Activity of Purkinje cells, parallel fibers, and climbing fibers in the developing rabbit cerebellum

A study has been made of the activity of Purkinje cells, parallel fibers, and climbing fibers in 120 rabbits aged from a few hours to 20 days. Spontaneous simple spike activity due to activation by the mossy fiber-granular cell-Purkinje cell system has been found, and also a complex spike related to direct activation by the climbing fibers from the first few hours of life. Statistical analysis of unit discharges shows that there is a general linear increase in their frequency with age. The presence of a propagated spike in the parallel fibers and of complex discharges in the Purkinje cells in response to stimulation of afferent climbing fibers confirms that a large part of the cerebellar synaptic system is operative from the time of birth.     

The role of climbing fibers in the development of Purkinje cell dendrites

Destruction of the inferior olivary nucleus was performed in newborn kittens in order to study the role of climbing fibers in the postnatal development of the cerebellum. In the three kittens which survived for 40–45 days after the unilateral destruction at 2 days old, histological examination demonstrated the lack of dendritic arborization in many Purkinje cells in the cerebellar cortex contralateral to the lesion. The olivectomized kittens showed cerebellar symptoms which became conspicous when the kittens started to walk. The results reveal an important role of climbing fibers in the development of Purkinje cell dendrites.     

Origin of the climbing fibers activated by glossopharyngeal nerve stimulation in the frog

The origin of climbing fibers activated by electrical stimulation of the frog's glossopharyngeal (IXth) nerve was investigated using histological and electrophysiological technique. At the molecular layer near the Purkinje cell layer, where the maximum negative cerebellar field potential could be recorded following electrical stimulation of the IXth nerve, horseradish peroxidase (HRP) was iontophoretically injected through the tip of the recording micropipette. The HRP labeled cells were seen in the contralateral inferior olive (IO). In some cases, a small number of HRP-labeled cells were seen in the ipsilateral IO. Labeled cells were not found in the other areas of the brain stem. After electrolytic lesion of the contralateral IO, the negative cerebellar field potential which would be recorded in the molecular layer following electrical stimulation of the IXth nerve had almost ceased. These results demonstrate that the climbing fibers activated by the IXth nerve stimulation have their origin in the contralateral IO.     

Identification under the electron microscope of climbing fibers and their synaptic contacts

Summary An attempt is made to identify, under the electron microscope, the climbing fibers of the cerebellum (in the cat) and their synaptic contacts with Purkinje cells and other cortical neurons. — Two kinds of axonal profiles, having synaptic contacts with primary and secondary dendrites of Purkinje neurons, can be recognized: One being terminal fibers densely packed with neurofilaments, having mainly contacts de passage with the dendrite surface, with small accumulations of synaptic vesicles at the presynaptic side of the contact. The others are rather knob-shaped contacts filled with synaptic vesicles and poor in neurofilaments. In chronically isolated folia, in which only local neurons and their processes have survived, all filamentous profiles have disappeared while vesicular ones are not appreciably reduced in number. It is inferred from this, that the neurofilamentous profiles correspond to climbing fibers, whereas the vesicular ones could be the endings of outer stellate axons, recurrent Purkinje axon collaterals, or ascending basket axon collaterals. — Similar two kinds of axon-terminal profiles are found in synaptic contact with Golgi and basket cell bodies. As in chronically isolated folia only the vesicular profiles survive, it is inferred that the climbing fiber has axo-somatic terminals on Golgi cells and basket cells as well. Previous information of this kind, gained with the light microscope and with degeneration studies, is thus substantiated with the aid of the electron microscope. The vesicular presynaptic profiles on Golgi and basket neurons are in the first case certainly and in the second with high probability endings of recurrent Purkinje axon collaterals. — The few axosomatic synapses found on outer stellate neurons may also be terminals of climbing fibers, but degeneration evidence for this is not conclusive. — The observations are summarized and evaluated from the functional point of view in a diagram, with consideration to recent physiological information on the function of climbing fibers.     

Multiple climbing fibers signal to molecular layer interneurons exclusively via glutamate spillover

Spillover of glutamate under physiological conditions has only been established as an adjunct to conventional synaptic transmission. Here we describe a pure spillover connection between the climbing fiber and molecular layer interneurons in the rat cerebellar cortex. We show that, instead of acting via conventional synapses, multiple climbing fibers activate AMPA- and NMDA-type glutamate receptors on interneurons exclusively via spillover. Spillover from the climbing fiber represents a form of glutamatergic volume transmission that could be triggered in a regionalized manner by experimentally observed synchronous climbing fiber activity. Climbing fibers are known to direct parallel fiber synaptic plasticity in interneurons, so one function of this spillover is likely to involve controlling synaptic plasticity.     

The climbing fibers of the cerebellar cortex,their origin and pathways in cat

Summary The sources and pathways of the climbing fibers to the cerebellar posterior vermis were studied with combined electrophysiological and anatomical methods in cats.Recording from identified cerebellar Purkinje cells, monosynaptic climbing fiber (CF) responses have been obtained both for stimulation of the inferior olive (IO) and various parts of the brain stem (BS). CF responses were found to be of three types, IO only, BS only or both IO and BS. However the responses to BS stimulation were very few in number in comparison with IO or IO and BS types of responses. The latencies of the responses were shorter for the BS cases consistent with their distance from the cerebellum.A comparison of latencies and the relative responsiveness of the different area of the brain stem which were studied, indicate that part of the CF ascend through the pontine region and enter the cerebellum by way of the medium and superior peduncles. This finding is confirmed by the results of anatomical studies in which degenerating fibers were found in the molecular layer (using the Nauta technique) after lesion of the brachium pontis but not after lesions of the medial portion of the pons. Similarly, injection of radioactive leucine into the pontine nuclei failed to show any labeled fibers in the molecular layer.Horseradish peroxidase (HRP) was injected into localized regions of the posterior vermis after total bilateral destruction of the inferior peduncles. Large numbers of positive, marked cells were still found in the inferior olive.It is concluded that nearly all, if not all, the climbing fibers originate in the inferior olive and that they ascend to the cerebellum by way of all the peduncles.     

Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex

By means of a semi-automatic image analyzer plugged into an Apple II computer and suitable computer programs it is possible to analyze transmitter-identified nerve terminals. Thus, a densitometric approach is applied on the original photograph followed by systematic sampling which is carried out by means of a grating of circles. This procedure allows the study quantitatively of density and intensities of different types of densely packed tyrosine hydroxylase (TH) immunoreactive nerve terminals of the nuc. caudatus putamen. It is shown that the islandic TH immunoreactive nerve terminals have a higher density, but a TH content similar to the diffuse types of TH immunoreactive nerve terminals in the nuc. caudatus putamen.     

Interrelations of basket cell axons and climbing fibers in the cerebellar cortex of the rat

Summary An analytical study was undertaken with both electron microscopy and the rapid Golgi method in order to clarify the interrelations of climbing fibers, basket cell axons, and Purkinje cell dendrites. The two fibers are readily distinguished in electron micrographs by means of their differing content of microtubules and neurofilaments, the packing density of synaptic vesicles, and the disposition of their synaptic junctions on the Purkinje cell dendrite. Climbing fibers are generally thin and contain many microtubules. They give off attenuated collaterals, whose rounded varicosities are densely packed with vesicles and which form en passant synapses with clusters of thorns projecting from the major Purkinje dendrites. In contrast, basket axons are relatively thick and contain many neurofilaments. By means of slight dilatations containing loosely aggregated vesicles, the axon and its collaterals form numerous synapses en passant with the smooth dendritic shafts and the perikaryon of the Purkinje cell. Climbing fibers and basket cell axons run along parallel with each other but without forming axo-axonic synapses as they ascend over the surface of the Purkinje dendrites. Both fibers form especially elaborate intertwined festoons at the branching points of the major dendrites. The kinds of synapses found are described in detail, and the functional implications are discussed.The hypothesis is developed that the dendritic thorn is a device for isolating the subsynaptic membrane from electrical events in the rest of the dendrite at the cost of reducing the effectiveness of the synapse. This principle is incorporated in the Purkinje dendrite—parallel fiber synapses, in which an individual fiber can be expected to have little importance. The disadvantage of using thorns as postsynaptic surfaces can be mitigated by clustering them and increasing the number of thorns contacted by each presynaptic terminal. This method is utilized at the junctions between the climbing fiber and the Purkinje dendrite to produce one of the most powerful excitatory synapses known. It is furthermore suggested that the elaborate plexus of climbing fibers and basket cell axons synapsing in the crotches of branching dendrites is strategically located to control the flow of information in the Purkinje cell dendritic tree.Supported by U.S. Public Health Service Research Grant NS03659 and Training Grant NS05591 from the National Institute of Neurological Diseases and Stroke.Postdoctoral trainee in Anatomy under Training Grant GM906 from the National Institute of General Medical Sciences.     

Differential role of granule cells in the specification of synapses between climbing fibers and cerebellar Purkinje cells in the rat

The effect of X-irradiation of the cerebellum during postnatal development on the establishment of olivo-cerebellar connections was investigated in the rat. Treatment with 950 R during the first week led to a persistance in the adult of multiple innervation of Purkinje cells (PC) by climbing fibers (CF). On the contrary, the same dose given during the second week was uneffective to prevent the establishment of the one-to-one relationship between CF and PC. These findings suggest that the regression of the multiple innervation mainly depends on early formed granule cells.     

Immunocytochemical localization of tubulin and the high molecular weight microtubule-associated protein 2 in Purkinje cell dendrites deprived of climbing fibers

The modifications in the localization of tubulin and the high molecular weight microtubule-associated protein 2 were studied in the cerebellum after partial denervation. Both proteins were localized in 40 micron sections using monoclonal antibodies against beta-tubulin (clones Tu9B and Tu12) or microtubule-associated protein 2 (clones AP9 and AP13), and polyclonal antisera against alpha- and beta-tubulin or microtubule-associated protein 2, visualized with the immunoperoxidase method of Sternberger [Sternberger (1979) Immunocytochemistry; Sternberger and Sternberger (1983) Proc. natn. Acad. Sci. U.S.A. 80 6126-6130] or a biotin-avidin system. The destruction of the inferior olive was performed in adult male rats by electrocoagulation or by intraperitoneal administration of 3-acetylpyridine. One day after chemical destruction of the inferior olive, anti-microtubule-associated protein 2 staining with either of the monoclonal antibodies or with the polyclonal antiserum was almost identical to that observed in the cerebellum of non-denervated animals. Specific staining was intense in the cell somata and dendrites and absent in myelinated tracts and in parallel fibers. However, 3 days after the lesion anti-microtubule-associated protein 2 staining showed a clear decrease, both in the proximal and the distal portions of thick secondary and tertiary dendritic trunks of the Purkinje cell. The intensity of the staining was also considerably reduced in the fine dendritic ramifications. By 8 days post-lesion, microtubule-associated protein 2 immunoreactivity began to increase, but only in the portions of the dendrites deprived of the climbing fibre; on the contrary, low immunoreactivity was found in the fine dendritic ramifications which are contacted by normal parallel fibers; microtubule-associated protein 2 immunoreactivity increased considerably by 11 days post-lesion, giving a pattern quite similar to that of non-denervated Purkinje cells. The alterations in microtubule-associated protein 2 immunoreactivity were also accompanied by a dramatic decrease in the immunostaining for tubulin, beginning on day-3 post-lesion and lasting until day-15 post-lesion. These changes were observed with either the monoclonal antibodies against beta-tubulin or with the polyclonal antiserum against alpha- and beta-tubulin. The changes in both molecules were also observed in animals in which the inferior olive was destroyed by electrocoagulation, ruling out the possibility of a direct action of 3-acetylpyridine on dendritic microtubular proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Motor unit recruitment and rate coding in response to fatiguing shoulder abductions and subsequent recovery

The purpose of the present study was to investigate motor unit (MU) recruitment and firing rate, and the MU action potential (MUAP) characteristics of the human supraspinatus muscle during prolonged static contraction and subsequent recovery. Eight female subjects sustained a 30° shoulder abduction, requiring 11–12% of maximal voluntary contraction (MVC), for 30 min. At 10 and 30 min into the recovery period, the shoulder abduction was repeated for 1 min. The rating of perceived exertion for the shoulder region increased to “close to exhaustion” during the prolonged contraction, and the surface electromyography (EMG) recorded from the deltoid and trapezius muscles showed signs of local muscle fatigue. From the supraspinatus muscle, a total of 23,830 MU firings from 265 MUs were identified using needle electrodes. Of the identified MUs, 95% were continuously active during the 8-s recordings, indicating a low degree of MU rotation. The mean (range) MU firing rate was 11.2 (5.7–14.5) Hz, indicating the relative force contribution of individual MUs to be larger than the overall mean shoulder muscle load. The average MU firing rate remained stable throughout the prolonged abduction, although firing rate variability increased in response to fatigue. The average concentric MUAP amplitude increased by 38% from the beginning (0–6 min) to the end (24–29 min) of the contraction period, indicating recruitment of larger MUs in response to fatigue. In contrast, after 10 min of recovery the average MU amplitude was smaller than seen initially in the prolonged contraction, but not different after 30 min, while the MU firing rate was higher during both tests. In conclusion, MU recruitment plays a significant role during fatigue, whereas rate coding has a major priority during recovery. Furthermore, a low degree of MU rotation in combination with a high relative load at the MU level may imply a risk of overloading certain MUs during prolonged contractions. Accepted: 6 June 2000     

Early terminal degeneration of cerebellar climbing fibers after destruction of the inferior oliver in the rat. Synaptic relationships in the molecular layer

Summary The cerebellar molecular layer in adult rats has been studied with the electron microscope at several early and consecutive survival times following 3-acetylpyridine intoxication. Climbing fiber (CF) terminals underwent a fast process of electron-dense degeneration which became apparent from 16 hours onwards. A small proportion of degenerating terminals were depleted of vesicles and filled with a dark flocculent and granular homogeneous matrix. Microtubular changes in degenerating CF tendrils were observed. CF terminals were found in relation with every Purkinje cell in normal animals and completely disappeared within 72 hours after the treatment. CF synapses were found on Purkinje dendritic and somatic thorns, sometimes also on the dendritic shafts or even on the Purkinje soma. Convincing evidence of synaptic contacts of CF varicosities on either basket or stellate cells could not be obtained. CF synapses with Golgi II cell dendrites in the molecular layer were described. Decrease in the number of post-synaptic dendritic thorns normally assigned fo CF synapses was observed consequential to CF anterograde degeneration. The observations are consistent with previous conclusions drawn from light microscopic studies that the clearing up of CF debris in the molecular layer is completed within the short time of three days, and that the inferior olive seems to be the only source of CFs.     

Role of climbing fibers in determining the spatial patterns of activation in the cerebellar cortex to peripheral stimulation: an optical imaging study

The spatial patterns of activation in the rat cerebellar cortex evoked by ipsilateral face stimulation were mapped using optical imaging based on the pH sensitive dye, Neutral Red. The aims of the study were to characterize the optical responses evoked by peripheral stimulation and test the hypothesis that the resultant parasagittal banding is due to climbing fiber activation. In the anesthetized rat Crus I and II of the cerebellar cortex were stained with Neutral Red. Epi-fluorescent changes produced by a train of stimuli (5-10s and 4-20 Hz) to the ipsilateral face were monitored in time using a fast, high resolution charge-coupled device camera. The patterns of activation were quantified using a two-dimensional fast Fourier transform analysis that removed signals with high spatial frequencies and minimized the contribution of horizontal structural elements (i.e. blood vessels). The dominant spatial pattern of activation evoked by face stimulation was that of parasagittal bands. The bands were highly frequency-dependent and were elicited most strongly by stimulus frequencies in the range of 6-8 Hz. There was a large fall-off in the response for frequencies above and below. The optical signal evoked by face stimulation built up over a period of 10s and then gradually decayed. Within a folium the individual parasagittal bands exhibited some frequency and temporal specificity. Stimulation of the contralateral inferior olive also resulted in the activation of parasagittal bands with characteristics similar to the bands evoked by face stimulation, including a preferred stimulus frequency which peaked at 10 Hz. Injection of lidocaine into the contralateral inferior olive blocked the parasagittal bands evoked by ipsilateral face stimulation, while control injections of saline had no effect.The results confirm that a parasagittal banding pattern is a dominant feature of the functional architecture of the cerebellar cortex. The parasagittal banding pattern observed with Neutral Red is due primarily to the activation of climbing fiber afferents. The frequency tuning of the responses, with the preference for peripheral stimuli of 6-8 Hz, is in agreement with previous findings that the inferior olive is inherently rhythmic. These observations support the hypothesis that inferior olivary neurons are dynamically coupled into groups that activate parasagittal bands of Purkinje cells in the cerebellar cortex. The frequency tuning also supports the hypothesis that the climbing fiber system is involved with timing. Activation of this afferent system may require stimuli with appropriate frequency content and stimuli synchronized to the rhythmicity of the inferior olive.     

Internal models of eye movement in the floccular complex of the monkey cerebellum

Internal models are a key feature of most modern theories of motor control. Yet, it has been challenging to localize internal models in the brain, or to demonstrate that they are more than a metaphor. In the present review, I consider a large body of data on the cerebellar floccular complex, asking whether floccular output has features that would be expected of the output from internal models. I argue that the simple spike firing rates of a single group of floccular Purkinje cells could reflect the output of three different internal models. (1) An eye velocity positive feedback pathway through the floccular complex provides neural inertia for smooth pursuit eye movements, and appears to operate as a model of the inertia of real-world objects. (2) The floccular complex processes and combines input signals so that the dynamics of its average simple spike output are appropriate for the dynamics of the downstream brainstem circuits and eyeball. If we consider the brainstem circuits and eyeball as a more broadly conceived “oculomotor plant,” then the output from the floccular complex could be the manifestation of an inverse model of “plant” dynamics. (3) Floccular output reflects an internal model of the physics of the orbit where head and eye motion sum to produce gaze motion. The effects of learning on floccular output suggest that it is modeling the interaction of the visually-guided and vestibular-driven components of eye and gaze motion. Perhaps the insights from studying oculomotor control provide groundwork to guide the analysis of internal models for a wide variety of cerebellar behaviors.