Specificity and labeling properties of anti-aldolase C antibodies. A, Western blots using antibodies against aldolase C. In comparing Coomasie staining of standard proteins (Prestained precision protein standards; 162-0372; Bio-Rad, Hercules, CA) and sample proteins of the cerebellum, a single identical band (39 kDa) was recognized in the Western blots of sample proteins by purified antibodies 69075 (b; 250 ng/ml) and 69076 (c; 125 ng/ml). The results with 12% SDS-polyacrylamide gel are shown here. B, Concentration-dependent blocking by the antigen peptide of immunohistological labeling of populations of Purkinje cells located in multiple zones in lobules VIII and IX. Adjacent coronal sections were incubated with one of the primary antibodies against aldolase C (20 ng/ml 69705, top row; 10 ng/ml 69076, bottom row) and various concentrations (51.2, 12.8, 3.2, 0.8, and 0 μg/ml; from left to right) of the peptide. C, High-magnification photomicrograph of a labeled Purkinje cell. The somata and dendrites as well as axons and axonal terminals are labeled by the anti-aldolase C antibody. D-F, Photomicrographs of coronal sections of the cerebellum at the caudal (D), intermediate (E), and rostral (F) levels labeled with the antibody 69076. Lines show the continuity of positive compartments across folia. I-X, Lobules I-X; a-c, sublobules a-c; CP, copula pyramidis; Cr I, crus I of ansiform lobule; CrII, crus II of the ansiform lobule; sim, simple lobule.

Reconstruction of aldolase C compartments through out the cerebellar cortex. Continuity of the compartments in the rostrocaudal direction was determined by accurate tracing of individual compartments on serial sections and then plotted on a map of the unfolded cerebellar cortex (see Materials and Methods). Several new compartments were discovered (marked by asterisks prefixed to their proposed names) beyond those identified previously by Hawkes and Leclerc (), Voogd et al. (), and Voogd and Ruigrok (). A positive compartment and its laterally neighboring negative compartment usually have the same name with different suffixes (+, positive compartment; -, negative compartment). The intensity of the gray color approximately corresponds to the intensity of labeling. The dotted line on the left indicates the rostrocaudal boundary of the compartment pattern (see Results). Arrowheads indicate areas in which the molecular layer is absent, one of which in the caudal apex of lobule VIc-crus I approximately coincided with the rostrocaudal border. I-X, Lobules I-X; a-c, sublobules a-c; CP, copula pyramidis; Cr I, crus I of ansiform lobule; Cr II, crus II of ansiform lobule; DPFL, dorsal paraflocculus; FL, flocculus; Par, paramedian lobule; pf, primary fissure; Sim, simple lobule; VPFL, ventral paraflocculus.

Narrow band-shaped longitudinal olivocerebellar projection connects corresponding rostral and caudal aldolase C compartments. A, Photomicrographs of several coronal sections showing that the narrow band of the distribution of labeled climbing fibers (asterisks) is parallel with and belongs to an aldolase C compartment. The brackets in each panel indicate positive compartments. All labeled climbing fibers were located in the medial portion of rostral compartment 1- or caudal compartment 1-, hereafter classified as 1-(med)//1- (see Results). B, C, Photomicrograph (B) and plot of the injection site in the inferior olive (C; horizontal representation of the left inferior olive) (the same experiment as shown in A). D, Distribution of all labeled climbing fibers in the experiment shown in A (red) and three other experiments (green, blue, violet) plotted in an unfolded representation of the cerebellar cortex. The colored squares indicate locations of individual labeled climbing fibers. The colored circles indicate the approximate areas of the photographs in A, E, F, and G. The dotted transverse line indicates the rostrocaudal boundary. E-G, Photomicrographs of double staining of aldolase C and climbing fibers in the three experiments plotted in green, blue, and violet in D. Labeled climbing fibers are indicated by circles, the colors of which correspond to those in D. The injection sites of these experiments were located in the lateral subnucleus b of the caudal part of the MAO (c-MAO; E), the rostral subnucleus b of the c-MAO (F), and the rostral part of the MAO (r-MAO; G). I-X, Lobules I-X; a-c, sublobules a-c; CP, copula pyramidis; Sim, simple lobule.

Mapping of labeled climbing fibers and summarized topographic scheme of the olivocerebellar projection belonging to group I. This group consisted of projections from caudal subnucleus a and the intermediate and rostral subnucleus a of the caudal part of the MAO (c-MAO) to 1+//1+ and 2+//3+, respectively, from the rostral part of the MAO (r-MAO) to 4+//5+; from the ventral lamella of the PO (v-PO), except for the caudomedial part, to 5+//6+; and from the dorsal lamella of the PO (d-PO) to 6+//7+. A-C, Plots of labeled climbing fibers in 19 injections terminating in 1+//1+ and 2+//3+ (A), 4+//5+ (B), and 5+//6+ and 6+//7+ (C). Labeled climbing fibers in all of these experiments (A-C) were nearly aligned in a single narrow band in a pair of linked rostral and caudal aldolase C compartments, except for one case (B; red), in which some outside climbing fibers were seen. D, Putative olivocerebellar topography within group I.

Mapping of labeled climbing fibers and summarized topographic scheme of the olivocerebellar projection belonging to group II. This group consisted of projections from subnucleus c of the caudal part of the MAO (c-MAO) to a+//2+, c+//4b+, and d+//5a+; from subnucleus β (BETA) to a+//2+; and from the DMCC, caudal dorsomedial subnucleus of the PO (DM), and caudomedial ventral lamella of the PO (v-PO) to 2b+//4+. A, Plots of climbing fibers in eight injections terminating in a+//2+ labeled with injections into the subnucleus β and caudal subnucleus c of the c-MAO. Two injections (red and light green) were spread to both the c-MAO and subnucleus β. B, Plots of climbing fibers in five injections terminating in a+//2+, c+//4b+, and d+//5a+ and retrogradely labeled olivary neurons in three experiments (yellow green, green, and purple). Plots of injection sites and retrogradely labeled neurons in the inferior olive were classified into medial and lateral ones and drawn separately to improve visibility. The yellow injection was spread to the subnucleus β, presumably explaining the labeled climbing fibers in lobules VIII-IX in this case. C, Plots of climbing fibers in five injections terminating in 2b+//4+ and retrogradely labeled olivary neurons in two experiments (light blue and purple circles). Labeled climbing fibers in each anterograde labeling experiment (A, B) were nearly aligned in a single narrow band in a pair of linked rostral and caudal aldolase C compartments with few, if any, outside climbing fibers, except for a few cases (A, blue and light blue; B, blue) in which climbing fibers were distributed in multiple compartments. D, Putative olivocerebellar topography within group II.

Mapping of labeled climbing fibers and summarized topographic scheme of the olivocerebellar projection belonging to group III. This group consisted of projections from subnucleus b of the caudal part of the MAO (c-MAO) to 1-(med)//1-, 1-(lat)/a-//2-, 2a-//3-, 2b-//4a-, c-//4b-, d-//5a-, and 3b-//e2-. A, Plots of labeled climbing fibers in seven injections terminating in 1-(med)//1-, 1-(lat)/a-//2-, and 2a-//3-. B, Plots of labeled climbing fibers in seven injections terminating in 2b-//4a-, c-//5a-, d-//5a-, and 3b-//e2-. Labeled climbing fibers in each anterograde injection in A and B were nearly aligned in a single narrow band with few, if any, outside climbing fibers. C, Putative olivocerebellar topography within group III determined from the results shown in A and B and 13 other cases. b, Subnucleus b.

Mapping of labeled climbing fibers and summarized topographic scheme of the olivocerebellar projection belonging to group IV. This group consisted of projections from the dorsal fold of the DAO (d-DAO), ventral fold of the DAO (v-DAO), and dorsomedial subnucleus of the PO (DM) to 2-//4-, b+//f+, b-//f-, 3+//e+, 3-//e1-, 4-//5-, and 5-//6-, and possibly to 3b+//e2+. A, Plots of labeled climbing fibers in nine injections terminating mainly in compartments 2-//4-, b+//f+, b-//f-, and 3+//e+. Two injections (green and light green) were spread to the d-DAO and v-DAO. B, Plots of labeled climbing fibers in four injections in the intermediate v-DAO, terminating in several compartments, including 3-//e1- (yellow). C, Plots of labeled climbing fibers in five injections in the medial v-DAO, terminating mainly in 4-//5- and also in caudal 5a-. D, Plots of labeled climbing fibers in four injections in the DM, terminating in 5-//6- and in caudal 5-. Labeled climbing fibers in 9 of 22 experiments were virtually aligned in a single narrow band, if a small number of outside climbing fibers can be ignored. E, Putative olivocerebellar topography within group III determined from the results shown in A and B.

Organization of aldolase C compartments and topographic olivocerebellar projection based on the five groups in the present study. A, Topographic organization explained with orientation axes. Areas belonging to each of the five groups (green, blue, yellow, red, and gray for groups I-V, respectively) were mapped on the aldolase C compartment pattern of the cerebellar cortex (left) and on the solid schemes of the inferior olive (right). Arrows with dark colors indicate the mediolateral direction in each group in the cerebellar cortex and the orientation axes in the inferior olive. The orientation axes correspond to the medial to lateral direction in the cerebellar cortex in terms of the topography of the projection. Group II has two orientation axes, corresponding to the projection to the central cortical area from the rostral and lateral subnucleus c (violet) and to the projection to the rostral and caudal cortical areas from the caudal subnucleus c, subnucleus β, DMCC, caudal dorsomedial subnucleus of the PO (DM), and caudomedial ventral lamella of the PO (v-PO) (blue). This panel is based on the data shown in Figures , , , . Group V (gray) consists of the flocculus and nodulus and a few areas in the hemisphere innervated by the DC and VLO (). B, A schematic of major inputs to the inferior olive reported so far sorted according to the groups classified in the present study. Question mark indicates that the specific inputs in those areas have not been clarified. See Discussion for details. Origins of major afferents to the inferior olive are arbitrarily colored. APP, Area parafascicularis prerubralis; DCN, dorsal column nucleus; IO, inferior olive; ND, nucleus of Darkschewitsch; NOT, nucleus of the optic tract; RN, red nucleus; SC, superior colliculus; VN, vestibular nucleus. C, Correspondence between the aldolase C-based five-group olivocerebellar organization in the present study and conventional olivocerebellar zones (zones A-D). Colors indicate the same groups as in panels A and B. I-X, Lobules I-X; a-c, sublobules a-c or subnuclei a-c; BETA, subnucleus β; c-MAO, caudal part of the MAO; CP, copula pyramidis; Cr I, crus I of ansiform lobule; Cr II, crus II of ansiform lobule; d-DAO, dorsal fold of the DAO; d-PO, dorsal lamella of the PO; DC, DC of Kooy; DM, dorsomedial subnucleus of the PO; DMCC, DMCC subnucleus; DPFL, dorsal paraflocculus; FL, flocculus; Param, paramedian lobule; r-MAO, rostral part of the MAO; Sim, simple lobule; v-DAO, ventral fold of the DAO; v-PO, ventral lamella of the PO; VPFL, ventral paraflocculus.