Cerebellar target neurons provide a stop signal for afferent neurite extension in vitro.

The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.     

Postnatal maturation of cerebellar mossy and climbing fibers: transient expression of dual features on single axons

We have studied the form and fine structure of developing afferent axons in postnatal mouse cerebellum, before and during the formation of synaptic connections. In slices of fresh brain, bundles of axons were injected with horseradish peroxidase (HRP), and individual axons were examined in the light and electron microscopes. At birth, before formation of cortical layers, axons with growing tips are rare in the peduncular tracts but instead ramify throughout the cerebellar anlage. All axons have similar structures; they branch infrequently and terminate in bud-like tips and/or small growth cones. Growth cones contain small and large vesicles in the flank and small vesicles in filopodia. Typical mossy and climbing fiber branching patterns and bouton shapes are recognizable after postnatal day (P) 5, even though fibers are still intermingled in a plexus beneath the newly formed Purkinje cell layer. Climbing fiber-like axon arbors are highly branched and covered with small foliate growing tips that contact Purkinje cells. Mossy fiber-like branches have large irregular expansions that give rise to long filopodia and resemble growth cones seen in vitro. The flanks of these growth cones contact granule cell dendrites and form glomeruli typical of mossy fibers, whereas the filopodia make primitive contacts or are associated with coated vesicles in adjacent profiles. A novel finding is the occurrence during the second postnatal week of many single axons that simultaneously have the morphology and synaptic connections of both climbing and mossy fibers. These "combination" axons have some branches that extend into the granule cell layer and others that enter the Purkinje cell layer, with the shape and synaptic connections of terminals on each branch type corresponding to the respective layer. Climbing fiber-like branches, including those on combination fibers, extend over several adjacent Purkinje cells. Combination fibers are rare in late postnatal or adult stages. These results suggest that long after arrival in the cerebellum, afferent axons have similar elementary forms and overlap in their projections. Mature axonal forms are not exhibited until cellular layers develop. During a limited period of postnatal maturation, some axons have dual morphologies and synaptic relations with appropriate and inappropriate partners. These aspects of cerebellar axonal development, particularly the transient exuberant branching onto two types of target cells, offer a valuable opportunity to examine, in developing cerebellum, the sorting out of afferents and the formation of specific synaptic connections.     

The development of synaptic contacts in the cerebellum of Macaca mulatta

The maturation of various cerebellar cortical cells, the appearance of afferent fibers to the cerebellum, and the development of synaptic contacts in the cerebellar cortex and deep nuclei was investigated in the fetal macaque. Ultrastructural studies were done on cerebellum obtained from fetuses at 75, 100, 125 and 150 days after conception to interrelate the temporal development of these three systems. At 75 days, synaptic contacts were seen on somas and axons of neurons in the deep cerebellar nuclei, and climbing fibers formed pericellular baskets around Purkinje cells. By 100 days the climbing fibers synapsed with somatic spines of the Purkinje cells, and mossy fiber endings were present in the internal granule cell layer. Synaptic contacts were also seen on dendritic processes of neurons in the deep cerebellar nuclei at this time. In the 125 day cerebellum, Golgi cells were identified for the first time and climbing fibers and parallel fibers made synaptic contact with both Purkinje and Golgi cells. At 150 days parallel fibers made synaptic contact with superficial stellate cells and mature cerebellar glomeruli had appeared. At this stage, axosomatic contacts of climbing fibers on the soma of Purkinje cells had disappeared. The relationship of these anatomical observations to possible functional activity is discussed.     

Early climbing fiber interactions with Purkinje cells in the postnatal mouse cerebellum.

The time and place of initial contacts between afferent axons and their target cells are not known for most regions of the mammalian CNS. To address this issue, we have selectively visualized afferent climbing fiber axons together with their synaptic targets, Purkinje cells, in postnatal mouse cerebellum. Climbing fibers were orthogradely labeled by injection of rhodamine isothiocyanate into their brainstem source, the inferior olivary nucleus. Purkinje cells were localized with an antibody to a calcium-binding protein, calbindin D-28k (CaBP), in the same section or in adjacent sections. A novel view of the olivocerebellar projection and the morphology of climbing fiber arbors prior to the well-known "nest" stage has emerged from this analysis. At birth, climbing fibers project into the zone of Purkinje cells, before these cells have aligned into a monolayer. During this phase, climbing fibers have simple morphologies consisting of relatively unbranched terminal arbors and small tapered growing tips. Purkinje cells are arranged 3-6 cells deep and have tufted dendrites and relatively smooth somata. By postnatal days 3-4, climbing fibers branch over several adjacent Purkinje cell perikarya, which are still organized in a band several cells thick. From postnatal days 5-7, when climbing fibers subsequently make focused nests on individual cells, Purkinje somata are smoother and form a more distinct monolayer. Up to this time, however, climbing fibers continue to associate with Purkinje perikarya, even though Purkinje cell dendrites have emerged and branched extensively. By postnatal days 8-10, climbing fiber terminals climb onto the trunk of the relatively mature Purkinje dendritic tree. At birth, mossy fibers originating from the pontine nuclei resemble immature climbing fibers in that they also have a simple unbranched morphology and growing tips, but project only so far as the internal granule cell layer. Occasional individual fibers reach into the Purkinje zone both at postnatal day 0 and postnatal day 4, confirming that the fibers formerly described as "combination fibers" (Mason and Gregory, S4. J. Neurosci, 4:1715-1735) can be mossy in origin. These data demonstrate that climbing fibers project among Purkinje cells earlier than suspected, before these afferents begin to arborize and form pericellular nests. Our observations are not in accord with the view derived from autoradiographic tracing studies that as in other cortical areas, climbing afferents wait in the vicinity of Purkinje cells in the early neonatal period, then advance onto these cells in synchrony with Purkinje cell alignment into a monolayer and dendritic maturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogenesis of enkephalinergic afferent systems in the opossum cerebellum

Enkephalin (ENK) immunoreactive climbing fibers, mossy fibers and a beaded plexus of axons are present in the adult opossum's cerebellar cortex. We have used the indirect antibody peroxidase-antiperoxidase technique to study the ontogeny of enkephalinergic axons in the cerebellum of pouch young opossums from postnatal day (PD) 1 to PD 83. On PD 1, ENK axons are present in the intermediate layer of the cerebellar anlage. At PD 18, after a period of 'waiting', ENK fibers form clusters throughout the cerebellar cortex primarily within the nascent Purkinje cell layer. By PD 40, axon terminals with a climbing fiber phenotype circumscribe Purkinje cells; immature mossy fiber rosettes are present within the internal granule cell layer. A third axon phenotype, beaded ENK fibers can be distinguished on PD 68. Between PD 40 and PD 68, the distributions of ENK climbing and mossy fibers overlap in vermal lobules II-VIII and X, whereas in the hemispheres climbing fibers predominate. However, by PD 83, ENK positive climbing fibers are no longer evident in lateral folia. These results indicate that early arriving ENK axons are present before the differentiation of their cellular targets. Further, a transient appearance of ENK in discrete populations of developing climbing fibers suggests several developmental events: (1) cell death in the inferior olive, (2) collateral regression, or (3) a transient expression of this peptide, that may be characteristic of this chemically defined system of axons.     

Congruence of mossy fiber and climbing fiber tactile projections in the lateral hemispheres of the rat cerebellum

We have examined the spatial relationship between the mossy fiber and climbing fiber projections to crus IIa in the lateral hemispheres of the rat cerebellum. Experiments were performed in ketamine/xylazine anesthetized rats using extracellular recordings and high-density micromapping techniques. Responses were elicited using small, tactile stimuli applied to the perioral and forelimb regions at a rate of 0.5 Hz. In our first series of experiments we demonstrate that the primary (i.e., strongest) receptive field for a single Purkinje cell's complex spike is similar to the primary receptive field of the granule cells immediately subjacent to that Purkinje cell. In our second series of experiments we demonstrate that the granule cell region most strongly activated by a particular peripheral stimulus is immediately subjacent to the Purkinje cells whose complex spikes are also activated most strongly by the same stimulus. The region of climbing fibers activated by a localized peripheral stimulus is "patchy"; it clearly does not conform to the notion of a continuous microzone. These results support original observations first reported in the 1960s using evoked potential recording techniques that the mossy fiber and climbing fiber pathways converge in cerebellar cortex. However, we extend this earlier work to show that the two pathways converge at the level of single Purkinje cells. Many cerebellar theories assume that mossy fiber and climbing fiber pathways carry information from different peripheral locations or different modalities to cerebellar Purkinje cells. Our results appear to contradict this basic assumption for at least the tactile regions of the lateral hemispheres.     

Embryonic Purkinje Cells Grafted on the Surface of the Cerebellar Cortex Integrate in the Adult Unlesioned Cerebellum

The presence of an injury or the selective degeneration of specific neuronal populations is commonly assumed to be a necessary prerequisite for the survival and the integration of grafted neurons in the recipient brain. In the present study we have placed solid grafts of cerebellar anlage in the fourth ventricle of adult rats, in close contact with the host cerebellar cortex, to assess the capacity of embryonic Purkinje cells to interact with adult neurons and integrate in the unlesioned cerebellar cortex. Numerous grafted Purkinje cells are indeed able to leave the implant and migrate into the host molecular layer, where they develop adult structural features. In addition, such cells are able to elicit the growth of host climbing fibre sprouts which end in newly formed arborizations impinging upon their dendritic trees. Climbing fibre collateral branches also penetrate the implant to innervate Purkinje cells which have not migrated in the host cerebellum. These results show that embryonic Purkinje cells are able to survive and integrate in an adult unlesioned cerebellar cortex. In addition, adult olivary axons respond to the increased size of the target population by expanding their terminal domain to innervate grafted Purkinje cells.     

Changes in the responses of cerebellar nuclear neurons associated with the climbing fiber response of Purkinje cells

Previous studies demonstrated that climbing fiber activity produces a short-term increase in the responsiveness of Purkinje cells to mossy fiber inputs. This led to the hypothesis that there are concomitant alterations in the discharge of cerebellar nuclear neurons. This series of experiments was initiated to test this hypothesis in simultaneously recorded Purkinje cell-nuclear cell pairs in related regions of the cerebellar cortex and nuclei. In decerebrate cats 50 pairs of Purkinje cells and nuclear neurons were identified and simultaneously recorded during spontaneous activity and during peripheral inputs. Auto-correlograms of nuclear cell activity and cross-correlograms of the simple spike and nuclear cell activity triggered on the occurrence of spontaneous complex spikes demonstrated little correlation between these events and the discharge of nuclear neurons. To examine the effect of evoked climbing fiber inputs on the Purkinje cell simple spike and nuclear cell responses, square wave mechanical stimuli which modulated the discharge of both cells of a pair were applied to the forepaw. A separation technique was used to construct one histogram illustrating the responses of the nuclear neuron and Purkinje cell in trials in which the peripheral stimulus evoked a climbing fiber input to the Purkinje cell and another histogram showing their responses in trials in which no climbing fiber input was activated. Using this separation technique it was shown that the amplitude of most Purkinje cell responses increased by 120-1200% in trials in which climbing fiber inputs were activated. The response amplitude of 68% of the nuclear cells was modified for these pairs. Most changes in nuclear cell responses were increases ranging from 120-220%. These changes were felt to reflect the action of many Purkinje cells converging on the isolated nuclear neuron. The modulation of the nuclear neuron was not due only to the effect of the related Purkinje cell, since the gain change of the Purkinje cell and nuclear cell of each pair was not correlated (r = 0.01). The discussion of these findings emphasizes that the increased responses of the nuclear cell are most likely produced by the intracortical action of the climbing fiber system on the responsiveness of Purkinje cells to mossy fiber inputs. Climbing fiber collateral input to nuclear neurons also may contribute to the changes in the nuclear cell responses observed in these experiments.     

Ultrastructural evidence for compensatory sprouting of climbing and mossy afferents to the cerebellar hemisphere after ipsilateral pedunculotomy in the newborn rat

Unilateral section of the inferior and middle cerebellar peduncles was performed in rats at postnatal days 1 or 2. The ultrastructure of the cerebellar hemispheric cortex ipsilateral to the lesion was examined 3 months later. The absence of contralateral inferior olive and of ipsilateral middle peduncle, together with a marked regression of the contralateral pontine gray, were indicative of successful pedunculotomy. In spite of a relative atrophy of the hemisphere, its cytological structure was qualitatively normal. Mossy and climbing fibers were present and their terminal varicosities disclosed normal features. The density of climbing fiber terminals was reduced compared to control cerebellum, whereas the density of mossy terminals seemed unchanged. Subsequent to the reduction of climbing afferents two subclasses, or types, of Purkinje cells were present: A “normal” type characterized by its climbing fiber innervation and a “hyperspiny” type devoid of climbing fiber. In some of the adult rats pedunculotomized at birth, section of the contralateral peduncles was performed 24 hours before fixation. Terminal degeneration of climbing and mossy fibers was observed in the neonatally deprived hemisphere, providing the proof that these fibers result from a compensatory transcommissural sprouting of afferents destined to the contralateral hemicerebellum. These results demonstrate that the cerebellar cortex neonatally deprived of its main afferents can be innervated by climbing and mossy fibers through a process of transcommissural sprouting. Although the newly formed synapses maintain their target specificity, a functional reorganization must occur because of the altered distribution of both systems of afferents.     

Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. I. Postsynaptic potentials

The development of the synaptic responses of intracerebellar nuclei neurons was studied in the rat by the use of thick sagittal cerebellar slices maintained in vitro. It has been shown that functional excitatory synapses are present on these neurons from birth, probably due to climbing and/or mossy fiber collaterals; functional inhibitory synapses, due to monosynaptic projections of Purkinje cell axons onto intracerebellar nuclei, are present as early as postnatal day 2; and a more complex pattern of synaptic responses, including short latency excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs), longer latency IPSPs, and late depolarizing responses, can be elicited in nuclear neurons as early as postnatal day 3, indicating an early development of some complete functional cerebellar circuits involving the intracerebellar nuclei.     

Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1.

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.     

Synaptic 5'-nucleotidase is transient and indicative of climbing fiber plasticity during the postnatal development of rat cerebellum

The transient appearance of 5'-nucleotidase, an adenosine-producing ecto-enzyme, was studied during specific stages of postnatal synaptogenesis in the rat cerebellum. For ultrastructural detection of 5'-nucleotidase activity, an enzyme-cytochemical technique was used. Between postnatal days 4 and 6, enzymatic reaction product was present in the synaptic clefts of climbing fibers containing the perisomatic spines, apical cones and emerging dendrites of Purkinje cells (CF-PC synapses). Labeled parallel fiber synapses were observed on dendritic shafts of cerebellar interneurons. At postnatal days 9 and 12, enzyme-positive parallel fiber terminals were in addition numerous on the spines of peripheral Purkinje branchlets, and gradually disappeared thereafter. Between postnatal days 8 and 15, labeling of perisomatic CF-PC contacts persisted. In contrast, climbing fiber synapses on Purkinje dendrites were only occasionally labeled. Between postnatal days 18 and 21, synaptic reaction product was restricted to mossy fibers. At the same time, association of 5'-nucleotidase with glial profiles was prominent throughout the cerebellar layers. In adult cerebellum (from 24 days onwards) all synapses were devoid of enzymatic activity. Throughout development, basket, stellate and Golgi cell synapses were devoid of enzymatic activity. We conclude that 5'-nucleotidase is present in excitatory cerebellar synapses during part of their generation period. The transient nature of this phenomenon suggests that 5'-nucleotidase may serve as a novel, cytochemical marker for a specific state of synaptic maturation, and in particular for climbing fiber plasticity. A role of 5'-nucleotidase in purinergic neuromodulation and cellular contact formation could be significant in these processes.     

Postsynaptic Targets of Purkinje Cell Terminals in the Cerebellar and Vestibular Nuclei of the Rat

The cerebellar and vestibular nuclei consist of a heterogeneous group of inhibitory and excitatory neurons. A major proportion of the inhibitory neurons provides a GABAergic feedback to the inferior olive, while the excitatory neurons exert more direct effects on motor control via non-olivary structures. At present it is not clear whether Purkinje cells innervate all types of neurons in the cerebellar and vestibular nuclei or whether an individual Purkinje cell axon can innervate different types of neurons. In the present study, we studied the postsynaptic targets of Purkinje cell axons in the rat using a combination of pre-embedding immunolabelling of the Purkinje cell terminals by L7, a Purkinje cell-specific marker, and postembedding GABA and glycine immunocytochemistry. In the cerebellar nuclei, vestibular nuclei and nucleus prepositus hypoglossi Purkinje cell terminals were found apposed to GABAergic and glycinergic neurons as well as to larger non-GABAergic, non-glycinergic neurons. In the cerebellar and vestibular nuclei individual Purkinje cell terminals innervated both the inhibitory and excitatory neurons. Both types of neurons were contacted not only by GABAergic Purkinje cell terminals but also by GABA-containing terminals that were not labelled for L7 and by non-GABAergic, non-glycinergic terminals that formed excitatory synapses. Glycine-containing terminals were relatively scarce (<2% of the GABA-containing terminals) and frequently contacted the larger non-GABAergic, non-glycinergic neurons. To summarize, Purkinje cell axons evoke their effects through different types of neurons present in the cerebellar and vestibular nuclear complex. The observation that individual Purkinje cells can innervate both excitatory and inhibitory neurons suggests that the excitatory cerebellar output system and the inhibitory feedback to the inferior olive are controlled simultaneously.     

Cerebellar responses to teleceptive stimuli in alert monkeys.

Discharges of single Purkinje cells in the intermediate and lateral zones of the cerebellar cortex and of neurons in the interpositus and dentate nuclei were recorded in alert monkeys during the presentation of intense auditory and visual stimuli. Concomitant monitoring of the electromyogram (EMG) demonstrated that these stimuli evoked characteristic startle responses in most instances. Firing patterns of cerebellar nuclear cells to auditory stimuli could be categorized into four types, the most common of which consisted of a short-latency acceleration of discharge, followed by a decrease in activity, and in most cells by a later period of facilitation. Simple spike discharge patterns of Purkinje cells consisted largely of prolonged increases or decreases in firing rate, although more complex patterns were seen. In almost 50% of the Purkinje cells tested, complex spikes were evoked by the auditory stimuli. Comparison of simple spike responses of Purkinje cells and of the discharges of cerebellar nuclear cells to auditory and visual inputs revealed that, except for a longer latency, the discharge pattern evoked by flash stimuli was identical to that evoked by sound in all instances. By contrast, in about one-third of the Purkinje cells with related complex spike discharge, complex spikes were evoked by stimuli of only a single modality. Comparison of the times of changes in nuclear and Purkinje cell activity suggests that the initial change in nuclear cell discharge was due to an increase in mossy fiber activity, while the subsequent decrease resulted from Purkinje cell inhibition evoked by mossy and climbing fiber inputs. The absence of increases in nuclear cell discharge at the time of most decreases in Purkinje activity indicates that removal of Purkinje inhibition does not have a major effect on the discharge rates of individual nuclear cells. The data also suggest that excitation of nuclear cells via climbing fiber collaterals played only a minor role in influencing their discharge. Since most EMG changes occurred after or at about the same time as the initial changes in cerebellar discharge, it is unlikely that the initial changes in cerebellar activity were a result of feedback from contracting muscles. It is proposed that the similar discharge patterns of cerebellar neurons to auditory and visual input results from a convergence of these inputs on a structure which projects to the cerebellum as mossy fibers.     

Ontogenesis of cerebellar afferents identified by cholecystokinin-like immunoreactivity.

The present account provides a developmental timetable for the maturation of cholecystokinin (CCK)-positive fibers in the cerebellar cortex and cerebellar nuclei of the opossum. CCK-positive fibers are in the cerebellar peduncle by postnatal day (PD) 1, however they wait until PD 7 to penetrate the cerebellar anlage. Between PD 7 and PD 20 the fibers wait again in the medullary core of the cerebellum. After PD 20, there are 2 distinct patterns of CCK localization within the overlying cortical layers. The first pattern develops between PD 20-26 when CCK puncta are present in restricted foci within the Purkinje cell layer of the anterior lobe vermis. They distribute in 4 parasagittal bands, 2 on either side of the midline, that extend from the primary fissure rostrally into the anterior lobe of the cerebellum. By PD 33 two additional parasagittal bands are present in the posterior lobe vermis. The vast majority of these CCK puncta are transient in nature as all but a few disappear by PD 84. Those that remain progress through a series of developmental stages characteristic of climbing fiber ontogeny. These climbing fibers persist in lobules V, VII and VIII of the adult cerebellum. Further, there is a transient expression of CCK-immunoreactivity within inferior olivary neurons. These observations support the interpretation that the transient population of CCK-IR puncta are immature climbing fiber axons derived from the inferior olive. The second pattern of CCK localization is evident between PD 30-33, the time when granule cells first can be recognized in a histologically distinct internal granule cell layer (IGL). Between PD 30 and PD 68 there is a differential pattern of distribution of CCK-IR profiles within the lobules of the cerebellum. Initially, CCK-IR axons are only present in the anterior vermis where they are aligned in register with the bands of CCK puncta in the Purkinje cell layer. CCK-IR puncta are not present in the posterior lobe vermis or hemispheres until later stages of development. Further, a sagittal organization is not evident in either of these latter 2 areas. Initially, CCK-IR profiles in the IGL cannot be identified as mossy fibers based on their terminal morphology. When they first enter the IGL they appear as punctate elements. Over time they become increasingly more complex in shape and between PD 68-84 develop morphological characteristics of adult mossy fiber rosettes. The cerebellar nuclei can be distinguished histologically by PD 18, but CCK-IR fibers are not evident among these neurons until PD 36 which corresponds to about the time they can be visualized in the IGL. In addition, CCK-IR cell bodies first appear in the cerebellar nuclei between PD 26-30; these are present in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization of visual mossy fiber projections and zebrin expression in the pigeon vestibulocerebellum

Extensive research has revealed a fundamental organization of the cerebellum consisting of functional parasagittal zones. This compartmentalization has been well documented with respect to physiology, biochemical markers, and climbing fiber afferents. Less is known about the organization of mossy fiber afferents in general, and more specifically in relation to molecular markers such as zebrin. Zebrin is expressed by Purkinje cells that are distributed as a parasagittal array of immunopositive and immunonegative stripes. We examined the concordance of zebrin expression with visual mossy fiber afferents in the vestibulocerebellum (folium IXcd) of pigeons. Visual afferents project directly to folium IXcd as mossy fibers and indirectly as climbing fibers via the inferior olive. These projections arise from two retinal recipient nuclei: the lentiformis mesencephali (LM) and the nucleus of the basal optic root (nBOR). Although it has been shown that these two nuclei project to folium IXcd, the detailed organization of these projections has not been reported. We injected anterograde tracers into LM and nBOR to investigate the organization of mossy fiber terminals and subsequently related this organization to the zebrin antigenic map. We found a parasagittal organization of mossy fiber terminals in folium IXcd and observed a consistent relationship between mossy fiber organization and zebrin stripes: parasagittal clusters of mossy fiber terminals were concentrated in zebrin‐immunopositive regions. We also describe the topography of projections from LM and nBOR to the inferior olive and relate these results to previous studies on the organization of climbing fibers and zebrin expression. J. Comp. Neurol. 518:175–198, 2010. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.     

Ins and Outs of Cerebellar Modules

The modular concept of cerebellar connections has been advocated in the lifetime work of Jan Voogd. In this concept, a cerebellar module is defined as the conglomerate of one or multiple and non-adjacent, parasagittally arranged zones of Purkinje cells, their specific projection to a well-defined region of the cerebellar nuclei, and the climbing fiber input to these zones by a well-defined region of the inferior olivary complex. The modular organization of these olivo-cortico-nuclear connections is further exemplified by matching reciprocal connections between inferior olive and cerebellar nuclei. Because the different regions of the cerebellar nuclei show highly specific output patterns, cerebellar modules have been suggested to constitute functional entities. This idea is strengthened by the observation that anatomically defined modules adhere to the distribution of chemical markers in the cerebellar cortex suggesting that modules not only differ in their input and output relations but also may differ in operational capabilities. Here, I will briefly review some recent data on the establishment of cerebellar modules in rats. Furthermore, some evidence will be shown suggesting that the other main afferent system (i.e., mossy fibers), at least to some extent, also adheres to the modular organization. Finally, using retrograde transneuronal tracing with rabies virus, some evidence will be provided that several cerebellar modules may be involved in the control of individual muscles.     

Developing mossy fiber terminal fields in the rat cerebellar cortex may segregate because of Purkinje cell compartmentation and not competition

Many mossy fiber afferent projections to the rat cerebellar cortex terminate in parasagittal bands. In particular, the anterior lobe ver-mis of the cerebellum contains alternating bands of mossy fibers from the spinal cord and external cuneate nuclei. The cerebellar cortical efferents, the Purkiwe cells, are also organized in parasagittal bands. These can be revealed by immurkochemical staining for the antigen zebrin II, which is selectively expressed by bands of Purkinie cells. In some cases, the boundaries between mossy fiber terminal fields align with identified transitions between zebrin⁺/^? sets of Purkinie cells, whereas others are located within apparently homogeneous Purkinie cell compartments. Two theories can explain the terminal-field topography: In one view, mossy fiber terminals segregate during development, because growth cones from different sources compete for common territory. Alternatively, mossy fiber growth cones directly recognize chemically distinct target territories, and activitydependent mechanisms play only minor roles. To explore these issues, two sets of experiments were performed. First, the terminal-field map of the neonatal spinocerebellar projection was compared to the Purkinie cell compartmentation as revealed by anticalbindin immunocytochemistry. Second, subsets of spinocerebellar mossy fiber afferents were ablated early in postnatal development, and the consequences for the neighboring cuneocerebellar terminal fields were mapped in the adult with reference to the zebrin II⁺/^? compartments. These experiments revealed no evidence that competitive interactions constrain the mossy fiber terminal-field distribution but, rather, suggest that the organization of the mossy fiber projections follows the oompartmentation of the Purkinie cells. © 1995 Wiley-Liss, Inc.     

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.