any cell labeling in MAO. Suchpooling produced no significant expansion of the NIP terminal terri-tory. Conventions and abbreviations as in Figure 2.



Fig. 6. Distributions of olivary and cerebellar nuclear labeling intwo animals in each of which two injection sites in different foliagenerated olivary labeling restricted to rMAO. A: Findings from ex-periment 19 in which green tracer was injected into folium 4 and red

tracer into folium 2. B: Findings from experiment 20 in which greentracer was injected into folium 1 and red tracer into folium 3. Forfurther details, see text. Conventions and abbreviations as in Figure2.



and more dorsal parts of the NIP territory for the foliummay not have been labeled.

Cases with retrograde cell labelingrestricted to vlPO

In five cases in five animals (cases 3R, 7R, 15G, L16R,26R; see Table 2), labeled olive cells were entirely confined

to vlPO; two cases are illustrated in Figure 7A,B. In viewof the widespread acceptance of the view that it is the Dzones that receive their climbing fibers from PO (see In-troduction and Discussion for References) and because theinjection sites were close beside the C2 zone, we interpretthese five as D1 zone cases. Table 1 and Figure 4 show thatthe injection sites in these cases (open circles in Fig. 4) lie

Fig. 7. Example cases in which distributions of olive cell labelingwere located within vlPO. A,B: Two cases (respectively, 7R and 3R) inwhich olivary labeling was entirely restricted to vlPO. C: The cell

labeling in vlPO in cases 26R and 26G in which the injection siteswere both in folium 3 with the red injection 1.4 mm rostral to thegreen. Conventions and abbreviations as in Figure 2.



rostral to but near the filled circles representing the C2zone sites (and note that three of the sites, 11 (15G), 13(3R), and 14 (L16R), were in folia in which, in the sameanimal, more caudal sites yielded labeling confined torMAO). Collectively, these cases involved folia 1, 2, and 3and indicate that in these folia a D1 zone lies rostral to theC2 zone. From Figure 4 and the data in Table 1 a conser-vative estimate for the rostrocaudal extent (i.e., the width)of the zone is �1.5 mm. However, when other sites aretaken into consideration, in which vlPO was labeled alongwith other olivary subnuclei (see below), it is probable thatthe zone is up to a millimeter wider.

Regarding the cortico-nuclear projections, reference toTable 2 (see also Fig. 7A,B) shows that in three cases(cases 3R, L16R, 26R) labeled terminations were entirelyconfined to NL. In a fourth (case 7R), they were similarlyconfined, apart from a very small subsidiary terminationfield nearby in rostroventral NIP. In the remaining case(case 15G) no terminal labeling was detected, but labeledstem axons could be traced to the nuclear region where allentered NL via its dorsal border.

The pooled findings in these cases are shown by thefilled areas in Figure 8. The olivary labeling occupiesmuch of vlPO except for its medial part and the cortico-nuclear labeling extends across the lateral three-quartersof NL and is centered at each mediolateral level in thecaudoventral quadrant of the nucleus. For explanation ofthe vertically hatched areas, see the legend of Figure 8.

Fine-grain topographical organizationwithin the D1 zone in crus I

Olivo-cerebellar localization. The ventral lamellaspans 11 rostrocaudal levels spaced 0.25 mm apart andamong the nine cases with any appreciable cell labelingwithin it (�25% of total, see Table 2), there were six in whichthe olivary territory spanned from 9–11 levels and threewith a span of 6–8 levels. The mediolateral width of eachterritory varied somewhat at different rostrocaudal levelsbut was 1.0 mm or less at its widest except in one case (2G;1.5 mm); some territories were considerably narrower (e.g.,0.2 mm and 0.4 mm in cases 8G and 1R, respectively).Essentially, therefore, the territories were rostrocaudallyoriented “columns” of cells, albeit bowed laterally where thelamella reaches its greatest width (cf. Fig. 7A).

With regard to the possibility of any “folium-based”localization in vlPO, there were several instances in whichinjection sites confined to one folium labeled cells at al-most every rostrocaudal level, and the representations ofdifferent folia therefore overlapped almost completely inthe rostrocaudal plane. Moreover, in animal 21 the (red)labeling from folium 4 was virtually identical in its ros-trocaudal extent to the (green) labeling from folium 2: it istherefore highly unlikely that any localization exists of akind in which different folia are represented at differentrostrocaudal levels in the lamella.

By contrast, there were two cases, 2G (not shown) and3R (see Fig. 7B), in which the vlPO labeling extendedfurther medially than in any other case. Case 3R was theonly case in which the injection site was confined to folium1 and 2G was the only other in which this folium wasinvolved (together with folium 2). When compared withthe other cases (e.g., case 7R; folium 2; see Fig. 7A), thesetwo were therefore compatible with a topography in whichmedial to lateral in the folial chain is mapped medial tolateral in vlPO. Such an arrangement also received sup-

port from case 8G (folium 3; not shown) in which labeledcells were confined to a narrow column along the extremelateral edge of vlPO at the junction with dlPO. However,among the other cases with substantial labeling in vlPO(15G, folium 2; L16R, folium 3 and 1R, folium 4) the cellcolumns all closely resembled that in case 7R. Moreover,although in animal 21 the red site in folium 4 generated arather patchy, discontinuous column of labeled cells, thesedid not differ systematically in their mediolateral locationfrom the column labeled from the green site in folium 2. Itis possible, therefore, that the underlying pattern is one inwhich a fairly medially located territory for folium 1 isaccompanied by (and partly overlapped by) a commonlateral column for folia 2, 3, and 4.

With regard to any localization relating to differentparts of the width of the D1 zone, cases 26G and R werethe most informative; the former was a “mixed” case (seeabove) mainly involving the D1 zone and the latter a“pure” D1 zone case. There was some overlap between thetwo injection sites but the former was centered furthercaudally in the folium and labeled rMAO as well as vlPO,so it presumably extended further medially across thewidth of the D1 zone. In vlPO, as may be seen in Figure7C, the green- and red-labeled cell columns were similarlylocated in the mediolateral plane but the green columnwas shifted caudally relative to the red so that overlapwas restricted to levels 9.75–10.25 (where a substantialproportion of cells were double labeled). This findingstrongly supports a topography in which medial and lat-eral in the D1 zone are represented respectively caudallyand rostrally in vlPO. Further support came from case 8Gin which the site, restricted to one folium and presumablyincluding the lateral part of the D1 zone (because therewas substantial labeling in dlPO as well as in vlPO), led tolabeling at rostral but not caudal levels in vlPO.

Cortico-nuclear localization. As previously stated,there were appreciable differences between cases in the lo-cation of the labeled termination fields within NL. Thesedifferences were in all three planes (i.e., mediolateral, ros-trocaudal, and dorsoventral) and they strongly suggest thepresence of some form(s) of cortico-nuclear localization.

With regard to the representation of different folia, it wasclear that in the case involving folium 1 (3R), the termina-tion fields in NL were either centered more dorsally or ex-tended further dorsally than in the cases involving morelateral folia. In addition, in experiment 21 (not shown), bothrostrally and caudally in NL the green termination fields(arising from folium 2) were at all mediolateral levels cen-tered more dorsally than the red (arising from folium 4). Wesuggest, therefore, that for both D zones medial to lateral inthe folial chain is mapped dorsal to ventral in the ventralhalf of NL, albeit in overlapping fashion.

Evidence was also found for a cortico-nuclear localiza-tion for the medial versus the lateral parts of the zone.Thus, cases 26G and R were instructive: of the two injec-tion sites the red was the more rostral in the folium and itproduced, in the caudal half of NL, terminal labeling thatextended laterally almost to the lateral extremity of NL(i.e., to level 2). By comparison the green tracer was lesswell transported but it was nevertheless clear that moremedial nuclear levels were targeted: most labeled stemaxons and preterminal branches were at levels 6–9, cours-ing ventrally where the caudal part of NL and the rostralpart of NIP adjoin. These findings indicate that lateral tomedial in the zone is mapped lateral to medial in caudal NL.



Fig. 8. Total extent of the olivary and cerebellar nuclear territoriesdefined by pooling cases in which there was olivary labeling in vlPO.The filled and vertically hatched areas together include all those partsof vlPO and NL in which labeling was present. Filled territories relateto those cases (n � 5; see open circles in Fig. 4) in which olivary celllabeling was entirely restricted to vlPO; for one of these (7R) a verysmall termination area rostroventral in NIP and very close to caudalNL is not shown. Vertical hatching shows the expansions of theterritories that resulted from including the vlPO and NL labelingfrom all cases with any label in vlPO. Note that this involved nine

additional (“mixed”; see text) cases but in vlPO the only significantexpansions were produced by case 2G (medially at levels 9.5–11.0)and case 1R (laterally at levels 10.25–10.75); in NL the only signifi-cant expansions were produced by case 2G. Note that for the “addi-tional” cases olivary labeling other than in vlPO and nuclear labelingother than in the caudal half of NL is omitted. The only cases in whichterminations in the rostral half of NL were omitted were case 10G(which had two separate fields, one in rostral, the other in caudal NL)and cases 21R and 21G, in which multiple injections were made alongthe folial length. Conventions and abbreviations as in Figure 2.



Cases with retrograde cell labelingrestricted to dlPO

Five cases (7G, 9G, 10CTB, 11CTB, 12R; see Table 2)had olivary labeling entirely restricted to dlPO. In onecase (9G) the number of neurons was small but in theothers they ranged from 345 (7G) to 720 (11CTB). Twocases are shown in Figure 9. In all five cases cortico-nuclear labeling was entirely restricted to NL, where itwas centered in the rostroventral quadrant of the nucleus.

Reference to Table 1 and to Figure 4 shows that theinjection sites (filled squares in Fig. 4) were all rostrallylocated, the most caudal (case 9 (11CTB)) being centered1.5 mm from the rostral end of the injected folium. Weinterpret these cases as showing that the most rostralparts of folia 1, 2, and 3 are occupied by a D2 zone receiv-ing its climbing fibers exclusively from dlPO, projectingonly to NL and likely to be at least 1.5 mm wide.

The “pooled” territories in dlPO and NL for these fivecases are shown in Figure 10 by the filled areas. Note that

the NL territory does not overlap with that in Figure 8 forthe vlPO group of cases, suggesting that the D1 and D2zones in crus I project differentially, respectively targetingthe caudal and rostral parts of the ventral part of thenucleus. In this regard the vlPO case 7R (see Fig. 7A) andthe dlPO case 7G (see Fig. 9A) are of special interestbecause, although the two injection sites were centeredonly 1.9 mm apart in one folium in the same animal, therewas no overlap between their NL territories. For the ver-tically hatched areas, see the legend to Figure 10.

Fine-grain topographical organizationwithin the D2 zone in crus I

Olivo-cerebellar localization. In each of the ninecases with heavy involvement of dlPO, a “column” of cellswas labeled that was substantially longer than it was wideand in five cases the length was such that labeled cellswere present at all but one or two of the 10 rostrocaudallevels spanned by dlPO (excluding the ventrolateral out-

Fig. 9. Distributions of olivary and cerebellar nuclear labeling in two cases in which olivary labelingwas entirely restricted to dlPO. A,B: Cases 7G and 11CTB, respectively. Conventions and abbreviationsas in Figure 2.



Fig. 10. Total extent of the olivary and cerebellar nuclear territo-ries defined by pooling cases in which there was olivary labeling indlPO. The filled and vertically hatched areas together include allthose parts of dlPO and NL in which labeling was present. Filledterritories relate to those cases (n � 5; see filled squares in Fig. 4) inwhich olivary cell labeling was entirely restricted to dlPO. Verticalhatching shows the expansions of the territories that resulted fromincluding all cases with any label in dlPO. Note this involved sevenadditional cases but in dlPO the only significant expansions wereproduced by case 21R (at levels 9.5–10.0) and 11R (at levels 10.0–10.5

and 11.25); in NL significant expansions of terminal labeling camefrom cases 21R, 11R, 10G, and 21G. For the “additional” cases olivarycell labeling other than in dlPO and nuclear terminal labeling otherthan in the rostral half of NL is omitted. Note that the only cases inwhich terminations in the caudal half of NL were omitted were case10G (which had two separate fields, one in rostral, the other in caudalNL), cases 21R and 21G, in which multiple injections were made alongthe folial length, and case 26G, in which most olivary labeling was invlPO and all NL labeling was in the caudal half. Conventions andabbreviations as in Figure 2.



growth). With respect to any “folial” localization, the ter-ritories labeled from different folia overlapped veryheavily in the rostrocaudal plane, including the two terri-tories in experiment 21. Therefore, as in the case of vlPO,there was no evidence for representation of the differentfolia at different rostrocaudal levels in the lamella.

However, when case 12R in which the injection site wasconfined to folium 1 is compared with case 11R with a sitein folia 2 and 3, the former territory clearly lies medial tothe latter. Moreover, a similar relationship is evidentwhen case 12R is compared with the two cases in Figure 9,both with sites in folium 2. These findings are compatiblewith a topography in which medial to lateral in the folialchain is mapped medial to lateral in the olive subnucleus.However, among the cases involving folia 2, 3, and 4 thecolumns overlapped very heavily in the mediolateral di-mension (as might be expected given that the folium 2territories in Fig. 9 extended laterally as far as the junc-tion with vlPO) and those involving injections in two folia(11R, 10CTB) or three (10G) were little if any wider thanthose involving a single folium (7G, Fig. 9A; 11CTB, Fig.9B,G). In addition, in animal 21 the columns for folium 2and folium 4 were almost identically located. Therefore, itis possible for dlPO as for vlPO that a “folium 1-related”territory centered near the middle of the width of thelamella is flanked laterally by a common column for folia2, 3, and 4.

With regard to the width of the D2 zone, cases 11R (notshown) and 11CTB (Fig. 9B) both involved the same fo-lium (although 11R also involved an adjacent folium) andalthough the two sites partly overlapped, site 11CTB ex-tended substantially further caudally; in dlPO the two cellcolumns also partly overlapped but the CTB-labeled cellcolumn extended substantially further rostrally, indicat-ing a localization opposite to that for the D1 zone, i.e., onein which medial to lateral across the width of the zone aremapped rostral to caudal within dlPO. Such a topographywas also suggested by cases 1R and 8G.

Cortico-nuclear localization. With regard to the rep-resentation of different folia, it was clear that in thosecases involving folium 1 (12R) the termination fields in NLwere either centered more dorsally or extended furtherdorsally than in the cases involving more lateral folia. Inaddition, in experiment 21 (not shown) both rostrally andcaudally in NL the green termination fields (arising fromfolium 2) were at all mediolateral levels centered moredorsally than the red (arising from folium 4). We suggest,therefore, that for both D zones medial to lateral in thefolial chain is mapped dorsal to ventral in the ventral halfof NL, albeit in overlapping fashion.

A similar pattern to that described for the D1 zone for acortico-nuclear localization for the medial versus lateralparts of the zone emerged: in animal 11 the red injectionsite in folia 2 and 3 was centered further rostrally than theCTB site in folium 2 and, correspondingly, in the rostralpart of NL the red terminal field extended further later-ally than the CTB field.

In summary, for both D zones there was evidence fortwo patterns of cortico-nuclear localization in one of whichthe mediolateral sequence of folia was mapped (in over-lapping fashion) in the dorsal to ventral dimension withinthe ventral half of NL and in the other of which themediolateral dimension within each zone was mapped inthe medial to lateral dimension within NL.

DISCUSSION

Zones and compartments in crus I

The present study is the most detailed anatomical in-vestigation to date of the olivo-cerebellar and cortico-nuclear connections of crus I of the ansiform lobule in thecat. Evidence was obtained for the presence from rostral tocaudal within crus I of zones D2, D1, and C2, with a narrowC3 zone present in the medialmost folium (folium 1, seebelow). Each zone was found to receive its olivo-cerebellarinput respectively from dlPO, vlPO, rMAO, and rDAO,and provide cortico-nuclear output respectively to ros-troventral NL, caudoventral NL, ventral NIP, and NIA. Afine-grain topographical localization was also foundwithin the D2, D1, and C2 zones, both in terms of inter-and intrafolial patterns of olivo-cortical and cortico-nuclear projections. Our results therefore support andextend the conclusions of some previous studies, but differsignificantly from others.

In particular, the location defined in the present studyfor the C2 cortical zone agrees quite well with that pro-posed by Rosina and Provini (1982, see their fig. 12) butwe deduce that the zone is wider, occupying most of thecaudal half of each folium. Strikingly, the cortico-nucleartermination fields were entirely confined to NIP, in accordwith the concept (see, e.g., Voogd and Bigare, 1980) of a C2compartment related to rMAO and NIP. This is in agree-ment with more limited bidirectional tracer results wehave reported previously for the medialmost folium of crusI (Edge et al., 2003), and for portions of the anterior lobeand paramedian lobule electrophysiologically identified asparts of the C2 zone (Trott et al., 1998b). By contrast,tracer studies in the rat (Panto et al., 1998, 2001) haveindicated that the C2 zone in the paraflocculus and inlobulus simplex provides, in addition to its main projectionto NIP, a subsidiary projection terminating elsewhere inthe cerebellar nuclei (e.g., for lobulus simplex, in parts ofNL). There may be a species difference here because noevidence of such cortico-nuclear divergence was found inany of our cases. Alternatively, the labeling in multiplecerebellar nuclei described by Panto et al. could have beendue to collateral labeling of mossy fibers, as suggested byVoogd et al. (2003).

With regard to the possible presence of an A zone, wealso differed from Dietrichs and Walberg (1980) in neverfinding labeling in nucleus fastigius (nor in caudal MAO),even though some sites involved the caudal ends of folia 1and 2. Nor did we confirm the inclusion by Rosina andProvini (1982) of a narrow C1 zone caudal to C2. The onlycase with many labeled cells in rDAO was case 2R, and byanalogy with the anterior lobe this labeling might arisefrom either the C3 or the medial C1 zone (see, e.g., Trottand Apps, 1991). However, the injection site location sug-gested an origin in a narrow extension of the C3 zoneinterpolated in folium 1 between the C2 and D1 zones, aninterpretation consistent with other findings (Rosina andProvini, 1982; Edge et al., 2003). This identification is alsosupported by the fact that the olivary cell column ex-tended to the rostral pole of DAO and in the anterior lobethe lateral C3 is the only zone innervated from an rDAOterritory reaching so far rostrally. Case 2R also involvedabundant terminal labeling in NIA and, interestingly, thiswas located in a caudolateral region partly overlappingwith the most lateral part of the cortico-nuclear territorypreviously demonstrated for the C3 zone in lobule V of the



anterior lobe (see Trott et al., 1990, 1998a; see also Edge etal., 2003). Note that our failure to find a C1 zone occurreddespite the fact that we found appropriate rDAO labelingin an unpublished case with a site in nearby lobulussimplex. This lobule was included in the injection sites ofRosina and Provini (1982), so we suggest that the C1 zoneis present there (as in rat, cf. Pardoe and Apps, 2002) butdoes not continue into crus I.

Our cases included five with olivary labeling entirelyconfined to vlPO and two with label confined to vlPO plusrMAO. The locations of the relevant sites, which collec-tively involved all four folia of crus I, leave little doubt thata vlPO-related region lies rostral to the C2 zone in thesefolia (although with an rDAO-related region interpolatedin folium 1). An identification as the D1 zone seems justi-fied by its juxtaposition to the C2 zone and the finding thatits nuclear target is within NL (cf. Rosina and Provini,1982; Edge et al., 2003).

In fact, in all seven cases (see Table 2) in which 69% ormore of the labeled olive cells were in vlPO, when terminallabeling was present within NL it was always confined tothe caudal half of the nucleus at middle and/or ventraldorsoventral levels (cf. Fig. 8). The nuclear target of the D1zone in crus I is therefore the caudoventral quadrant ofNL across all its width except perhaps the most medialpart of the nucleus (levels 9–11).

There were five cases with olivary labeling confined todlPO plus two with over 90% in dlPO (see Table 2). In allthese cases, which collectively involved folia 1, 2, and 3,the cortico-nuclear termination fields were entirely re-stricted to NL. All the sites were very rostral in the foliaand were denoted as D2 sites in Results. Appreciable la-beling in dlPO was also generated in two “mixed” cases(1R, 21R), with sites located rostrally in folium 4, so it isclear that the D2 zone is well represented in all the foliaand, as the zonal hypothesis would predict, always in thesame (most rostral) parts.

Among the seven sites centered in the D2 zone, terminallabeling was confined to middle and ventral levels of therostral half of NL (cf. Fig. 10) except in case 10G, where,given that some labeled cells were present in vlPO, a smalladditional field in caudal NL was probably attributable tospread of the injection site into the D1 zone. Collectively,the sites gave terminal labeling at all 11 mediolaterallevels in NL and in three individual cases at seven or morelevels (maximum 9, in case 11R). We therefore concludethat the sole nuclear target of the D2 zone in crus I is therostroventral quadrant of NL across its entire width.There was no overlap between the D2 and D1 terminationfields, even in experiment 7, where a D2 and a D1 site werecentered only 1.9 mm apart along the length of a folium.Such differential projection of the two D zones is impor-tant support for the hypothesis that the olivo-cortico-nuclear compartments are discrete. Overall, these find-ings accord well with those of Dietrichs and Walberg(1980) and are in partial agreement with the deduction byVoogd and Bigare (1980), based on retrograde transport ofHRP, and on other evidence, that the cerebellar nuclearstages of the D1 and D2 compartments are respectively thecaudolateral and rostromedial parts of NL. The main dif-ference relates to the D2 zone in that the present studysuggests that in crus I the D2 projects to the whole me-diolateral extent of NL.

Extent of the olivary and cerebellar nuclearterritories of the crus I zones

It is clear from anterograde tracing studies of the olivo-cerebellar projection, from retrograde tracing in the Pur-kinje cells (e.g., Voogd and Bigare, 1980), and from elec-trophysiological studies (e.g., Jorntell et al., 2000) that thecerebellar cortical zones are present in “buried” parts ofthe folia as well as in the upper parts to which our injec-tion sites were confined. It is therefore certain that sub-stantial portions of each zone remained uninjected in thepresent study, and therefore possible that the C2, D1, andD2 zones may each receive climbing fibers from more oftheir corresponding olivary subnucleus and project tomore of their corresponding cerebellar nucleus than isindicated by Figures 5, 8, and 10.

However, there is evidence to suggest that the extent ofany underestimations of the crus I olivary territories isunlikely to be large. This involves comparison with thestudies by Rosina and Provini (1982) and Kotchabhakdi etal. (1978), most of whose injection sites were large, withheavy involvement of buried areas of cortex. If the illus-trated cases of these authors are pooled, the resultantterritory within rMAO appears to lie entirely within thatshown in Figure 5—indeed, the match as to size andlocation is remarkable. For vlPO and dlPO the correspond-ing territories are again very similar to ours in Figures 8and 10, although in some of their cases labeling extendedto the medial borders of the lamellae. However, this maybe attributable, except in one case (case 789R of Kotchab-hakdi et al., 1978), to the involvement of lobulus simplexin the injection sites.

For the cerebellar nuclear territories in Figures 5, 8,and 10 our findings are less easily compared with those ofDietrichs and Walberg (1980) because of presentationaldifferences, but those authors appear to have found, likeus, that crus I injections labeled substantially less thanthe whole of NIP and NL. On present evidence, therefore,it appears very probable that the “overall” territories inFigures 5, 8, and 10 come close to defining the position andextent of those for the whole of crus I.

Fine-grain localizations within crus I

At their longest, the folia in the paravermal part of thecat anterior lobe are typically �7–8 mm long and withinthis span at least six zones are recognized (from medial tolateral, the C1, C2, C3, D1, Y, and D2 zones). By compari-son, the crus I folia in the same species are as long orlonger and most accommodate only a C2, a D1, and a D2zone. These three zones, and particularly C2, are thereforesignificantly wider than in the anterior lobe. This, to-gether with the availability of different tracers useable inparallel in the same animal, and our production of numer-ous small injection sites, has allowed us to make observa-tions indicative, in both projections studied and in allthree zones, of two orthogonally intersecting topographi-cal localizations.

Collectively, our findings suggest the hypothesis sum-marized in Figure 11, in which, for each zone, the unbro-ken arrows represent a pattern in which folia more medialin crus I (and therefore more rostral in the folial chain) arerepresented more medially (or in the case of rMAO morecaudomedially) in the corresponding olivary subnucleusand more dorsally in the corresponding cerebellar nuclearterritory. A folial organization within the olivocerebellar



projections to the C2, D1, and D2 zones in the anterior lobeand the paramedian lobule have also been reported in catsby Brodal and Kawamura (1980). However, the folial pat-tern we determined for crus I is also envisaged as coexist-ing with another, shown by the broken arrows, in whichprogressively more rostral parts of each zone are repre-sented progressively more laterally in the correspondingcerebellar nuclear territory and for the D2, D1, and C2zones, respectively, more caudally in dlPO, more rostrallyin vlPO, and more rostromedially in rMAO. As alreadynoted, rostral and caudal in the folium can, given theorder of the zones and the sagittal orientation of the folia,be equated respectively with lateral and medial in thezone.

It must be emphasized that the weight of evidence givenin the Results is greater for some parts of the hypothesisthan for others. For both the “zone-width” and folial pat-tern, the evidence was strongest for the C2 zone. For the Dzones, the most important observations were that sitesconfined to a single folium frequently labeled a “column” ofolive cells extending throughout much of the rostrocaudalextent of the corresponding PO lamella. Also, that sitesinvolving the most medial folium labeled olivary and NLterritories that extended respectively further mediallyand further dorsally than those for more lateral folia.

The localizations in Figure 11 have important implica-tions (see below) for the concept that cortical microzonesand olivo-cortico-nuclear microcomplexes are fundamen-tal functional units of the cerebellum, and close agreementto our summary diagram can be obtained from the olivo-cerebellar findings of Rosina and Provini (1982), Kotchab-hakdi et al. (1978), and Edge et al. (2003). With regard tothe cortico-nuclear projections from crus I, less informa-tion is available in the cat, but data broadly consistentwith Figure 11 can be found in the studies by Dietrichsand Walberg (1980) and Edge et al. (2003).

Microzones and microcomplexes?

In the present experiments no electrophysiological re-cordings were made of any responses mediated via theclimbing fibers and we were obviously therefore unable toobtain direct evidence in any of the zones for corticalmicrozones and olivo-cortico-nuclear microcomplexes ofthe kind that have been defined in detail within the ante-rior lobe (e.g., Andersson and Oscarsson, 1978a,b; Ekerotet al., 1991a,b; Garwicz et al., 1992; Garwicz and Ekerot,1994) and often thought to be a feature present through-out the cerebellum (e.g., Ito, 1984, 2000). However, anolivo-cerebellar topography in which different parts of thewidth of a cortical zone are innervated from differentgroups of olivary neurons is an essential prerequisite fortheir existence, and to that extent our findings stronglysupport the microzonal hypothesis. If such units existwithin crus I, their olivary territories should be parallel tothe unbroken arrows in Figure 11, and it is obvious thatthis relationship with the heavily overlapping “folial” rep-resentation would be well suited to generating microzonesextending across several folia in the manner well-documented for the anterior lobe. Note also that the ter-ritories in rMAO and PO occupied by cell labeling arisingfrom injections into the ansiform lobule overlap with thosedescribed for other “lobular” regions of the cerebellum.This facilitates the existence of microzones extendingacross more than one lobule, like, for example, those that

are represented in both the anterior lobe and the parame-dian lobule (see Apps, 2000).

With regard to microcomplexes, we again have no directevidence, but projection of different parts of the width of azone to different parts of the zone’s cerebellar nuclearterritory is a necessary precondition and the orthogonalintersection in NIP and NL of this (mediolateral) localiza-tion with the (dorsoventral) “folial” and “lobular” localiza-tions is strikingly parallel in principle to the arrangementin the olivary subnuclei.

Branching of olivo-cerebellar axons

There is abundant evidence in the cat (see, e.g., Faberand Murphy, 1969; Armstrong et al., 1973a–c; Brodal etal., 1980; Ekerot and Larson, 1982; Rosina and Provini,1983, 1987; Apps et al., 1991; Apps, 2000; Sugihara et al.,1999, 2001) that olivo-cerebellar axons branch extensivelyand it is probable in cats that each gives rise, on average,to around 10 climbing fibers. Branching is most frequentin the parasagittal plane so as to link different rostrocau-dal levels of the same zone. Purkinje cells innervated bythe same olive cell must belong to the same microzone ormultizonal microcomplex (cf. Apps and Garwicz, 2005) sothis form of branching is, at the least, consistent with theexistence of microzones and may help create them (andmay help determine their rostrocaudal extent).

In the present study there were two animals (19, 20) inwhich fluorescent tracers were injected into the C2 zone intwo different crus I folia and a third (21) in which the C2,D1, and D2 zones were all injected in two folia. These casesoffered the opportunity of detecting interfolial branchingof the olivo-cerebellar axons via double-labeling of theparent olive cell bodies. In all three animals some double-labeled cells were present in rMAO, but their numberswere relatively small (respectively 5, 3, and 8% of the cellslabeled from the less effective site). In animal 21, double-labeled cells were also present in vlPO and dlPO, again inrelatively small numbers (1.3 and 6%). It seems, therefore,that “longitudinal” branching occurs, but that only a lim-ited number of the cells innervating the crest of one foliumalso provide an axonal branch to the crest of the next butone folium. This does not, of course, preclude branches toother folia either within crus I or in other more distantcerebellar regions.

A different approach to investigating branching of theolivo-cerebellar axons has recently been taken by Sugi-hara et al. (1999, 2001), who reconstructed the entiretrajectories of individual axons labeled as a result of in-traolivary microinjections of biotinylated dextran amine.Although these studies were carried out in the rat andmostly involved regions other than crus I, several injec-tions were made into rMAO and dlPO that labeled axonsproviding climbing fibers to crus I folia. In all cerebellarregions studied, including crus I, all branches of eachparent axon were confined to a single very narrow (0.2–0.3mm) longitudinal band of cortex, suggesting that axonalbranching is indeed an important contributor to the for-mation of microzones.

If the results of this elegant investigation can be extrap-olated (at least in their fundamentals) to the cat, then theyharmonize well with those of the present study. Togetherthe two studies indicate that microzones do exist in crus I,that some extend into other lobules, and that they resultfrom a combination of axonal branching patterns of thekind demonstrated by Sugihara et al. (2001) with the



Fig. 11. Summary diagram of the patterns of localization deducedfor the olivo-cerebellar and cortico-nuclear projections of crus I. Solidand broken arrows, respectively, represent the “folial” and “zone-width” patterns referred to in the text; note the orthogonal intersec-tions between the two. Note also that in both the inferior olive andcerebellar nuclei the folial localizations appear to involve heavy over-laps between the representations (“territories”) of successive folia; invlPO and dlPO the territorial overlaps for folia 2, 3, and 4 may becomplete or nearly so (see text). Enclosed areas in rMAO and NIP,

vlPO and caudal NL, dlPO and rostral NL are the overall crus Iterritories shown in Figures 5, 8, and 10, respectively, inclusive ofadditions from “mixed” cases (see text); alternate levels in NL areomitted for clarity. Note that the territories occupied by cell or termi-nal labeling are not exclusively related to crus I; other studies implyvery substantial overlaps with territories for other “lobular” regions ofthe cerebellar cortex (see text). Conventions and abbreviations as inFigure 2.

zone-width and the heavily overlapping “folial” and “lob-ular” topographies evidenced by our findings. Neverthe-less, the point made by Sugihara et al. (2001) that muchremains to be elucidated remains true. For example, nei-ther study permits precise specification of the dimensionsof the olivary equivalent of a microzone, although wesuggest that (at least in rMAO, vlPO, and dlPO) the unitinvolved is a localized population of cells distributed as amicrocolumn, with its long axis orientated parallel to theunbroken arrows in Figure 11 (cf. Apps, 1990). Dependingon the length of such a microcolumn, the degree of disper-sion of the axonal branches of the cells involved, and thelocation of the microcolumn relative to the folial and lob-ular topographies, microzones with a range of lengths anda variety of positions relative to the length of the wholezone could readily arise. However, the degree of variabil-ity actually present, the precise functions of the microzo-nal units, and the developmental mechanisms responsiblefor the evident intricacies of the adult pattern all remainto be investigated.

Comparative implications

The lateral cerebellum reaches its largest size and com-plexity in primates and man. Although the precise zonalorganization of olivo-cortico-nuclear projections remainscontroversial, there are strong indications that projectionzones corresponding to the D1 and D2 zones also exist inthe human cerebellum (Voogd, 2003, 2004). The cortico-nuclear targets of these zones in the dentate nucleus,namely, its caudoventral (for D1 zone) and rostromedial(for D2 zone) subdivisions are likely to be very similar tothose reported in the present study in the cat.

The cerebral cortical connections of the dentate nucleusand afferents to the principal olive have been studied inprimates. Caudoventral dentate includes projections, viathe thalamus, to regions of the prefrontal cortex, whereasrostromedial dentate includes projections to motor andpremotor areas of the cerebrum (Hoover and Strick, 1999;Middleton and Strick, 2000). Moreover, the ventral la-mella of the principal olive receives afferents, via theparvocellular red nucleus, from the prefrontal cortex,whereas the dorsal lamella receives input from motor andpremotor cortex (see Voogd, 2003, for further details).Recent interest in the cerebellum has focused on the pos-sibility that it may be involved in nonmotor activities,including cognition (e.g., Leiner et al., 1986; Schmah-mann, 1991). Given the key role of the prefrontal cortex inhigher-order operations the contribution of the cerebellumin such processes could be linked (in a range of species) tothe ventral lamella of the principal olive, the caudoventraldentate, and the D1 zone.

ACKNOWLEDGMENT

We thank Ms. R. Bissett and Ms. C. Everard for experttechnical assistance.

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olivo-cortico-nuclear localizations within crus i of the cerebellum

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