Yoshikazu Shinoda

Molecular, Topographic, and Functional Organization of the Cerebellar Cortex: A Study with Combined Aldolase C and Olivocerebellar Labeling

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.

Morphology of Single Olivocerebellar Axons Labeled With Biotinylated Dextran Amine in the Rat

The morphology of olivocerebellar (OC) axons originating from the inferior olive (IO) was investigated in the rat by reconstructing the entire trajectories of single axons that had been labeled with biotinylated dextran amine. Virtually all of the OC axons entered the cerebellum through the inferior cerebellar peduncle (ICP) contralateral to the IO, with a few exceptions. Although most OC projection was contralateral, a few axons projected bilaterally by crossing the midline within the cerebellum. Collaterals of OC axons could be classified into thick branches and thin collaterals. Thick branches of each OC axon (6.1 ± 3.7/OC axon, mean ± SD for n = 16 axons) terminated as climbing fibers (CFs) on single Purkinje cells (PCs) in a one‐to‐one relationship. Besides terminal arborization around PC thick dendrites, CFs had terminals that surrounded a PC soma, fine branchlets that extended transversely in the molecular layer, and thin retrograde collaterals that re‐entered the PC and granular layers. Innervation of a single PC by two CFs originating from the same axon was seen, although infrequently. Concerning thin collaterals, about half of the OC axons had one or only a few collaterals terminating in the white matter of the ICP, most had 1 to 6 collaterals terminating in a single cerebellar nucleus, and all had 3 to 16 collaterals that terminated mainly in the granular layer, but occasionally in the cerebellar white matter and the PC layer. Some swellings of thin collaterals touched somata of presumed Golgi cells and PCs. No OC axons terminated solely in the ICP, cerebellar nucleus or granular layer without giving rise to CFs. J. Comp. Neurol. 414:131–148, 1999.

Projection Patterns of Single Mossy Fibers Originating from the Lateral Reticular Nucleus in the Rat Cerebellar Cortex and Nuclei

Projection of neurons in the lateral reticular nucleus (LRN) to the cerebellar cortex (Cx) and the deep cerebellar nuclei (DCN) was studied in the rat by using the anterograde tracer biotinylated dextran amine (BDA). After injection of BDA into the LRN, labeled terminals were seen bilaterally in most cases in the vermis, intermediate zone, and hemisphere of the anterior lobe, and in various areas in the posterior lobe, except the flocculus, paraflocculus, and nodulus. Areas of dense terminal projection were often organized in multiple longitudinal zones. The entire axonal trajectory of single axons of labeled LRN neurons was reconstructed from serial sections. Stem axons entered the cerebellum through the inferior cerebellar peduncle (mostly ipsilateral), and ran transversely in the deep cerebellar white matter. They often entered the contralateral side across the midline. Along the way, primary collaterals were successively given off from the transversely running stem axons at almost right angles to the Cx and DCN, and individual primary collaterals had longitudinal arborizations that terminated as mossy fibers in multiple lobules of the Cx. These collaterals arising from single LRN axons terminated bilaterally or unilaterally in the vermis, intermediate area, and sometimes hemisphere, and in different cerebellar and vestibular nuclei simultaneously. The cortical terminals of single axons appeared to be distributed in multiple longitudinal zones that were arranged in a mediolateral direction. All of the LRN axons examined (n = 29) had axon collaterals to the DCN. All of the terminals observed in the DCN and vestibular nuclei belonged to axon collaterals of mossy fibers terminating in the Cx. J. Comp. Neurol. 411:97–118, 1999.

Molecular, Topographic, and Functional Organization of the Cerebellar Nuclei: Analysis by Three-Dimensional Mapping of the Olivonuclear Projection and Aldolase C Labeling

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.

Functional compartmentalization in the flocculus and the ventral dentate and dorsal group y nuclei: An analysis of single olivocerebellar axonal morphology

The cerebellar cortex consists of multiple longitudinal bands defined by their olivocerebellar projection. Single olivocerebellar axons project to a narrow longitudinal band in the cerebellar cortex and to the cerebellar nucleus with their axon collaterals. This olivocortical and olivonuclear organization is related to the functional compartmentalization of the cerebellar system. To reveal the detailed morphologic organization in the flocculus and the cerebellar and vestibular nuclei, we examined olivocerebellar projection by reconstructing the entire trajectories of 19 single olivofloccular axons and by anterograde mapping with biotinylated dextran in the rat. The flocculus was composed of 12 longitudinal band‐shaped compartments that subdivided 5 previously described zones. These longitudinal bands were innervated differentially by the caudal and rostral portions of the dorsal cap (DC) and the ventrolateral outgrowth (VLO) and the rostral pole of the medial accessory olive. Single olivofloccular axons with an average of 5.1 climbing fibers usually projected to a single longitudinal band in the flocculus and to the ventral dentate or dorsal group y nucleus with their collaterals. DC neurons projected moderately to the rostrolateral portion of the ventral dentate nucleus, whereas VLO neurons projected densely to the medial portion of the ventral dentate nucleus and the dorsal group y nucleus with rostrocaudal topography. DC and VLO neurons did not project to the vestibular nuclei, although floccular Purkinje cells projected to the vestibular, ventral dentate, and dorsal group y nuclei. The fine morphologically identified longitudinal bands and topographic olivonuclear projections were correlated with previous electrophysiologically defined functional zones in the flocculus and inferior olive. J. Comp. Neurol. 470:113–133, 2004.

Morphology of single neurones in the cerebello-rubrospinal system

Axonal branching patterns of physiologically identified cerebellar nucleus neurones and rubrospinal neurones were investigated in the cat with intra-axonal injection of horseradish peroxidase and 3-dimensional reconstruction on serial sections. Axons of dentate and interpositus neurones projected to the VL nucleus of the thalamus and on their way, several axon collaterals were given off from the stem axons to the red nucleus. Axon terminals of interpositus neurones terminated as a sagittal sheet of arborizations in the red nucleus. Their terminal boutons made apparent contact with cell bodies and proximal dendrites of rubrospinal neurones. In rubrospinal axons, multiple axon collaterals were identified at different segments of the cervical spinal cord.

Synaptic organization of the cerebello-thalamo-cerebral pathway in the cat. II: Input-output organization of single thalamocortical neurons in the ventrolateral thalamus

Input-output neural organization of single thalamocortical (T-C) neurons in the ventrolateral nucleus (VL) of the thalamus was investigated using an intracellular recording technique in the anesthetized cat. Stimulation of the dentate (DN) and the interpositus (IN) nuclei produced monosynaptic unitary EPSPs of large amplitude in T-C neurons projecting to the motor cortex or area 6 over the entire mediolateral region of VL. The thalamic projections from DN and IN are very wide and there is a considerable overlap between the dentate and the interpositus projection areas in VL. And in this overlapping area, a considerable number of T-C neurons (50%) receive inputs from both DN and IN. More than 40% of T-C neurons were antidromically activated from widely separated electrodes in the motor cortex, indicating that the cortical arbolization of single T-C neurons is very wide and the number of these neurons with widely divergent projections is considerably large.

Excitatory inputs to cerebellar dentate nucleus neurons from the cerebral cortex in the cat

Summary1. In anesthetized cats, we investigated excitatory and inhibitory inputs from the cerebral cortex to dentate nucleus neurons (DNNs) and determined the pathways responsible for mediating these inputs to DNNs. 2. Intracellular recordings were made from 201 DNNs whose locations were histologically determined. These neurons were identified as efferent DNNs by their antidromic responses to stimulation of the contralateral red nucleus (RN). Stimulation of the contralateral pericruciate cortex produced excitatory postsynaptic potentials (EPSPs) followed by long-lasting inhibitory postsynaptic potentials (IPSPs) in DNNs. The most effective stimulating sites for inducing these responses were observed in the medial portion (area 6) and its adjacent middle portion (area 4) of the precruciate gyrus. Convergence of cerebral inputs from area 4 and area 6 to single DNNs was rare. 3. To determine the precerebellar nuclei responsible for mediation of the cerebral inputs to the dentate nucleus (DN), we examined the effects of stimulation of the pontine nucleus (PN), the nucleus reticularis tegmenti pontis (NRTP) and the inferior olive (IO). Systematic mapping was made in the NRTP and the PN to find effective low-threshold stimulating sites for evoking monosynaptic EPSPs in DNNs. Stimulation of either the PN or the NRTP produced monosynaptic EPSPs and polysynaptic IPSPs in DNNs. Using a conditioning-testing paradigm (a conditioning stimulus to the cerebral peduncle (CP) and a test stimulus to the PN or the NRTP) and intracellular recordings from DNNs, we tested cerebral effects on neurons in the PN and the NRTP making a monosynaptic connection with DNNs. Conditioning stimulation of the CP facilitated PN- and NRTP-induced monosynaptic EPSPs in DNNs. This spatial facilitation indicated that the excitatory inputs from the cerebral cortex to DNNs are at least partly relayed via the PN and the NRTP. 4. Stimulation of the contralateral IO produced monosynaptic EPSPs and polysynaptic IPSPs in DNNs. These monosynaptic EPSPs were facilitated by conditioning stimulation of the CP, strongly suggesting that the IO is partly responsible for mediating excitatory inputs from the cerebral cortex to the DN. A comparison was made between the latencies of IO-evoked IPSPs in DNNs and the latencies of IO-evoked complex spikes in Purkinje cells. Such a comparison indicated that the shortest-latency IPSPs evoked from the IO were not mediated via the Purkinje cells and suggested the pathway mediated by inhibitory interneurons in the DN. 5. The functional significance of the excitatory inputs from the PN and the NRTP to the DN is discussed in relation to the motor control mechanisms of the cerebellum.

Thalamic terminal morphology and distribution of single corticothalamic axons originating from layers 5 and 6 of the cat motor cortex

We investigated the axonal morphology of single corticothalamic (CT) neurons of the motor cortex (Mx) in the cat thalamus, using a neuronal tracer, biotinylated dextran amine (BDA). After localized injection of BDA into the Mx, labeled CT axons were found ipsilaterally in the thalamic reticular nucleus (TRN), the ventroanterior–ventrolateral complex (VA‐VL), the central lateral nucleus (CL), the central medial nucleus, and the centromedian nucleus, but with the primary focus in the VA‐VL. The terminals in the VA‐VL formed a large laminar cluster, which extended approximately in parallel with the internal medullary lamina. The laminar organization mirrored morphologic features of single CT axons. We reconstructed the trajectories of 25 single CT axons that arose from layer V (16 axons) or layer VI (9 axons) and terminated in the VA‐VL. Terminals of single CT axons that originated from both layer V and layer VI were confined within a laminar structure about 700 μm thick, suggesting the existence of laminar input organization in the VA‐VL. Otherwise, the two groups of the CT axons showed contrasting features. All of the CT axons derived from layer VI gave rise to a few short collaterals to the TRN and then formed extensive arborization with numerous small, drumstick‐like terminals in the VA‐VL. On the other hand, the CT axons arising from layer V gave rise to collaterals whose main axons descended into the cerebral peduncle. Each collateral projected to the VA‐VL or CL without projection to the TRN and formed a few small clusters of giant terminals. The two groups of CT neurons in the same cortical column had convergent rather than segregated termination in the VA‐VL. However, the terminals of layer VI CT neurons were distributed diffusely and widely in the VA‐VL, whereas the terminals of layer V CT neurons were much more focused and surrounded by the terminals of the former group. These contrasting features of the two types of CT projections appear to represent their different functional roles in the generation of motor commands and control of movements in the Mx. J. Comp. Neurol. 437:170–185, 2001.

Vestibular projection to the periarcuate cortex in the monkey.

Vestibular inputs to the cerebral cortex are important for spatial orientation, body equilibrium, and head and eye movements. We examined vestibular input to the periarcuate cortex in the Japanese monkey by analyzing laminar field potentials evoked by electrical stimulation of the vestibular nerve. Laminar field potential analysis in the depths of the cerebral cortex showed that vestibular-evoked potentials consisted of early-positive and late-negative potentials and early-negative and late-positive potentials in the superficial and deep layers of the periarcuate cortex, respectively, with latencies of 4.8-6.3 ms, suggesting that these potentials were directly conveyed to the cortex through the thalamus. These potentials were distributed continuously in the fundus, dorsal and ventral banks of the spur and the bottom of the junctional part of the arcuate sulcus and spur. This vestibular-projecting area overlapped the cortical distribution of corticovestibular neurons that were retrogradely labeled by tracer injection into the vestibular nuclei (previously reported area 6 pa), and also the distribution of smooth pursuit-related neurons recorded in the periarcuate cortex including area 8 in a trained monkey. These results are discussed in relation to the function of vestibular information in control of smooth pursuit and efferents of the smooth pursuit-related frontal eye field.