The effect of SK channel modulators on the simple spike firing frequency in discharge of cerebellar Purkinje cells in laboratory mice

The activity of cerebellar Purkinje cells is studied as affected by CyPPA, a positive modulator of small-conductance calcium-activated potassium channels type 3 and 2 (SK3/SK2), and NS309, an activator of small- and intermediate-conductance calcium-activated potassium channels (IK/SK), in male two-month-old laboratory mice. CyPPA decreases the simple spike firing frequency in the discharge of Purkinje cells by an average of 25% 1 hour after application of 1mM of the compound. An application of 100 μM of NS309 reduces the simple spike firing frequency by an average of 47% during the same period. These results confirm the hypothesis that SK channels may be involved in the downregulation of simple spike firing frequency in Purkinje cells. The frequency-regulating effect of NS309 is stronger, suggesting that IK/SK channels play a decisive role in the regulation of Purkinje cell spiking activity. Since an increase of simple spike firing frequency in these cells is symptomatic of many locomotor activity disorders, e.g., spinocerebellar ataxia, the substances studied or their functional analogues might be of medicinal interest.     

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.     

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.     

The multiple roles of Purkinje cells in sensori-motor calibration: to predict, teach and command

Neurophysiological recordings in the cerebellar cortex of awake-behaving animals are revolutionizing the way we think about the role of Purkinje cells in sensori-motor calibration. Early theorists suggested that if a movement became miscalibrated, Purkinje cell output would be changed to adjust the motor command and restore good performance. The finding that Purkinje cell activity changed in many sensori-motor calibration tasks was taken as strong support for this hypothesis. Based on more recent data, however, it has been suggested that changes in Purkinje cell activity do not contribute to the motor command directly; instead, they are used either as a teaching signal, or to predict the altered kinematics of the movement after calibration has taken place. I will argue that these roles are not mutually exclusive, and that Purkinje cells may contribute to command generation, teaching, and prediction at different times during sensori-motor calibration.     

Mapping the development of cerebellar Purkinje cells in zebrafish

The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1174–1188, 2015     

Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cells

Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an alpha-nNOS knockout mutant (nNOSalpha(-/-)) to reduce the production of NO. Analysis of the double mutants showed that blocking alpha-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRdelta2(+/Lc):nNOS(-/-) double mutant Purkinje cells, which suggests that the failure to rescue GluRdelta2(+/Lc):nNOS(-/-) Purkinje cells may be explained by the induction of alternative nNOS isoforms.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.     

Microglia promote the death of developing Purkinje cells

The loss of neuronal cells, a prominent event in the development of the nervous system, involves regulated triggering of programmed cell death, followed by efficient removal of cell corpses. Professional phagocytes, such as microglia, contribute to the elimination of dead cells. Here we provide evidence that, in addition to their phagocytic activity, microglia promote the death of developing neurons engaged in synaptogenesis. In the developing mouse cerebellum, Purkinje cells die, and 60% of these neurons that already expressed activated caspase-3 were engulfed or contacted by spreading processes emitted by microglial cells. Apoptosis of Purkinje cells in cerebellar slices was strongly reduced by selective elimination of microglia. Superoxide ions produced by microglial respiratory bursts played a major role in this Purkinje cell death. Our study illustrates a mammalian form of engulfment-promoted cell death that links the execution of neuron death to the scavenging of dead cells.     

Light and dark Purkinje cells (light microscopic and microautoradiographic studies]

The authors deal with the phenomenon of dualism of pale and dark ganglion cells in general, the phenomenon which, since FLEMMING (1882), still remains an actual problem of neurohistology. They deal with Purkinje cells from a special aspect with the aim to demonstrate the dualism through various staining methods. They directed their attention also to the question of the influence of perfusion and immersion fixation and length of staining with luxol-fast-blue on the production of luxol-positive (chromophilic) Purkinje cells. The authors have found that these methodologic circumstances do not influence the production of luxol-positive Purkinje cells, so it can be hardly spoken about an artifact. Examinations with the labelled leucine have shown that the increased metabolic activity can be observed in those Purkinje cells which in HE-picture are seen as pale ones. In the dark Purkinje cells leucine granulations are located on the surface of plasmatic membrane and they follow to some distance the main dendrite of those cells. The microautoradiographic method evidences for the increased leucine metabolism of the pale Purkinje cells as well.     

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.     

Cumulative effects of alcohol (ethanol) on the activity of Purkinje cells in the cat cerebellum

The effects of three consecutive injections of 2 ml/kg 30% alcohol (ethanol, i.v.) on the activity of identified cerebellar Purkinje cells were investigated during experiments on adult cats anesthetized by a Nembutal-chloralose mixture. It was found that repeated alcohol injections exert a cumulative effect on the firing rate of these cells. The alcohol-induced rise in Purkinje cell firing rate, produced by excitation of the mossy fibers, was generally accompanied by a reduction in that of cells responding to excitation of climbing fibers. The inhibitory pause occurring after complex discharges in cerebellar Purkinje cells grew shorter with successive alcohol injections.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 74–80, January–February, 1987.     

Maturation of the activity of cerebellar Purkinje cells and the lift reaction

In experiments on guinea pigs (from newborn to adults), studies have been made on the extensor, support and lift reactions, as well as on the activity of cerebellar Purkinje cells in the same animals. First signs of immature lift, extensor and support reactions were observed already 12th after birth. At this period, mean discharge frequency in Purkinje cells was significantly lower than in the adult animals, reaching 11.5 +/- 1.2 imp/s for simple spikes and 0.45 +/- 0.05 imp/s for complex ones. Complete maturation of lift, extensor and support reactions takes place to the beginning of the 2nd week (8-9 days) of postnatal life. Within this period, significant changes in the activity of Purkinje cells were observed: mean discharge frequency of simple and complex spikes increased correspondingly up to 17.9 +/- 2.3 and 1.48 +/- 0.25 imp/s. At the same time, the mean discharge frequency in Purkinje cells, the average duration of inhibition pause, and the response latency became more stable.     

Signal processing in cerebellar Purkinje cells

Mechanisms and functional implications of signal processing in cerebellar Purkinje cells have been the subject of recent extensive investigations. Complex patterns of their planar dendritic arbor are analysed with computer-aided reconstructions and also topological analyses. Local computation may occur in Purkinje cell dendrites, but its extent is not clear at present. Synaptic transmission and electrical and ionic activity of Purkinje cell membrane have been revealed in detail, and related biochemical processes are being uncovered. A special type of synaptic plasticity is present in Purkinje cell dendrites; long-term depression (LTD) occurs in parallel fiber-Purkinje cell transmission when the parallel fibers are activated with a climbing fiber innervating that Purkinje cell. Evidence indicates that synaptic plasticity in Purkinje cells is due to sustained desensitization of Purkinje dendritic receptors to glutamate, which is a putative neurotransmitter of parallel fibers, and that conjunctive activation of a climbing fiber and parallel fibers leads to desensitization through enhanced intradendritic calcium concentration. A microzone of the cerebellar cortex is connected to an extracerebellar neural system through the inhibitory projection of Purkinje cells to a cerebellar or vestibular nuclear cell group. Climbing fiber afferents convey signals representing control errors in the performance of a neural system, and evoke complex spikes in Purkinje cells of the microzone connected to the neural system. Complex spikes would modify the performance of the microzone by producing LTD in parallel fiber-Purkinje cell synapses, and consequently would improve the overall performance of the neural system. The primary function of the cerebellum thus appears to be endowing adaptability to numerous neural control systems in the brain and spinal cord through error-triggered reorganization of the cerebellar cortical circuitry.     

Reciprocal bidirectional plasticity of parallel fiber receptive fields in cerebellar Purkinje cells and their afferent interneurons

The highly specific relationships between parallel fiber (PF) and climbing fiber (CF) receptive fields in Purkinje cells and interneurons suggest that normal PF receptive fields are established by CF-specific plasticity. To test this idea, we used PF stimulation that was either paired or unpaired with CF activity. Conspicuously, unpaired PF stimulation that induced long-lasting, very large increases in the receptive field sizes of Purkinje cells induced long-lasting decreases in receptive field sizes of their afferent interneurons. In contrast, PF stimulation paired with CF activity that induced long-lasting decreases in the receptive fields of Purkinje cells induced long-lasting, large increases in the receptive fields of interneurons. These properties, and the fact the mossy fiber receptive fields were unchanged, suggest that the receptive field changes were due to bidirectional PF synaptic plasticity in Purkinje cells and interneurons.     

Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.     

Isolation and morphology of single Purkinje cells from the porcine heart

Purkinje cells were isolated from both ventricles of young adult domestic pigs and examined by transmitted light or laser scanning confocal microscopy. Purkinje cells in free running Purkinje fibres were organised in multicellular strands where individual cells were tightly connected end-to-end and closely side-to-side. After isolation, single cells gradually lost the elongated appearance and became more rounded, but the cell membrane remained smooth and undamaged. The contractile material was not very dense and was seen most clearly in the submembraneous area. Staining of the cell membrane with the lipophilic fluorescent (lye di-8-ANNEPS, and visualization with confocal microscopy, confirmed that the cell surface membrane was smooth without blebs. This staining also showed that Purkinje cells had no transversal tubules. We reconstructed the three-dimensional geometry of the Purkinje cells and determined the cell size. The average values were 62 +/- 9 microm for length, 32 +/- 3 microm for width, and 41 +/- 4 microm for depth (n = 7). Calculated cross-section area and volume were 1047 +/- 167 microm2 and 47 +/- 14 pl. Compared to ventricular cells, the morphology of the Purkinje cells reflects their specific role in impulse conduction.     

Postnatal development of the shape of cerebellar Purkinje cells in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals—guinea pigs—was performed using Nissl’s procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.     

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.     

Characteristics of activity of "fast" and "slow" Purkinje cells of the cerebellum

In the guinea pig cerebellar cortex, three types of Purkinje cells were identified according to the properties of complex spikes: fast, intermediate, and slow cells. Fast Purkinje cells have following properties as compared with slow Purkinje cells: (i) salient components with short intervals in complex impulses (on the average, five components with a period of about 2 ms versus two components with a period of about 4 ms); (ii) a short duration of simple spikes (in the average, 2.13 +/- 0.53 ms versus 3.9 +/- 0.65 ms) and a quick restoration of their amplitude after preceding simple spikes (in the mean, 2.83 +/- 0.75 ms versus 11.0 +/- 2.82 ms); and (iii) a more pronounced rebound in the auto-correlation histogram of simple spikes (3.09 +/- 2.12 versus 1.45 +/- 0.36) and a short-latency excitation of simple spikes after complex spikes (2.81 +/- 1.64 versus 1.26 +/- 0.52). A decrease of interspike intervals in simple spike activity of all Purkinje cells was revealed (5.25 +/- 2.71 ms versus 9.71 +/- 3.48 ms in activity fragments without complex spikes). It is supposed that the properties of complex spikes depend on the type of Purkinje cells and may be one of the basic factors determining the interactions between the inputs of climbing and parallel fibers in Purkinje cells.