Synaptic localization of 5'-nucleotidase activity in the cerebellar cortex in adult rats irradiated by X rays after birth

5'-nucleotidase cytochemistry was performed in the cerebellum of adult rats exposed previously to a single postnatal X-irradiation which maintains until adulthood the normally transient multiple innervation of cerebellar Purkinje cells by climbing fibers. 5'-nucleotidase activity persists in climbing fiber synapses and other asymmetrical synapses while it is transient during normal development. Therefore, in the X-irradiated rat, an immature stage of the excitatory synapses persists which, for the climbing fibers, co?ncides with or precedes their involution during normal development.     

Extent of multiple innervation of purkinje cells by climbing fibers in the olivocerebellar system of weaver,reeler, and staggerer mutant mice

Multiple innervation of cerebellar Purkinje cells (PCs) by climbing fibers (CFs) has been described recently in adult weaver, reeler, and staggerer mutant mice, instead of the monoinnervation found in normal adults. In the present study, the extent of this multiple innervation was estimated by two methods, using both evoked and spontaneous activity of the olivocerebellar system. Concordant values were obtained: the mean number of CF collaterals per PC was between 3.5 and 4 in weaver and staggerer and close to 3.2 for the multiply innervated PCs of reeler mice. These values are of the same order of magnitude as those for the transient multiple innervation in developing rats (Mariani and Changeux, 1981a, b).     

Eph receptors are involved in the activity-dependent synaptic wiring in the mouse cerebellar cortex

Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex.     

Roles of phospholipase Cbeta4 in synapse elimination and plasticity in developing and mature cerebellum

The beta isoforms of phospholipase C (PLCbetas) are thought to mediate signals from metabotropic glutamate receptor subtype 1 (mGluR1) that is crucial for the modulation of synaptic transmission and plasticity. Among four PLCbeta isoforms, PLCbeta4 is one of the two major isoforms expressed in cerebellar Purkinje cells. The authors have studied the roles of PLCbeta4 by analyzing PLCbeta4 knockout mice, which are viable, but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple climbing fiber innervation is clearly impaired in the rostral portion of the cerebellar vermis, where PLCbeta4 mRNA is predominantly expressed in the wild-type mice. In the adult, long-term depression is deficient at parallel fiber to Purkinje cell synapses in the rostral cerebellum of the PLCbeta4 knockout mice. The impairment of climbing fiber synapse elimination and the loss of long-term depression are similar to those seen in mice defective in mGluR1, Galphaq, or protein kinase C. Thus, the authors' results strongly suggest that PLCbeta4 is part of a signaling pathway, including the mGluR1, Galphaq and protein kinase C, which is crucial for both climbing fiber synapse elimination in the developing cerebellum and long-term depression induction in the mature cerebellum.     

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.     

Serotonin fiber innervation of cerebellar cell suspensions intraparenchymally grafted to the cerebellum ofpcd mutant mice

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of Purkinje cell degeneration (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss inpcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11–13 normal mouse embryos and were intraparenchymally grafted into the cerebellum ofpcd mutants either directly or after pre-treatment with 5, 7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca²⁺-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells. On the other hand, in grafts pre-treated with 5,7-DHT, no 5-HT cell bodies were seen in the grafted cerebellum; 5-HT fibre innervation of the grafts occurred, but it appeared to be slightly less robust compared to situations of co-grafted 5-HT cell bodies. These findings suggest that host 5-HT fibers are able to provide afferent innervation to donor cerebellar tissue; the presence of co-grafted 5-HT cells may augment such an innervation.Special issue dedicated to Dr. Morris H. Aprison.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal x-irradiation

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilateral forlimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 ± 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Axonal motility and its modulation by activity are branch-type specific in the intact adult cerebellum

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

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal X-irradiation.

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilpateral forelimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 +/- 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Glutamate-immunoreactive climbing fibres in the cerebellar cortex of the rat

The climbing fibre system, one of the two main excitatory inputs to the cerebellar cortex, is anatomically and physiologically well characterized, while the nature of its neurotransmitter is still a matter of debate. We wished to determine whether glutamate-immunoreactive profiles with the morphological characteristics of climbing fibres could be found in the rat cerebellar cortex. For this purpose, a monoclonal anti-glutamate antibody has been used in combination with a sensitive postembedding immunoperoxidase method on semithin sections or in combination with a postembedding immunogold method on ultrathin sections. At the light microscopic level, climbing fibres appeared as strongly stained fibrous profiles, chains of interconnected varicosities or heavily labelled dots of various sizes, often in close apposition to principal Purkinje cell dendrites. At the electron microscopic level, certain labelled varicosities or more elongated profiles resembling climbing fibre terminals were in synaptic contact with dendritic spines of Purkinje cells. Quantitative analysis of gold particle densities showed that such elements were about three to four times more heavily labelled than their postsynaptic partners. The results obtained in this study demonstrate that at least a subset of climbing fibres and their terminals contain relatively high levels of glutamate-like immunoreactivity and provide additional evidence for a role of glutamate as transmitter in these cerebellar afferents.     

Development of nerve connections under the control of neurotrophic factors: parallels with consumer-resource systems in population biology

The development of connections between neurons and their target cells involves competition between axons for target-derived neurotrophic factors. Although the notion of competition is commonly used in neurobiology, the process is not well understood, and only a few formal models exist. In population biology, in contrast, the concept of competition is well developed and has been studied by means of many formal models of consumer-resource systems. Here we show that a recently formulated model of axonal competition can be rewritten as a general consumer-resource system. This allows neurobiological phenomena to be interpreted in population biological terms and, conversely, results from population biology to be applied to neurobiology. Using findings from population biology, we have studied two extensions of our axonal competition model. In the first extension, the spatial dimension of the target is explicitly taken into account. We show that distance between axons on their target mitigates competition and permits the coexistence of axons. The model can account for the fact that in many types of neurons a positive correlation exists between the size of the dendritic tree and the number of innervating axons surviving into adulthood. In the second extension, axons are allowed to respond to more than one neurotrophic factor. We show that this permits competitive exclusion among axons of one type, while at the same time there is coexistence with axons of another type innervating the same target. The model offers an explanation for the innervation pattern found on cerebellar Purkinje cells, where climbing fibres compete with each other until only a single one remains, which coexists with parallel fibre input to the same Purkinje cell.     

Patterned Neuroprotection in the Inpp4awbl Mutant Mouse Cerebellum Correlates with the Expression of Eaat4

The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4a^wbl mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4a^wbl mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.     

Cerebellar complex spikes multiplex complementary behavioral information

Purkinje cell (PC) discharge, the only output of cerebellar cortex, involves 2 types of action potentials, high-frequency simple spikes (SSs) and low-frequency complex spikes (CSs). While there is consensus that SSs convey information needed to optimize movement kinematics, the function of CSs, determined by the PC’s climbing fiber input, remains controversial. While initially thought to be specialized in reporting information on motor error for the subsequent amendment of behavior, CSs seem to contribute to other aspects of motor behavior as well. When faced with the bewildering diversity of findings and views unraveled by highly specific tasks, one may wonder if there is just one true function with all the other attributions wrong? Or is the diversity of findings a reflection of distinct pools of PCs, each processing specific streams of information conveyed by climbing fibers? With these questions in mind, we recorded CSs from the monkey oculomotor vermis deploying a repetitive saccade task that entailed sizable motor errors as well as small amplitude saccades, correcting them. We demonstrate that, in addition to carrying error-related information, CSs carry information on the metrics of both primary and small corrective saccades in a time-specific manner, with changes in CS firing probability coupled with changes in CS duration. Furthermore, we also found CS activity that seemed to predict the upcoming events. Hence PCs receive a multiplexed climbing fiber input that merges complementary streams of information on the behavior, separable by the recipient PC because they are staggered in time.

Purkinje cell (PC) discharge, the only output of cerebellar cortex, involves both high-frequency simple spikes and low-frequency complex spikes; the function of the latter, determined by a PC’s climbing fibre input, remains controversial. This study shows that PCs receive a multiplexed climbing fibre input that merges complementary streams of information relevant for behaviour.     

Neuronal expression of macrophage colony stimulating factor in Purkinje cells and olfactory mitral cells of wild-type and cerebellar-mutant mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.     

Diminished climbing fiber innervation of Purkinje cells in the cerebellum of myosin Va mutant mice and rats

Myosin Va is an actin-based molecular motor that is involved in organelle transport and membrane trafficking. Here, we explored the role of myosin Va in the formation of synaptic circuitry by examining climbing fiber (CF) innervation of Purkinje cells (PCs) in the cerebella of dilute-neurological (d-n) mice and dilute-opisthotonus (dop) rats that have mutations in dilute-encoded myosin Va. Anterograde labeling of CFs with biotinylated dextran amine (BDA) revealed that they arborized poorly and that their tips extended only half way through the thickness of the molecular layer (ML) in adult d-n mice. Using immunohistochemistry specific for vesicular glutamate transporter 2 (VGluT2) to visualize CF synaptic terminals, we found that during development and in adulthood, these terminals did not ascend as far along the proximal shaft dendrites of PCs in d-n mice and dop rats as they did in normal animals. An irregular distribution of BDA-labeled bulbous varicosities and VGluT2 spots along CF branches were also noted in these animals. Finally, VGluT2-positive CF terminals were occasionally localized on the PC somata of adult d-n cerebella. These phenotypes are consistent with our electrophysiological findings that CF-mediated excitatory postsynaptic currents (EPSCs) were significantly smaller in amplitude and faster in decay in adult d-n mice, and that the regression of multiple CFs was slightly delayed in developing d-n mice. Taken together, our results suggest that myosin Va is essential for terminal CF extension and for the establishment of CF synapses within the proper dendritic territories of PCs.     

Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the adult staggerer mutant mouse

Intracellular recordings from Purkinje cells (PC) in the cerebellum of adult staggerer mutant mice revealed that the orthodromic response of PCs to juxtafastigial (JF) stimulation closely resembled a climbing fiber response (CFR). However, for most of the PCs studied, these responses were graded in a stepwise manner when the stimulus strength was increased. The underlying excitatory synaptic potentials (EPSPs) had the typical shape of EPSPs mediated through climbing fibers (CFs), but their size fluctuated in discrete steps, the highest one reaching the firing level. In the same PCs, the size of the spontaneous EPSPs fluctuated in a similar fashion and the frequency of each step was in the range of CF-mediated EPSPs. These results strongly suggest that in staggerer mice several CFs synapse with each PC instead of a single CF as in normal adults. Furthermore, the activation through some of these CFs does not reach the firing level of the corresponding PC.     

Long-term changes in the activity of Purkinje cells and efferent cerebellar neurons following bilateral destruction of the inferior olive

The spontaneous discharge frequency of Purkinje cells and neurones of the cerebellar nuclei was evaluated in rats after complete bilateral destruction of their inferior olive with 3-acetylpyridine, performed one day to six months before. The deafferentation from the climbing fibers produced an increased inhibitory action of the Purkinje cells on their target neurones, lasting at least for one week. A relative compensation took place progressively during the first month, but the normal activity of the circuit did not recover even after six months.     

Differentiation and innervation of the atrioventricular bundle and ventricular Purkinje system in sheep heart

The development of the atrioventricular bundle (AVB) and ventricular Purkinje system and their innervation have been studied in fetal sheep from 27 to 140 days gestation (term is 147 days). The AVB initially consisted of a primordium, which lacked innervation and was composed of small, relatively undifferentiated myocytes. Differentiation of Purkinje-like cells within the AVB began near its distal end and extended towards the atrioventricular node (AVN). Differentiation of the ventricular Purkinje system extended distally from the region of bifurcation of the AVB from cells that were indistinguishable from the working myocardium and continuous with the AVB primordium. Differentiation of Purkinje-like AVB cells was complete by 46 days gestation but Purkinje fibres were still differentiating within the ventricular wall at 60 days gestation. The main morphological changes included a large increase in cell size and organization into strands, development of characteristic glycogen-filled regions containing many intermediate filaments and early development of myofibrillar M lines compared to the working myocardium. The differentiation of AVB cells and the ventricular Purkinje system preceded their innervation. The AVB became innervated earlier than ventricular Purkinje fibres, intimate contacts between proximal AVB cells and nerve axons being present at 60 days gestation. Nerve fibres were present in the septomarginal band at this time, however, en passant associations with ventricular Purkinje fibres were rarely observed until 140 days gestation and intimate contacts were not present at any stage. Although the AVB and ventricular Purkinje system of adult sheep are composed of morphologically similar cells, the present study demonstrates that they differ in origin and their mode of differentiation as well as timing and intimacy of innervation. Innervation is not part of the developmental mechanism leading to the differentiation of Purkinje fibres. No primordium of the ventricular Purkinje system could be identified, suggesting that the mechanism of differentiation of ventricular Purkinje fibres involves recruitment from early working myocardium.     

Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells

The receptor tyrosine kinase TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), are critically important for growth, survival and activity-dependent synaptic strengthening in the central nervous system. These TrkB-mediated actions occur in a highly cell-type specific manner. Here we report that cerebellar Purkinje cells, which are richly endowed with TrkB receptors, develop a normal morphology in trkB-deficient mice. Thus, in contrast to other types of neurons, Purkinje cells do not need TrkB for dendritic growth and spine formation. Instead, we find a moderate delay in the maturation of GABAergic synapses and, more importantly, an abnormal multiple climbing fiber innervation in Purkinje cells in trkB-deficient mice. Thus, our results demonstrate an involvement of TrkB receptors in synapse elimination and reveal a new role for receptor tyrosine kinases in the brain. Electronic Supplementary Materials Supplementary Materials is available in the online version of this article at     

Activity of the climbing fiber innervating various Purkinje cells

Low-amplitude potentials (10-130 microV) related to the action of a distant branch of the climbing fiber, which elicits complex spikes of the reference Purkinje cell were revealed by means of potential averaging synchronously with complex spikes of Purkinje cells in 10 out of 255 paired records of cerebellar Purkinje cells activity and extracellular field potentials at interelectrode distances of 200-1500 microns. These potential waves had a stable form in independent sets of data. In 3 out of 10 cases, the low-amplitude potentials included a slow (about 100 ms in duration) component. In one case, both test and reference electrodes recorded both simple and complex spikes of different Purkinje cells so that complex spikes of both cells were practically synchronous (conditional probability of complex spikes p = 0.97, onset time difference 0.54 ms). Thus for the first time in cerebellar physiology both simple and complex spikes activity of two Purkinje cells controlled by the same climbing fiber was recorded.