The Ferdinando Rossi Memorial Lecture: Zones and Stripes-Pattern Formation in the Cerebellum.

The cerebellum has a complex architecture-highly reproducible and conserved through evolution. Cerebellar architecture is organized around the Purkinje cell. Purkinje cells in the mouse cerebellum come in many different subtypes, identifiable by expression markers, sensitivity to mutation, etc. These are organized first into five "transverse zones," each of which is further subdivided into dozens of reproducible "stripes." This arrangement serves as the scaffolding to organize afferent topography and restrict the distribution of excitatory and inhibitory interneurons. This brief review will survey some of the mechanisms that lead to the formation of this elaborate pattern during cerebellar development. Pattern formation in the cerebellar cortex is a multistage process that begins early in development with the generation of the various Purkinje cell subtypes, and matures through the dispersal of Purkinje cell clusters into stripes. Two developmental processes will be discussed in particular: the mechanisms that lead to Purkinje cell subtype specification (i.e., how different kinds of Purkinje cells are made) and the role played by Purkinje cell migration in pattern formation (i.e., how these Purkinje cell subtypes end up in a reproducible array of stripes).

New insights on vertebrate olivo-cerebellar climbing fibers from computerized morphological reconstructions.

Characterization of neuronal connectivity is essential to understanding the architecture of the animal nervous system. Specific labeling and imaging techniques can visualize axons and dendrites of single nerve cells. Two-dimensional manual drawing has long been used to describe the morphology of labeled neuronal elements. However, quantitative morphometry, which is essential to understanding functional significance, cannot be readily extracted unless the detailed neuronal geometry is comprehensively reconstructed in three-dimensional space. We have recently applied an accurate and robust digital reconstruction system to cerebellar climbing fibers, which form highly dense and complex terminal arbors as one of the strongest presynaptic endings in the vertebrate nervous system. Resulting statistical analysis has shown how climbing fibers morphology is special in comparison to other axonal terminals. While thick primary branches may convey excitation quickly and faithfully to the far ends, thin tendril branches, which have a larger bouton density, form the majority of presynaptic outputs. This data set, now publicly available from NeuroMorpho.Org for further modeling and analysis, may constitute the first detailed and comprehensive digital reconstruction of the complete axonal terminal field with identified branch types and full accounting of boutons for any neuronal class in the vertebrate brain.

Probabilistic identification of cerebellar cortical neurones across species.

Despite our fine-grain anatomical knowledge of the cerebellar cortex, electrophysiological studies of circuit information processing over the last fifty years have been hampered by the difficulty of reliably assigning signals to identified cell types. We approached this problem by assessing the spontaneous activity signatures of identified cerebellar cortical neurones. A range of statistics describing firing frequency and irregularity were then used, individually and in combination, to build Gaussian Process Classifiers (GPC) leading to a probabilistic classification of each neurone type and the computation of equi-probable decision boundaries between cell classes. Firing frequency statistics were useful for separating Purkinje cells from granular layer units, whilst firing irregularity measures proved most useful for distinguishing cells within granular layer cell classes. Considered as single statistics, we achieved classification accuracies of 72.5% and 92.7% for granular layer and molecular layer units respectively. Combining statistics to form twin-variate GPC models substantially improved classification accuracies with the combination of mean spike frequency and log-interval entropy offering classification accuracies of 92.7% and 99.2% for our molecular and granular layer models, respectively. A cross-species comparison was performed, using data drawn from anaesthetised mice and decerebrate cats, where our models offered 80% and 100% classification accuracy. We then used our models to assess non-identified data from awake monkeys and rabbits in order to highlight subsets of neurones with the greatest degree of similarity to identified cell classes. In this way, our GPC-based approach for tentatively identifying neurones from their spontaneous activity signatures, in the absence of an established ground-truth, nonetheless affords the experimenter a statistically robust means of grouping cells with properties matching known cell classes. Our approach therefore may have broad application to a variety of future cerebellar cortical investigations, particularly in awake animals where opportunities for definitive cell identification are limited.

Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this process

Fontes alternativas de células ß têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células ß-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células ß. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células ß e no processo de diferenciação ß-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoß-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células ß-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células

New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic ß-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to ß-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in ß-cells and in ß-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to ß-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic ß-cells and possibly being related to maintenance of cell viability and ß-cell maturation

Digital morphometry of rat cerebellar climbing fibers reveals distinct branch and bouton types.

Cerebellar climbing fibers (CFs) provide powerful excitatory input to Purkinje cells (PCs), which represent the sole output of the cerebellar cortex. Recent discoveries suggest that CFs have information-rich signaling properties important for cerebellar function, beyond eliciting the well known all-or-none PC complex spike. CF morphology has not been quantitatively analyzed at the same level of detail as its biophysical properties. Because morphology can greatly influence function, including the capacity for information processing, it is important to understand CF branching structure in detail, as well as its variability across and within arbors. We have digitally reconstructed 68 rat CFs labeled using biotinylated dextran amine injected into the inferior olive and comprehensively quantified their morphology. CF structure was considerably diverse even within the same anatomical regions. Distinctly identifiable primary, tendril, and distal branches could be operationally differentiated by the relative size of the subtrees at their initial bifurcations. Additionally, primary branches were more directed toward the cortical surface and had fewer and less pronounced synaptic boutons, suggesting they prioritize efficient and reliable signal propagation. Tendril and distal branches were spatially segregated and bouton dense, indicating specialization in signal transmission. Furthermore, CFs systematically targeted molecular layer interneuron cell bodies, especially at terminal boutons, potentially instantiating feedforward inhibition on PCs. This study offers the most detailed and comprehensive characterization of CF morphology to date. The reconstruction files and metadata are publicly distributed at NeuroMorpho.org.

Microlesions of the inferior olive reduce vestibular modulation of Purkinje cell complex and simple spikes in mouse cerebellum.

Cerebellar Purkinje cells have two distinct action potentials: complex spikes (CSs) are evoked by single climbing fibers that originate from the contralateral inferior olive. Simple spikes (SSs) are often ascribed to mossy fiber-granule cell-parallel fiber inputs to Purkinje cells. Although generally accepted, this view lacks experimental support. Vestibular stimulation independently activates primary afferent mossy fibers and tertiary afferent climbing fibers that project to the uvula-nodulus (folia 8-10). CSs and SSs normally discharge antiphasically during sinusoidal roll-tilt. When CSs increase, SSs decrease. We tested the relative independence of these pathways in mice by making electrolytic microlesions of the two inferior olivary nuclei from which vestibular climbing fibers originate; the ß-nucleus and dorsomedial cell column. This reduced vestibular climbing fiber signaling to the contralateral folia 8-10, while leaving intact vestibular primary and secondary afferent mossy fibers. We recorded from Purkinje cells and interneurons in folia 8-10, identified by juxtacellular labeling with Neurobiotin. Microlesions of the inferior olive increased the spontaneous discharge of SSs in contralateral folia 8-10, but blocked their modulation during vestibular stimulation. The vestibularly evoked discharge of excitatory cerebellar interneurons (granule cells and unipolar brush cells) was not modified by olivary microlesions. The modulated discharge of stellate cells, but not Golgi cells, was reduced by olivary microlesions. We conclude that vestibular modulation of CSs and SSs depends on intact climbing fibers. The absence of vestibularly modulated SSs following olivary microlesions reflects the loss of climbing fiber-evoked stellate cell discharge.

Receptive-field structure of optic flow responsive Purkinje cells in the vestibulocerebellum of pigeons.

Neurons sensitive to optic flow patterns have been recorded in the the olivo-vestibulocerebellar pathway and extrastriate visual cortical areas in vertebrates, and in the visual neuropile of invertebrates. The complex spike activity (CSA) of Purkinje cells in the vestibulocerebellum (VbC) responds best to patterns of optic flow that result from either self-rotation or self-translation. Previous studies have suggested that these neurons have a receptive-field (RF) structure that "approximates" the preferred optic flowfield with a "bipartite" organization. Contrasting this, studies in invertebrate species indicate that optic flow sensitive neurons are precisely tuned to their preferred flowfield, such that the local motion sensitivities and local preferred directions within their RFs precisely match the local motion in that region of the preferred flowfield. In this study, CSA in the VbC of pigeons was recorded in response to a set of complex computer-generated optic flow stimuli, similar to those used in previous studies of optic flow neurons in primate extrastriate visual cortex, to test whether the receptive field was of a precise or bipartite organization. We found that these RFs were not precisely tuned to optic flow patterns. Rather, we conclude that these neurons have a bipartite RF structure that approximates the preferred optic flowfield by pooling motion subunits of only a few different direction preferences.

Complementary stripes of phospholipase Cbeta3 and Cbeta4 expression by Purkinje cell subsets in the mouse cerebellum.

Transverse boundaries divide the cerebellar cortex into four transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. Several molecules believed to mediate long-term depression at the parallel fiber-Purkinje cell synapse are known to be expressed in stripes. We have therefore explored the distributions of phospholipase Cbeta3 and phospholipase Cbeta4, key components in the transduction of type 1 metabotropic glutamate receptor-mediated responses. The data reveal that both phospholipase Cbeta isotypes are expressed strongly in the mouse cerebellum in subsets of Purkinje cells. The two distributions are distinct and largely nonoverlapping. The pattern of phospholipase Cbeta3 expression is unique, revealing stripes in three of the four transverse zones and a uniform distribution in the fourth. In contrast, phospholipase Cbeta4 appears to be confined largely to the Purkinje cells that are phospholipase Cbeta3-negative. PLCbeta3 is restricted to the zebrin II-immunopositive Purkinje cell subset. Not all zebrin II-immunoreactive Purkinje cells express PLCbeta3: in lobules IX and X it is restricted to that zebrin II-immunopositive subset that also expresses the small heat shock protein HSP25. PLCbeta4 expression is restricted to, and coextensive with, the zebrin II-immunonegative Purkinje cell subset. These nonoverlapping expression patterns suggest that long-term depression may be manifested differently between cerebellar modules.

Spontaneous electrical activity and dendritic spine size in mature cerebellar Purkinje cells.

Previous experiments have shown that in the mature cerebellum both blocking of spontaneous electrical activity and destruction of the climbing fibres by a lesion of the inferior olive have a similar profound effect on the spine distribution on the proximal dendrites of the Purkinje cells. Many new spines develop that are largely innervated by parallel fibers. Here we show that blocking electrical activity leads to a significant decrease in size of the spines on the branchlets. We have also compared the size of the spines of the proximal dendritic domain that appear during activity block and after an inferior olive lesion. In this region also, the spines in the absence of activity are significantly smaller. In the proximal dendritic domain, the new spines that develop in the absence of activity are innervated by parallel fibers and are not significantly different in size from those of the branchlets, although they are shorter. Thus, the spontaneous activity of the cerebellar cortex is necessary not only to maintain the physiological spine distribution profile in the Purkinje cell dendritic tree, but also acts as a signal that prevents spines from shrinking.

Effect of simple spike firing mode on complex spike firing rate and waveform in cerebellar Purkinje cells in non-anesthetized mice.

Cerebellar Purkinje cells receive two different excitatory inputs from parallel and climbing fibers, causing simple and complex spikes, respectively. Purkinje cells present three modes of simple spike firing, namely tonic, silent and bursting. The influence of complex spike firing on simple spike firing has been extensively studied. However, it is unknown whether and how the simple spike firing mode may influence complex spike waveform and firing rate in vivo. We studied complex spike firing during tonic and silent mode periods in non-anesthetized mice. We found that complex spike firing rate is not influenced by simple spike firing modes, but that the complex spike waveform is altered following high frequency simple spike firing. This alteration is a specific decrement of the second depolarizing component of the complex spike. We demonstrate that the amplitude of the second depolarizing component is inversely proportional to the simple spike firing rate preceding the complex spike and that this amplitude is independent of previous complex spike firing. This waveform modulation is different from previously reported modulation in paired-pulse depression and refractoriness.

Purkinje cell activity during learning a new timing in classical eyeblink conditioning.

During classical eyeblink conditioning, animals acquire adaptive timing of the conditioned response (CR) to the interstimulus interval (ISI) between the conditioned stimulus (CS) and the unconditioned stimulus (US). To investigate this coding of the timing by the cerebellum, we analyzed Purkinje cell activities during acquisition of new timing after we shifted the ISI. Decerebrate guinea pigs were conditioned to an asymptotic level of learning using a delay paradigm with a 250-ms ISI. A 350-ms tone and a 100-ms electrical shock were used as the CS and US, respectively. As reported previously in other species, Purkinje cells in the simplex lobe exhibited three types of responses to the CS: excitatory, inhibitory, or a combination of the two. After we increased the ISI to 400 ms, the frequency of the CR stayed at an asymptotic level, but the latency of the CR peak became gradually longer. Two types of cells were observed, based on changes in the nature of their response to the CS; one changed its type of response in parallel with learning the new timing, while the other did not. There was no correlation between the type of response before and after we changed the ISI. In some cells, the peak latency of activities became longer or shorter, while the type of response did not change. These results suggest that some Purkinje cells code the timing of the CR, but do not play a consistent role in shaping the CR over a range of ISIs.

Functional significance of climbing-fiber synchrony: a population coding and behavioral analysis.

Population coding and behavioral approaches were taken toward analyzing the functional significance of the climbing-fiber system. Analyses of neuronal interaction using the joint peristimulus time histogram showed that given a low rate of firing, the climbing-fiber system organizes itself to fire synchronously during movement--a feature that bears little or no relationship to the modulation in firing rate during movement or whether olivary neurons respond to a sensory stimulus. Moreover, the climbing-fiber system avoids synchrony during a passive sensory response but actively makes a transition into synchrony as a movement is initiated. Thus, from a functional viewpoint, the active feature of the climbing-fiber system to organize into synchronously firing cell ensembles is uniquely motor. Analyses of behaving rats without an inferior olive revealed that the climbing-fiber system optimizes the timing of skilled movement by reducing reaction time and the interval between repetitive movements by 100 milliseconds. Finally, using classical delay eyeblink conditioning, it was found that the inferior olive is essential for learning about rapid sequences of events but not the same event sequence when given more slowly. It was concluded that the climbing-fiber system exerts its function through synchrony, which provides a 100 ms advantage in movement speed and the ability to learn about events that are rapidly presented in time.

Abnormal dispersion of a purkinje cell subset in the mouse mutant cerebellar deficient folia (cdf).

Purkinje cells of different molecular phenotypes subdivide the cortex of the cerebellum both rostrocaudally into parasagittal bands and mediolaterally into transverse zones. Superimposed on the Purkinje cell compartmentation, the cerebellar cortex is pleated into a reproducible array of lobes and lobules. During cerebellar development, Purkinje cell bands are formed through the rostrocaudal dispersal of embryonic clusters, triggered primarily by a Reelin-dependent signaling pathway. In the naturally occurring mouse mutant cerebellar deficient folia (cdf), there is a failure of Purkinje cell dispersion that results in widespread Purkinje cell ectopia in the adult. The ectopia is restricted primarily to that subset of Purkinje cells that does not express zebrin II/aldolase C and that forms ectopic clusters in among the cerebellar nuclei. Most Purkinje cells that express zebrin II are located normally in a monolayer. Thus, the cerebellum of cdf mutants has a failure of Purkinje cell dispersion that is confined primarily to a zebrin II-negative (zebrin II(-)) subpopulation. Despite the Purkinje cell ectopia, the parasagittal band organization of the cerebellum is still clear. The shortening of the cortex is distributed evenly over all lobules, with the result that transverse expression boundaries are relocated with respect to the lobules and fissures. The number of Purkinje cells in the cdf/cdf cerebellum is similar to the number in littermate controls. Therefore, it appears that the lesion in cdf results in the failure of a zebrin II(-) Purkinje cell subset to disperse either due to a cell intrinsic defect or due to an abnormal interaction between the Purkinje cells and either granule cells or afferent inputs.

Morphometric analysis of dendritic tree of cerebellar Purkinje-cells.

Basic neural processes of sensorimotor adaptation can be observed by cellular-level studies on cerebellar cortex, both by electrophysiological and morphological means. In earlier studies we demonstrated double (sometimes triple or quadruple) rhythmic prespike activity patterns in dendritic microelectrode records taken from cerebellar Pc. By their active and passive interactions, the actual input pattern will turn into an arrhythmic output spiking, realizing a nonlinear and phase-sensitive integration. This curious complex spike-generating process can give rise to novel cerebellar functional models. The acting membrane dynamics rely not just upon the ionic current machinery but also upon the specific micromorphology of the dendritic tree, especially that of the branching areas. Although, statistical analyses on Pc dendrites generally followed the graph-theory, elaborated abstract parameters for complexity and hierarchy, and denied realistic geometry. Thus a novel specific morphometric study should be carried out, first applied to the main branching sites.

Projections from the medial column of the inferior olive to different classes of rotation-sensitive Purkinje cells in the flocculus of pigeons.

In the flocculus of pigeons, as in other species, there are two major types of Purkinje cell responses to rotational optokinetic stimuli. One type prefers rotation about the vertical axis (VA neurons) whereas the other prefers rotation about an horizontal axis oriented at 135 degrees ipsilateral azimuth (H-135 neurons). In this study, we injected the retrograde tracer cholera toxin subunit B into the VA and H-135 zones in attempt to determine the origin of inferior olive inputs. We found that VA and H-135 zones received input from the caudal and rostral margins of the medial column of the inferior olive, respectively. There is a similar pattern of connectivity in mammalian species.

Multiple subclasses of Purkinje cells in the primate floccular complex provide similar signals to guide learning in the vestibulo-ocular reflex.

The neural "learning rules" governing the induction of plasticity in the cerebellum were analyzed by recording the patterns of neural activity in awake, behaving animals during stimuli that induce a form of cerebellum-dependent learning. We recorded the simple- and complex-spike responses of a broad sample of Purkinje cells in the floccular complex during a number of stimulus conditions that induce motor learning in the vestibulo-ocular reflex (VOR). Each subclass of Purkinje cells carried essentially the same information about required changes in the gain of the VOR. The correlation of simple-spike activity in Purkinje cells with activity in vestibular pathways could guide learning during low-frequency but not high-frequency stimuli. Climbing fiber activity could guide learning during all stimuli tested but only if compared with the activity present approximately 100 msec earlier in either vestibular pathways or Purkinje cells.

On the heterogeneity of Purkinje neurons in vertebrates. Cytochemical and morphological studies of chromatin during eel (Anguilla anguilla L.) life cycle.

The relationship between the heterogeneity of Purkinje cell chromatin and the functional involvement of cerebellum has been analyzed comparing two stages of eel life cycle (yellow eel and silver eel) which are characterized by a different degree of swimming activity. The DNA content and the chromatin condensation have been studied by means of microdensitometry (Feulgen-DNA, Feulgen-DNA/area), microfluorometry (DNA after intercalation with Propidium Iodide at low and high concentration) and electron microscopy. In the transition from the yellow eel to the silver eel, the Purkinje cell heterogeneity undergoes variations. In the silver eel, which shows a higher swimming activity, high Feulgen-DNA and Propidium Iodide-DNA (H2c to 4c) were more frequent and a greater heterogeneity in the chromatin condensation was observed. In particular, the changes were more evident in the lobus vestibulolateralis; mainly in this area, which receives inputs from the lateral line, the Purkinje cells are scattered in the molecular layer. On the contrary, in the yellow eel these large cells occupy the zone near or within the granular layer. The data suggest a relationship between the degree of heterogeneity of Purkinje cells and the afferent systems of the cerebellar areas examined. The changes of cerebellar cytoarchitectonics may be expression of the higher degree of the swimming activity of silver eel.

Discharges of Purkinje cells in the paravermal part of the cerebellar anterior lobe during locomotion in the cat.

Extracellular recordings were made from 124 Purkinje cells in the paravermal part of lobule V of the cerebellum in cats walking steadily at a speed of 0.5 m/s on a moving belt. All cells tested had a tactile receptive field from which simple spikes could be evoked and 96% of these were on the ipsilateral forelimb. Seventy-six of the cells were also studied whilst the animals sat or lay quietly without movement. Complex spikes were discharged at 1-2/s and these were accompanied by simple spikes in fifty-nine cells (78%); in the remaining cells there were no or few simple spikes. The over-all mean discharge rate (including both types of spike) was 37.8 +/- 27 impulses/s (+/- S.D.). During locomotion all cells discharged both types of spike and the over-all mean rate was 57.6 +/- 29 impulses/s (+/- S.D.). In all cells but one, the frequency of the simple spikes was modulated rhythmically in time with the stepping movements but the phasing relative to the step cycle varied widely between cells. Peak rates also varied widely, the average being 91.5 +/- 44 impulses/s (+/- S.D.). Most cells (63%) generated one period of accelerated discharge per step but others generated two (35%) or three (2%) such periods. Despite the individual variations in discharge timing the population as a whole was considerably more active during the swing than the stance phase of the step cycle in the ipsilateral forelimb (68 impulses/s as compared with 49 impulses/s on average). Thirty-four cells were electrophysiologically identified as lying in the c1 zone of the cortex and twenty-five as being in the c2 zone (nomenclature of Oscarsson, 1980). During locomotion, the population activity in the two zones differed slightly: activity in the c1 population was phase advanced by approximately one-tenth of the step cycle. The results are discussed, with particular emphasis on the finding that population activity in the Purkinje cells of the c1 zone fluctuated during the step cycle in parallel with that in the part of nucleus interpositus to which they project.

[Analysis of the population of Purkinje's cells in the cerebellar cortex of dogs subjected to heart arrest]. / Izuchenie populiatsii kletok Purkin'e kory mozzhechka sobak, perenesshikh ostanovku sistemnogo krovoobrashcheniia.

The population of Purkinje's cells (PC) was studied in the cerebellum cortex of 5 mongrel dogs that suffered 12-minute circulatory arrest caused by electric trauma followed by complete external recovery of the neurological status. Morphometric analysis revealed that 2 weeks after resuscitation the total number of PC per mm of their layer length remains unchanged, while the composition of the population drastically changes. In all the zones of the cerebellum studied, the number of PC having different morphological changes increases, whereas the number of light PC was significantly decreased. The result of the changes was that 2 weeks after clinical death, dark cells constituted half the normal (morphologically unchanged) population of PC. Meanwhile in the control group, dark PC constituted 1/3 of the population. Cytophotometry revealed that in both groups of animals (the intact and with a history of clinical death) the dark PC are characterized by the increased content of nucleic acid, as compared to that of the light PC. It is suggested that the increase in the relative number of the dark PC in the cerebellum cortex of dogs after systemic circulation arrest is related to the transition of intracellular physiological regeneration to the reparative one. Therefore, both pathological and compensatory processes are seen in the dog cerebellum cortex 2 weeks after resuscitation. It was disclosed that PC of the lateral zone of the cerebellum hemisphere are most vulnerable, while those of the medial zone (vermis cerebelli) are most viable.