Harmaline-induced climbing fiber activation causes amino acid and peptide release in the rodent cerebellar cortex and a unique temporal pattern of Fos expression in the olivo-cerebellar pathway

Cerebellar climbing fibers have a unique relationship with the dendritic tree of cerebellar Purkinje cells and have been proposed as a key input in establishing long-term plastic changes in the cerebellar cortex. Although both glutamate and aspartate and a number of neuropeptides have been implicated as climbing fiber-released neurotransmitters/neuromodulators, the in vivo release of these substances during climbing fiber stimulation remains to be demonstrated. In the present study, climbing fibers were activated with harmaline and rats or mice were implanted with a microdialysis probe or a microperfusion probe, respectively, to measure amino acid or peptide release. Additional rats were euthanized at various timepoints post-harmaline injection and Fos immunocytochemistry was used to visualize the activation pattern of the inferior olive, cerebellar cortex and deep nuclei over time. Fos expression was first detected in the inferior olive at 15 min post-harmaline injection followed by expression in the deep cerebellar nuclei (30 min) and then in the cerebellar cortex (1 h). Between 2 and 6 h Purkinje cells expressing Fos were found in variable numbers in both the vermal and paravermal regions and there was a distinct parasagittal-banding pattern in the vermal region. Of several amino acids measured following harmaline administration only glutamate and aspartate levels increased significantly in the first dialysate sample compared to preharmaline levels and their release was blocked by prior lesion of the inferior olive. Citrulline also increased following climbing fiber stimulation, but this occurred in the second and third dialysate samples and may reflect nitric oxide production. Four peptides were examined in cerebellar microperfusates following climbing fiber stimulation. Only corticotropin releasing factor (CRF), calcitonin gene related peptide (CGRP) and bradykinin were significantly increased compared to pre-harmaline levels. These results suggest that glutamate, aspartate, CRF and CGRP are released from climbing fibers during activation of the olivocerebellar system.     

Depression of the efferent activity of the cerebellar nuclei following destruction of the inferior olive

The spontaneous discharge frequency of the fastigial and interpositus nuclei was evaluated in three experimental conditions in Rat: (a) in the "intact" animal; (b) in animals with total and selective destruction of the inferior olive, depriving the Purkinje cells of their afferent climbing fiber; (c) in animals having inferior olive destruction and cryocoagulation of the cerebellar cortex, destroying Purkinje cells innervating the neurones of the fastigial and interpositus nuclei. Unit activity was high in group (a) (32.9 +/- 22.9/s); it was markedly reduced in group (b) (1.1 +/- 1.3/s); it was higher in group (c) than in group (a) (43.7 +/- 25.5/s). Suppression of the inferior olive thus increases the Purkinje cell inhibitory action upon neurones of the cerebellar nuclei.     

Effect of injury to the lateral reticular nucleus and nuclei of the inferior olive on electrical responses of the cerebellar cortex evoked by vagus nerve stimulation in cats

The role of the lateral reticular nucleus and nuclei of the inferior olive in the formation of cerebellar cortical evoked potentials in response to vagus nerve stimulation was determined in experiments on 28 cats anesthetized with chloralose and pentobarbital. After electrolytic destruction of the lateral reticular nucleus, in response to vagus nerve stimulation, especially ipsilateral, lengthening of the latent period and a decrease in amplitude of evoked potentials were observed; after bilateral destruction of this nucleus, evoked potentials could be completely suppressed. It is concluded that the lateral reticular nucleus relays interoceptive impulses in the vagus nerve system on to the cerebellar cortex. Additional evidence was given by the appearance of spike responses of Purkinje cells, in the form of mainly simple discharges, to stimulation of the vagus nerve. After destruction of the nuclei of the inferior olive, the latent period and the number of components of evoked potentials in response to vagus nerve stimulation remained unchanged but their amplitude was reduced. The role of the nuclei of the inferior olive as a regulator of the intensity of the flow of interoceptive impulses to the cerebellum is discussed.N. I. Pirogov Medical Institute, Vinnitsa. Translated from Neirofiziologiya, Vol. 9, No. 3, pp. 290–299, May–June, 1977.     

On the cerebellum and motor learning

A critical review of the role of the cerebellum in motor learning is presented. Specifically, the hypothesis that the climbing fibers that issue from the inferior olive serve to modify the responsiveness of cerebellar Purkinje cells is evaluated. It is concluded that there is no convincing evidence, at this time, to support the view that a long-term modification of Purkinje cell activity is either the basis of motor learning or an authentic mechanism of cerebellar function. An alternative view, based on the biophysical, anatomical and ensemble properties of olivary neurons, suggests an important role for the olivocerebellar system in the coordination of movements. Future work in this interesting area of neuroscience will distinguish these two hypotheses.     

Spatial and temporal changes in natural and target deprivation‐induced cell death in the mouse inferior olive

The survival of inferior olive neurons is dependent on contact with cerebellar Purkinje cells. There is evidence that this dependence changes with time. Because inferior olivary axons, called climbing fibers, already show significant topographical ordering in cerebellar target zones during late embryogenesis in mice, the question arises as to whether olive neurons are dependent on target Purkinje cells for their survival at this early age. To better characterize this issue, inferior olive development was studied in two transgenic mouse mutants, wnt‐1 and L7ADT, with embryonic and early postnatal loss of cerebellar target cells, respectively, and compared to that in the well‐studied mutant, Lurcher. Morphological criteria as well as quantitative measures of apoptosis were considered in this developmental analysis. Survival of inferior olive neurons is observed to be independent of Purkinje cells throughout embryogenesis, but dependence begins immediately at birth in both wild types and mutants. Thereafter, wild types and mutants show a rapid increase in olive cell apoptosis, with a peak at postnatal day 4, followed by a period of low‐level, but significant, apoptosis that continues to at least postnatal day 11; the main difference is that apoptosis is quantitatively enhanced in the mutants compared to wild types. The multiphasic course of these effects roughly parallels the known phases of climbing fiber synaptogenesis. In addition, despite significant temporal differences among the mutants with respect to absolute numbers of dying cells, there are common spatial features suggestive of distinct intrinsic programs linking different olivary subnuclei to their targets. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 18–30, 2000     

A model of the cerebellum in adaptive control of saccadic gain

 We review data showing that the cerebellum is required for adaptation of saccadic gain to repeated presentations of dual-step visual targets and thus, presumably, for providing adaptive corrections for the brainstem saccade generator in response to any error created by the open-loop saccadic system. We model the adaptability of the system in terms of plasticity of synapses from parallel fibers to Purkinje cells in cerebellar cortex, stressing the integration of cerebellar cortex and nuclei in microzones as the units for correction of motor pattern generators. We propose a model of the inferior olive as an error detector, and use a ‘window of eligibility’ to insure that error signals that elicit a corrective movement are used to adjust the original movement, not the secondary movement. In a companion paper we simulate this large, realistic network of neural-like units to study the complex spatiotemporal behavior of neuronal subpopulations implicated in the control and adaptation of saccades. Received: 25 November 1994/Accepted in revised form: 6 February 1996     

Spatial and temporal changes in natural and target deprivation-induced cell death in the mouse inferior olive

The survival of inferior olive neurons is dependent on contact with cerebellar Purkinje cells. There is evidence that this dependence changes with time. Because inferior olivary axons, called climbing fibers, already show significant topographical ordering in cerebellar target zones during late embryogenesis in mice, the question arises as to whether olive neurons are dependent on target Purkinje cells for their survival at this early age. To better characterize this issue, inferior olive development was studied in two transgenic mouse mutants, wnt-1 and L7ADT, with embryonic and early postnatal loss of cerebellar target cells, respectively, and compared to that in the well-studied mutant, Lurcher. Morphological criteria as well as quantitative measures of apoptosis were considered in this developmental analysis. Survival of inferior olive neurons is observed to be independent of Purkinje cells throughout embryogenesis, but dependence begins immediately at birth in both wild types and mutants. Thereafter, wild types and mutants show a rapid increase in olive cell apoptosis, with a peak at postnatal day 4, followed by a period of low-level, but significant, apoptosis that continues to at least postnatal day 11; the main difference is that apoptosis is quantitatively enhanced in the mutants compared to wild types. The multiphasic course of these effects roughly parallels the known phases of climbing fiber synaptogenesis. In addition, despite significant temporal differences among the mutants with respect to absolute numbers of dying cells, there are common spatial features suggestive of distinct intrinsic programs linking different olivary subnuclei to their targets.     

Delayed reverberation through time windows as a key to cerebellar function

We present a functional model of the cerebellum comprising cerebellar cortex, inferior olive, deep cerebellar nuclei, and brain stem nuclei. The discerning feature of the model being time coding, we consistently describe the system in terms of postsynaptic potentials, synchronous action potentials, and propagation delays. We show by means of detailed single-neuron modeling that (i) Golgi cells can fulfill a gating task in that they form short and well-defined time windows within which granule cells can reach firing threshold, thus organizing neuronal activity in discrete `time slices', and that (ii) rebound firing in cerebellar nuclei cells is a robust mechanism leading to a delayed reverberation of Purkinje cell activity through cerebellar-reticular projections back to the cerebellar cortex. Computer simulations of the whole cerebellar network consisting of several thousand neurons reveal that reverberation in conjunction with long-term plasticity at the parallel fiber-Purkinje cell synapses enables the system to learn, store, and recall spatio-temporal patterns of neuronal activity. Climbing fiber spikes act both as a synchronization and as a teacher signal, not as an error signal. They are due to intrinsic oscillatory properties of inferior olivary neurons and to delayed reverberation within the network. In addition to clear experimental predictions the present theory sheds new light on a number of experimental observation such as the synchronicity of climbing fiber spikes and provides a novel explanation of how the cerebellum solves timing tasks on a time scale of several hundreds of milliseconds. Received: 23 July 1999 / Accepted in revised form: 31 August 1999     

Reduction of GABAB receptor binding induced by climbing fiber degeneration in the rat cerebellum

When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABAB receptor binding determined with 3H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the Bmax of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABAB sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABAA and benzodiazepine receptor binding labelled with 3H-muscimol and 3H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABAB sites but neither GABAA nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals.     

Signals from the cerebellum guide the pathfinding of inferior olivary axons

During development, inferior olivary axons cross the floor plate and project from the caudal to the rostral hindbrain, whence they grow into the cerebellar plate. We have investigated the axon guidance signals involved in the formation of this projection in vitro. When the cerebellar plate was grafted ectopically along the margin of the hindbrain in organotypic cultures, inferior olivary axons could pathfind to the ectopic cerebellum, establishing a topographically normal projection. Following rostrocaudal reversal of a region of tissue in the axon pathway between the inferior olive and the cerebellum, olivary axons still navigated towards the cerebellum. Moreover, olivary axons could cross a bridging tissue explant (spinal cord) to reach a cerebellar explant. In collagen gel cultures of inferior olive explants, olivary axon outgrowth increased significantly in the presence of cerebellar explants and axons deflected towards the cerebellar tissue. These results show that the cerebellum is a source of diffusible axon guidance signals for olivary axons. We also found that, in organotypic cultures, olivary axons which had crossed the floor plate showed an increased tendency to respond to cerebellar cues. Taken together, these results indicate that the cerebellum is the source of cues that are chemoattractant and growth-promoting for inferior olivary axons; prior exposure to the floor plate increases responsiveness to these cues.     

The rat olivocerebellar system visualized in detail with anterograde PHA-L tracing technique, and sprouting of climbing fibers demonstrated after subtotal olivary lesions

The rat olivocerebellar climbing fiber system has been investigated at the light and electron microscopic level with anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) tracing. From PHA-L Injections in different parts of the inferior olive labelled axons could be traced to the contralateral cerebellum. Arriving in the deep cerebellar white matter, the olivocerebellar axons ran around and through the cerebellar nuclei. Plexuses of labelled terminal fibers appeared in the cerebellar nuclei, and the density of this innervation was estimated to 1-4 million varicosities per mm3. Ultrastructurally, these boutons engaged in asymmetric synapses with small dendrites. Bundles of labelled fibers continued into the folial white matter, and terminated as climbing fibers in sagittal zones of the cerebellar cortex. Both the cortical and nuclear terminations of the olivocerebellar system are strictly topographically organized. The plasticity of climbing fibers was studied after partial lesions of the inferior olive induced by 3-acetylpyridine. One to 6 months after the lesion, surviving climbing fibers demonstrated extensive sprouting. The newly formed axons originated from parent climbing fiber plexuses, grew in the direction of parallel fibers, and formed terminal plexuses around several neighbouring Purkinje cells. As normal climbing fiber terminals, these terminals formed asymmetric synapses with spines of proximal Purkinje cell dendrites, and evidence by Benedetti et al. (1983) shows that the regenerated innervation is electrophysiologically functional. It is suggested that denervated Purkinje cells release a trophic substance, which stimulate surviving climbing fibers to sprouting, axonal growth and synapse formation.     

Effects of unilateral lesion of the inferior olive on L-[3H] aspartate receptors binding in synaptic membranes of cat cerebellar cortex

In adult cats, local injection of kainic acid (KA) in the inferior olive (IO) of one side, from which the crossed olivocerebellar projection originates, produced asymmetric postural and motor deficits, attributed to selective damage of the olivary neurons. Since aspartate is one of the putative transmitters of the olivocerebellar fibers, experiments were performed to find out whether 6-8 days after injection of KA within the IO of one side produced changes in aspartate receptors binding in different zones of the cerebellar cortex. In particular, binding in the contralateral zones of the cerebellar cortex was referred to proteins contained in membrane suspensions and compared with the control values obtained in the same experiments from the ipsilateral zones. Binding of L-[3H] aspartate decreased on the average to 53.4% of the control value in the medial zone and to 86.1% of the control value in the intermediate and lateral zones of the cerebellar cortex. This reduction varied in different experiments according to the side of the injection, in agreement with the well known pattern of regional distribution of the olivocerebellar projection within the cerebellar cortex. These findings favour aspartate as the putative neurotransmitter of the climbing fibers. The demonstration that binding of aspartate decreased in the cerebellar cortex of one side, 6-8 days after injection of KA in the corresponding IO, indicates that plastic events occur at this level following destruction of the olivocerebellar pathway. In particular, the reduced binding can be attributed either to a decrease in number of the postsynaptic receptor sites for aspartate or to a decreased affinity of this amino acid for the corresponding receptors. These findings, however, do not exclude that an hypersensitivity by denervation may occur at the level of individual Purkinje cells when they are deprived of the climbing fibers input. In order to answer this question further experiments are required to find out how the binding for aspartate is modified at increasing time intervals after the olivary lesion.     

Functional localisation in the cerebral cortex and cerebellum: lessons from the past

One of the basic questions about the working of the brain is the extent to which its various functions are localised. In the nineteenth century great advances were made in the study of localisation. The control of speech, movement, and vision was identified with specific regions of the cerebral cortex. Although since the nineteenth century lesions of the cerebellum have been known to produce impaired movement, there has been rather little progress towards answering more detailed questions about the functions of the cerebellum and cerebellar localisation. The experts are still not agreed on what the cerebellum does or how and where it does it. Three examples are given of functions which probably are mediated by the cerebellum; adaptation of the vestibulo-ocular reflex, classical conditioning of the nictitating membrane response, and adaptation of saccadic eye movements. In all three cases the control of these functions has been localised to a specific region of the cerebellar cortex and/or nuclei. The success of localisation studies in the cerebral cortex can serve as a guide. Continued experimentation directed at the question of localisation should prove a fruitful approach to understanding more about the functions of the cerebellum.     

Interaction between unit responses of the cat cerebellar cortex during paired stimulation of the forelimbs

Interaction between spike responses of 41 cerebellar cortical neurons to electrical stimulation of the two forelimbs with different intervals between stimuli were studied in cats anesthetized with chloralose and pentobarbital. The responsiveness of neurons with a phasic type of discharge to testing stimulation of the limb was reduced for 300–500 msec or longer after conditioning stimulation of the other limb. Interaction between the responses was less clear in neurons with a tonic type of response. Interaction was absent or was summating in character if the stimuli were applied at the same times. Only if the intertrial intervals were 50–150 msec was regular inhibition of the responses of tonic type to the testing stimulus observed. It is postulated that the nucleus of the inferior olive participates in the interaction between phasic unit responses during simultaneous stimulation of the two limbs or to stimulation separated by short intervals (under 30 msec). With longer intervals between stimuli, interaction between responses of either type is connected with involvement of the lateral reticular nucleus. In the process of interaction competitive relations may develop between responses caused by impulses reaching neurons of the cerebellar cortex along climbing and mossy fibers.     

Rearrangement of parameters of efferent activity in generators of cyclic motor response produced by electrical stimulation of cerebellar inputs in cats

Rearrangement of the parameters of scratch and locomotor generators produced by electrical stimulation of the inferior olive and nucleus reticularis lateralis as well as the cerebellar fastigial nucleus and nucleus interpositus was investigated in decerebrate immobilized cats. Results showed that a comparable rearrangement of the time course of activity in both locomotor and scratch generators was produced by altering the nature of signals proceeding along mossy and climbing fibers alike. Maximum rearrangement of scratch and locomotor generator activity, as induced by electrical activation of the inferior olive and lateral reticular nucleus, is observed during the first half of flexor half-center operation in these generators. The scratch (unlike the locomotor) generator typically shows considerably rearranged efferent activity following electrical activation of nuclei of the cerebellum and cerebellar afferents. The article discusses mechanisms of cerebellar origin which may be responsible for exerting a corrective action on scratch and locomotor generators during change in the phase and amplitude parameters of cerebellar input signals.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 2, pp. 131–140, March–April, 1992.     

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.     

Attenuation of activity-induced increases in cerebellar blood flow by lesion of the inferior olive

We sought to define the contribution of the climbing fibers (CF), one of the major inputs to Purkinje neurons, to the increase in cerebellar blood flow (BFcrb) produced by activation of the cerebellar cortex. The neurotoxin 3-acetylpyridine was used to lesion the inferior olive, the site from which the CF originate. Crus II, an area of the cerebellar cortex that receives sensory afferents from the perioral region, was activated by low-intensity stimulation of the upper lip (5-25 V and 4-16 Hz) in sham-lesioned and lesioned mice. BFcrb was recorded in crus II using a laser-Doppler flow probe. The increase in BFcrb produced by harmaline, an alkaloid that activates the CF, was abolished in lesioned mice (P > 0.05 vs. BFcrb before harmaline, n = 6), attesting to the effectiveness of the lesion. In sham-lesioned animals, upper lip stimulation increased BFcrb in crus II by 25 +/- 2% (25 V and 10 Hz, n = 6). The rise in BFcrb was attenuated by 63 +/- 7% (25 V and 10 Hz) in lesioned mice (P < 0.05, n = 6). In contrast, the increase in BFcrb produced by hypercapnia was not affected (P > 0.05). These data suggest that CF are responsible for a substantial portion of the increase in BFcrb produced by crus II activation. Thus the hemodynamic response evoked by functional activation of the cerebellar cortex reflects, in large part, CF activity.     

Electron-microscopic immunocytochemistry of 5-hydroxytryptamine in the ascidian endostyle

Summary Corticotropin releasing factor (CRF)-immunoreactive (IR) perikarya, visualized by the indirect immunoperoxidase method in colchicine-pretreated cats, were localized in many discrete regions of the medulla oblongata. They were found mainly in the dorsal aspect and midline of the medulla oblongata, and more rostrally in the ventrolateral portion. Our results also demonstrated CRF-IR neurons in the rostrocaudal extent of the inferior olive, probably projecting to the cerebellar cortex via thick axons visualized along the lateral edge of the medulla. CRF-IR olivary cells were also found in the pontine cat from which the forebrain was removed, but neither in hypophysectomized nor adrenalectomized cats.     

Activity of neurons of the red nucleus after destruction of the inferior olive in the rat

The spontaneous frequency of discharge of the red nucleus neurones has been evaluated in the rat before and after total and bilateral destruction of the inferior olive by 3-acetylpyridine. The suppression of the inferior olive increasing the cerebellar inhibition, produces, as a consequence, a consequence, a disfacilitation of the activity of the red nucleus neurones. The process persists for a few days, then a progressive compensation takes place. A month later the average frequency of discharge is recovered but a normal unit activity and motor behaviour are not restaured.     

Alien hand syndrome: neural correlates of movements without conscious will

Background

The alien hand syndrome is a striking phenomenon characterized by purposeful and autonomous movements that are not voluntarily initiated. This study aimed to examine neural correlates of this rare neurological disorder in a patient with corticobasal degeneration and alien hand syndrome of the left hand.

Methodology/Principal Findings

We employed functional magnetic resonance imaging to investigate brain responses associated with unwanted movements in a case study. Results revealed that alien hand movements involved a network of brain activations including the primary motor cortex, premotor cortex, precuneus, and right inferior frontal gyrus. Conscious and voluntary movements of the alien hand elicited a similar network of brain responses but lacked an activation of the inferior frontal gyrus. The results demonstrate that alien and unwanted movements may engage similar brain networks than voluntary movements, but also imply different functional contributions of prefrontal areas. Since the inferior frontal gyrus was uniquely activated during alien movements, the results provide further support for a specific role of this brain region in inhibitory control over involuntary motor responses.

Conclusions/Significance

We discuss the outcome of this study as providing evidence for a distributed neural network associated with unwanted movements in alien hand syndrome, including brain regions known to be related to movement execution and planning as well as areas that have been linked to inhibition control (inferior frontal gyrus) and experience of agency (precuneus).