Aldolase C (zebrin) expression in Purkinje cells reveals stripe-shaped compartments in the cerebellar cortex. However, it is not clear how these compartments are related to cerebellar functional localization. Therefore, we identified olivocerebellar projections to aldolase C compartments by labeling climbing fibers with biotinylated dextran injected into various small areas within the inferior olive in rats. Specific rostral and caudal aldolase C compartments were linked in an orderly manner by common olivocerebellar projection across the rostrocaudal boundary on lobule VIc-crus Ib. Based on the localization of the olivary origins of projection to similar compartments, the compartments and olivocerebellar projections could be sorted into five groups: group I, positive compartments extending from the posterior lobe to the anterior lobe innervated by the principal olive and some neighboring areas; group II, positive compartments localized within the posterior lobe innervated by several medial subnuclei; group III, vermal and central negative compartments innervated by the centrocaudal medial accessory olive; group IV, negative and lightly positive compartments in the hemisphere and the rostral and caudal pars intermedia innervated by the dorsal accessory olive and some neighboring areas; group V, the flocculus and nodulus. The olivocerebellar topography within each group was simple and suggests an "orientation axis" within the concerned parts of the inferior olive. Furthermore, parts of the inferior olive in each group receive specific afferent inputs, indicating a close relationship between aldolase C compartments and functional localization. Thus, the five-group scheme we propose here may integrate the molecular, topographic, and functional organization of the cerebellum.

The olivocerebellar system is known to generate periodic synchronous discharges that result in synchronous (to within 1 msec) climbing fiber activation of Purkinje cells (complex spikes) organized in parasagittally oriented strips. These results have been obtained primarily in anesthetized animals, and so the question remains whether the olivocerebellar system generates such patterns in the awake animal. To this end, multiple electrode recordings of crus 2a complex spike activity were obtained in awake rats conditioned to execute tongue movements in response to a tone. After removal of all movementand tone-related activity, the remaining data were examined to characterize spontaneous complex spike activity in the alert animal. Spontaneous complex spikes occurred at an average firing rate of 1 Hz and a clear Ϸ10 Hz rhythmicity. Analysis of the autocorrelograms using a rhythm index indicated that the large majority of Purkinje cells displayed rhythmicity, similar to that in the anesthetized preparation. In addition, the patterns of synchronous complex spike activity were also similar to those observed in the anesthetized preparation (i.e., simultaneous activity was found predominantly among Purkinje cells located within the same parasagittally oriented strip of cortex). The results provide unequivocal evidence that the olivocerebellar system is capable of generating periodic patterns of synchronous activity in the awake animal. These findings support the extrapolation of previous results obtained in the anesthetized preparation to the waking state and are consistent with the timing hypothesis concerning the role of the olivocerebellar system in motor coordination.

1. The role of gamma-aminobutyric acid (GABA) on the pattern generation properties of neuronal ensembles in the olivocerebellar system was studied utilizing multiple electrode recordings of complex spikes (CSs) from rat crus 2a Purkinje cells (PCs). Initially multiple electrode experiments were combined with microinjections of picrotoxin into the inferior olive (IO). To corroborate the picrotoxin findings, the cerebellar nuclei, a major source of the GABAergic terminals in the IO, were chemically lesioned with the use of microinjections of kainic acid and N-methyl-D-aspartate. Both procedures generated comparable results. 2. After intraolivary picrotoxin injection there was an increase in the average firing rate, synchrony, and rhythmicity of spontaneous CS activity. In addition, the neuronal oscillation frequency tended to shift to lower frequencies. 3. The spatial distribution of synchronous CS activity in control conditions displayed a predominantly rostrocaudal orientation. Injection of picrotoxin to the IO disrupted this rostrocaudal organization and led to synchronous CS activity among PCs throughout crus 2a. Similar effects were observed relating to the distribution of CSs evoked via the "climbing fiber reflex," in which antidromic activation of the climbing fibers is followed by a return excitation that is mediated by the gap junctions between olivary neurons. 4. Chemical lesions of the cerebellar nuclei resulted in increased CS average firing rates. The effect of the lesions on CS synchronicity was similar to that following the picrotoxin injections, but greater in magnitude. In contrast to the olivary picrotoxin injections, the cerebellar nuclear lesions did not lead to an enhanced CS rhythmicity. 5. Bilateral recordings from left and right crus 2a demonstrated significant interhemispheric synchronization of CS activity, consistent with a previous report. Both unilateral olivary injections of picrotoxin and unilateral cerebellar nuclear lesions resulted in increased synchronization of CS activity between the left and right crus 2a. 6. We conclude that the cerebellar nucleoolivary projection to the olivary glomeruli modulates the effective electrotonic coupling between olivary neurons, and thereby carves out ensembles of neurons whose activity is synchronized. Thus these two nuclei may form the basis for a flexible and sophisticated motor coordination system able to help generate the many distinct movements that organisms are capable of performing.

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.

The functional partitioning of the cerebellar cortex depends on the projection patterns of its afferent and efferent neurons. However, the entire morphology of individual projection neurons has been demonstrated in only a few classes of neurons in the vertebrate CNS. To investigate the contribution of the projection pattern of individual olivocerebellar axons to the cerebellar functional compartmentalization, we labeled individual olivocerebellar axons, which terminate in the cerebellar cortex as climbing fibers, with biotinylated dextran amine injected into the inferior olive in the rat, and completely reconstructed the entire trajectories of 34 olivocerebellar axons from serial sections of the cerebellum and medulla. Single axons had seven climbing fibers on average, which terminated at similar distances from the midline in a single or in multiple lobules. Cortical projection areas of adjacent olivary neurons were clustered as narrow but separate longitudinal segments and often innervated by collaterals of single neurons. Comparison of the cerebellar distribution of olivocerebellar axons arising from different sites within a single olivary subnucleus indicated that slightly distant neurons projected to complementary sets of such segments in a single longitudinal band. Several of these longitudinal bands formed a so-called parasagittal zone innervated by a subnucleus of the inferior olive. Single olivocerebellar axons projected rostrocaudally to segments within a single band but did not project mediolaterally to multiple bands. These results suggest fine substructural organization in the cerebellar compartmentalization that may represent functional units.

Aldolase C (zebrin II) is expressed in Purkinje cells aligned in complicated longitudinal stripe-shaped compartments. The tight link between these aldolase C compartments and the topographic olivocerebellar projection to them has made it possible to identify each compartment as a target of a specific subarea of the inferior olive and thus as a functionally distinct entity in the rat. However, it is unknown whether the overall organization of aldolase C compartments is preserved in other mammals. In this study, we tried to clarify this organization in the mouse, which is more useful in genetic studies than the rat, by identifying each aldolase C compartment in terms of the olivocerebellar projection pattern. First, aldolase C compartments were reconstructed from serial sections throughout the cerebellar cortex. Aldolase C and olivocerebellar climbing fibers were then doubly labeled by small injections of biotinylated dextran amine into various areas of the inferior olive. Climbing fibers were topographically distributed on a specific linked pair of aldolase C compartments in the rostral and caudal cerebellum. The overall relationship between aldolase C compartments and the topographic olivocerebellar projection to them in the mouse was similar to that in the rat, except for some minor differences, suggesting that the aldolase C compartments and olivocerebellar projection are organized according to a common fundamental organization in the mouse and rat. This allowed the unequivocal identification of all aldolase C compartments in the mouse by referring to the definition and nomenclature in the rat.

3. Multiple-electrode recording of spontaneous Purkinje cell CS activity was employed to study the spatial extent of CS synchronicity in the cerebellar cortex. Recordings of CS were obtained from Purkinje cells located on the surface and along the walls of lobule crus 2a. The rostrocaudal band-like distribution of simultaneous (within 1 ms) CS activity in Purkinje cells extended down the sides of the cerebellar folia to the deepest areas recorded (1-6-2-6 mm deep). As shown in previous experiments, the distribution of simultaneous CS activity did not extend significantly (500 ,um) in the mediolateral axis of the cerebellar cortex.4. In two animals a detailed determination of the length of the olivocerebellar fibre bundles was performed by staining the fibres with PHA-L injected into the contralateral inferior olive. This measurement included fibre bundles terminating in twenty-six different areas, ranging from the tops of the various folia to the bottoms of the fissures in both the hemisphere and the vermis. There was a 47-5 % difference between the length of the longest measured fibre bundle (15-8 mm, terminating in lobule 6b, zone A) and the length of the shortest measured fibre bundle (8-3 mm, terminating in the cortex at the base of the primary fissure, zone D), after correction for tissue shrinkage. To attain an isochronous conduction time the conduction velocities for these two fibre bundles were calculated to be 4-22 m/s and 2-37 m/s, respectively. * Present address: Department of Physiology, Tokyo Medical and Dental University School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. MS 1708I. SUGIHARA, E. J. LANG AND R. LLINAS 5. By interpolating between measured points a simple formula was derived to estimate the average length of olivocerebellar fibres terminating in any given area of the cerebellar cortex, excluding the paraflocculus, the flocculus and the most lateral regions of the hemisphere.6. We investigated the most likely mechanisms by which conduction velocity variations with length could result in global isochronicity. We found that longer branches tended to have thicker diameters than shorter branches, indicating more rapid conduction velocities; however, other parameters such as internodal distance could not be unambiguously determined.7. We conclude that the isochronicity of the climbing fibre activation of Purkinje cells is the result of a differential conduction velocity in the climbing fibre system such that longer fibres conduct faster than shorter ones. Thus while the cerebellar cortex is a deeply folded structure, the conduction time for the climbing fibre system is tuned such that the cortex functions, in the time domain, as an unfolded surface.

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