Maturation of evoked climbing fiber input to rat cerebellar Purkinje cells (I.)

Summary An analysis of evoked responses of Purkinje cells in developing rat cerebellum to climbing fiber input was conducted to determine which identifying properties of this afferent system are established early in development and which specific features mature with age. Rat pups at various ages were anesthetized with 0.5% halothane and unit recordings made with glass micropipettes. By the third postnatal day, electrical stimulation of the sensorimotor cortex and limbs at low stimulation rates (<1/sec) could elicit distinct burst responses appearing at long latencies (180 msec), indicating that pathways of both ascending and descending climbing fiber systems are intact early in cerebellar cortical development. A distinctive feature maturing over the first 1–1.5 weeks was the characteristic of the all-or-none burst response since before about day 11 the interspike interval, amount of inactivation, and number of spikes in evoked burst responses all varied from stimulation to stimulation. Mean latencies decreased from 180 msec at day 3 to 50 msec by day 10, but did not achieve the adult value of 20 msec until the fourth week. Typically, climbing fiber responses could only follow at stimulation rates of less than 0.2/sec at day 3, but by day 12 could follow up to 10/sec, which is the same as in the adult. The data indicate that the climbing fiber system establishes connectivity from diverse sources and exhibits identifying characteristics similar to the adult early in cerebellar development. Most aspects of the maturation of transmission can be explained if there were a decrease in the time scale of function of the synapses involved, mainly those in the inferior olive.Supported by N.I.H. Grant 5-R01-6M00133 and N.S.F. Grant GB 43301     

The distribution to the cerebellar anterior lobe of the climbing and mossy fiber inputs from the plantar and palmar cutaneous afferents

Summary Systematic examination has been made of the potentials evoked in the ipsilateral anterior lobe by single Group II volleys in different branches of cutaneous nerves to the fore-paw and hind-paw of the cat. Field potentials evoked by the mossy and climbing fiber inputs have been recorded along microelectrode tracks arranged so that there has been a comprehensive study through the whole branching foliated structure. In a previous investigation it was shown that large cutaneous nerves of the forelimb and hindlimb have wide fields of action for both the mossy fiber and climbing fiber inputs. In this present investigation it was found that small cutaneous nerves have more localized distributions within these wide fields. This discriminative distribution is exhibited for Group II volleys in the subdivisions of the nerves providing innervation to the palmar and plantar foot pads. It thus appears from this somatotopic investigation that there are pathways to the cerebellum sufficiently specific to give information about the part of the foot that is being stimulated in natural movements.     

Discharge patterns of Purkinje cells activated through the climbing fiber system by stimulation of somatic and visceral afferents

Extracellular recordings were obtained from single Purkinje cells (PC) in the intermediate part of the lobule V and VI of the cerebellar cortex in cats anaesthetized with Nembutal. A number of PC responded with a complex spike (CS) to stimulation of both the superficial radial nerve (SRN) and the vagus nerve (VN). By suppressing the transmission of mossy fiber (MF) inputs through manipulation of the level of anaesthesia, attention was paid to the simple spike (SS) activity after the CS. Modality-specific differences were found in the length of the post-CS pause, the presence of a post-pause rebound, the effect of pre-CS SS firing rate on pause duration and in the frequency and regularity of post-pause SS discharge. We concluded that these differences arouse from the location of the PC within the climbing fiber (CF) sagittal strip and from the differential activation of the inhibitory interneurons. We propose that modification of SS activity following a CS represents a possible means of information transmission by the CF system.     

Maturation of evoked mossy fiber input to rat cerebellar Purkinje cells (II)

Summary The aim of this work was to establish a time sequence for the functional maturation of the mossy fiber afferent system to the cerebellum, in order to place it in the context of overall cerebellar cortical development. Rat pups at various ages were anesthetized with 0.5% halothane, and Purkinje cell activity was monitored extracellularly as limbs were electrically stimulated. The results showed that Purkinje cells can receive input from the periphery via the mossy fiber system at least by the seventh postnatal day, which is relatively early in overall cerebellar development. It is clear that synaptic transmission begins soon after the mossy fiber terminals and granule cell dendrites are in physical proximity. Initially, such input has a long latency (66 msec), is easily fatigued (at 10/sec), and has a prolonged duration of excitatory effect on Purkinje cells (27 msec). These and other functional parameters become mature by the third postnatal week (6–9 msec latency, following frequency above 20/sec), which is well before overall cerebellar cortical cytological development is completed. The maturation of many functional parameters of the cerebellar afferent systems and the acquisition of new motor behaviors are shown to emerge in parallel. Motor function appears to correlate with the establishment of a mature time scale of operation of the cerebellar circuitry.Supported by N. I. H. Grant 5-R01-GM00133 and N.S.F. Grant GB 43301     

Activation of mossy and climbing fiber pathways to the cerebellar cortex by stimulation of the fornix in the rat

Summary The fornix of the rat was electrically stimulated with bipolar concentric electrodes to determine the properties of single unit responses in Purkinje cells of the cerebellar cortex. Both climbing (CF) and mossy fiber (MF) pathways were activated by fornix stimulation. MF responses were indicated by single or double spike responses appearing at latencies of 5–10 ms. The MF spike responses, as quantified by histogram analysis, were further identified by appearance of graded responses with increasing stimulus strength and by following at frequencies up to and greater than 20/s. CF responses were identified by characteristic complex all-or-none burst responses with latencies usually between 10 and 20 ms and with following frequencies at no faster than 10/s. Experiments which involved movement of the stimulating electrode and production of lesions around it established that the activated fiber system was within the dorsal fornix and not in adjacent areas. The results indicate that hippocampal and other limbic areas can influence the cerebellar cortex by direct mossy and climbing fiber pathways, as has been demonstrated for other afferents. It is further suggested that motor patterns linked to hippocampal activity may be regulated by this system.This work was supported by N.S.F. Grant No. 77-01174 awarded to Dr. Donald J. Woodward, and an award from the Biological Humanics Foundation     

Gravity responses of Purkinje cells in the nodulus

Summary In cats, either decerebrated or under chloralose anaesthesia, Purkinje cells (P-cells) of the cerebellar nodulus have been examined with the animal under static lateral tilt (roll±20°). The cell activity was extracellularly recorded and both simple and complex spike discharge patterns were studied.In 20 cells out of a population of 198, simple spike firing was found to be affected by static roll. Ten cells had an -type response, 8 a -type, while only single examples of and activations were found.Out of 67 Purkinje cells tested for complex spike activation, 5 were found to be sensitive to static roll, 4 with an or response and one with a response.The results are to be attributed to pure otolith activation and show that this input is able to modulate P-cell activity in the nodulus through both the mossy fibre and the climbing fibre systems.     

Organization of climbing fiber input from mechanoreceptors to lobule V vermal cortex of the cat

Summary The somatotopic organization of the climbing fiber (CF) projections to the vermal cortex of lobule V of the cat was revealed by low threshold natural stimulation of mechanoreceptors. Extracellular single-unit recordings were made from 554 Purkinje cells in cats anesthetized with sodium pentobarbital. Forty-nine percent of the CF responses were elicited by cutaneous stimulation of the forelimb (62%), hindlimb (25%), or upper back and neck (13%). The topographical arrangement consisted of a 1 mm wide medial zone and a 1–1.5 mm wide lateral zone. In the medial zone, the CF responses were mainly nonresponsive to any cutaneous stimulation except in the caudomedial portion of the lobule where the upper back, neck or ears were represented in a narrow parasagittally oriented strip. The lateral zone contained a mixture of CF responses representing projections from different portions of the ipsilateral forelimb and hindlimb. Although CF responses connected with the forepaw or hindpaw predominated throughout all parts of the lateral zone, the more medial portions of this zone contained larger receptive fields involving the more proximal areas of the limb whereas the lateral part of the zone had smaller receptive fields representing the distal regions, particularly the ventral forepaw surface. Cells with similar receptive fields were often grouped together, but adjacent skin areas were not necessarily represented in adjacent cortical patches. Thus, the cutaneous projections to this lobule terminated in a patchy or mosaic fashion.Supported by NIH grant S-R01-NS-02289     

Topographical investigations on the climbing fiber inputs from forelimb and hindlimb afferents to the cerebellar anterior lobe

Summary Volleys in group I and II fibers of muscle nerves and group II fibers of cutaneous, joint and fascial nerves have evoked CF responses in the anterior lobe of the cerebellum. In the pars intermedia there is a fairly sharp somatotopic localization of the forelimb CF responses to the Vth lobule (Larsell) and the hindlimb to the IVth and IIIrd lobules. In the vermis there is much more admixture, with the hindlimb-evoked responses tending to dominate in the lateral vermis of the Vth lobule, and the forelimb more medially. In the IVth and IIIrd lobules forelimb responses were rare and were never large. In the medial vermis up to 1–1.7 mm from the midline there were no CF-evoked responses from the limb nerves. These distributions of CF-evoked responses are remarkably different from those reported by Oscarsson, and consideration is given to the factors responsible for this discrepancy.A more detailed examination was made of the CF-evoked responses from a large variety of hindlimb and forelimb nerves. Observations were made along many tracks usually arranged in a transverse plane, and it was found that between different recording sites along the same track or along adjacent tracks, there was a great deal of variation in the relative magnitudes of the CF-evoked responses from the different nerves. These distributions have an ill-defined patchy character so that at any focus there is opportunity for the most diverse kinds of piecemeal integration.These findings on the CF-input are considered in relationship to the mossy fiber input. It is pointed out that the pathways conveying CF-input to the cerebellum have a level of discriminative input adequate for the operation of fine control.     

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.     

Analysis of electrical potentials evoked in the cerebellar anterior lobe by stimulation of hindlimb and forelimb nerves

Summary Responses were evoked in the anterior lobe of the cerebellum by volleys in group I and II fibers of forelimb and hindlimb nerves — cutaneous, muscular, joint and fascial. These responses have been observed along microelectrode tracks that traverse the whole depth of the anterior lobe. These tracks have been identified in histological sections, and the recording sites along these tracks have been determined.It has been shown that there are many distinguishing features for the responses produced by the two types of afferent input to the cerebellum: climbing fibers and mossy fibers.The depth profiles are of particular importance in the differentiation of the CF and MF responses, and they correspond to those already determined for the exposed surface areas of the cerebellar cortex. As would be expected from the distribution of synapses by the CF fibers to the Purkinje cell dendrites, there is a maximum extracellular negativity deep in the molecular layer with sources superficial and deep thereto.In contrast, the mossy fiber input produces a powerful synaptic excitation in the granular layer, which is recorded there as a negative wave (N₂). The mossy fiber input by sequential relay also produces a negative wave (N₃) in the molecular layer. This wave is distinguished from the CF-evoked negative wave because it is not reversed in the fissura and the adjacent superficial molecular layer.An important distinguishing feature of the MF- and CF-evoked responses is that the latencies of the former are shorter by 6–12 msec for forelimb nerves and by 9–15 msec for hindlimb nerves. It is thus possible to measure the sizes of the MF and CF responses in the same traces.Another distinguishing feature is the failure of the CF responses with stimulus frequencies of 5–15/sec, whereas the MF-evoked potentials are well maintained above 15/sec. Also CF-evoked responses show much more size and latency variance than the MF-evoked responses, and often the facilitation of two or three volleys is required in order to evoke a stable CF response.By utilizing these various tests it is always possible to distinguish between the CF- and the MF-evoked responses recorded along the microelectrode tracks in the anterior lobe.The authors wish to express their grateful thanks to Mr. Lionel M. Davies who was responsible for the re-installation of Canberra equipment in Chicago and for its operational servicing.     

Development and migration of Purkinje cells in the mouse cerebellar primordium

Summary The mode of Purkinje cell migration in the mouse cerebellar primordium was examined immunohistochemically, by marking Purkinje cells with anti-spot 35 antibody and labeling them with 5-bromodeoxyuridine. The cells migrated radially from the neuroepithelium of the fourth ventricle towards the cortical surface between the 13th and 17th days (E13–E17) of gestation. Regional differences in the migratory process were evident: the final settlement of the Purkinje cells proceeded earlier in the lateral and posterior parts of the primordium, exhibiting latero-medial and posteroventral-anterodorsal diminishing sequences. To elucidate the factors involved in the migration, the arrangement of radial glial fibers, and expression of the cell adhesion molecule, tenascin, were examined immunohistochemically with the monoclonal antibody 1D11, a marker for both immature and mature astroglia, and an anti-tenascin antibody. At E14, 1D11-immunopositive fibers were seen to extend from the ventricle to the pial surface, and the cell bodies of immature glia migrated after E15 towards the cortex, shortening the radial processes whose end-feet were attached to the pia mater. Tenascin, which possesses a neuron-glial adhesiveness, was also expressed on the radial fibers during the migration of the Purkinje cells. The fibers were closely apposed to the migratory Purkinje cells, and their arrangement and orientation accorded with the migratory direction of the Purkinje cells. Further, changes in the molecular species of antigens detected by both the 1D11 and anti-tenascin antibodies were observed by immunoblotting analysis during the course of cerebellar development. These findings suggest that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthday     

Are the climbing fibres essential for the Purkinje cell inhibitory action?

Summary Following an almost total chemical lesion of the inferior olive, the inhibitory control of the cerebellar Purkinje cells upon their target neurones, mainly in Deiters nucleus, remains practically unchanged. This result is at variance with some recent findings having important implications in the cerebellar theories of motor learning.This work has been supported in part by the C.N.R.     

Long-term effects of 3-acetylpyridine-induced destruction of cerebellar climbing fibers on Purkinje cell inhibition of vestibulospinal tract cells of the rat

Summary The inhibitory action of Purkinje cells on vestibulospinal tract (VST) cells was examined in rats deprived of climbing fibers with 3-acetylpyridine (3-AP) intoxication. In order to resolve discrepancies raised in previous studies with various means, special efforts were devoted to directly estimate Purkinje cell inhibition at synaptic levels by using intracellular recording, to avoid sampling bias by using a systematic survey of VST cells in each rat, and to evaluate the time-dependence of the effects of climbing fiber deafferentation by regular testing at 10 day intervals until 160 days after 3-AP intoxication. As compared with 661 VST cells impaled in 15 control rats, 1771 VST neurons impaled in 29 3-AP-treated rats revealed four basic changes in the monosynaptic inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons in the white matter of the cerebellar anterior lobe. First, the rate of IPSP occurrence among VST cells was 0.64 in control rats; at more than 10 days after 3-AP intoxication it decreased gradually, down to 0.37–0.38 at the 70th–81st days, and thereafter increased up to 0.53 by the 160th day. The rate of IPSP occurrence varied considerably between the rostral and caudal regions, and also between the dorsal and ventral divisions of the VST cell population, but its reduction after 3-AP intoxication occurred approximately in parallel in all divisions. Second, IPSPs evoked with standard 500 A pulse stimuli were smaller in size on and after day 10. The reduction of IPSP size was by as much as 53% of control values at the 70th–101st days in the dorsal division, but no significant change occurred in the ventral division of the VST cell population. Third, the latency of the IPSPs was prolonged by about 0.25 ms on and after day 10. Analysis of the relationship between the IPSP latency and the dorsoventral location of VST cells in the medulla suggests that the major cause for the prolongation of IPSP latency is an increased synaptic delay at Purkinje cell axon terminals. Fourth, the cerebellar stimulation threshold for evoking IPSPs was almost always below 100 A in control rats, but values of 100–250 A were common after the 40th day. Thus, climbing fiber deafferentation exerts long-term influences on excitability of Purkinje cell axons, and on the connectivity and synaptic transmission from Purkinje cell axons to VST cells.On leave from the Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand     

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.     

Spontaneous activity of the Purkinje cells in the pigeon cerebellum

Summary In intact and decerebrate pigeon, both under barbiturate anaesthesia, the spontaneous activity of single Purkinje cells has been analysed.The average frequency of the simple spikes was similar to that described in mammals and was essentially the same in the intact and decerebrate preparations. The average frequency of the complex spikes was higher than that found in mammals and it was significantly depressed in the decerebrate pigeon as compared with the intact one. The amount of the depression, however, was less than that described in mammals.For the simple spikes a correlation was found between the mean interspike interval and both the standard deviation and the percentage of intervals at the mode. The correlation indicates that as the frequency increases the firing tends to become more regular. The same conclusion applies to the complex spikes, although in this case the only significant correlation was between the mean interval and standard deviation.Interval histograms of simple spikes were usually unimodal, but were occasionally also bimodal in both intact and decerebrate pigeons. Interval histograms of complex spikes were usually multimodal with many peaks at regular intervals, the highest peak not necessarily being the first one. The duration of the basic interval varied in different cells from 65 to 130 ms.The probability of firing of the simple spikes is always reduced following the appearance of a complex spike. The duration of the pause is related to the frequency of the simple spikes; the higher the frequency, the shorter the pause.     

Acetylcholinesterase activity in certain glomeruli and Golgi cells of the granular layer of the rat cerebellar cortex

Summary The Karnovsky method for the precise demonstration of acetyl and butyryl cholinesterase was applied to perfusion-fixed rat cerebellar vermis, and the results were examined by light and electron microscopy. Heavier concentrations of acetylcholinesterase activity were demonstrated in lobules IX and X than elsewhere in the cerebellar cortex. Acetylcholinesterase was found in the intercellular clefts and in relation to the synaptic membranes of mossy fiber and climbing fiber terminals in the granular layer of the uvula and nodulus. Within these lobules there were also similar endings of both the mossy and climbing fibers that showed no reaction. Acetylcholinesterase activity was found in the nuclear envelope and the cisternae of the rough endoplasmic reticulum of most Golgi cell perikarya and on the surface membranes of their axon terminals. Reasons for the selective distribution of acetylcholinesterase are discussed.Supported in part by USPHS Training Grant NS 5591 and Research Grant NS 3659 from the National Institute of Neurological Diseases and Stroke, and USPHS Grant EY-00361 from the National Eye Institute. Work carried out during Special Research Fellowship USPHS Grants 1F10-NB1890 and 2F11-NB1890 to Dr. Brown.     

Somatosensory representation of the climbing fiber system in the rostral intermediate cerebellum

Summary The fine grain organization of the climbing fiber (CF) representation for the intermediate cortex of lobules III, IV, and Va was examined. Data were obtained from cats anesthetized with sodium pentobarbital. Through extracellular recording techniques, CF responses were identified in 814 Purkinje cells; 74% were elicited by mechanical stimulation of various body surfaces. Of the CF responses elicited by stimulation, 60% involved the ipsilateral hindlimb, 36% represented parts of the forelimb, and only 4% represented areas of the face, tail, or abdomen. Hindlimb representation predominated in lobules III and IV, and forelimb representation was mainly confined to lobule Va. No distinct parasagittal zones that involved all three lobules were identified. In general, the organization for most sublobules could be best described as a mixture of patches of forelimb or hindlimb representations. Within the patches there was considerable diversity of receptive field types, particularly for the extremities. The receptive fields involving the distal phalanges were generally smaller than those for the proximal areas, but a range in field sizes was evident for both distal and proximal regions. More than half of either the forelimb or the hindlimb representation was limited to the distal paw areas; the two middle toes received the most frequent representation. Representations of the proximal limb areas were generally centered around either the wrist or the heel, or around the elbow or knee, but many of these receptive fields also extended to the phalanges. The multiple representation of various areas of forelimb and hindlimb throughout the rostral intermediate cortex was consistent with the type of organization that had been identified in other regions of the anterior lobe, although each cortical area contained a unique proportional CF representation of various body areas.     

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.