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The Journal of Neuroscience, September 5, 2007, 27(36):9696-9710; doi:10.1523/JNEUROSCI.1579-07.2007
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Behavioral/Systems/Cognitive
Molecular, Topographic, and Functional Organization of the Cerebellar Nuclei: Analysis by Three-Dimensional Mapping of the Olivonuclear Projection and Aldolase C Labeling
Izumi Sugihara andYoshikazu Shinoda Department of Systems Neurophysiology, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo 113-8519, Japan
Abstract
Top
Abstract
Introduction
The olivocerebellar climbing fiber projection pattern is closely correlated with the pattern of aldolase C expression in cerebellar Purkinje cells. Based on this expression pattern, the olivocerebellar projection can be classified into five "groups" of functional compartments. Each group originates from a subarea within the inferior olive that projects to multiple cortical stripes of Purkinje cells, all of which are either aldolase C positive or aldolase C negative. However, no equivalent compartmental organization has been demonstrated in the cerebellar nuclei (CN). Thus, in the CN of the rat, we systematically mapped the location of olivonuclear projections belonging to the five groups and determined their relationship to the expression of aldolase C in Purkinje cell axonal terminals.The CN were divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts. The olivonuclear terminations from the five groups projected topographically to five separate compartments within the CN that partly crossed the traditional boundaries that define the fastigial, interposed, and dentate nuclei. Each compartment had mostly uniform cytoarchitecture and the same aldolase C expression (either positive or negative) that was found in the corresponding olivocortical projection. These results suggest a new view of the organization of the CN whereby the pattern of olivonuclear terminations links portions of different CN together. We propose that each compartment in the CN, along with its corresponding olivary subarea and cortical stripes, may be related to a different aspect of motor control.
Key words: climbing fiber; inferior olive; aldolase C; zebrin; biotinylated dextran amine; rat
Introduction
The cerebellar nuclei (CN), the major source of the cerebellar output, are conventionally divided into the fastigial (medial), interposed, and dentate (lateral) nuclei (Brodal, 1981; Voogd, 2004
In the cerebellar cortex, longitudinal compartmentalization beyond the conventional three divisions has been revealed by the demonstration of zones A–D based on cholinesterase labeling in the cerebellar white matter and the topography of the corticonuclear and olivocortical projections (Voogd, 1967
The compartmentalization in the cerebellar cortex suggests that an equivalent fine compartmentalization may exist in the CN, because Purkinje cells project topographically onto the CN (Buisseret-Delmas and Angaut, 1993
In the present study, olivary axons were labeled by a small injection of tracer into various areas of the IO in the rat. In each case, we classified the olivocortical projection pattern of the labeled olivary neurons into one of the five groups by mapping labeled climbing fibers within cortical aldolase C compartments. We then mapped the location of labeled terminals of olivonuclear collaterals that arose from the same labeled olivary neurons within the CN. A new three-dimensional mapping technique was used to facilitate the comparison of the positional relationship among mapped areas, because the positional relationship is not easy to grasp from the conventional maps used in previous studies (Buisseret-Delmas and Angaut, 1993
Materials and Methods
Tracer injection and histological procedure. Mapping of the olivonuclear termination area was performed in a total of 76 Long–Evans adult rats (Kiwa Laboratory Animals, Wakayama, Japan), which included 48 rats that were used in our previous study on cortical compartmentalization (Sugihara and Shinoda, 2004Briefly, 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 min starting 1 h after the initial dose, as required. Biotinylated dextran amine [BDA; D-1956, 10,000 molecular weight (MW) or D-7135, 3000 MW; Invitrogen, Eugene, OR] was pressure injected at various locations in the IO (
0.004 µl of 10% solution in saline) for anterograde labeling. Single injections were made in the left and right IO of each animal because the olivocerebellar projection is almost entirely contralateral. The injected tracer spread to a diameter of 0.1–0.2 mm. After a survival period of 6–9 d, the rats were anesthetized in the same way as in the first operation, but with 1.5 times larger dose of ketamine and xylazine. They were perfused intracardially with PBS, followed by fixative containing 5% paraformaldehyde, 3% sucrose, and 50 mM phosphate buffer, pH 7.4. Eighty-micrometer-thick serial frozen sections were cut from the cerebellum and medulla. Horizontal sections were obtained from 11 rats, and coronal sections were cut from 65 rats.
The immunohistological procedures used to double label BDA (black reaction product) and aldolase C (brown reaction product) with diaminobenzidine have been described previously (Sugihara and Shinoda, 2004
Six Long–Evans adult rats were used solely for labeling of aldolase C in the CN. These adult rats were anesthetized, perfused, and fixed in the same way as described above. Serial frozen sections of the cerebellum and medulla were cut in the horizontal, parasagittal, and coronal planes. The immunohistological procedures used to label aldolase C black with diaminobenzidine have been described previously (Sugihara and Shinoda, 2004
Mapping nuclear termination areas on the three-dimensional template of the CN. To make standardized three-dimensional maps for the CN of the rat, four Long–Evans adult rats were anesthetized, perfused, and fixed in the same way as described above. In each animal, a cubic block containing the CN was dissected from the cerebellum and medulla. The block was embedded in gelatin, and coronal, parasagittal, or horizontal 40-µm-thick frozen serial sections were cut. The sections were mounted and stained with thionine. The outlines of the CN and adjacent structures were depicted in every section with a camera lucida to make our own atlas of the CN in coronal, parasagittal, and horizontal planes in these rats. These outlines were digitized using two-dimensional graphics software (Illustrator; Adobe Systems). The outlines in serial sections were drawn in different "layers" of the software. The positions and tilts of the drawings of serial sections were adjusted so that all sections fit each other when superimposed. The Illustrator graphic files were then imported into three-dimensional graphics software (Rhinoceros; Robert McNeel & Associates, Seattle, WA). Outlines in different sections were shifted in the direction of the z-axis by a distance equivalent to the thickness of the section to reconstruct the CN in the three-dimensional space of the software. The three images of the reconstructed CN based on the coronal, parasagittal, and horizontal sections were superimposed on each other in the software. Slight adjustments (magnification, rotation, shearing, shifting) were made to the images to obtain the best fit among the three images of the CN. We took this final image to be an optimum, standardized three-dimensional image of the CN, which we then used for mapping terminal locations.
To map the terminations (swellings) of olivocerebellar projection within the CN, the following procedure was followed. When a histological section contained labeled terminals, its relative position within the CN was determined. For coronal sections, the most caudal part of the posterior interposed nucleus (PIN) and the most rostral part of the anterior interposed nucleus (AIN) were used to mark the limits of the CN, and the distance of the section from these limits defined its relative position. The dorsal pole of the dorsolateral protuberance (DLP) and the most rostral corner of the fourth ventricle were used for the same purpose for horizontal sections. The sections corresponding to the relative position and adjacent positions in the standardized CN were displayed on the computer monitor. The histological section was superimposed on these standard sections by using a camera lucida apparatus attached to the microscope, and the standardized section that gave the best fit for a given section was selected and defined the position of histological section. Higher magnifications were then used in the microscope to see the terminal swellings of the olivocerebellar axons, which were marked on the standardized section that were digitally magnified. The area in which axon terminals were located was carefully circumscribed by drawing a curve within Rhinoceros. The same procedures were repeated for all sections that contained nuclear terminations from the same injection. The "loft" command in Rhinoceros was then used to construct a solid shape that enclosed these terminals and thereby defined the termination region of the labeled axons (see Fig. 2, compare C, D).
The procedures for mapping injection sites in the horizontal representation of the IO and labeled climbing fibers in the unfolded cerebellar cortex have been described previously (Sugihara and Shinoda, 2004
In the figures in this paper, all brain drawings for which injections were made into the left IO were reversed about the midline, so that all climbing fibers were plotted in the left cerebellar cortex and nuclei. The anatomical terms for the CN corresponded to those used by Voogd (2004)
Results
Aldolase C compartments in the CN
Aldolase C (zebrin II) is expressed in a population of Purkinje cells that are arranged in a stripe pattern in the cerebellar cortex (Brochu et al., 1990
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Figure 1. Photomicrographs of aldolase C compartments in the CN. A, B, Parasagittal (Aa–Ag, medial to lateral) and coronal (Ba–Bd, caudal to rostral) sections of the CN labeled with anti-aldolase C antibody. Dotted curves indicate the contour of the CN and borders between aldolase C-positive and -negative areas within the nuclei. Asterisks indicate bundles of labeled Purkinje cell axons approaching the CN. The calibration bar in Bd applies to all panels in A and B. C, Photomicrograph of axonal terminals of aldolase C-positive Purkinje cells in the CN labeled with anti-aldolase C antibody. Dark dots of
Thus, we defined regions of the CN (and vestibular nuclei) as aldolase C positive or negative based on the presence or absence of labeled Purkinje axon terminals. If one just considered overall labeling, the distinction or contrast between the aldolase C-positive and -negative areas was less clear than in the cerebellar cortex; however, it was noted that often axons of labeled Purkinje cells may pass through an otherwise unlabeled area without forming terminals. Therefore, we based our definition of positively and negatively stained CN regions exclusively on terminal labeling. With this criterion, clearly demarcated compartments emerged in the CN.Parasagittal sections showed that the overall staining pattern in the interposed and fastigial nuclei was positively stained areas located caudoventrally and negatively stained regions located rostrodorsally with a clear boundary in each nucleus (Fig. 1Aa–Af). In coronal sections, the ventral parts of the CN were generally aldolase C positive, consistent with the findings in parasagittal sections.
Regarding individual CN, the fastigial nucleus (FN) was aldolase C positive in the ventrocaudal portion and aldolase C negative in the rostrodorsal portion (Fig. 1Aa–Ac,Ba–Bd). The DLP was aldolase C negative, except for its caudoventral neck (Fig. 1Ba,D). The interstitial cell group (ICG) (Buisseret-Delmas et al., 1998
The AIN was generally aldolase C negative (Fig. 1Ac–Ae,Bb–Bd). The most medial and caudal portion of the AIN, which is called the dorsomedial crest (DMC), stained weakly for aldolase C (Fig. 1Bb). However, this area was considered to be the aldolase C negative in this study, because the labeling was so weak compared with the other aldolase C-positive areas. The dorsolateral hump (DLH), which protrudes rostrodorsally and usually is considered to be the most lateral part of the AIN, was aldolase C negative (Fig. 1Af,Bc). The caudal pole (CP), which is located caudal to the DLH and protrudes caudally, was aldolase C positive (Fig. 1Af,Ba). The CP may be considered to belong to the dentate nucleus (DN; see below, Group I and Discussion).
The PIN was aldolase C negative in the small dorsomedial portion (Fig. 1Ad,Ba) and aldolase C positive in the rest (Fig. 1Ad,Ae,Ba,Bb). Thus, aldolase C labeling can delineate the boundary between the lateral PIN and the AIN (Fig. 1Ae).
The entire DN was aldolase C positive (Fig. 1Af,Ag,Bb,Bc). Among the aldolase C-positive areas in the CN, the ventral DN and ventral PIN were the most densely labeled (Fig. 1Ad–Ag,Ba–Bc).
We also analyzed aldolase C labeling of the vestibular nucleus, some parts of which were innervated by nuclear collaterals of olivocerebellar projection (see the next paragraph). The dorsal Y nucleus (infracerebellar nucleus), which receives the projection of olivocerebellar axons innervating the flocculus (Sugihara et al., 2004
Several patches within the central part of the lateral and inferior vestibular nuclei were strongly aldolase C positive and seemed to consist of dense bundles of aldolase C-labeled axons that projected to the medial vestibular nucleus (Fig. 1Bc). Among all of these vestibular nuclei, an olivary projection has been demonstrated in the dorsal part of the LVN as well as in the dorsal Y nucleus (Ruigrok and Voogd, 2000
Nuclear termination of olivocerebellar projection
More than 90% of olivocerebellar axons have collaterals terminating in the CN (Sugihara et al., 1999
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Figure 2. Nuclear termination of the olivocerebellar projection. A, Photomicrograph of two bilateral injection sites of BDA (white and black arrowheads) in the IO. The one on the right side (black arrowhead) is the origin for the terminals shown in B and C. B, Photomicrographs of axonal terminals in a nuclear termination area. Inset, One of the retrogradely labeled neurons in the CN, which were seen near the axonal terminals (arrowhead) when the injection was larger. C, Camera lucida drawing of all swellings of axonal terminals in serial sections (80 µm thick). Their location (arrowheads and dotted curves) was determined under low power (4x objective), and individual swellings were mapped under medium power (20x). D, Illustration of the three-dimensional mapping of the nuclear termination area. Curves were drawn in three-dimensional software (Rhinoceros) to circumscribe most of the swellings in serial sections (a). The loft command of Rhinoceros was used to form a three-dimensional closed surface that represents the entire termination area (b).
In most cases (n = 80 of 123 injections), labeled climbing fibers were distributed within a single aldolase C compartment. To find the nuclear termination area, a bundle of labeled olivocerebellar axons was traced proximally from the cerebellar cortex toward the cerebellar peduncle. At some point near or inside the CN, several thin collaterals were given off from their stem axons. These thin collaterals ran for variable distances of <100 to >500 µm and finally terminated in the CN by making a sparse arborization with swellings, as reported previously (Sugihara et al., 1999The swellings of traced nuclear collaterals were distributed within a single circular area that was 150–300 µm in diameter on individual sections (Fig. 2C). A few swellings were occasionally separated from the main high-density area. When reconstructed in three-dimensional space, however, the nuclear termination area was generally elongated in a certain direction so that it looked cuneiform shaped rather than spherical (Fig. 2D). The length of the cuneiform along its long axis was
Olivonuclear organization conformed to the five-group scheme
We previously reported that the aldolase C compartments in the cerebellar cortex and the olivocerebellar projections to these compartments could be classified into five groups (groups I–V), based on clustering of the olivary origin of the topographic olivocerebellar projection to similar compartments (Table 1) (Sugihara and Shinoda, 2004
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Table 1. Definition of groups I–V and presumed major input to the IO of each group
To determine whether the olivonuclear projection also obeys this scheme, we sorted the 123 IO injection cases into the five groups based on the compartment in which the labeled climbing fibers terminated as was done previously (Sugihara and Shinoda, 2004
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Figure 3. Clustering in the CN of termination areas of nuclear collaterals of the olivary projections sorted into five groups based on their cortical projection patterns. A, Mapping of 123 injection sites on the dorsal view of individual subnuclei of the right IO. Dorsal subnuclei were shifted to the right. B, Mapping of labeled climbing fibers in aldolase C compartments on the unfolded scheme of the cerebellar cortex. Each injection was classified into one of five groups (see Results) based on its projection pattern to aldolase C compartment by referring to the five-group scheme of the olivocerebellar projection [Sugihara and Shinoda (2004)
Group IV areas (i.e., nuclear termination areas of olivocerebellar axons, the climbing fibers of which terminated in a group IV cortical compartment) (Fig. 3Ca,Cb, red) were best observed in rostral and dorsal views of the CN. Group IV areas included regions located in the dorsal portion of the LVN, the entire AIN (including the DLH), and the area between the AIN and LVN. These areas were continuous with one another and thus formed a single cluster.Group III areas were best observed from the dorsal view (Fig. 3Cb, yellow). Group III areas included the entire rostrodorsal surface of the FN, the DLP, the dorsal aspect of the ICG, and the dorsomedial PIN. These areas were continuous with each other and formed a single cluster that extended mediolaterally from the most medial FN to the PIN.
Most group II areas could be observed in the caudal view. Most were distributed in the caudal FN and ICG (Fig. 3Cc, blue). These areas extended to the ventral FN between group I and III areas, as seen in the ventral view (Fig. 3Cd, blue between green and yellow). The other group II areas were distributed laterally in the ventral PIN (Fig. 3Cc, blue, Cd, blue curve). These two clusters nearly touched each other at the caudoventral junction between the PIN and ICG.
Group I areas could be observed with a ventral view (Fig. 3Cd), in which the group II areas in the ventral PIN were removed for clarity. Group I areas were distributed in the ventral FN, ventral ICG, PIN, and DN (Fig. 3Cd, green). These group I areas were continuously arranged from the most medial to the most lateral CN. Group V areas were distributed in the ventral DN and in the adjacent dorsal Y nucleus (infracerebellar nucleus) (Fig. 3Cd, gray), as reported previously (Sugihara et al., 2004
In summary, the termination areas of each group were clustered in specific, non-overlapping portions of the CN. The clusters tended to be elongated transversely, mostly parallel to each other. Thus, our data suggest that the five-grouped organization, which was originally proposed with regard to aldolase C compartments and the olivocerebellar projection pattern in the cerebellar cortex, seem to be applicable to the CN. Therefore, in the following sections, we will describe the detailed topographic organization of the olivonuclear projection in each group.
Group I
Cortical group I area has been defined as darkly labeled aldolase C-positive compartments that extend rostrally to the anterior lobe beyond the primary fissure (1+//1+, 2+//3+, 4+//5+, 5+//6+, and 6+//7+) in the cerebellar cortex (Fig. 3B, green). They are innervated by the ventrolateral parts of subdivisions of the IO [subnucleus a of the caudal parts of the medial accessory olive (c-MAO); the rostral part of the medial accessory olive (r-MAO); the ventral lamella of the principal olive (v-PO), except for the caudomedial part; and the dorsal lamella of the principal olive (d-PO)] (Fig. 3A, green) (Sugihara and Shinoda, 2004The caudal part of subnucleus a, the most lateral part of the c-MAO, which innervates 1+//1+ in lobules I–VI and VIII–IX in the cortex (Sugihara and Shinoda, 2004
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Figure 4. Olivonuclear projection pattern of group I. A–D, Three-dimensional mapping of nuclear termination areas of collaterals of olivocerebellar axons labeled by BDA injections in subnucleus a of the c-MAO (A; n = 8; specific colors for individual injections), the r-MAO (B; n = 11), the v-PO (C; n = 7), and the d-PO (D; n = 11). The leftmost subpanels show the injection sites in the dorsal view of the right IO. The three subpanels on the right show caudal, dorsal, and medial (lateral for D) views of the mapped termination areas depicted within the coronal, horizontal, or parasagittal contours of the nuclei (wire frames), respectively, at the same levels as where the termination areas were located. Olivocerebellar neurons in these areas were classified into group I, based on the injection sites in the olive and their distribution of labeled climbing fibers in the cortex (see Results for details). E, Summary of the topographic olivonuclear projection of group I (as indicated with green) depicted in the solid scheme of the IO (left) and the CN (right). Black and white arrows in A–E indicate the primary and second orientation axes, respectively, to show topographic correspondence between the IO and CN. The cortical aldolase C compartments that the main axons of the depicted olivonuclear projection innervate are indicated as 1+//1+ and so on.
The r-MAO (Fig. 4B), which innervates 4+//5+ in the cortex (Sugihara and Shinoda, 2004The v-PO, except for its caudomedial portion (Fig. 4C), which innervates 5+//6+ in the cortex (Sugihara and Shinoda, 2004
The d-PO (Fig. 4D), which innervates 6+//7+ in the cortex (Sugihara and Shinoda, 2004
In summary, the topography of group I olivonuclear collaterals was highly correlated with the olivocortical topography. To describe the olivocerebellar topography within each group, a hypothetical sequential arrangement (designated "orientation axis") of olivary subareas has been proposed (Sugihara and Shinoda, 2004
Group II
Cortical group II area has been defined as darkly labeled aldolase C-positive compartments that do not extend rostrally beyond the primary fissure (a+//2+, 2b+//4+, c+//4b+, and d+//5a+) in the cerebellar cortex. They are innervated by several medial subnuclei and adjacent areas in the IO [subnucleus c of the c-MAO, subnucleus ß, dorsomedial cell column subnucleus (DMCC), caudal parts of the dorsomedial group of the v-PO (DM), and caudomedial part of the v-PO] (Sugihara and Shinoda, 2004Concerning the nuclear projection of group IIa (Fig. 5A), subnucleus ß projected to the central and caudoventral FN and ventral ICG. This area is located dorsocaudal to the area innervated by group I (see above). Caudal subnucleus ß projected to the medial part of the caudoventral FN (Fig. 5A, red, brown, orange, yellow), whereas rostral subnucleus ß projected to the lateral part of the caudoventral FN and the ventral ICG (Fig. 5A, yellow-green, light green, green, cyan). This topography could be explained by the orientation axis that ran from caudal subnucleus ß to rostral subnucleus ß (Sugihara and Shinoda, 2004
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Figure 5. Olivonuclear projection pattern of group II. A–D are depicted in the same format as in Figure 4. A–C, Three-dimensional mapping of nuclear termination areas of collaterals of olivocerebellar axons labeled by BDA injections in subnucleus ß including caudal subnucleus c of the c-MAO (A; n = 8), the DMCC, caudal DM and caudomedial v-PO (B; n = 7), and medial and lateral subnucleus c of the c-MAO (C; n = 9). D, Summary of the topographic olivonuclear projection of group II (as indicated with cyan and blue; cyan, from subnucleus ß including caudal subnucleus c of the c-MAO to the caudoventral FN and the ventral PIN; blue, from medial and lateral subnucleus c of the c-MAO to the centrocaudal FN) depicted in the solid scheme of the IO (left) and the CN (right). In the dorsomedial view of the CN (rightmost panel), the group III area, except for the DLP, was removed to show group II areas. Black arrows in A–D show the orientation axis to indicate topographic correspondence between the IO and CN.
Concerning the rest of group IIa (Fig. 5B), the DMCC (Fig. 5B, cyan, blue), caudal DM (purple, magenta), and caudomedial v-PO (salmon pink, pink), all of which innervate 2b+//4 in the cortex, projected to the most ventral sheet-shaped area of the PIN. The projection areas of group IIa in the FN and ventral ICG and in the ventral PIN did not seem to simply be contiguous to each other but were intercalated by the projection area of group I. The topography within the projection to the ventral PIN was not so clear, although the DMCC, caudal DM, and caudomedial v-PO seemed to project to the ventral PIN more or less medially, centrally, and laterally, respectively. The orientation axis has been hypothesized to run from the DMCC, through the caudal DM, and to the caudomedial v-PO (Sugihara and Shinoda, 2004Concerning the nuclear projection of group IIb, subnucleus c innervated the central FN, dorsal to the area where group IIa projected (Fig. 5C). In the central FN, the caudal part of medial subnucleus c, which projects to the most medial area in lobules VIb–VIc and VII in the cortex, projected most medially (Fig. 5C, red, brown), and the rostral part of medial subnucleus c, which innervates a+//2+ in lobules VIb–VIc and VII in the cortex, projected more laterally (Fig. 5C, yellow, light green, green). The rostral and caudal parts of lateral subnucleus c, which mainly innervates c+//4b+ and d+//5a+ in the cortex, respectively, projected further laterally to the dorsal (Fig. 5C, cyan, blue) and ventral (Fig. 5C, magenta, pink) portions of the caudal neck of the DLP, respectively. The orientation axis ran in the caudorostral direction in medial and then lateral subnucleus c in succession (Fig. 5C, leftmost panel) (Sugihara and Shinoda, 2004
In summary, the olivonuclear topography in group II could be primarily explained by assuming two subgroups (IIa and IIb) that were aligned along distinct but parallel orientation axes. Group IIa areas were further subdivided into the FN (plus ICG) part and the ventral PIN part. For each subgroup, the caudorostral arrangement of IO regions mostly could be mapped to a mediolateral sequence of CN regions (Fig. 5D, black arrows).
Group III
Cortical group III has been defined as aldolase C-negative compartments in the vermis and the central pars intermedia [1–(med)//2–, 1–(lat)/a–//2–, 2a–//3–, 2b–//4a–, c–//4b–, and d–//5a–)]. They are innervated by the central portion of the c-MAO (subnucleus b) (Sugihara and Shinoda, 2004
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Figure 6. Olivonuclear projection pattern of group III. A–C are depicted in the same format as in Figure 4. A, B, Three-dimensional mapping of nuclear termination areas of collaterals of olivocerebellar axons labeled by BDA injections in lateral subnucleus b of the c-MAO (A; n = 14; specific colors) and medial subnucleus b of the c-MAO (B; n = 14). C, Summary of the topographic olivonuclear projection of group III (as indicated with yellow and orange; yellow, from lateral subnucleus b to rostrodorsal FN and dorsal ICG; orange, from medial subnucleus b to DLP and dorsomedial PIN) depicted in the solid scheme of the IO (left) and the CN (right). In the right top subpanel, the DLP and dorsomedial PIN (orange areas) were removed to show yellow areas. Dotted yellow and orange curves indicate lateral extension of yellow and orange areas underneath the AIN. Black arrows in A–C show the orientation axis to indicate topographic correspondence between the IO and CN.
Concerning lateral subnucleus b (Fig. 6A), its caudal part, which innervates cortical compartment 1–(med)//1–, projected to the rostrodorsal aldolase C-negative area of the most medial part of the FN (Sugihara and Shinoda, 2004Concerning medial subnucleus b (Fig. 6B), its caudolateral part, which innervates 2b–//4a– in the cortex, projected to the rostral neck of the DLP (Fig. 6B, red, orange, brown, yellow). The caudomedial part of medial subnucleus b, which innervates c–//4b– in the cortex, projected to the apex of the DLP (Fig. 6B, yellow-green, green, dark green). The intermediate part of medial subnucleus b, which innervates d–//5a– in the cortex, projected to the caudal neck of the DLP close to the aldolase C-positive area (Fig. 6B, cyan, blue, dark blue). There was some degree of overlap among the above three nuclear projection areas. The rostral part of medial subnucleus b, which mainly innervates 3b–//e2– in the cortex, projected to the dorsomedial area in the PIN (Fig. 6B, lavender, magenta, violet, pink). Thus, the caudorostral orientation axis in medial subnucleus b corresponded to the mediolateral direction from the DLP to the dorsomedial PIN (Fig. 6C, arrows in the orange areas).
In summary, lateral and medial subnucleus b mostly projected to the ventral and dorsal areas of a combination of the rostrodorsal FN, dorsal ICG, and dorsomedial PIN, respectively. In either projection, more rostral areas in subnucleus b projected more laterally in the CN.
Group IV
Cortical group IV has been defined as aldolase C-negative compartments and their neighboring lightly positive compartments in the rostral and caudal portions of pars intermedia (2–//4–, b+//f+, b–//f–, 3+//e1+, 3–//e1–, and putatively 3b+//e2+) and all of the negative compartments in the hemisphere (4–//5– and 5–//6–) of the cerebellar cortex. They are innervated by the dorsal subnuclei of the IO [dorsal (d-DAO) and ventral (v-DAO) folds of the dorsal accessory olive and the central and rostral DM] (Sugihara and Shinoda, 2004The d-DAO (Fig. 7A), which innervates 2–//4– in the cortex, projected to the dorsal portion of the LVN and the gray matter scattered in the hilar white matter between the LVN and the medial AIN (Fig. 7A). These islands of gray matter were designated here as the anterior ICG (AICG), because they were located mostly anterior (rostral) to the ICG but also included the most rostral portion of the ICG [Buisseret-Delmas et al. (1993)
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Figure 7. Olivonuclear projection pattern of group IV. A–D are depicted in the same format as in Figure 4. A–C, Three-dimensional mapping of nuclear termination areas of collaterals of olivocerebellar axons labeled by BDA injections in the d-DAO (A; n = 7; specific colors), the caudal and lateral v-DAO (B; n = 10), the rostral and medial v-DAO and the central and rostral DM (C; n = 11). In A, the entire termination areas are depicted in the top part. The densest distribution of the terminals was often seen in the junction between the AIN and the LVN, designated here AICG. Areas of the densest termination in the AICG are depicted in the bottom part. D, Summary of the topographic olivonuclear projection of group IV (as indicated with pink and red; pink, from d-DAO to LVN and AICG; red, from v-DAO to AIN and DM) depicted in the solid scheme of the IO (left) and the CN (right). Black and white arrows in A–D show the primary and second orientation axes, respectively, to indicate topographic correspondence between the IO and CN.
It has been suggested that the lateral pole of the d-DAO innervates mainly b+//f+ in the cortex (Sugihara and Shinoda, 2004The caudolateral v-DAO (Fig. 7B), which mainly innervates 3+//e1+ in the cortex (Sugihara and Shinoda, 2004
The projections described above indicated that a two-dimensional topography was consistently observed throughout the projection from the v-DAO (and DM) to the AIN (Fig. 7B,C, black and white arrows). The more caudolateral and rostromedial v-DAO projected to the more medial and lateral AIN, respectively (Fig. 7B, light-green, green, red, and so on; C, blue, cyan, purple), whereas the more rostrolateral and caudomedial v-DAO innervates the more rostral (Fig. 7B, red, brown, yellow; C, green, blue) and caudal (Fig. 7B, cyan, blue, pink; C, red, brown, yellow, blue) AIN, respectively. The topographic projection from the central and rostral DM to the DLH could be simply understood by extending the topographic projection from the v-DAO to the AIN.
In summary, the topography of the group IV was characterized by assuming two orientation axes (Sugihara and Shinoda, 2004
Group V
Cortical group V area has been defined as the flocculus and nodulus in the cerebellar cortex, which are innervated by the dorsal cap subnucleus and ventrolateral outgrowth subnucleus (Sugihara et al., 2004Compartmentalization of the entire CN and its correspondence to the aldolase C expression pattern in the CN
To understand how the CN are divided into the termination areas of the five groups, three-dimensional schemes of the termination areas of each group (Figs. 4–7, bottom panels) were assembled into a single model of the CN, which was then cut at several parasagittal and coronal planes to show schematic parasagittal and coronal sections with group organization indicated by colors (Fig. 8). The schematic sections in Figure 8 were obtained roughly at the same levels as the photomicrographs of stained sections in Figure 1. Comparison of Figure 1, A and B, and Figure 8, A and B, indicated that the termination areas of groups I (green), II (blue and cyan), and V (gray) are located in an aldolase C-positive caudoventral part of the CN and those of groups III (yellow and orange) and IV (red and pink) are located in an aldolase C-negative rostrodorsal part of the CN. Indeed, such a relationship was noticed during mapping of nuclear termination areas in individual experiments, because brain sections were double labeled for BDA and aldolase C. The correspondence of termination areas of groups I, II, and V with aldolase C-positive areas was directly shown by comparing dorsocaudal views of the three-dimensional displays of these areas (Fig. 9). The correspondence between the five groups and aldolase C expression in the CN was consistent with that in the cerebellar cortex, because groups I, II, and V also occupied aldolase C-positive compartments in the cerebellar cortex (Sugihara and Shinoda, 2004
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Figure 8. Schematic summary of compartmentalization of the CN shown in parasagittal (Aa–Ag, medial to lateral) and coronal (Ba–Bd, caudal to rostral) sections of the left CN. Vertical bars in Bb (a–g) indicate approximate locations of parasagittal sections in Aa–Ag. Colors indicate groups (green, group I; cyan, group IIa; blue, group IIb; yellow and orange, group III; red and pink, group IV; gray, group V) as used in the bottom panels of Figures 4
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Figure 9. Three-dimensional reconstruction of the aldolase C-positive area (top) and termination areas of olivonuclear projections (bottom) that belong to groups I (green), II (blue), and V in the left CN shown in a dorsocaudal view for comparison. The wire frames are coronal contours of the CN. Group V termination areas are mostly hidden under group I areas in this view.
Concerning the FN, the group I area was located most ventrally (Fig. 8Aa,Ab,Bb, green), and the group IIa area was located next most ventrally (Fig. 8Aa–Ac,Ba–Bd, cyan). The group IIb area was located in the dorsocaudal FN (Fig. 8Aa,Ab,Ba,Bb, blue). These areas (groups I, IIa, and IIb) were aldolase C positive. Group III areas (Fig. 8Aa–Ac,Ba–Bd, yellow, orange) occupied the rostrodorsal portion and the DLP of the FN, which were aldolase C negative. Concerning the ICG, the group I area was located in the ventral part of the ICG (Fig. 8Ac,Ba,Bb, green), and the group III area was located in the dorsal part of the ICG (Fig. 8Ac,Ba,Bb, yellow).The entire AIN, including the DLH, belonged to group IV (Fig. 8Ac–Af,Bb–Bd, red) and was generally aldolase C negative, although the DMC had a slightly higher level of aldolase C labeling. In the PIN, group III was located in the aldolase C-negative dorsomedial portion (Fig. 8Ad,Ba,Bb, orange), and group IIa was located in the medial and rostral part of the most ventral portion (Fig. 8Ad,Ae,Ba,Bb, cyan). The rest of the PIN belonged to group I (Fig. 8Ad,Ae,Ba,Bb, green). Group IIa and I areas were aldolase C positive in the PIN. The entire DN, which was aldolase C positive, belonged to group I (Fig. 8Af,Ag,Bb–Bd, green), except for the most ventral and rostral portion, which belonged to group V (Fig. 8Af,Ag,Bb,Bc, gray). The CP, which was aldolase C positive, belonged to group I (Fig. 8Af,Ba, green). This finding further supported the notion that the CP belongs to the DN but not to the DLH or AIN.
The AICG and LVN, which were generally aldolase C negative, belonged to group IV (Fig. 8Ad,Bc,Bd, pink). The dorsal Y nucleus (infracerebellar nucleus), which was aldolase C positive, belonged to group V (Fig. 8Ae,Bc, gray).
Cytoarchitecture of the nuclear termination areas
Different parts of the CN differ with regard to the size and density of neurons (Voogd, 2004
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Table 2. Major olivocortical and olivonuclear topographic projection pattern in the rat
Group I areas contained medium-sized neurons in the ventral FN and ICG and large or middle-sized round neurons in the dorsal PIN and DN (Table 2). Group II areas contained small to medium-sized neurons, which were round or polygonal in the caudoventral FN and round or fusiform in the ventral PIN. The entire group III area in the rostrodorsal FN, dorsal ICG, and dorsomedial PIN contained large polygonal neurons. Cytoarchitecture in group IV areas showed more variation than in other areas. Group V areas contained small to middle-sized round or fusiform neurons.Overall, areas belonging to the same group sometimes showed, although not always, similar cytoarchitecture across different subnuclei. Furthermore, the borders between groups within the FN, ICG, PIN, and DN tended to coincide with the borders between areas with different cytoarchitecture. Specifically, the rostrodorsal "shell-shaped area" of the FN with large neurons (Voogd, 2004
Topographic olivonuclear organization across the groups in the FN
The organization of the CN as a whole and the orientation axes of the olivonuclear projection are summarized in Figure 10 and compared with the organization of the cerebellar cortex and the IO that was reported previously (Sugihara and Shinoda, 2004
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Figure 10. Schematics of the topography of five group-based compartmental organization of the right IO, left cerebellar cortex, and left CN of the rat. A, Subnuclei of the right IO in a dorsal view. In subpanels a–c, some subnuclei that are located more dorsally were omitted to show more ventral subnuclei (a, MAO; b, MAO, v-PO, DM, DMCC, BETA; c, MAO, v-PO, DM, DMCC, BETA, d-PO; d, the entire IO). B, Unfolded left cerebellar cortex. The group organization here is basically the same as in our previous report (Sugihara and Shinoda, 2004
In the AIN and dorsal main portions of the PIN and DN, which belonged to either group I or IV, the olivocerebellar topography was clearly organized in two-dimensional space. In contrast, the entire FN and ICG were divided into groups I–III and showed no comparable two-dimensionally organized topography. However, some topographic organization could be discerned across groups involved in the projection from the c-MAO to the FN and ICG. In the c-MAO, subnucleus a (green, group I), lateral subnucleus b (yellow, group III), medial subnucleus b (orange, group III), and subnucleus c (blue, group IIb) are located from the lateral to medial portions (Fig. 10Ab,Da, gray arrow). Subnucleus ß and caudal subnucleus c (cyan, group IIa) are then located dorsocaudally to medial subnucleus c. The above consecutive subareas of the c-MAO projected to different subareas in the FN, which were aligned sequentially from the ventral FN (green, group I), through the rostrodorsal FN (yellow, group III), the DLP (orange, group III), and the caudal neck of the DLP and the caudal FN (blue, group IIb), to the caudoventral FN (cyan, group IIa), as shown by the gray arrow in Figure 10Db. These findings indicated that the positional arrangement of groups was preserved in the projection from the c-MAO to the FN.
Discussion
