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

Projection of Reconstructed Single Purkinje Cell Axons in Relation to the Cortical and Nuclear Aldolase C Compartments of the Rat Cerebellum IZUMI SUGIHARA,1* HIROFUMI FUJITA,1 JIE NA,1,2 PHAM NGUYEN QUY,1 BING-YANG LI,2 1 AND DAISUKE IKEDA 1 Department of Systems Neurophysiology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 2 Laboratory of Brain and Cognitive Science, Shenyang Normal University, Shenyang 110034, China

ABSTRACT Although the overall topography of the cerebellar corticonuclear projection formed by Purkinje cell (PC) axons has been described, only a few studies have dealt with the organization of this projection at the level of individual PC axons. Thus, we reconstructed 65 single PC axons that were labeled with biotinylated dextran amine in the rat. We then analyzed the relationship between the projections of these PCs and the compartmentalization of the cerebellar cortex and nuclei based on the topography of olivocerebellar projection and aldolase C expression in PCs. After giving rise to short local recurrent collaterals near the soma, a PC axon formed a terminal arbor in a specific small area in the cerebellar nuclei (CN). The terminal arbors of vermal PCs were spread more widely than those of hemispheric PCs and sometimes 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 projected to the caudoventral and rostrodorsal parts of the CN, respectively, precisely in accordance with the compartmentalization of the cortex and nuclei. Mediolateral segregation and rostrocaudal convergence were seen between projections of separate PCs in a single aldolase C compartment. The results revealed a tight link between the projection patterns of individual PC axons, the topography of the olivocerebellar pathway, and the aldolase C expression pattern. Their overall correspondence seems to reflect a basic aspect of cerebellar organization, although some areadependent variation in the relationship of these three entities was also present. J. Comp. Neurol. 512:282–304, 2009. © 2008 Wiley-Liss, Inc.

Indexing terms: cerebellar cortex; cerebellar nucleus; vestibular nucleus; biotinylated dextran amine; zebrin; topography

The organization of the cerebellar system is characterized by the topography and compartmentalization of the connections of its input and output fibers. Among these fibers, the Purkinje cell (PC) axons constitute the sole output of the cerebellar cortex and the major input to the cerebellar nuclei (CN) to connect these two essential cerebellar structures, and thus significantly contribute to the organization of the cerebellar system (Brodal, 1981; Ito, 1984; Voogd, 2004). The cerebellar cortex has been longitudinally subdivided according to the arrangements of projecting axons and acetylcholinesterase activity (Voogd, 1967; Voogd and Bigare´, 1980). These subdivisions have been reflected or followed by the topographic projection patterns of olivocerebellar climbing fibers and PC axons. PCs in each subdivision (designated zones A, B, C1-3, and D0-2) are innervated by neurons in distinct subnuclei of the inferior olive and project to different areas of the CN (Groenewegen and Voogd, 1977; Azizi and Woodward, 1987; Buisseret-Delmas and Angaut, 1993).

© 2008 Wiley-Liss, Inc.

Much finer longitudinal compartmentalization of the cerebellar cortex has recently been evidenced by longitudinal stripe-shaped expression patterns of specific molecules, such as aldolase C (ⴝzebrin 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 relatively brain-specific isozyme of fructose-1,6-(bis)phosphate aldolases, which is involved in glycolysis (Mukai et al., 1991).

Grant sponsor: Japan Society for the Promotion of Science; Grant number: Grant-in-Aid for Scientific Research 20300137. *Correspondence to: Dr. Izumi Sugihara, Dept. of Systems Neurophysiology, Tokyo Medical and Dental University Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: isugihara.phy1@tmd.ac.jp Received 26 December 2007; Revised 18 June 2008; Accepted 23 September 2008 DOI 10.1002/cne.21889 Published online in Wiley InterScience (www.interscience.wiley.com).


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About 20 longitudinal compartments, in which PCs show positive, negative, or lightly positive expression of aldolase C, have been defined in the rat cerebellar cortex. A specific name such as 1ⴙ, 1ⴚ and so on (usually a numeral and/or a letter followed by a sign indicating positive or negative) has been given to each compartment. Tracing studies have identified specific olivocerebellar projection to each compartment and clarified the correspondence between the aldolase C compartments and zones A-D (Voogd et al., 2003; Sugihara and Shinoda, 2004; Sugihara and Quy, 2007). For example, aldolase C compartments 1ⴙ, 1ⴚ, 2ⴙ, 2ⴚ, 3ⴙ, 3ⴚ, and 4ⴙ belong to zone A in the caudal cerebellum. Furthermore, it has been suggested that aldolase C-negative and -positive areas mainly receive somatosensory and other (cerebral, tectal, vestibular, and visual) inputs, respectively, through the areaspecific inputs to the inferior olive and the topographic olivocerebellar projection (Sugihara and Shinoda, 2004). Thus, aldolase C compartments may represent fundamental functional organization of the cerebellar cortex. Recently, fine compartmentalization in the CN has also been demonstrated by tracing nuclear collaterals of olivocerebellar axons (Sugihara and Shinoda, 2007). According to this study, the CNs are primarily divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts, in contrast to the conventional mediolateral nuclear subdivisions. The aldolase C-positive and -negative areas in the CN are then further subdivided into multiple compartments based on the topographic olivonuclear projection. This aldolase-C compartmentalization within the CN has no trace of the longitudinal stripes that characterized cortical compartmentalization. These fine but different compartmentalizations in the cerebellar cortex and nuclei based on the olivocerebellar projection raise the question of whether PC projection is organized according to the same compartmentalizations. To answer this question, understanding the morphology of the axonal arbor

of single PCs is essential. Although the labeling of single PC axons has been reported in the paramedian lobule and anterior lobe (cat, Bishop et al., 1979), flocculus (rabbit, de Zeeuw et 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 patterns of PC axons have not been systematically studied in relation to cerebellar compartmentalization. Therefore, we sought to trace the entire trajectories of single PC axons to investigate the basic and general structural organization of their projection in relation to cortical and nuclear compartmentalizations by using biotinylated dextran amine as a tracer.

MATERIALS AND METHODS Tracer injection and histological procedure Reconstruction of PC axons was performed in 24 Long– Evans adult rats (Kiwa Laboratory Animals, Wakayama, Japan). All of the experimental animals in this study were treated according to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (2001 and 2002 editions). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (numbers 0040089, 0050041, 0060121, and 0070133). The anesthesia, surgical, and histological procedures were similar to those described previously (Sugihara et al., 1999, 2001). Briefly, the animals were anesthetized with an intraperitoneal injection of ketamine (130 mg/kg body weight) and xylazine (8 mg/kg). Atropine (0.4 mg/kg) was also given intraperitoneally. Supplemental doses of ketamine (13 mg/kg) and xylazine (1 mg/kg) were given every 30 minutes starting 1 hour after the initial dose, as required. Some animals were anesthetized with an intraperitoneal injection of pentobarbital so-

Abbreviations 4V I-X a-d AICG AIN BDA C cMAO CN Cop CP CrI CrII D d-Y das DC DCoN dDAO DLH DLP DM DMC DMCC DN DPFL dPO FL

Fourth ventricle Lobules I-X Sublobules a-d Anterior interstitial cell group Anterior interposed nucleus Biotinylated dextran amine Caudal Caudal part of the medial accessory olive Cerebellar nuclei Copula pyramidis Caudal pole crus I of ansiform lobule crus II of ansiform lobule Dorsal Dorsal Y nucleus Dorsal acoustic stria Dorsal cap of Kooy Dorsal cochlear nucleus Dorsal fold of the dorsal accessory olive Dorsolateral hump (of the AIN) Dorsolateral protuberance (of the FN) Dorsomedial group subnucleus Dorsomedial crest (of the AIN) Dorsomedial cell column subnucleus Dentate (lateral) nucleus Dorsomedial paraflocculus Dorsal lamella of the principal olive Flocculus

FN ICG icp IVN L LVN M MVN n7 Par PIN PBN PC pf R rMAO scp Sim sp5 SVN V, vvDAO VLO VPFL vPO X Y

Fastigial (medial) nucleus Interstitial cell group Inferior cerebellar peduncle Inferior vestibular nucleus Lateral Lateral vestibular nucleus Medial Medial vestibular nucleus Facial nerve Paramedian lobule Posterior interposed nucleus Parabrachial nucleus Purkinje cell Primary fissure Rostral Rostral part of the medial accessory olive Superior cerebellar peduncle Simple lobule Spinal trigeminal tract Superior vestibular nucleus Ventral Dorsal fold of the dorsal accessory olive Ventrolateral outgrowth Ventral paraflocculus Ventral lamella of the principal olive Nucleus X Nucleus Y


The Journal of Comparative Neurology 284 dium (90 mg/kg body weight). Atropine (0.4 mg/kg) was also given intraperitoneally. Supplemental doses of pentobarbital sodium (20 mg/kg) were given every 40 minutes starting 1 hour after the initial dose, as required. Biotinylated dextran amine (BDA, D-1956, 10,000 MW or D-7135, 3,000 MW; Molecular Probes, Eugene, OR; 10% solution in saline) was pressure-injected with a Picopump PV820 (WPI, Sarasota, FL) in the molecular layer at various locations in the cerebellar cortex (putative volume, 1–5 nL) for anterograde labeling. Spontaneous complex spike activities were recorded to locate the molecular layer through the injection pipette by using a 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 temporarily inactivated (Fig. 1A). Multiple injections (up to six) were made in separate locations throughout the cortex in each rat since it was not difficult to distinguish axons from different origins by following them. Some injections labeled one to several axons intensely. Other injections labeled no PC axons intensely or too many PC axons to trace individual ones. After a survival period of 6 – 8 days the rats were anesthetized as in the first operation but with a 1.5-times larger dose of anesthetics. They were perfused intracardially with phosphatebuffered saline (PBS) followed by fixative containing 5% paraformaldehyde, 2% sucrose, and phosphate buffer 50 mM (pH 7.4). Eighty-␮m-thick serial frozen sections were cut coronally from the cerebellum and medulla. BDA was visualized in black with an Elite ABC kit (PK6100, Vector Laboratories, Burlingame, CA). In most cases, aldolase C was then immunostained in brown. The immunohistological procedures that were used to double-label BDA (black reaction product) and aldolase C (brown reaction product) with diaminobenzidine have been described previously (Sugihara and Shinoda, 2004). The anti-aldolase C antibody used in this study was raised in our laboratory by immunizing a rabbit with a synthetic peptide that represented amino acids 322–344 from rat aldolase C (Sugihara and Shinoda, 2004). This antibody stains a single band on Western blot with rat cerebellar tissue and the addition of the immunizing peptide to the primary antibody solution abolishes immunostaining (Sugihara and Shinoda, 2004). Some sections were counterstained with thionine after they were mounted on glass slides. In three Long–Evans adult rats, BDA was injected in the fastigial (medial) nucleus (FN) by a procedure similar to that described above (diameter 0.2– 0.3 mm, volume 4 –13 nL) to label PCs retrogradely. The brains of these rats were cut parasagittally and treated for BDA visualization. Otherwise, the procedures for these brains were the same as those described above.

I. SUGIHARA ET AL. In seven Long–Evans adult rats, fluorescent (red and/or green) latex microspheres (Lumafluor, Naples, FL) were injected into the CN by a procedure similar to that described above (diameter 0.2– 0.3 mm, volume 4 –13 nL) to label PCs retrogradely. The surgery, survival, and histological procedures for these rats were the same as those for other rats. The brains of these rats were cut coronally and immunostained for aldolase C with diaminobenzidine. The sections were mounted on glass slides and photographed after being coverslipped using PBS. One Long–Evans adult rat was used for immunostaining of aldolase C in the cerebellar cortex to label recurrent collaterals of PC axons. This rat was anesthetized, perfused, and fixed in the same way as described above. Serial frozen sections of the cerebellum were cut coronally. The immunohistological procedures used to label aldolase C black with diaminobenzidine have been described previously (Sugihara and Shinoda, 2004).

Reconstruction of individual axons and photomicroscopic procedure Axonal trajectories of single-labeled olivocerebellar axons were reconstructed from serial coronal sections using a threedimensional imaging microscope (Edge R400; SNT Microscopes, Los Angeles, CA) equipped with a camera lucida apparatus. Cut ends of an axon on one section were connected properly to the corresponding cut ends of the same axon on the successive section (Shinoda et al., 1981; Sugihara et al., 1999; Wu et al., 1999). Only axons that were well labeled, isolated from other axons, and could be traced from the injection site to every end were considered to be completely reconstructed, whereas axons that could not be traced at any point on their pathway due to poor labeling or intermingling with other axons were regarded as “not fully reconstructed.” Reconstructions in the sectioning plane (coronal) were sometimes converted to those in another plane (parasagittal). In drawings of single-axon images, fibers and swellings were drawn thicker than scale for clarity, as is conventionally done in drawings of reconstructed fibers. Single-axon images in the present figures are depicted on a montage of drawings of sections for the BDA injection site and the center of the terminal arbor. These drawings contained contours of cerebellar structures and boundaries between areas with different aldolase C labeling intensities. The cerebellar lobules were defined according to Larsell (1952) and Voogd (2004). Aldolase C compartments in the cerebellar cortex were defined according to Sugihara and Shinoda (2004), who basically adopted 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, and its 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 1ⴚ in lobule IXc (B) and labeled PC axons running in the granular layer in the same compartment (1ⴚ) (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 labeled fibers and swellings in this panel belong to a single PC axon. Montage of five photographs with different focus depths. Arrowhead indicates the cut end 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 other figures, the shaded areas in the molecular layer of the cerebellar cortex indicate the aldolase C-positive compartments and the shaded areas in the CN indicate the aldolase C-positive caudoventral part. The terminal arbor of this axon belonged to the wide type. H: A compact-type terminal arbor of a reconstructed PC axon. Arrowhead indicates a collateral that extended rostrally by about 200 ␮m from the main terminal arbor. I: An elongated-type terminal arbor of another reconstructed PC axon. Filled circles in H and I indicate the stem axon. Scale bars ⴝ 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).


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Figure 1


The Journal of Comparative Neurology 286 was usually indicated by a numeral plus a sign, such as 5ⴙ. To indicate linked compartments located in the rostral and caudal cerebellum, their names were connected with a “//,” as in “4ⴙ//5ⴙ.” While we traced axons in the cerebellar nuclei, we also traced the contour of the CN and boundaries between aldolase C-positive and -negative areas in the CN (Sugihara and 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 LE and Illustrator software (Adobe, San Jose, CA). The software was used to adjust contrast and brightness, but no digital enhancements were applied. Sections of rats that had been injected with fluorescent latex microspheres were photographed with PBS and photographed with a fluorescent microscope and a color digital camera (BX51WI and DP-70, Olympus) with 4ⴛ, 10ⴛ, or 20ⴛ objectives. Weak brightfield illumination was applied to visualize the contour of the brain structures and aldolase C compartments 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 the right side of a coronal section were flipped about the vertical axis.

RESULTS Morphology of entire PC axons that were labeled anterogradely The injection of BDA created a darkly labeled spot in the molecular layer, which typically measured 0.04 – 0.10 mm in the transverse direction and about 0.08 – 0.20 mm in the longitudinal direction (Fig. 1B). A few or several PC axons were labeled by such an injection (Fig. 1C). We picked up one or more well-labeled axons in the folial white matter and started tracing toward the proximal and distal directions. We completely reconstructed 65 PC axons that were labeled in 43 injections in 24 rats in the present study. A reconstructed axon could always be traced back to the injection site. The origin of the axon from a PC soma (Fig. 1D) was visible unless the PC was not completely covered by the dark labeling of the tracer injection spot (23 of 65 axons). An axon usually gave rise to one or two recurrent collaterals near the PC soma (see below). A PC axon then descended through the folial white matter without any branching until it was close to the CN. A stem axon usually branched into two or occasionally three relatively thick (diameter ⬇0.8 ␮m) primary branches shortly before or after it entered the CN (Fig. 1G–I). These primary branches ran roughly parallel to each other in the same region of the CN for about 0.5–1 mm. Several thinner secondary branches (diameter ⬇0.5 ␮m) were given off from the primary branches or from the stem axon. Abundant short tertiary branches or branchlets (diameter ⬇0.3 ␮m) were given off from secondary branches. Many en-passant and terminal swellings (diameter: ⬇2–3 ␮m) were located on tertiary branches (Fig. 1F). The number of branchings ranged from 27 to 155 (mean and SD, 45.8 ⴞ 20.1, n ⴝ 65) per axon. The number of swellings ranged from 63 to 404 (121.9 ⴞ 53.4) per axon. Occasionally a short fine collateral (length, 1–2 ␮m) with a small satellite swelling (diameter, 1–1.5 ␮m) at the end extended from en-passant and terminal swellings. Overall, the

I. SUGIHARA ET AL. terminal arbor of a single axon resembled loose roots of a plant. Swellings of an axon were scattered along the entire length of the terminal arbor (Fig. 1F–I). However, some of the swellings of a single axon sometimes strategically surrounded the 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 aldolase C-positive (133.0 ⴞ 61.9, mean and SD, n ⴝ 38) was slightly greater than that of aldolase C-negative PCs (106.8 ⴞ 29.5, n ⴝ 27), and this difference was statistically significant in a single-factor analysis of variance (ANOVA) analysis (F ⴝ 4.20, d.f.(between) ⴝ 1, d.f.(residual) ⴝ 63, P ⴝ 0.045).

Variation in terminal arbor morphology A remarkable difference was observed in the shape and size of PC terminal arbors in the CN. Therefore, in the present study the terminal arbors in the CN were tentatively classified into elongated, wide, and compact types, although a few terminal arbors showed conformations intermediate between these types. This variation in the terminal arbor conformation was related to the location of the arbor within the CN. First, terminal arbors in most of the FN, except for those in the ventral FN and the dorsolateral protuberance (DLP) of the FN, were usually widely spread in multiple directions by more than 0.8 mm (wide type, Fig. 1G). Second, terminal arbors in the DLP, in the anterior interposed nucleus (AIN) and in most of the posterior interposed nucleus (PIN) and the dentate (lateral) nucleus (DN), except for those in their ventral regions, were relatively compact in size (less than 0.8 mm in spread) and dense with regard to swelling disposition (compact type, Fig. 1H). The overall conformation of compact-type terminal arbors showed some elongation in a direction that seemed equivalent to the direction of the hilus of the nuclei. Occasionally, one or a few thin collaterals extended for a long distance in this direction apart from the main compact part of the terminal arbor (Fig. 1H, arrowhead). Finally, terminal arbors in the ventral parts of the DN, PIN, and FN were often elongated in 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 extracerebellar targets (n ⴝ 6 axons, see below; also refer to floccular PCs in fig. 10 of Sugihara et al., 2004) would be classified as elongated-type or wide-type. The number of swellings in wide, compact, and elongated terminal arbors in the CN, 130.1 ⴞ 85.5 (mean and SD, n ⴝ 20), 117.1 ⴞ 30.0 (n ⴝ 31), and 114.5 ⴞ 26.0 (n ⴝ 8), respectively, were similar, and a single-factor ANOVA analysis found no statistically significant difference (F ⴝ 0.429, d.f.(between) ⴝ 2, d.f.(residual) ⴝ 56, P ⴝ 0.653).

Aldolase C compartment-specific PC projection (Groups I–IV) The cerebellar cortex is divided into about 20 longitudinal stripes that are alternately aldolase C-positive (or -lightly positive) and -negative (Hawkes and Leclerc, 1987; Sugihara and Shinoda, 2004; Sugihara and Quy, 2007). Previous studies have identified the pattern of olivocerebellar climbing fiber projection to each aldolase C stripe in the cortex (Voogd et al., 2003; Sugihara and Shinoda, 2004). On the other hand, the CN is subdivided into rostrodorsal aldolase C-negative and cau-


The Journal of Comparative Neurology SINGLE PURKINJE CELL AXONS doventral aldolase C-positive parts (Sugihara and Shinoda, 2007). This aldolase C subdivision is partially compatible with the conventional subdivision of the CN into the FN, AIN, PIN, and DN. However, the topography of the collateral projection of olivocerebellar axons to the CN has indicated a fine compartmentalization in each of the aldolase C-positive and -negative parts of the CN (Sugihara and Shinoda, 2007), which corresponds nearly completely to the cortical fine compartmentalization based on aldolase C stripes. To better understand the cortical compartmental organization and relate it to functional aspects, we have proposed that aldolase C-positive and -negative compartments can be classified into three (Groups I, II, and V) and two (Groups III and IV) groups, respectively (Fig. 2A; Sugihara and Shinoda, 2004, 2007). The lightly positive compartments and several negative compartments have been considered to be akin to each other, and have been included together in Group IV, since they both receive olivary projection from the same subnuclei. The fivegroup scheme can also be applied to the compartmentalization of the CN (Fig. 2B,C; Sugihara and Shinoda, 2007). Areas in the cortex and CN that belong to the same group are innervated by the same population of inferior olive neurons. Aldolase C is expressed in PCs, including their axonal terminals, but not in nuclear neurons (Sugihara and Shinoda, 2007). Therefore, the subdivision of the CN into rostrodorsal aldolase C-negative and caudoventral aldolase C-positive parts indicates that Purkinje cells in aldolase C-positive areas (Groups I, II, and V) in the cortex do not project much to aldolase C-negative areas (Groups III and IV) in the CN. However, much detail remains unclear regarding the corticonuclear PC projection. Therefore, we examined whether the topography of PC projection is also in accordance with the five-group scheme by analyzing axonal projections of PCs that belonged to Groups I to IV. Group V, flocculus and nodulus, was not considered in the present study. Cortical Group I has been defined as aldolase C-positive compartments that extend rostrally to the anterior lobe beyond the primary fissure (green areas in Fig. 2A). They correspond to zones C2, D1, and D2 and some areas in zone A, and are innervated by the ventrolateral parts of subdivisions of the inferior olive (Table 1), which mainly receive midbrain inputs (Sugihara and Shinoda, 2004). Axons of seven PCs that belong to Group I are shown in Figure 3. They projected to the

Figure 2. Five-group compartmentalization of the cerebellar cortex and nuclei based on the topography of the olivocortical and olivonuclear projections and aldolase C immunostaining. A: Unfolded scheme of the left cerebellar cortex. B,C: Three-dimensional scheme of the most ventral portions of the left CN (B) and the entire left CN (C) in the dorsocaudal view. Compartments that belong to each group are indicated by different colors: Group I (aldolase C-positive), green; Group II (positive), 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 are innervated by the same subareas of the inferior olive (see fig. 10 of Sugihara and Shinoda, 2007). Arrows represent a fine topographic correspondence within each group or subgroup, i.e., areas in the cortex and nuclei that are roughly overlaid by the same portion of the arrows (base to tip) receive divergent projection from a subarea of the inferior olive. Each arrow is colored in the same hue as the (sub)group to which it is related. These schemes are derived from figure 10A,C of Sugihara and Shinoda (2007) with slight modification.

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


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

1

Aldolase C compartment as major termination area (Sugihara and Shinoda, 2004). See Results for details. Voogd and Bigare´, 1980; Buisseret-Delmas and Angaut, 1993; Voogd et al., 2003; Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2004. 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 subarea that 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 ďŹ gure does not fully agree with the CN subarea listed in the same line in the table, which was obtained in the previous study of olivonuclear projection (Sugihara and Shinoda, 2007). 7 Speculated based on the results of the present study (Fig. 8D). 8 Sugihara et al., 2004. 2 3


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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 the drawings of the molecular layer and the cerebellar nuclei, the aldolase C-positive parts are indicated by shading. In the drawings of the cerebellar 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 and Shinoda, 2007). Origin, destination, and type of the terminal arbor of each axon: 1ⴙ in lobule II, ventromedial FN, elongated (A); medial 2ⴙ in lobule VIa, ventrolateral FN, wide (B); lateral 3ⴙ in lobule VIII, ventral ICG to caudal PIN, compact (C); 5ⴙ in crus IIa, central PIN, compact (D); 5ⴙ in copula pyramidis, caudal PIN, compact (E); 5ⴙ or 6ⴙ 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 circles indicate other Group I injections in which PC axons were reconstructed in this study. Scale bar ⴝ 500 ␮m.


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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 this study (open circles) were prepared in the same format as in Figure 3. Origin, destination, and type of the terminal arbor of each axon: aⴙ in lobule VIb, midcaudal FN, compact (A); medial 2ⴙ in lobule IXa, ventrocaudal FN, wide (B); lateral 2ⴙ in lobule VII, midcaudal FN, wide (C); lateral 2ⴙ in lobule IXc, ventrolateral FN, wide (D); 4ⴙ in lobule IXc, ventral PIN, elongated (E); 5aⴙ in crus IIb, caudal neck of the DLP, compact (F); cⴙ in crus Ia, central FN, compact (G). The drawing of the section containing the FN was shifted upward in A. Scale bar ⴝ 500 ␮m.

crus Ib or the dorsal paraflocculus, it was not straightforward to determine the compartments for the injection sites in these cases. The target areas of these axons generally coincided with the Group I areas in the CN (green areas in Fig. 2B,C). Furthermore, the topography was rather straightforward, at least in the mediolateral directions; PCs in more lateral stripes in the cortex projected more laterally in the CN (in the order of A to G in Fig. 3). This topography generally agreed with the topography of olivocortical and olivonuclear projections (Table 1). For example, olivocerebellar axons originating from the rostral part of subnucleus a of the caudal part of the medial

accessory olive innervate lateral compartment 3ⴙ in lobule VIII and the ventral ICG, which were the origin and target of the axon depicted in Figure 3C (line 6 in Table 1), respectively. Cortical Group II has been defined as aldolase C-positive compartments that do not extend rostrally beyond the primary fissure (cyan and blue areas in Fig. 2A). They correspond to most of zone A in lobules VI-IX and zone X-CX, and are innervated by several medial subnuclei and adjacent areas in the inferior olive (Table 1), which mainly receive vestibular and collicular inputs (Sugihara and Shinoda, 2004). Axons of six PCs that belonged to Group II are shown in Figure 4. They


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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 this study (open circles) were prepared in the same format as in Figure 3. Origin, destination, and type of the terminal arbor of each axon: 1ⴚ in lobule VIa, rostrodorsal and medial FN, wide (A); 1ⴚ in lobule IXc, rostrodorsal and medial FN, wide (B); 3ⴚ in lobule VIII, dorsal ICG, compact (C); cⴚ in crus Ia, dorsal DLP, compact (D); 4bⴚ in crus IIb, dorsal DLP, compact (E). Scale bar ⴝ 500 ␮m.

projected to the midcaudal FN from aⴙ in lobule VIb (Fig. 4A), to the ventrocaudal FN from 2ⴙ in lobule IXa (Fig. 4B), to the midcaudal FN from 2ⴙ in lobule VII (Fig. 4C), to the lateral FN from ventrolateral 2ⴙ in lobule IXc (Fig. 4D), to the ventral PIN from 4ⴙ in lobule IXc (Fig. 4E), to the caudal neck of the DLP from 5aⴙ in crus IIb (Fig. 4F), and to the central FN from cⴙ in crus Ia (Fig. 4G). The target areas of these axons generally coincided with the Group II areas in the CN (cyan and blue areas in Fig. 2B,C). Although the topography of the projection of these axons seemed complicated, it generally agreed with the topography of olivocortical and olivonuclear projections (Table 1). For example, olivocerebellar axons originating from the caudomedial part of the ventral lamella of the principal

olive nucleus innervate compartment 4ⴙ in lobule IX and the most ventral PIN, which were the origin and the target of the axon depicted in Fig. 4E (line 15 in Table 1), respectively. Cortical Group III has been defined as aldolase C-negative compartments in the vermis and the central pars intermedia (yellow and orange areas in Fig. 2A). They correspond to most of zone A in lobules I–V, some areas of zone A in other lobules and 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 midbrain inputs (Sugihara and Shinoda, 2004). Axons of five PCs that belonged to Group III are shown in Figure 5. They projected to the rostrodorsal and medial FN from 1ⴚ in lobule VIa


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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: bⴚ in lobule IIIa (A); 3ⴚ in lobule IIIb (B); fⴚ in lobule VIII (C); 4ⴚ in lobule IIIb (D); 3ⴚ in lobule V (E); 5ⴚ in lateral lobule III (F); 5ⴚ (satellite) in lobule V (G). All of these depicted axons terminated in the AIN and their terminal arbors were classified into the compact type. Scale bar ⴝ 500 ␮m.

(Fig. 5A), to the rostrodorsal and medial FN from 1ⴚ in lobule IXc (Fig. 5B), to the dorsal ICG from 3ⴚ in lobule VIII (Fig. 5C), and to the dorsal DLP from cⴚ in crus Ia and 4bⴚ in crus IIb (Fig. 5D,E). The target areas of these axons generally coincided with the Group III areas in the CN (yellow and orange areas in Fig. 2B,C). Furthermore, the topography of the projection of these axons generally agreed with the topography of olivocortical and olivonuclear projections (Table 1). For example, olivocerebellar axons originating from the rostral part of the lateral subnucleus b of the medial accessory olive innervate compartment 3ⴚ in lobule VIII and dorsal ICG, which were 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 compartments and neighboring lightly positive compartments in the rostral and caudal portions of pars intermedia, and all of the negative compartments in the hemisphere (pink and red areas in Fig. 2A). They correspond to zones B, C1, C3, and D0, and are innervated by the dorsal subnuclei of the inferior olive (Table 1), which mainly receive somatosensory inputs (Sugihara and Shinoda, 2004). Axons of six PCs that belonged to Group IV are shown in Figure 6. Injection sites were located at various positions within Group IV areas in the cerebellar cortex. PCs in all of these areas projected to the AIN, which also belonged to Group IV (red in Fig. 2C). Terminal arbors of these axons were compact and vertically organized in the dorsomedial-to-ventrolateral direction. More lateral PCs often project more laterally in the AIN (Fig. 6G) than more medial PCs (Fig. 6A–F). However, a PC in 4ⴚ

in lobule IIIB (Fig. 6D) projected more medially than a PC in 3ⴚ in lobule VId (Fig. 6E). Comparison of the olivary and PC projections indicated that the projections of most PC axons generally agreed with the topography of olivocortical and olivonuclear projections (Table 1). However, some discrepancy remained in the present results, as indicated by the figure numbers in the parentheses in Table 1 (see footnote 6 for Table 1). For example, compartment 4ⴚ in the rostral cerebellum and compartment 5ⴚ in the caudal cerebellum were speculated to be related to the lateral AIN based on olivary projection (line 28 in Table 1). Although the projection of a PC in caudal 5ⴚ to the lateral AIN (Fig. 7B) was consistent with this relationship, the projection of a PC in rostral 4ⴚ to the relatively medial AIN (Fig. 6D) was not. Therefore, we could not fully explain the topographic relationship of olivary and PC projections for Group IV in the present study. The above results indicated that the topography of the projection of PC axons was generally arranged in accordance with the compartmental organization of the cerebellar cortex and nuclei, which was originally based on the olivocortical and olivonuclear projections, except for a few cases (Table 1). Thus, the basic topography of the PC projections seemed to be generally interpreted by the five-group scheme, which was originally proposed based on the olivary projection. The projection patterns of all axons other than those depicted in Figures 3– 6 (see open circles in Figs. 3H, 4H, 5F, 6H) supported this conclusion (some of these axons are shown in other figures). Basically, with regard to the aldolase C compartmentalization, aldolase C-positive PCs (Groups I and II) pro-


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Figure 7. Projections of PCs located slightly separately within the same or different aldolase C compartment(s). A: Projection of PCs in compartments dⴚ and 4ⴙ 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 dⴚ and 4ⴙ. The location of the somata and axonal paths of these PCs indicated that these axons belonged to dⴚ and 4ⴙ. B: Projections of two PCs that were separated transversely but located in the same compartment. Two small injections were made in medial and lateral 5ⴚ in the apex of crus IIa. PC axons that originated from each of the injections were reconstructed. Blue axons indicate those that were partially reconstructed, except for the fine branches in the terminal arbor. The medial PCs projected to the lateral AIN while the lateral PCs projected to the junction between the DLH and lateral AIN. Scale bar ⴝ 500 ␮m.

jected to the caudoventral parts of the CN and aldolase C-negative (Groups III and IV) and lightly positive (Group IV) PCs projected to the rostrodorsal parts of the CN. This distinct projection underlay the aldolase C compartmentalization in the CN that has been reported previously (Sugihara and Shinoda, 2007). We counted the number of swellings in aldolase C-positive and -negative parts of the CN for each reconstructed axon. For 23 of 37 aldolase C-positive and 16 of 22 aldolase C-negative (including lightly positive) reconstructed PC axons that projected mainly to the CN, all swellings were located in the aldolase C-positive and -negative parts of the CN, respectively. For the rest of the axons (14 of 37 aldolase C-positive and 7 of 22 aldolase C-negative axons), a small fraction of the swellings (1.8 –34.7%, 14.3 ⴞ 10.4%, n ⴝ 21 axons) were located in the opposite parts of the CN (aldolase C-negative parts for aldolase C-positive axons and vice versa). On average for all reconstructed axons that terminated mainly in the CN (n ⴝ 59 axons), 4.8% of swellings were located in the opposite parts. These swellings were located near the boundary between the aldolase C-positive and -negative parts of the CN, 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 in the PC axonal projections. This seemed to be at least partially why the boundary itself is not as clear in the CN as in the cortex (Sugihara and Shinoda, 2007).

Nearby PCs had partially overlapping termination areas when they belonged to the same aldolase C compartment We 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 whether adjacent PCs that are located in a single aldolase C compartment would project to termination areas in the CN according to the same topographic principle. Therefore, we tried to reconstruct multiple axons for small cortical injections and compared their projections. In 11 injections we could completely reconstruct multiple (2 to 6) PC axons originating from an aldolase C compartment. Several examples of multiple reconstructed axons are shown in Figure 8. Three axons were reconstructed with an injection to 4ⴙ in crus Ic (Fig. 8A). They terminated in the ventral PIN. One axon also terminated in the caudal FN with part of the terminal arbor (Fig. 8A, blue); however, this does not contradict the scheme of Sugihara and Shinoda (2007), since the two termination areas of these axons, as well as their origin, belong to Group II, according to the nuclear compartmentalization of Sugihara and Shinoda (2007). In fact, the results suggest that the two separate termination areas could be functionally related to some extent. Three axons were reconstructed with an injection to 1ⴚ in lobule IXc (Fig. 8B). Although the termination areas of these axons were significantly separated, they were still located within the aldolase C-negative rostral FN. This part of the FN, as well as the origin of these axons, belonged to Group III. The distal part of the black axon seemed to slightly extend into aldolase C-positive areas (Fig. 8B, arrowhead). Two axons were reconstructed with an injection to 2ⴚ in lobule III (Fig. 8C). They mainly terminated in the lateral vestibular nucleus (LVN) and anterior interstitial cell group (AICG). The termination areas of these axons hardly overlapped, but were adjacent. The AICG (Sugihara and Shinoda, 2007) is the


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Figure 8. Projection patterns of adjacent PCs labeled by a single injection of BDA in an aldolase C compartment. A: Three PCs located in 4ⴙ 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 1ⴚ in lobule IXc. They all innervated the aldolase C-negative area in the rostral FN. However, the termination areas did not fully overlap. The axon colored in blue terminated mainly in more dorsocaudal areas than the other axons. C: Axons of two PCs located in 2ⴚ in the junction between lobules II and III. They innervated similar areas in the LVN. They also innervated the AICG. D: Two PCs in bⴙ in lobule IV innervated overlapping areas in the rostromedial AIN. Scale bar ⴝ 500 ␮m.

area located rostral to the ICG and ventral to the AIN. This divergent projection of PCs to the LVN and AICG is parallel to the olivary projection, since nuclear collaterals of olivary axons that innervate 2ⴚ in the anterior lobe project to both the LVN and AICG (Sugihara and Shinoda, 2007). Two axons were reconstructed with an injection to bⴙ in lobule 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 that a lightly positive compartment (bⴙ) is akin to an adjacent negative compartment (bⴚ), in which a PC also projected to the rostromedial AIN (Fig. 6A), as proposed previously based on the olivocortical projection (Sugihara and Shinoda, 2004). In sum, terminal arbors of adjacent PCs did not necessarily show tight overlap but were sometimes located within areas in the CN that could be considered to be the same or closely


The Journal of Comparative Neurology SINGLE PURKINJE CELL AXONS related functional compartments. In the DN (not shown), AIN, and PIN, however, they seemed to overlap more tightly than in the FN and LVN. This indicated that corticonuclear topography might be more precisely organized in the DN, AIN, and PIN (see Discussion). We next compared the projection of adjacent PCs that are located across the boundary of aldolase C compartments. Axons of two PCs labeled by an injection that was centered at the boundary between dⴚ and 4ⴙ in rostral crus Ia were reconstructed (Fig. 7A). Locations of the somata and path of the proximal part of axons indicated that one PC was located in compartment 4ⴙ (aldolase C-positive) and the other was located in compartment dⴚ (aldolase C-negative). While the aldolase C-positive PC innervated the centrodorsal PIN, which is aldolase C-positive, the other aldolase C-negative PC innervated the dorsomedial PIN and dorsal ICG, which was mostly aldolase C-negative (Fig. 7A, left and right axons, respectively). Although these results also support the idea that the PC projection is precisely related to the aldolase C compartmentalization in the cerebellar cortex (as was concluded from the results of the previous section), this example was the sole case that we could reconstruct PC axons in an injection across the compartmental boundary.

Projections from separate narrow zones within the same aldolase C compartments Andersson and Oscarsson (1978) have shown electrophysiologically that PCs arranged into separate narrow microzones within zone B in the lateral vermis receive different somatosensory inputs via olivocerebellar projection. A similar observation has been made in zone C3 in the pars intermedia by Ekerot et al. (1991). In the rat, PCs arranged in a narrow longitudinal band show significantly synchronous activity (Lang et al., 1996). This band often equates to an aldolase C compartment, but is sometimes narrower (Sugihara et al., 2007). With regard to anatomical studies, olivocerebellar climbing fibers originating from adjacent olivary neurons project to PCs arranged within a longitudinal band-shaped area (Sugihara et al., 2001). Such areas are generally much narrower than a single aldolase C compartment (Sugihara and Shinoda, 2004). These findings suggest that PCs that are separate in the mediolateral direction within a single aldolase C compartment may be functionally distinct and project differently to the CN. Small injections were made into mediolaterally separate areas in the same aldolase C compartment in the same lobule (medial and central 5ⴚ in crus IIb) and PC axons originating from each injection site were reconstructed (Fig. 7B). The axon originating from medial 5ⴚ formed a compact terminal arbor in the lateral AIN and the other axon from central 5ⴚ formed a compact terminal arbor more laterally in the junction between the AIN and DLH. These two terminal arbors were completely separate in the mediolateral direction (Fig. 7B, black axons). A few other axons were also labeled by these two injections, which were also reconstructed except for fine branches in the terminal arbor. The terminal arbor of each partially reconstructed axon overlapped that of the fully reconstructed axon that originated from the same injection site, and was also separate from that of the axons originating from the other injection site (Fig. 7B, blue axons).

295 These results suggested that fine mediolateral organization was present within the CN, especially in the dorsal main parts of the DN, AIN, and PIN, which reflected microzones in the cerebellar cortex by the precise topographic projection of PCs. Although we did not perform additional studies of similar adjacent microzonal anterograde labeling, the results of retrograde labeling (see below) supported this suggestion.

Convergent projections from the same aldolase C compartments in separate lobules Single olivocerebellar axons branch several times and project as climbing fibers to about seven PCs that are generally located in a longitudinal band-shaped area, often in adjacent and separate multiple lobules (Sugihara et al., 1999, 2001). There seems to be a rule that climbing fibers that originate from a single axon tend to innervate specific combinations of lobules (for example, simple lobule sublobule b, crus IIa, and crus IIb; see figs. 2A,B and 5b green of Sugihara et al., 2001; figs. 4C orange and 8D red and orange of Sugihara and Shinoda, 2004). With regard to the output of the cerebellar cortex, it is also possible that the projection of PCs located in different areas of the cerebellar cortex can converge in the CN. Indeed, Purkinje cells located in zones C1 and C3 in lobule V project to overlapping areas in the anterior interposed nucleus in the cat (Apps and Garwicz, 2000), which is an example of mediolateral convergence. However, the divergent olivocortical projection along with the general longitudinal organization of the cerebellar cortex (Groenewegen and Voogd, 1977; BuisseretDelmas and Angaut, 1993; Voogd, 2004) suggests that rostrocaudal convergence might be more common than mediolateral convergence in PC projection throughout the cerebellum. Here, we tried to find the correlate for such longitudinal convergence in the morphology of single PC projection. We reconstructed PC axons originating from two sites aligned on the same longitudinal compartment (medial 6ⴙ) in neighboring crus IIa and IIb in a rat (Fig. 9A–C). The axons projected with compact terminal arbors to the caudal pole (CP), which is the dorsocaudally protruding area in the junction between the DN and PIN (Voogd, 2004). Terminal arbors of these axons significantly overlapped each other, as seen in the frontal and lateral trajectories of the reconstructed axons (Fig. 9A,B), which indicated convergence of PC projections originating from crus IIa and IIb. Compartment 6ⴙ in the caudal cerebellar cortex (including crus IIa and IIb) is innervated by the ventral lamella of the principal olive, which also projects to the caudal DN including the CP (part of Group I, Sugihara and Shinoda, 2004, 2007). Therefore, the convergent corticonuclear topography of these axons paralleled the divergent olivocortical and olivonuclear topography. In other rats we reconstructed PC axons originating from the most lateral aldolase C-positive compartments (6ⴙ//7ⴙ) in simple lobule sublobule b and crus IIa (Fig. 9D–H). These compartments, 6ⴙ in the rostral cerebellum including simple lobule and 7ⴙ in the caudal cerebellum including crus II, have been considered to be linked or equivalent based on the spatial pattern of aldolase C compartments and olivocortical projection (Sugihara and Shinoda, 2004). These PC axons terminated in the lateral DN. The small termination areas of these axons nearly overlapped each other in their frontal trajectories (Fig. 9D,E). Although these axons were labeled in different rats, their lateral trajectories were drawn on the same


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

panel by carefully referring to the contour of the CN in cerebellar sections of the two rats (Fig. 9F,G). In their lateral trajectories, their terminal arbors were located in the rostrodorsal portion of the DN, with one of the crus IIa PC (Fig. 9G) slightly rostral to the other (Fig. 9F). Therefore, the results seem to support the possibility that PC axonal projections from the rostral and caudal cerebellum can converge to some extent in the CN. The linked compartments 6ⴙ//7ⴙ are innervated by the dorsal lamella of the principal olive, which also project to the lateral DN through nuclear collaterals (part of Group I, Sugihara and Shinoda, 2004, 2007). Therefore, the convergent corticonuclear topography of these axons would parallel the divergent olivocortical and olivonuclear topography. Similarly, target areas in the CN of PC axons projecting from the linked aldolase C compartments in separate lobules were

located close to each other between the cases depicted in Figure 5D,E. However, it was difficult to analyze the convergent PC projection by anterograde labeling and axonal reconstruction as shown in this section since aldolase C compartments are not visible during injection. Therefore, we performed retrograde labeling (below).

Convergent PC projections revealed by retrograde labeling To further examine the possibility of the convergence of PC axonal projections from multiple lobules, a small amount of fluorescent latex microspheres (Apps and Ruigrok, 2007) was injected into the CN, and the distribution of retrogradely labeled PCs was mapped (Fig. 10). It has been proposed that the putative transverse line in lobule VIc and crus Ib represents the rostrocaudal boundary of the cerebellar cortex


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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 1ⴚ 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 6ⴙ in the simple lobule (F, arrowheads) and rostral crus Ia, and in medial 7ⴙ 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 (ⴙ, ⴚ) in A indicate aldolase C-positive and -negative parts in the FN, respectively. The distributions of PCs were mapped on the scheme of aldolase C compartments in the unfolded cerebellar cortex (D,H). Dotted lines in D and H indicate the rostrocaudal boundary of the cerebellar cortex. Scale bars ⴝ 500 ␮m in B (applies to C), in F (applies to G).

based on the stripe pattern of aldolase C expression and the divergent olivocerebellar projection (dotted transverse lines in Fig. 10D,H; Sugihara and Shinoda, 2004). Therefore, the positional relationship of the distribution to the rostrocaudal boundary was primarily examined. With a small injection into the rostrodorsal FN (aldolase C-negative part, Fig. 10A), PCs were labeled mostly in central and relatively medial areas in 1ⴚ in lobule II-VIa (rostral cerebellar cortex, Fig. 10B,D) and 1ⴚ in lobule VIII and IXa (caudal cerebellar cortex, Fig. 10C,D). These compartments, rostral 1ⴚ and caudal 1ⴚ (1ⴚ//1ⴚ), have been considered to be linked compartments since they receive the divergent projection of the same olivocerebellar axons (Sugihara and Shinoda, 2004). The present results indicated that rostrocaudal convergence occurs in the PC projection from these linked compartments (1ⴚ//1ⴚ). In this case, the injection site in the rostrodorsal FN (Fig. 10A) coincided roughly with the termination area of the reconstructed axon of a PC in 1ⴚ//1ⴚ (Fig. 5A) and

that of nuclear collaterals of olivocerebellar axons projecting to 1ⴚ//1ⴚ (line 16 in Table 1). Another injection of fluorescent retrograde tracer was made into the dorsolateral DN (aldolase C-positive part, Fig. 10E). PCs were labeled in relatively medial 6ⴙ in simple lobule sublobules a and b and rostral crus Ia (rostral cerebellar cortex, Fig. 10F,H) and in relatively medial 7ⴙ in crus IIa-b and paramedian lobule (caudal cerebellar cortex, Fig. 10G,H), which indicated convergent projection of these PCs in the rostral and caudal cerebellar cortex. These compartments (6ⴙ//7ⴙ) receive divergent projection of the same olivocerebellar axons and are thus considered to be linked compartments (Sugihara and Shinoda, 2004). The injection site of this case in the dorsolateral DN (Fig. 10E) coincided roughly with the termination area of the reconstructed axons of PCs in 6ⴙ//7ⴙ (Fig. 9D–G) and that of nuclear collaterals of olivocerebellar axons projecting to 6ⴙ//7ⴙ (see Table 1).


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Therefore, these results not only indicated rostrocaudal convergence in the PC projection but also supported the conclusion in the earlier section that the topography of corticonuclear PC projection was closely linked to that of olivocerebellar projection. In six other injections of retrograde tracers into the CN, similar results were obtained. However, further studies will be needed to obtain a systematic understanding of the details of the convergence of PC projections. As an additional finding of the experiments shown in Figure 10, retrogradely labeled PCs were generally distributed not in the entire mediolateral range of a single aldolase C compartment, but only in a part of it. This supported the notion of microzonal organization in the topography of PC projection (see above).

Extracerebellar innervation of PC axons Cerebellar nuclei are the general targets of PC projection. However, PCs in some areas of the cerebellum project to structures outside of the cerebellum. PCs in the flocculus, nodulus, and parts of the uvula project to the medial vestibular nucleus, SVN, and dorsal and ventral Y nucleus, depending on which zones they belong to (Bernard, 1987; De Zeeuw et al., 1994; Wylie et al., 1994; Sugihara et al., 2004). In the anterior lobe of the cerebellum, PCs in the lateral vermis (zone B and a part of zone A that correspond to the lateral compartment 1ⴚ) project to the LVN (Buisseret-Delmas and Angaut, 1993; Voogd and Ruigrok, 2004). Some PCs in the cardiovascular responsive parts of the cerebellum including the median and paramedian anterior vermis and the lateral uvula and nodulus are reported to project to the parabrachial nucleus (PBN) (Sadakane et al., 2000; Nisimaru, 2004). However, the details of such extracerebellar projection have not been clearly reported. For example, it is not known whether such PC axons project solely to extracerebellar targets or whether they project to both the CN and extracerebellar targets, except for those from the flocculus and nodulus that have been reconstructed (De Zeeuw et al., 1994; Wylie et al., 1994; Sugihara et al., 2004). Therefore, we analyzed the trajectories of reconstructed PC axons that projected to structures outside of the cerebellum. Ten of 65 reconstructed PC axons, which did not include those from the flocculus or nodulus, had extracerebellar projections in the present study. All of these 10 axons originated from the vermis. In four of them projection to the CN was predominant and one or a few branches of the axon extended to the extracerebellar target. In the six other axons, projection to the extracerebellar area was predominant, but often retained some projection to the CN. The PC axons shown in Figure 8C, which originated from compartment 2ⴚ in lobule III, projected to the LVN. One of these axons also projected to the AICG, which is the junctional area between the AIN and LVN (Sugihara and Shinoda, 2007). The AICG and LVN are the common targets of nuclear collaterals of the olivocerebellar climbing fibers that project to compartment 2ⴚ (zone B) (Sugihara and Shinoda, 2007). The PC axons shown in Figure 11A originated from the lateral part of compartment 1ⴚ in lobule III. These axons projected to the rostrodorsal and lateral FN, ventral LVN, and the inferior vestibular nucleus (IVN). One of the two axons extended down to nucleus X, which is located between the IVN and inferior cerebellar peduncle. The IVN and nucleus X are innervated by olivary axons that also innervate lateral 1ⴚ

in the rostral cerebellum and part of the rostral aldolase C-negative FN (Sugihara and Shinoda, 2007). The axon shown in Figure 11B, which originated from the median aldolase C-positive area in apical lobule VII, terminated with a wide terminal arbor in the caudoventral aldolase C-positive area in the FN. One of the branches in the terminal arbor extended rostroventrally to the PBN. This axon had 10 terminals in the mediodorsal part of the PBN. PCs in median lobule VII respond to cardiovascular vagal signals in rabbits (Okahara and Nisimaru, 1991). The PBN is concerned with autonomic functions including cardiovascular regulation (Nisimaru, 2004; Saper, 2004). Therefore, this axon may be concerned with cardiovascular control. Although PC projection from lobule VII to the PBN has not been reported so far, PC projections to the PBN from other autonomic areas (median and paramedian anterior vermis and in the lateral uvula and nodulus) have been reported (Sadakane et al., 2000). The PC axon shown in Figure 11C, which originated from compartment 2ⴚ in lobule IXc, mainly projected to the caudomedial SVN. Additionally, this axon had collaterals that terminated in the rostral aldolase C-negative part of the FN (arrowhead in Fig. 11C). According to reports about olivary projection, the SVN is not innervated by olivary axons, while the rostral aldolase C-negative part of the FN is innervated by olivary axons that also innervate caudal 2ⴚ (Ruigrok and Voogd, 2000; Sugihara and Shinoda, 2004). The PC axon shown in Figure 11D, which originated from compartment 2ⴙ in lobule IXa, made a terminal arbor with 30 swellings in the caudal FN, but gave rise to two long branches. One of these projected to the rostrolateral SVN with 131 swellings. The other projected to the caudal nucleus Y with 49 swellings. Three other axons that originated from lobule IX (compartment 1ⴚ and 2ⴙ) extended one branch to the SVN or LVN with a few swellings, while projecting predominantly to the FN (two shown in Fig. 4B,D). In sum, some vermal axons had predominant extracerebellar projection and weak projection to the CN (Figs. 8C, 11A,C,D). Some other vermal axons had predominant projection to the CN and weak extracerebellar projection (Figs. 4B,D, 11B). Various subareas of the vestibular nucleus and the PBN are the extracerebellar targets of PC axons that have been reconstructed so far. Some extracerebellar targets receive the projection of collaterals of olivocerebellar axons and others do not (Table 1). No hemispheric PCs had extracerebellar targets in this study. Thus, the extracerebellar projection seems deeply involved in the functional organization of the cerebellar system in the vermis.

Recurrent cortical collaterals of PC axons The morphology of local recurrent collaterals of PC axons has been described in detail (Ramo´n y Cajal, 1911; ChanPalay, 1971; Hawkes and Leclerc, 1989). Indeed, their entire morphology has been reported based on intracellular horseradish peroxidase labeling in the cat (Bishop, 1982; O’Donoghue and Bishop, 1990). Here we reexamined the morphology of these collaterals as part of single axonal reconstruction in relation to the aldolase C compartmental pattern. With anterograde labeling the morphological isolation of local recurrent collaterals was often difficult because of the local spread and local concomitant labeling of cells other than PCs unless the injection was precisely localized in the molec-


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Figure 11. Extracerebellar projection of some PC axons. A: Two PCs located in lateral 1ⴚ of lobule III projecting to the rostrodorsal aldolase C-negative FN, 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 to the caudal aldolase C-positive FN and the PBN (arrowhead). C: A PC located in compartments 2ⴚ in lobule IXc projecting to the rostral FN (arrowhead) and the caudomedial SVN. D: A PC located in medial 2ⴙ in lobule IXa projecting to the caudal FN, rostrolateral SVN, and caudal nucleus Y. The drawing of the section containing the caudal nucleus Y was shifted downward to improve the view. Scale bar ⴝ 500 ␮m.


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Figure 12. Local recurrent collaterals of PC axons. A: Two cortical collaterals originating from a PC axon that was intensely labeled retrogradely by an injection of BDA into the FN. The photomicrograph is a montage with five different focuses in a parasagittal section. Arrowheads indicate collaterals 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 reaction in coronal sections. Recurrent collaterals of aldolase C-positive PCs innervated mainly aldolase C-positive areas (open arrowheads) and sometimes aldolase C-negative areas (filled arrowheads). D: Photomicrograph of a recurrent collateral of a BDA-labeled aldolase C-negative PC in 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 ⴝ 500 ␮m in A, A inset; 50 ␮m in B–D, D inset.

ular layer. The proximal part of the axon was isolated down to the soma of the PC enough to clearly identify the branching points of recurrent collaterals in 26 reconstructed PC axons that were labeled anterogradely. In the other reconstructed axons, the very proximal part was not clear enough to identify branching because of spread of the injected tracer into the adjacent granular and PC layers. To further examine recurrent collaterals, we performed retrograde labeling of PC axons by injecting BDA into the fastigial (medial cerebellar) nuclei. In strongly labeled PCs (n ⴝ 13 PCs in three rats), the stem axons were well visualized and recurrent collaterals, if present, could be fully reconstructed (Fig. 12A). Among these 39 PCs (26 anterogradely labeled and 13 retrogradely labeled PCs), 28 (71.8%) had one, 8 (20.5%) had two, and 3 (7.7%) had no recurrent collaterals. The average

number of recurrent collaterals was 1.1 (SD ⴝ 0.5). When a PC axon had two recurrent collaterals, they rose from the same branching point (4 of 8 PC axons) or from different branching points (4 of 8). We completely reconstructed 30 recurrent collaterals in 22 PCs (10 anterogradely labeled and 12 retrogradely labeled PCs). The branching points were located 60 – 210 ␮m (139 ⴞ 37 ␮m, mean ⴞ SD, n ⴝ 30) from the soma, which presumably corresponded to the first or second Ranvier’s node. These collaterals were mainly distributed in the PC layer and the superficial granular layer. The total number of swellings seen on a recurrent collateral, including its branches, ranged from 6 to 38 (26.7 ⴞ 7.9). The swellings that belonged to a single collateral were distributed within a range of 103 ⴞ 47 ␮m (mean ⴞ SD, n ⴝ 30) in the rostrocaudal direction and 82 ⴞ 75 ␮m in the mediolateral direction. The


The Journal of Comparative Neurology SINGLE PURKINJE CELL AXONS center of the distribution of the swellings was separated from the soma by 104 ⴞ 69 ␮m in the rostrocaudal direction and by 80 ⴞ 63 ␮m in the mediolateral direction. These differences in the direction were not statistically significant (P > 0.05, twosided t-test with paired samples). Thus, the recurrent collaterals did not tend to run in any clear specific direction. Innervation formed by individual recurrent collaterals in the rat seemed weaker than that in the cat (Bishop, 1982; O’Donoghue and Bishop, 1990). To study the relationship between aldolase C compartments and the projection of local collaterals, we visualized aldolase C-positive PCs including their axonal collaterals by immunostaining. A dense distribution of collaterals was seen in the PC layer and upper granular layer in the aldolase C-positive compartment (Fig. 12B,C). This agreed with the previous observation by Hawkes and Leclerc (1989). However, some aldolase C-positive collaterals were also seen within aldolase C-negative compartments (filled arrowheads in Fig. 12B,C). Such sparse distribution of aldolase C-positive fibers was seen in all aldolase C-negative compartments throughout the cerebellum. Concerning BDA-filled PC axon collaterals reconstructed in aldolase C-labeled cerebellar sections, in most cases (n ⴝ 9 of 10) they terminated in the same aldolase C compartment in which the PC soma was located. However, one recurrent collateral of the axon that originated from an aldolase C-negative PC that was located close to the boundary of an aldolase C-positive compartment innervated both aldolase C-negative and -positive compartments (Fig. 12D). We encountered similar divergent innervation to aldolase C-positive and -negative compartments for recurrent collaterals (n ⴝ 2) that could not be fully reconstructed. These results indicated that the projection of local recurrent collaterals was not exclusively specific to aldolase C compartments.

DISCUSSION The present study clarified the morphology of entire axons of single PCs located in identified aldolase C compartments in the rat cerebellum. Their projection patterns supported a precise corticonuclear topography that was generally consistent with the topography of the olivocerebellar pathway. Their projection patterns also showed some area-dependent variation. The significance and implications of these characteristics in cerebellar organization will be discussed.

Innervation of single PC axons The present study confirmed that PC axons have local recurrent collaterals and a nuclear main terminal arbor, and that they do not have any other branches that might project to other cortical areas in the middle of the axonal path. Projection of local recurrent collaterals was much weaker than projection to the CN and was not completely discriminative with regard to aldolase C compartments. The nuclear terminal arbor of a PC with an average 122 swellings in the present study in the rat may be comparable to 474 swellings per PC as estimated in a Golgi staining study in cats (Palkovits et al., 1977) and agrees with the strong synaptic input from PCs recorded in CN neurons electrophysiologically (Shinoda et al., 1987). An electron microscopic study has shown that CN neurons receive dense innervation of PC terminals at the soma as well as at dendrites (Chan-

301 Palay, 1973), although it is unclear whether these terminals belong to the same or different PCs. The finding that several swellings of an axon surrounded a single CN neuron suggests that a CN neuron receives strong synaptic input at the soma from a relatively small number of PCs. The number of CN neurons innervated by a single PC axon could not be determined directly in the present study.

Corticonuclear topography in terms of aldolase C labeling in the cortex and nuclei The cerebellar cortex is compartmentalized into some 20 longitudinal stripes which contain aldolase C-positive or -negative PCs (Brochu et al., 1990; Voogd et al., 2003; Sugihara and Shinoda, 2004), while the CN, in which only PC axons express aldolase C, are generally divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts (Sugihara and Shinoda, 2007). This has raised the hypothesis that aldolase C-positive and -negative PCs project distinctively to the caudoventral and rostrodorsal parts of the CN, respectively. Furthermore, it has been proposed that, in both 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). The projections of the reconstructed single PC axons in the present study were generally consistent with this five-group scheme (Table 1). Thus, PC projection and olivonuclear projection seem to be based on a common principle of cerebellar compartmentalization, which is represented partially in the aldolase C expression pattern. This notion suggests that the further systematic and detailed analysis of topography of corticonuclear PC projection will help refine the current understanding of cerebellar compartmentalization. For example, the compartmental and topographic organization within the AIN or Group IV is still obscure (footnotes 6 and 7 for Table 1) and, furthermore, the relationship between the anatomical organization and functional or somatotopic organization (Ekerot et al., 1995; Dum and Strick, 2003; Dimitrova et al., 2006) is also ambiguous in the CN. In the conventional scheme, the cerebellar nuclei are subdivided into the FN, AIN, PIN, and DN, which are innervated by the vermis (zone A), parts of the pars intermedia (zones C1 and C3), other parts of the pars intermedia (zone C2), and the hemisphere (zone D) (Voogd and Bigare´, 1980; Brodal, 1981; Buisseret-Delmas and Angaut, 1993; Voogd, 2004). The corticonuclear topography obtained in the present study, which considered finer compartmentalization, generally agrees with the conventional scheme. However, when we look at the present results in detail, several exceptions to the conventional scheme are obvious. For example, a lateral area in vermal lobule IX projects to the PIN (compartment 4ⴙ, zone X-CX, Fig. 4E). In the central cerebellum, some areas in the pars intermedia project to the FN including the DLP (compartments cⴙ, Fig. 4G; cⴚ, Fig. 5D; 4bⴚ, Fig. 5E, and 5aⴙ, Fig. 4F; all belonging to lateral zone A), while another area in the lateral vermis located more medially than the former projects to the PIN (compartment 4ⴙ in crus Ic, putatively zone X-CX, Fig. 8A). All of these unusual projections are actually consistent with the aldolase C expression-based five-group scheme (Table 1).


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Variation in the spatial conformation of PC terminal arbors may be indicative of different organizations in the CN Three types of PC terminal arbors were seen in different areas in the CN in the present study. This suggests another aspect of the organization of the CN. In the dorsal main part of the DN, PIN, and AIN and probably in the dorsolateral protrusion (DLP) of the FN, the terminal arbors were dense and compact and were oriented in the dorsoventral direction (dorsolateral to ventromedial direction in the DN), which was roughly equivalent to the direction from the surface to the hilus located in the center of the CN. Therefore, in these areas the CN may be composed of columnar or cuneiform functional entities (units) arranged radially or parallel. Such radial organization was first reported by Chan-Palay (1973) in the DN. A fine mediolateral topographic segregation of axonal projection seen in these areas (Fig. 7B) supports such organization. Thus, these areas in the CN seem to be organized most regularly in the CN with regard to PC projection. Interestingly, the olivonuclear projection to these parts of the CN also has columnar or cuneiform termination areas and is regularly arranged with a two-dimensional topography (Sugihara and Shinoda, 2007). In most of the aldolase C-positive and -negative areas in the FN, terminal arbors were more widely spread than in other areas. Furthermore, the direction of the elongation of terminal arbors was not consistent among them, although they are most frequently elongated in the mediolateral and caudorostral directions. Therefore, these areas may not be as regularly organized as the other areas (see above). In the ventral parts of the DN, PIN, and FN, terminal arbors were remarkably elongated in the mediolateral direction. This suggested that functional compartmentalization is organized more or less transversely in these areas. These ventral areas were innervated by compartments 1ⴙ and 2ⴙ in the vermis, compartments 4ⴙ in lobule IX and paraflocculus. Projection from the flocculus to the dorsal Y nucleus and ventrorostral DN (Sugihara et al., 2004) was similarly elongated. An analysis of olivonuclear projection also suggested that the ventral parts of the DN, PIN, and FN may be considered to be organized distinctly from the dorsal main parts of these subnuclei (Sugihara and Shinoda, 2007). In sum, based on the different structures of the terminal arbor, some parts of the CN seem to differ from other parts with regard to organization. Subdivisions of the CN defined in this way do not directly correspond to those based on aldolase C compartmentalization. Thus, further studies will be needed to elucidate the organization of the CN.

Extracerebellar projections of PCs Some axons of vermal PCs extended beyond the CN to other structures. These extracerebellar projections seemed to be organized according to the cortical compartments, similar to the PC projections to the CN. The lateral vermal area that projects to the dorsal part of the LVN (zone B; Groenewegen and Voogd, 1977; Ito, 1984; Buisseret-Delmas and Angaut, 1993) has been identified as compartment 2ⴚ (Sugihara et al., 2004; Voogd and Ruigrok, 2004). It has recently been reported that PCs in lateral compartment 1ⴚ in the anterior lobe also project 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 X from this area. In relation to this finding, collaterals of olivary axons that innervate lateral compartment 1ⴚ project to nucleus X and the IVN (Sugihara and Shinoda, 2007). Our study also showed projection from median lobule VII to the PBN. A previous study has shown that a different area (the lateral nodulus and uvula) projects to the PBN (Sadakane et al., 2000). Further analysis of the extracerebellar projections of PCs in relation to their somatotopy or function will be required to understand the output system of the vermis.

Variable conformation of the olivo-corticonuclear loop The results in our present and previous studies (Sugihara and Shinoda, 2004, 2007) generally agree with the hypothesis that the whole cerebellar system is formed by the parallel assembly of an olivo-cortico-nuclear loop (microcomplex or modules) (Ito, 1984; Apps and Garwicz, 2005; Pijpers et al., 2005). However, a close look at the morphology of PC axons indicates that the formation of such loops is not uniform among areas in the cerebellum, suggesting some areadependent differentiation in the organization of the cerebellum. An essential part of the olivo-cortico-nuclear loop consists of three putative projections which interconnect precisely: a population of PCs and nuclear collaterals of the olivary axons that innervate these PCs project to the same nuclear neurons. The nucleo-olivary projection may be added to the loop as an additional component (Ruigrok and Voogd, 1990). A possible exception is that extracerebellar targets of some PCs may not be innervated by olivary axons. The PBN that is innervated by some vermal PCs (present study and Sadakane et al., 2000), the SVN that is innervated by PCs in the flocculus, uvula, and nodulus (Bernard, 1987; de Zeeuw et al., 1994; Sugihara et al., 2004; and present study), and the medial vestibular nucleus that is innervated by some PCs in the flocculus and nodulus (Bernard, 1987; de Zeeuw et al., 1994) may not receive the innervation of olivary axons (Sugihara and Shinoda, 2007). The LVN (and AICG) that is innervated by compartment 2ⴚ (Fig. 8C; Voogd and Ruigrok, 2004) and the IVN (including nucleus X) that is innervated by compartment 1ⴚ (Fig. 11A) receive innervation from collaterals of the olivary axons that innervate compartments 2ⴚ and 1ⴚ, respectively (Sugihara and Shinoda, 2007). Additionally, it is unclear whether the entire termination area of a wide terminal arbor in the FN or an elongated terminal arbor in the ventral PIN, ventral DN, and dorsal Y (this study and Sugihara et al., 2004) is completely innervated by the same population of olivary axons, since the termination of the olivonuclear projection is generally localized (Ruigrok and Voogd, 2000; Sugihara and Shinoda, 2007). Thus, the organization of the olivo-cortico-nuclear loop has some variations in the FN and ventral CN. On the other hand, a typical olivo-cortico-nuclear loop seems to be formed in the rest of the CN (dorsal main parts of the AIN, PIN, and DN), in which a PC has a compact and dense terminal arbor. In these areas the basic topographic conformation of the olivo-cortico-nuclear loop seem to have a one area-to-multiple areas-to-one area relationship, since olivary axons show a rostrocaudally divergent (branching) projection to the cortex (Sugihara et al., 2001; Pijpers et al., 2006) and PC axons in rostrocaudally separate cortical areas converge to


The Journal of Comparative Neurology SINGLE PURKINJE CELL AXONS the specific area in the CN (Figs. 9, 10), where the collaterals of olivary axons also project to (Table 1).

ACKNOWLEDGMENT The authors thank Dr. E.J. Lang for reading the article.

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Projection of Reconstructed Single Purkinje Cell Axons ...