Olivocerebellar Axons Research Articles

Part I: The Complex Spikes as One of the Cerebellar Secrets

The olivocerebellar tract has unique morphological, physiological, and developmental properties. Olivocerebellar axons are the source of multiple climbing fibers (CFs). The synapse between CFs and the Purkinje neuron is one of the most powerful excitatory in the central nervous system. Complex spikes are composed of an initial large amplitude spike followed by spikelets. The spatiotemporal patterns of complex/simple spikes complement the rate coding to enhance the accuracy of motor and cognitive processing, and to improve predictions related to internal models. Understanding the role of complex spikes is essential in clarifying how the cerebellar cortex contributes to learning, motor control, cognitive tasks, and the processing of emotions. This Cerebellar Classic is devoted to the pioneering work of Eccles, Llinás, and Sasaki on complex spikes using intracellular recordings from Purkinje neurons.

Compartmentalization of the chick cerebellar cortex based on the link between the striped expression pattern of aldolase C and the topographic olivocerebellar projection.

The avian cerebellum is organized into multiple longitudinal stripes defined by expression profiles of aldolase C (zebrin II) in Purkinje cells. The relationship between the aldolase C striped pattern and the olivocerebellar projection pattern is crucial in understanding cerebellar functional compartmentalization. We identified all aldolase C stripes across all lobules with the serial section alignment analysis method and then looked at this relationship by anterograde and retrograde labeling of olivocerebellar axons in the chick cerebellum. Aldolase C stripes were generally consistent and continuous from lobule I through VII and to the medial part of lobules VIII-IXb. The dorsal and ventral lamellas (DL, VL) of the inferior olive projected to the stripes in these areas with a simple mediolateral topographic relation. A few aldolase C stripes appeared at the lateral edge of lobules VI-VIII. Several more stripes were added in the lateral parts of lobules IXa-IXb and IXc-X. The medial column (MC) of the inferior olive projected to the stripes in lobules VIII-X, including the added lateral stripes, with a complex topographic relation. Sharp boundaries between aldolase C-positive and -negative stripes often accompanied a gap in the Purkinje cell layer and bordered topographically distinct groups of axons. Although the compartmental organization of the chick cerebellum is comparable to that of the mammalian cerebellum, several significant differences in the organization suggest partly separate evolutionary lineages of the mammalian and avian cerebella. We propose that rostral lobules may be evolved by rostral extension of medial stripes from caudal lobules in the avian cerebellum.

A large-scale physiological model of Inferior Olive neurons reveals climbing fiber intra-burst frequency depends on Olivocerebellar axon morphology

We have constructed a large-scale structural model of the Inferior Olive. Using this model as input to our neural tissue simulator, we constrained a multi-compartment (>1000 compartments) model of an olivary neuron, including dendrites, a cell body, and axon. Our physiological model derives from Schweighofer et al. [1], with changes to its several channel conductances (Na, K, Cah, Cal, KCa, and h) targeted to neuronal branches in order to replicate olivary oscillations in our more detailed structure, which includes a full-scale olivocerebellar axon and climbing fibers. Our implementation also computes detailed Ca2+ balance solved in parallel with membrane potential and channel currents for each compartment. Furthermore, a larger circuit model comprising these neurons includes glomeruli subject to anatomical constraints on neuronal touch detection, and targets to them a collection of gap junctions, which contribute to both Ca2+ balance and electrotonic coupling. Previous work has demonstrated that spike generation and bursting properties of olivary neurons depend on axonal lengths [2], but these lengths varied only from 0-300 μm in the slice preparation studied. Our structural model, inspired by measures of real Olivocerebellar axons [3], employs a 3-D mesh of the Purkinje cell layer of rat cerebellum to constrain axonal morphology and length (Figure 1A, B). This method generates a distribution of axonal lengths that more closely resembles the in vivo distribution, which is on the order of millimeters. Figure 1 A. Short axon (grey) Olivo-cerebellar neuron. Full dendritic morphology (inset) is downsampled to create dendritic morphology (green). B. Identical dendrites with long axon. C. Short axon bursts. D. Long axon bursts. With our simulator, we explored how preserving precise dendritic morphology, channel distributions, and gap junctional coupling, while varying axonal lengths according to our distribution, affects model dynamics. We demonstrate that the intra-burst spike frequency and number depend on these variations in axonal lengths from 1-5 mm (Figure 1C, D). Therefore, we argue that axons should not be considered structural constraints on only microcircuit connectivity in neural tissue simulation, but also on the dynamics of neuronal spike generating systems.

Morphology of the olivocerebellar projection of the chick: An axonal reconstruction study

The projection pattern of the olivocerebellar (OC) axons, which terminate mainly as climbing fibers (CFs) in the cerebellar cortex, tightly reflects the compartmental and developmental organization of the cerebellum as revealed by mapping and reconstruction studies in the rat. The avian cerebellum is well lobulated and longitudinally compartmentalized like the mammalian cerebellum. However, the projection pattern of the OC axons has not been studied in detail for most areas of the avian cerebellum. In the present study, we reconstructed labeled chick OC axons resulting from a small focal injection of biotinylated dextran amine into the inferior olive to investigate their morphological characteristics, and to determine their relationship to the general morphology of the chick cerebellum. Labeled CFs were distributed basically in a single longitudinally elongated narrow band-shaped area in lobules I-VIII, but in multiple, transversely widened, band-shaped areas in lobules IX-X. Three of the four reconstructed OC axons terminated in a single longitudinally band-shaped area in lobules IXa-c, whereas the other one terminated in multiple mediolaterally separated areas in lobule IXc, which is part of the flocculus. Single OC axons branched into 14 CFs on average. Two CFs occasionally merged to form a single terminal arbor. Axons also had thin, non-CF collaterals that projected either to a cerebellar nucleus or to the cortex. The results indicate that the morphological characteristics of OC axons, including branching and termination, are basically conserved between the chick and the rat.

Branching patterns of olivocerebellar axons in relation to the compartmental organization of the cerebellum

A single olivocerebellar (OC) axon gives rise to about seven branches that terminate as climbing fibers (CFs). Branching patterns of an OC axon, which are classified into local, transverse, and longitudinal types, are highly organized, in relation to the longitudinal molecular (aldolase C or zebrin II) compartmentalization and the transverse lobulation of the cerebellum. Local branching is involved in forming a narrow band-shaped functional subarea within a molecular compartment. On the other hand, transverse and longitudinal branchings appear to be involved in linking mediolaterally separated molecular compartments and rostrocaudally separated lobular areas, respectively. Longitudinal branching occurs frequently between equivalent molecular compartments of specific combinations of lobules. These combinations include lobule V-simple lobule and crus II-paramedian lobule in the pars intermedia and hemisphere, and lobules I–V and lobule VIII in the vermis. The longitudinal branching pattern not only fits with mirror-imaged somatosensory double representation of the body in the pars intermedia, but it also suggests a general rostrocaudal link exists for the whole cerebellum across the putative rostrocaudal boundary in lobule VIc-crus I. Molecular compartments of the cerebellar cortex originate from the Purkinje cell (PC) clusters that appear in the late embryonic stage, when the immature OC projection is formed. Some clusters split rostrocaudally across crus I during the development of cortical compartments, which would result in longitudinal branching of OC projection across crus I. Supposing that the branching pattern of OC axons represents an essential organization of the cerebellum, longitudinal branching suggests a functional and developmental links between the rostral and caudal cerebellum across lobule VIc-crus I throughout the cerebellar cortex.

Structural plasticity of climbing fibers and the growth-associated protein GAP-43

Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, CFs undergo a regressive modification when their target is deleted. In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.

Morphology of single olivocerebellar axons in the denervation–reinnervation model produced by subtotal lesion of the rat inferior olive

An olivocerebellar axon branches into about seven climbing fibers (CFs) as well as several non-CF thin collaterals that terminate mainly in the cerebellar nuclei and in the granular layer. Systemic administration of 3-acetylpyridine (3-AP) induces degeneration of inferior olive neurons. A small number of inferior olive neurons survive the 3-AP treatment and grow up to make compensatory reinnervation on nearby Purkinje cells in their CFs. To elucidate the correlation between denervation–reinnervation processes and morphology of the whole axonal arbor, we reconstructed axonal trajectories of inferior olive neurons in the 3-AP-treated rat in the present study. The number of CFs per axon (average and S.D., 1.6±1.1, n=11 axons) was significantly smaller in the 3-AP-treated rat than that in the untreated rat (about seven), indicating that inferior olive neurons that have a smaller number of CFs are more resistant to 3-AP neurotoxity. Predominant growth of CFs, as well as sprouting of thin collaterals in the cerebellar nuclei and in the granular layer, was observed in the 3-AP-treated rat. A CF was larger in size when an axon had one CF than when an axon had multiple CFs. CFs that were much smaller than the normal CF (“small CFs” in contrast to other “large CFs”) were occasionally encountered at the end of thin collaterals and may be new CFs formed from non-CF thin collaterals. The results suggest that the capacity of surviving axons to reinnervate is dependent on targets and possibly limited by neuronal metabolism and axonal flow.

Synaptic action of the olivocerebellar system on cerebellar nuclear spike activity.

Cerebellar output is necessary for the ideal implementation of many nervous system functions, particularly motor coordination. A key step toward understanding the generation of this output is characterizing the factors that shape the activity of the cerebellar nuclei (CN). There are four major sources of synaptic input that modulate CN activity; collaterals of climbing and mossy fibers are two, and the remaining two are provided by Purkinje cell (PC) axons in the form of simple spikes (SSs) and complex spikes (CSs). Most hypotheses of cerebellar function focus on SSs as the primary determinant of CN activity. However, it is likely that CSs also cause significant direct effects on CN activity, something that is rarely considered. To explore this possibility, we recorded from synaptically connected PC-CN neuron cell pairs in rats. Cross-correlograms of CS and CN activity from such recordings demonstrate that spontaneous CSs have a strong inhibitory effect on CN activity, apparently sufficient, in some cases, to trigger changes in the intrinsic excitability of the CN neuron that long outlast the underlying CS-mediated GABAergic IPSP. Furthermore, many CS-CN correlograms show an initial excitatory response, demonstrating the ability of climbing fiber collaterals to significantly excite CN neurons. A substantial fraction (24%) of correlograms displayed an excitation-inhibition sequence, providing evidence that a CN neuron often receives collaterals from the same olivocerebellar axons as innervate the PCs projecting to it. Thus, excitation followed by inhibition appears to be a hard-wired response pattern of many CN neurons to olivocerebellar activity.

Axonal Motility and Its Modulation by Activity Are Branch-Type Specific in the Intact Adult Cerebellum

We performed two-photon in vivo imaging of cerebellar climbing fibers (CFs; the terminal arbor of olivocerebellar axons) in adult mice. CF ascending branches innervate Purkinje cells while CF transverse branches show a near complete failure to form conventional synapses. Time-lapse imaging over hours or days revealed that ascending branches were very stable. However, transverse branches were highly dynamic, exhibiting rapid elongation and retraction and varicosity turnover. Thus, different branches of the same axon, with different innervation patterns, display branch type-specific motility in the adult cerebellum. Furthermore, dynamic changes in transverse branch length were almost completely suppressed by pharmacological stimulation of olivary firing.

Brain-derived neurotrophic factor induces post-lesion transcommissural growth of olivary axons that develop normal climbing fibers on mature Purkinje cells

In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, post-lesion plasticity may generate alternate paths providing models to investigate factors that promote reinnervation to appropriate targets. Following unilateral transection of the neonatal rat olivocerebellar pathway, axons from the remaining inferior olive reinnervate the denervated hemicerebellum and develop climbing fiber arbors on Purkinje cells. However, the capacity to recreate this accurate target reinnervation in a mature system remains unknown. In rats lesioned on day 15 (P15) or 30 and treated with intracerebellar injection of brain-derived neurotrophic factor (BDNF) or vehicle 24 h later, the morphology and organisation of transcommissural olivocerebellar reinnervation was examined using neuronal tracing and immunohistochemistry. In all animals BDNF, but not vehicle, induced transcommissural olivocerebellar axonal growth into the denervated hemicerebellum. The distribution of reinnervating climbing fibers was not confined to the injection sites but extended throughout the denervated hemivermis and, less densely, up to 3.5 mm into the hemisphere. Transcommissural olivocerebellar axons were organised into parasagittal microzones that were almost symmetrical to those in the right hemicerebellum. Reinnervating climbing fiber arbors were predominantly normal, but in the P30-lesioned group 10% were either branched within the molecular layer forming a smaller secondary arbor or were less branched, and in the P15 lesion group the reinnervating arbors extended their terminals almost to the pial surface and were larger than control arbors ( P < 0.02). These results show that BDNF can induce transcommissural olivocerebellar reinnervation, which resembles developmental neuroplasticity to promote appropriate target reinnervation in a mature environment.

Isochrony in the olivocerebellar system underlies complex spike synchrony

In a recent issue of The Journal of PhysiologyBaker & Edgley (2006) published results which they interpreted as showing a non-uniform olivocerebellar conduction time in the rat. Baker and Edgley used the climbing fibre reflex to measure conduction time to different points along a folial wall. This reflex is initiated by electrical stimulation of the cerebellar white matter, which triggers antidromic spikes in olivocerebellar axons that invade the inferior olive (IO). Current generated by these spikes spreads to neighbouring IO neurones via the gap junctions that electrically couple IO neurones (Llinas et al. 1974). This spreading excitation can trigger orthodromic spikes that return to the cerebellar cortex and trigger Purkinje cell complex spikes (CSs). Baker and Edgley found that climbing fibre reflex latency varied with recording electrode depth, and inferred from this that conduction time between the IO and cerebellar cortex varies systematically with cortical location. This study conflicts with results by us and others that indicate a near uniform olivocerebellar conduction time in rats and turtles (Sugihara et al. 1993; Lang & Rosenbluth, 2003; Ariel, 2005). Below we discuss several troubling issues with this new study, but first we would like to make the point that spontaneous CS synchrony patterns almost necessitate a uniform olivocerebellar conduction time.

Organization and remodeling of the olivocerebellar climbing fiber projection

Climbing fibers, terminal portions of the axons of inferior olive neurons, form strong synaptic connections to Purkinje cells in an exclusive one-to-one relationship. This projection is established during development by drastic reshaping in each climbing fiber and in overall axonal arborization. Early climbing fibers form loose 'creeper'-type terminal arbors that seem to make weak contact with many Purkinje cells in the first postnatal week. The terminal arbor then becomes focused on a single Purkinje cell with the aggregation of swellings ('transitional' type), and eventually tightly surrounds the Purkinje cell soma ('nest' type) in the second postnatal week. The terminal arbor is then displaced upward to the stem of the apical dendrite of the Purkinje cell ('capuchon' or 'hood') and eventually to the proximal portion of the dendritic tree (mature climbing fiber). Single-axon morphology in rats has shown that olivocerebellar axons in the creeper stage branch more frequently and have many more climbing fibers than those in adults. The climbing fibers that originate from an axon are largely organized into microzones as in adults. Concomitant with this remodeling of climbing fibers, the number of climbing fibers per olivocerebellar axon is significantly decreased by the putative retraction of climbing fibers during development from the creeper to nest stage. Due to additional retraction after the nest stage, an olivocerebellar axon in an adult has about seven climbing fibers. The above morphological remodeling and retraction during development can be closely compared to the changes in climbing fiber-Purkinje cell synaptic interaction observed in rats and mice. Generation and aggregation of the swellings in the terminal arbor between the creeper and nest stages are correlated with maturation of the synaptic connection. The decrease in climbing fibers in the same and following periods is correlated with the elimination of overabundant synapses to establish one-to-one connections.

Purkinje cell spinogenesis during architectural rewiring in the mature cerebellum

Spines can grow and retract within hours of activity perturbation. We investigated the time course of spine formation in a model of plasticity involving changes in brain architecture where spines of a dendritic domain become innervated by a different neuronal population. Following a lesion of rat olivocerebellar axons, by severing the inferior cerebellar peduncle, new spines grow on the deafferented proximal dendrite of the Purkinje cells (PCs) and these new spines become innervated by parallel fibres (PFs) that normally contact only the distal dendrites. The varicosities of climbing fibre (CF) terminal arbors disappear within 3 days of the lesion. Spine density in the proximal dendritic domain begins to rise within 3 days and continues to increase towards a plateau at 6-8 days. In 'slow Wallerian degeneration' mice, in which axonal degeneration is delayed, climbing fibre varicosities virtually disappear at 14 rather than 3 days. Spine density in the proximal dendritic domain is similar to control Purkinje cells up to 14 days and increases significantly 18 days postlesion. The delayed spinogenesis in the latter mutant is the result of a persistence of the climbing fibre presynaptic structure in the absence of activity. Therefore, climbing fibre activity itself is not directly responsible for the suppression of spine formation, but suppression mechanisms tend to become weaker as long as the structural dismantling of the presynaptic varicosities proceeds. Thus, spinogenesis is guided by two different mechanisms; a rapid one related to changes in homotypic remodeling and a slower one, which requires the removal of a competitive afferent.

Microzonal projection and climbing fiber remodeling in single olivocerebellar axons of newborn rats at postnatal days 4–7

An adult olivocerebellar axon ramifies into about seven climbing fibers that innervate single Purkinje cells arranged in a longitudinal microzone. To clarify the developmental basis of this projection, individual olivocerebellar axons were labeled with biotinylated dextran amine injected into the inferior olive in rats at postnatal days 4-7. The entire trajectories of single olivocerebellar axons and single terminal arbors of climbing fibers were reconstructed from serial sections of the cerebellum and medulla. Single axons ramified into climbing fibers that terminated in a narrow band-shaped area comparable to the adult microzone. This indicated that olivocerebellar microzones are predetermined. Terminal arbors of climbing fibers were remodeled from loose creeper type, through intermediate transitional type, into dense nest type. Each olivocerebellar axon had some 100 nascent climbing fibers in the creeper stage, whereas each axon had about 10 climbing fibers and about as many atrophic climbing fibers in the nest stage. This decrease indicated that overabundant nascent climbing fibers degenerate concomitantly with the remodeling of remaining climbing fibers. Atrophic terminal arbors and non-climbing fiber thin collaterals were considered the intermediate forms of degenerating climbing fibers. This remodeling and degeneration of climbing fibers may be related to the electrophysiological regression of climbing fiber-Purkinje cell synapses. The remodeling of climbing fibers occurred earliest in lobules VIII (caudal part) and IXa-b, and then in lobules IXc and X. The more developed granular layer in these areas compared to other areas suggests that the cortical environment triggers climbing fiber remodeling.

Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

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.

A novel morphological technique to investigate a single climbing fibre synaptogenesis with a Purkinje cell in the developing mouse cerebellum: DiI injection into the inferior cerebellar peduncle

To study morphologically the relationship between climbing fibre and Purkinje cell in the developing mouse cerebellum, we established a novel tract tracing using injection of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the inferior cerebellar peduncle, the half point of olivocerebellar projection. In this tracing method, only a certain number of climbing fibres were labelled with DiI and they revealed the single-fibre resolution, individually. These technical advantages enabled us to follow the projection of a climbing fibre to a Purkinje cell at the light microscopic level. To further investigate how a single labelled olivocerebellar axon interacts with a Purkinje cell, we introduced a photoconversion method into this tracing method and successfully observed the photo-oxidized climbing fibre terminals at the electron microscopic level. At postnatal days 7 and 9, a single DiI-labelled climbing fibre arborized around some adjacent Purkinje cell bodies in a distinguishable nest. At this pericellular nest stage, we first demonstrated that the terminal arborization stemmed from a single climbing fibre formed synapses simultaneously on both a soma and dendrites of a Purkinje cell. This finding suggests that the pericellular nest may be such an efficient form that a single climbing fibre innervates a Purkinje cell at both perisomatic and peridendritic sites. Thus, we succeeded in establishing an effective tracing method to investigate a single climbing fibre synaptogenesis with a Purkinje cell both at the light and electron microscopic levels.

Target-Specific Innervation of Embryonic Cerebellar Transplants by Regenerating Olivocerebellar Axons in the Adult Rat

The reestablishment of topographically organized connections is a necessary prerequisite to obtain a full anatomical repair following brain injury. One system where such an issue can be addressed is the olivocerebellar system, where, normally, clusters of inferior olive neurons project to neurochemically heterogeneous Purkinje cell compartments defined by the expression of cell-specific markers, such as zebrin II. To assess whether adult injured olivocerebellar axons that regenerate into cerebellar transplants are able to establish target-specific innervation of grafted Purkinje cells, we made surgical transections in the white matter of adult rat cerebella and placed solid grafts from the embryonic cerebellar anlage into the lesion site. The transplanted tissue developed highly organized minicerebella, in which Purkinje cells were distributed into distinct clusters of zebrin II-immunopositive or -immunonegative neurons, mimicking the cortical compartments present in the normal adult cerebellum. Olivocerebellar axons, labeled by biotinylated dextran amine tracing, regenerated into the transplants where they formed discrete patches made of several terminal arbors impinging upon Purkinje cell dendrites. Among 401 such climbing fiber patches, 96% exclusively innervated Purkinje cells of either phenotype and stopped at the border of the zebrin II+/− Purkinje cell clusters, whereas only 4% were extended across this boundary and innervated both zebrin II-positive and -negative Purkinje cells. The results obtained support the view that the embryonic cerebellar tissue provides target-specific information that can be decoded by ingrowing adult olivocerebellar axons in order to establish appropriate innervation patterns with zebrin II-positive or -negative Purkinje cell compartments.

The Entire Trajectories of Single Olivocerebellar Axons in the Cerebellar Cortex and their Contribution to Cerebellar Compartmentalization

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.

Olivocerebellar Climbing Fibers in the Granuloprival Cerebellum: Morphological Study of Individual Axonal Projections in the X-Irradiated Rat

Elimination of cerebellar granule cells early during postnatal development produces abnormal neural organization that retains immature characteristics in the adult, including innervation of each Purkinje cell by multiple climbing fibers from the inferior olive. To elucidate mechanisms underlying development of the olivocerebellar projection, we studied light-microscopic morphology of single olivocerebellar axons labeled with biotinylated dextran amine in adult rats rendered agranular by a single postnatal X-irradiation. Each reconstructed olivocerebellar axon gave off approximately 12 climbing fibers, approximately twice as many as in normal rats. Terminal arborizations of climbing fibers made irregular tufts in most areas, whereas they were arranged vertically in a few mildly affected areas. Each climbing fiber terminal arborization innervated only part of the dendritic arbor of a Purkinje cell, and multiple climbing fibers innervated a single Purkinje cell. These climbing fibers originated either from the same olivocerebellar axon (pseudomultiple innervation) or from distinct axons (true multiple innervation). Abundant non-climbing fiber thin collaterals projected to all cortical layers. Although the longitudinal pattern of the zonal olivocerebellar projection was generally observed, lateral branching, including bilateral projections, was relatively frequent. These results suggest that the granule cell-parallel fiber system induces several important features of olivocerebellar projection: (1) organization of the climbing fiber terminal arborization tightly surrounding Purkinje cell dendrites, (2) elimination of pseudo- and true multiple innervations establishing one-to-one innervation, (3) retraction of non-climbing fiber thin collaterals from the molecular layer, and (4) probable refinement of the longitudinal projection domains by removing aberrant transverse branches.