Embryonic Purkinje Cells Grafted on the Surface of the Adult Uninjured Rat Cerebellum Migrate in the Host Parenchyma and Induce Sprouting of Intact Climbing Fibres

By grafting solid pieces of cerebellar anlage onto the surface of the adult rat cerebellum, we have investigated the problem of the interactions between embryonic and adult neurons in an intact brain. A few days after grafting, embryonic astrocytic processes crossed the graft-host interface and radiated into the recipient molecular layer. Several grafted Purkinje cells also migrated into the host brain along such processes as well as adult Bergmann glia. Adult climbing fibres, labelled by means of Phaseolus vulgaris leucoagglutinin (PHA-L), sprouted new collateral branches which terminated on embryonic Purkinje cells at both extra- and intraparenchymal levels. No sign of activation of host astroglia or microglia was evident in the host cerebellum in relation to these processes. Embryonic Purkinje cells which migrated into the host cerebellum developed an adult-like morphology. Intraparenchymal grafts of neocortical embryonic tissue induced conspicuous growth of host olivary axons, characterized by a pattern which was different from that observed following cerebellar grafts. By contrast, when neocortical tissue was placed onto the surface of the recipient cerebellum, graft-host interactions were limited and climbing fibre sprouting was rarely seen. These results show that (i) supernumerary Purkinje cells can penetrate and settle in the adult intact cerebellar cortex, (ii) adult climbing fibres are able to innervate these new targets in the absence of any injury or activation of non-neuronal cells of the adult brain, and (iii) in the absence of damage to the adult brain, the plasticity of adult olivary axons is specifically elicited and controlled by embryonic Purkinje cells.     

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

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

Postsynaptic Currents and Short-term Synaptic Plasticity in Purkinje Cells Grafted onto an Uninjured Adult Cerebellar Cortex

It has been shown recently that embryonic Purkinje cells grafted extraparenchymally into an intact cerebellum, in the absence of any sign of damage, are able to migrate into the host molecular layer where they receive a climbing fibre innervation. Using the same technique, we investigated the development of the electrophysiological properties of the synapses between the grafted cells and their main afferents. Purkinje cells either in the graft or having migrated into the molecular layer of the host were recorded using the whole-cell patch-clamp method in acutely prepared slices 17–112 days after grafting. Spontaneous postsynaptic currents with a single-exponential decay and mediated by GABA_A receptors were very similar to those described in normal Purkinje cells. Excitatory postsynaptic currents (EPSCs) evoked by climbing fibre and by parallel fibre stimulation were blocked by an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate antagonist, and displayed the linear current-voltage relation typical of postnatal Purkinje cells. The attainment of normal functional properties by the adult axons at the newly formed synaptic sites was shown by the expression of short-term facilitation of parallel fibre EPSCs and of short-term depression of climbing fibre EPSCs. The grafted Purkinje cells showed climbing fibre polyinnervation 17-20 days after grafting which evolved to monoinnervation at 23-45 days, confirming the completion of the developmental programme up to maturation. Our experiments support the view that the adult intact brain is able to accept and integrate an additional number of neurons which show fully mature electrophysiological properties which are electrophysiologically indistinguishable from those of the host neurons.     

Early Development of Olivocerebellar Projections in the Fetal Rat Using CGRP Immunocytochemistry

The expression of calcitonin gene-related peptide (CGRP) immunoreactivity in certain inferior olivary neurons is transient and developmentally regulated. Labelled neurons begin to appear at embryonic day 16 (E16), and reach their maximal extent by postnatal day 2 (P2). The extinction of the labelling occurs between P13 and P16. Expression of CGRP immunoreactivity is also observed in a few cerebellar fibres from E17, when axons in the restiform bundle begin to enter medially the cerebellar parenchyma. Their maximal extent is reached by P6, and thereafter they slowly disappear following a precise pattern, although fibre extinction is not complete. The spatio-temporal changes in the olivary distribution of the labelled neurons and the changes in the cerebellar labelled fibres follow the known pattern of topographic arrangement of the olivocerebellar system in adult rats. Moreover, the developmental phases of the CGRP-labelled fibres in postnatal rats correspond to those known for climbing fibre phenotypic acquisition. Thus, CGRP immunocytochemistry identifies in the fetal rat a subset of inferior olivary neurons and their corresponding cerebellar climbing fibres. Using this approach, we have analysed some of the initial events leading to the formation of the olivocerebellar projection, and obtained the following information: (i) Olivocerebellar axons are not randomly distributed in the restiform bundle before they enter the cerebellum. (ii) In the presence of a large spectrum of choices at the surface of the rostral half of the cerebellar plate the labelled olivary axons begin to enter the cerebellum at a precise medial point to abut a region composed solely of migrating Purkinje cells, and establish contacts with their targets before these neurons reach their final cortical location. (iii) From E18 to E19, the bundle of labelled fibres loses its superficial location, being bypassed by migrating Purkinje cells, to occupy a region corresponding to the prospective white matter. This translocation is coincident with the occurrence of a second axonal entry point, somewhat more lateral than the previous one, and with the appearance of a new lateral stripe of labelled fibres. (iv) Both the early and the late appearing labelled stripes remain confined from the time of their formation in precise cerebellar territories, indicating that only some clusters of Purkinje cells are contacted by the CGRP fibres. The results obtained imply that there is neither a waiting period nor an initial phase of randomness in the formation of the olivocerebellar projection map. This absence of chaotic cerebellar invasion, and the high selectivity of the entry points, suggest that the orientation of CGRP-positive olivocerebellar fibres towards their targets is regulated by positional information shared between subsets of olivary neurons and clusters of Purkinje cells. The result of this process would be the formation of a precocious coarse topography that would need further refinement.     

The development of synaptic contacts in the cerebellum of Macaca mulatta

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

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. II. Development of synaptic responses: an in vitro study

Solid pieces of cerebellar primordia from 12-day-old C57Bl embryos were implanted in the cerebellar vermis of 3-4-month-old "Purkinje cell degeneration" mutant mice. Ten to 22 days after grafting, mutant mice were sacrificed, and synaptic responses of grafted Purkinje cells were studied by intracellular recordings performed in 400 microns thick sagittal slices in vitro. As early as 10 days after transplantation, grafted Purkinje cells have already completed their migration from the implant into the host molecular layer. Accordingly, inhibitory as well as excitatory responses were already elicited in these cells by electrical stimulation of the host subcortical white matter. Furthermore, a transient stage of multiple innervation of Purkinje cells by climbing fibers exists between 10 and 15 days after grafting, as revealed by the stepwise variation in amplitude of the climbing fiber-mediated excitatory postsynaptic potentials recorded before 15 days after grafting. Thirteen days after transplantation, typical all-or-none climbing fiber-mediated responses, parallel fiber-mediated excitatory postsynaptic potentials, and inhibitory postsynaptic potentials were also already present. Finally, normal adult-type synaptic responses were observed in all tested cells 15 to 17 days after grafting. Together with the companion paper (Sotelo et al., 1990), these results demonstrate that grafted Purkinje cells are able to impose on host afferents a pattern of synaptogenesis which closely follows that occurring during normal development, in particular, the transient stage of multiple innervation of Purkinje cells by climbing fibers.     

New insight on the factors orienting the axonal outgrowth of grafted Purkinje cells in the pcd cerebellum.

Despite Purkinje cell replacement, leading to the repair of the cortical circuit of the pcd mouse cerebellum grafted with E12 cerebellar primordium, the reestablishment of the corticonuclear projection only occurs for some Purkinje cells and in a small percentage of grafted mice. In order to assess the importance of: (1) competition between host and grafted deep nuclei, and (2) the distance between the implants and the host deep nuclei, new grafted experiments have been performed. In the latter, solid grafts were taken from E13 or E14 donor embryos after removal of the region containing the postmitotic deep nuclear neurons, and randomly positioned at various cerebellar depths. With cortical implants, the absence of donor nuclear neurons is not sufficient to allow the axons of the grafted Purkinje cells that have invaded the host molecular layer to escape the confinement of this layer. The molecular/granular layer interface appears as an almost impassable obstacle, and the granule cell layer as a nonpermissive milieu. With grafts located between the host deep nuclei and the 4th ventricle (deep grafts), the grafted Purkinje cells project massively to the host nuclei, but they are unable to leave the implant and, therefore, they are not integrated in the deficient cortical circuit. Finally, when the grafts positioned in the central white matter (intermediate grafts) disrupt the integrity of the host granule cell layer, some of the grafted Purkinje cells invade the host molecular layer, while most of them remain within the implant. Some axons of the cortically integrated Purkinje cells, using the nearby graft as a bridge, seem able to innervate the host deep nuclei. The latter, in addition, receive a massive projection from the nonintegrated Purkinje cells. These results emphasize the ability of grafted Purkinje cells to specifically innervate their target host neurons, when either there is proximity, or when a permissive microenvironment for their axonal outgrowth is created by embryonic grafted cortical cerebellar neurons, filling the gap between the molecular layer and the deep nuclei of the host.     

The cerebellar component of Friedreich’s ataxia

Lack of frataxin in Friedreich’s ataxia (FRDA) causes a complex neurological and pathological phenotype. Progressive atrophy of the dentate nucleus (DN) is a major intrinsic central nervous system lesion. Antibodies to neuron-specific enolase (NSE), calbindin, glutamic acid decarboxylase (GAD), and vesicular glutamate transporters 1 and 2 (VGluT1, VGluT2) allowed insight into the disturbed synaptic circuitry of the DN. The available case material included autopsy specimens of 24 patients with genetically defined FRDA and 14 normal controls. In FRDA, the cerebellar cortex revealed intact Purkinje cell somata and dendrites as assessed by calbindin immunoreactivity. The DN, however, displayed severe loss of large NSE-reactive neurons. Small neurons remained intact. Labeling of Purkinje cells, basket fibers, Golgi neurons, and Golgi axonal plexuses with antibodies to GAD indicated normal intrinsic circuitry of the cerebellar cortex involving γ-aminobutyric acid (GABA). In contrast, the DN displayed severe loss of GABA-ergic terminals and formation of GAD- and calbindin-reactive grumose degeneration. The surviving small GAD-positive DN neurons provided normal GABA-ergic terminals to intact inferior olivary nuclei. The olives also received normal glutamatergic terminals as shown by VGluT2-reactivity. VGluT1-immunocytochemistry of the cerebellar cortex confirmed normal glutamatergic input to the molecular layer by parallel fibers and the granular layer by mossy fibers. VGluT2-immunoreactivity visualized normal climbing fibers and mossy fiber terminals. The DN, however, showed depletion of VGluT1- and VGluT2-reactive terminals arising from climbing and mossy fiber collaterals. The main functional deficit underlying cerebellar ataxia in FRDA is defective processing of inhibitory and excitatory impulses that converge on the large neurons of the DN. The reason for the selective vulnerability of these nerve cells remains elusive.     

The reconstruction of cerebellar circuits.

Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit.     

Immature chemodifferentiation of purkinje cell synapses revealed by 5′-nucleotidase ecto-enzyme activity in the cerebellum of the reeler mouse

During postnatal development of the rodent cerebellum, a transient enzyme activity of ecto-5′-nucleotidase has been shown in the asymmetrical synapses of Purkinje cells. The alterations of the afferent circuitry and microenvironment of the ectopic Purkinje cells present in the cerebellum of the reeler mutant mouse could enlighten parameters that influence the synaptic 5′-nucleotidase activity of these cells. Ecto-enzyme cytochemistry reveals intense 5′-nucleotidase activity in 43% of synapses of the Purkinje cells throughout the cortex and the core of the reeler cerebellar vermis, although the molecular layer displays large areas with less than 1% of labelled synapses. However, enzymatic labelling is found in considerably more Purkinje cells synapses (73%) throughout the granular layer and the subcortical mass. Climbing fiber synapses of monoinnervated Purkinje cells are labelled by 5′-nucleotidase activity in the molecular layer, as well as asymmetrical synapses made on the subjacent ectopic Purkinje cells by the multiple climbing fibers and by the heterologous afferences. The non-innervated dendritic spines of these cells are also labelled, suggesting that 5′-nucleotidase activity at postsynaptic sites of reeler Purkinje cells does not depend on the presynaptic innervation. Rather, 5′-nucleotidase enzyme activity is enhanced at theses sites when the Purkinje cells have not achieved chemodifferentiation but have conserved immature wiring, i.e., low parallel fiber and multiple climbing fiber inputs. Synapse 29:279–292, 1998. © 1998 Wiley-Liss, Inc.     

Exposure to Kainic Acid Mimics the Effects of Axotomy in Cerebellar Purkinje Cells of the Adult Rat

We have investigated the long-term structural changes which affect Purkinje cells exposed to a single dose of kainic acid. Following intraparenchymal injection of the excitotoxin in the cerebellar cortex (1 μ1 of a 1 mg/ml solution), Purkinje cells which survived within the lesioned area or close to its edges showed remarkable axonal abnormalities, involving the formation of torpedoes, hypertrophy of recurrent collaterals and atrophy of the corticofugal portion of the axon. In addition, their dendritic trees were often affected by conspicuous regressive alterations. The climbing fibres contacting these Purkinje cells were characterized by thick perisomatic plexuses, whereas their peridendritic branches were atrophic. The dendrites innervated by such atrophic olivary arbours were studded with huge numbers of newly formed spines. These alterations were already present a few days after kainic acid administration and persisted for the total period of observation of 6 months after the lesion. The remarkable similarity between the abnormalities of Purkinje cells exposed to kainic acid and those observed after axotomy indicates that in these two conditions common mechanisms determine analogous long-lasting modifications in the affected neurons. It is proposed that kainic acid-induced intracellular calcium overload disrupts cytoskeletal components and impairs axonal transport, thus depriving the affected Purkinje cells of retrograde trophic influences from their target neurons. As a consequence the affected neurons undergo long-lasting regressive modifications and compensatory remodelling phenomena.     

Characterization in vivo of bilaterally branching pontocerebellar mossy fibre to Golgi cell inputs in the rat cerebellum

Golgi cells regulate the flow of information from mossy fibres to the cerebellar cortex, through a mix of feedback and feedforward inhibitory actions on granule cells. The aim of the current study was to examine mossy fibre input to Golgi cells, in order to assess their impact on switching Golgi cells into feedforward behaviour. In urethane-anaesthetized rats, extracellular recordings were made from Golgi cells in Crus II ( n  =   18). Spikes were evoked in all Golgi cells by microstimulation within the contralateral hemispheral cortex, via branches of mossy fibres that terminate in both cerebellar hemispheres. The latencies of these responses were very short, consistent with a monosynaptic mossy fibre contact [average onset latency 2.3 ± 0.1 ms (SEM)]. The same stimuli had no measurable effect on spike responses of nearby Purkinje cells ( n  =   12). Systematic mapping in the contralateral cerebellar hemisphere (Crus Ib, IIa, IIb and the paramedian lobule) usually revealed one low-intensity stimulus 'hotspot' (12–35 μA) from which short-latency spikes could be evoked in an individual Golgi cell. Microinjections of red and green retrograde tracers (latex beads, ∼50–150 nL injection volume) made at the recording site and the stimulation hotspot resulted in double-labelled neurons within the pontine nuclei. Overall, this suggests that subsets of pontine neurons supply mossy fibres that branch to both hemispheres, some of which directly target Golgi cells. Such an arrangement may provide a common feedforward inhibitory link to temporally couple activity on both sides of the cerebellum during behaviour.     

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. I. Development of reciprocal graft-host interactions

In this paper, we have morphologically studied the developmental events underlying the neuronal replacement, 3-21 days after grafting. Despite their abnormal environment, Purkinje cell progenitors proceed with their proliferation in the grafted neuroepithelium, with a time window similar to that characterizing proliferation of this neuronal class in control mouse embryos. Only postmitotic Purkinje cells leave the grafts and migrate to the host molecular layer following stereotyped pathways. These neurons invade the host molecular layer, either through a tangential migration under the pial basal lamina from the graft/host interface or breaking locally the latter, and passing directly from the lateral swellings of the graft lying on the surface of the host folia. Whatever the pathway for host invasion, the migrating Purkinje cells penetrate radially and/or obliquely into the host molecular layer until their inward-oriented processes attain the molecular/granular layer interface, which occurs about 7 days after grafting. At the end of their migration, the grafted Purkinje cells with bipolar shapes and long and smooth processes begin to build up their ultimate dendritic trees. This dendritogenesis proceeds with constructive and regressive processes, passing through the same three developmental phases described by Ramón y Cajal (Trab. Lab. Invest. Biol. Univ. Madrid 24:215-251, 1926) for control Purkinje cells (phase of the fusiform cell, phase of the stellate cell with disoriented dendrons, and phase of orientation and flattening of the dendrites). In the grafted cerebella, the duration of the second and third phases is somewhat shorter than during normal cerebellar ontogenesis. Synaptogenesis between adult host axons and grafted Purkinje cells starts when the latter attain their second phase of dendritic development. Somatic filopodia emerging from grafted Purkinje cells begin, 10-11 days after grafting, to be synaptically contacted by axonal sprouts of the host climbing fibers resulting, 2 days later, in the formation of pericellular nests. Synaptogenesis between slender dendritic spines and host parallel fibers, together with that of axon terminals from host molecular layer interneurons and the smooth surface of the grafted Purkinje cell somata, begin earlier than in control mouse development, being almost simultaneous with climbing fiber/Purkinje cell synaptogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parallel fibre receptive fields of Purkinje cells and interneurons are climbing fibre-specific

In cats decerebrated at the intercollicular level, the cutaneous parallel fibre receptive fields of Purkinje cells, molecular layer interneurons and Golgi cells in the cerebellar C3 zone were delineated by natural stimulation of the skin during extracellular unitary recordings. The locations of these receptive fields were compared with the climbing fibre receptive field of the local Purkinje cell and with the receptive fields of other neurons located along a beam of parallel fibres. The parallel fibre receptive fields of these neurons were highly specific to the local climbing fibre receptive field. In Purkinje cells, the parallel fibre receptive fields were located outside the climbing fibre receptive field of the same cell. In contrast, the parallel fibre receptive fields of interneurons were similar to the receptive field of the locally terminating climbing fibres. In both types of neurons, the parallel fibre receptive fields were small and had distinct borders. The location on the skin of the parallel fibre receptive fields differed conspicuously between neighbouring Purkinje cells and between neighbouring interneurons along a beam as well as between Purkinje cells and interneurons in the same electrode tracks. The remarkable specificity between the parallel fibre receptive fields in Purkinje cells and interneurons and the receptive field of the local climbing fibre is most easily explained by different forms of parallel fibre synaptic plasticity.     

The cerebellum of the frog Rana ridibunda. An electron microscopic study

An electron microscopic study of neuronal types and different synaptic contacts has been made in the cerebellum of the frog Rana ridibunda. The Purkinje cells have a pear-shaped cell body and in their cytoplasm the organelles show a special arrangement because of the great amount of microtubules they contain. The granule cells are small, rounded neurons with a large nucleus surrounded by a thin rim of cytoplasm. The stellate cells are interneurons of the molecular layer whose large nuclei show a single finger-like invagination of its nuclear envelope. The afferent tracts to the cerebellum end either as climbing fibers or mossy fibers. The axon terminals of climbing fibers are large and the synaptic complexes exhibit all the features of a type-I Gray synapse. The mossy fibers reach the granular layer and synapses between them and granule cell dendrites are by far the most abundant. The parallel fibers establish synaptic contacts on the spines arising from the spiny branchlet units of the Purkinje cells and with the perikaryon and dendrites of stellate cells. The stellate cell axons cross the molecular layer and establish type-II Gray synapses on the Purkinje cells.     

Development of cholinesterase histochemical staining in cerebellar cortex: transient expression of "nonspecific" cholinesterase in Purkinje cells of the nodulus and uvula.

Patterns of "nonspecific" cholinesterase (ChE) and acetylcholinesterase (AChE) activity were studied in developing rat cerebellar cortex by enzyme histochemistry and light and electron microscopy. Three types of ChE histochemical reaction product were observed in cerebellar cortex: (i) ChE is found in capillary endothelium throughout the cerebellum. Capillary ChE staining is present by the time of birth and continues into adulthood. (ii) ChE is found in radial glial fibers and their parent cell bodies, the Golgi epithelial cells. Radial glial fiber staining is mot intense during the first 3 weeks of postnatal life. (iii) ChE is found in Purkinje cells of the nodulus and ventral uvula. No ChE staining of Purkinje cells was seen in other parts of the cerebellum. ChE staining of Purkinje cells appears to be transient, first appearing at Postnatal Day 2 (P2), reaching peak intensity at P7-9, and decreasing to adult levels by P16. AChE activity displays a pattern markedly different from ChE, with staining in deep cerebellar nuclei, in putative mossy fiber terminals, and in Golgi neurons of cerebellar cortex. No evidence was found for transient AChE staining in Purkinje cells in any part of the cerebellum. The function of transiently expressed ChE activity in developing Purkinje neurons is unknown, but may be related to reorganization of cerebellar cortical circuitry associated with growth of mossy fiber afferents.     

Cerebellar efferent neurons in teleost fish

In tetrapods, cerebellar efferent systems are mainly mediated via the cerebellar nuclei. In teleosts, the cerebellum lacks cerebellar nuclei. Instead, the cerebellar efferent neurons, termed eurydendroid cells, are arrayed within and below the ganglionic layer. Tracer injections outside of the cerebellum, which retrogradely label eurydendroid cells demonstrate that most eurydendroid cells possess two or more primary dendrites which extend broadly into the molecular layer. Some eurydendroid cells mostly situated in caudal portions of the cerebellum have only one primary dendrite. The eurydendroid cells receive inputs from the Purkinje cells and parallel fibers, but apparently do not receive inputs from the climbing fibers. Eurydendroid cells of the corpus cerebelli and medial valvula project to many brain regions, from the diencephalon to the caudal medulla. A few eurydendroid cells in the valvula project directly to the telencephalon. About half of the eurydendroid cells are aspartate immunopositive. Anti-GABA and anti-zebrin II antibodies that are known as markers for the Purkinje cells in mammals also recognize the Purkinje cells in the teleost cerebellum, but do not recognize the eurydendroid cells. These results suggest that the eurydendroid cells receive GABAergic inputs from the Purkinje cells. This relationship between the eurydendroid and Purkinje cells is similar to that between the cerebellar nuclei and Purkinje cells in mammals. The eurydendroid cells of teleost have both dissimilar as well as similar features compared to neurons of the cerebellar nuclei in tetrapods.     

Simple and Complex Spike Firing Patterns in Purkinje Cells During Classical Conditioning

Classical blink conditioning is known to depend critically on the cerebellum and the relevant circuitry is gradually being unravelled. Several lines of evidence support the theory that the conditioned stimulus is transmitted by mossy fibers to the cerebellar cortex whereas the unconditioned stimulus is transmitted by climbing fibers. This view has been dramatically confirmed by recent Purkinje cell recordings during training with a classical conditioning paradigm. We have tracked the activity of single Purkinje cells with microelectrodes for several hours in decerebrate ferrets during learning, extinction, and relearning. Paired peripheral forelimb and periocular stimulation, as well as paired direct stimulation of cerebellar afferent pathways (mossy and climbing fibers) causes acquisition of a pause response in Purkinje cell simple spike firing. This conditioned Purkinje cell response has temporal properties that match those of the behavioral response. Its latency varies with the interstimulus interval and it responds to manipulations of the conditioned stimulus in the same way that the blink does. Complex spike firing largely mirrors the simple spike behavior. We have previously suggested that cerebellar learning is subject to a negative feedback control via the inhibitory nucleo-olivary pathway. As the Purkinje cell learns to respond to the conditioned stimulus with a suppression of simple spikes, disinhibition of anterior interpositus neurons would be expected to cause inhibition of the inferior olive. Observations of complex spike firing in the Purkinje cells during conditioning and extinction confirm this prediction. Before training, complex spikes are unaffected or facilitated by the conditioned stimulus, but as the simple spike pause response develops, spontaneous and stimulus-evoked complex spikes are also strongly suppressed by the conditioned stimulus. After extinction of the simple spike pause response, the complex spikes reappear.