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Noel G. Carlson - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological o
2016Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, L. Clardy, Ikuo Tsunoda, Noel G. CarlsonAbstract:Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Pur-kinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic Cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat Cerebellar Slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in dis-ease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo anti-body to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody bind-ing; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, in-cubation of rat Cerebellar Slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microgli
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anti yo antibody uptake and interaction with its intracellular target antigen causes purkinje cell death in rat Cerebellar Slice cultures a possible mechanism for paraneoplastic Cerebellar degeneration in humans with gynecological or breast cancers
PLOS ONE, 2015Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Stacey L Clardy, Noel G. CarlsonAbstract:Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic Cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat Cerebellar Slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat Cerebellar Slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.
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Comparison of Purkinje cell cytotoxicity produced by anti-Yo antibodies with cytotoxicity produced by commercial and patient antibodies reactive with other Purkinje cell cytoplasmic proteins.
2015Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Stacey L Clardy, Noel G. CarlsonAbstract:Rat Cerebellar Slice cultures were incubated for 72 hours with either 1) sera from patients with anti-Yo antibodies; 2) commercial antibodies to calbindin, calmodulin, or PCP-2/L7; 3) anti-Purkinje cells antibodies from the three neurologically normal patients studied, whose sera labeled Purkinje cell cytoplasm but did not react with Yo antigens; or 4) normal human IgG. Cultures were quantified for cell death as indicated by uptake of SYTOX dyes. Extensive Purkinje cell death was seen in cultures incubated with all 4 anti-Yo sera (data for 2 anti-Yo sera not shown). In contrast, cell death was not observed in cultures incubated with any of the three commercially obtained antibodies reactive with intracellular anti-Purkinje cell proteins nor with sera from Patients 1–3, despite extensive Purkinje cell uptake. Cultures incubated with normal human IgG exhibited only faint antibody accumulation by Purkinje cells and no detectable Purkinje cell death. Statistical significance between groups was determined by non-parametric Mann-Whitney ANOVA. Death in cultures incubated with anti-Yo antisera was statistically significantly greater than that seen in cultures incubated with commercial antisera, sera from patients 1–3, or normal human IgG.
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comparative uptake and cytotoxicity of anti hu and anti ri antibodies in rat Cerebellar Slice cultures p02 161
Neurology, 2013Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Noel G. CarlsonAbstract:OBJECTIVE: To evaluate neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in organotypic cultures of rat cerebellum. BACKGROUND: The paraneoplastic autoantibodies, anti-Hu and anti-Ri, recognize intracellular antigens present in essentially all neurons. At autopsy, brains of patients with anti-Hu antibody show neuronal destruction. In contrast, anti-Ri antibody is less clearly associated with neuronal death, and patients with anti-Ri antibodies may respond to treatment. The role of antibodies in causing paraneoplastic neurological injury has not been defined. However, we have previously demonstrated that anti-Yo antibodies, associated with paraneoplastic Cerebellar degeneration, accumulate in Purkinje cells in Slice cultures of rat cerebellum and that antibody accumulation was followed by cell death. The present study was conducted to determine 1) whether anti-Hu and anti-Ri antibodies are also taken up by neurons in this culture system; 2) whether uptake is restricted to Purkinje cells, and 3) whether uptake of either antibody is followed by cell death. DESIGN/METHODS: Rat Cerebellar Slice cultures were incubated with either anti-Hu or anti-Ri antibodies and were followed over time. TUNEL and FLICA methods were used to detect apoptosis. The dead cell stain, SYTOX green, was used to detect non-apoptotic cell death. RESULTS: Anti-Hu and anti-Ri antibodies accumulated not only in Purkinje cells but also in multiple other neuronal populations. Accumulation of anti-Hu antibody was followed by apoptosis involving multiple neuronal populations: this became detectable within 72-96 hours. In contrast, as compared to controls, cells accumulating anti-Ri antibodies showed no evidence of apoptosis or necrotic cell death over a period of 200 hours. CONCLUSIONS: Uptake of anti-Hu and anti-Ri antibodies was observed in viable neurons throughout the cerebellum and was not restricted to Purkinje cells. While anti-Hu antibody appears to be specifically cytotoxic, anti-Ri antibody did not affect neuronal viability and may predominantly cause neuronal dysfunction rather than death. Supported by: United States Department of Veterans Affairs. Disclosure: Dr. Greenlee has received personal compensation for activities with American Academy of Neurology and Oliver Maner LLP. Dr. Greenlee has received personal compensation in an editorial capacity for Medlink. Dr. Clawson has nothing to disclose. Dr. Wood has nothing to disclose. Dr. Hill has nothing to disclose. Dr. Tsunoda has nothing to disclose. Dr. Carlson has nothing to disclose.
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purkinje cell death after uptake of anti yo antibodies in Cerebellar Slice cultures
Journal of Neuropathology and Experimental Neurology, 2010Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Noel G. CarlsonAbstract:Paraneoplastic Cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of Cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in Slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic Cerebellar degeneration.
Daniel L Alkon - One of the best experts on this subject based on the ideXlab platform.
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pairing specific long term depression of purkinje cell excitatory postsynaptic potentials results from a classical conditioning procedure in the rabbit Cerebellar Slice
Journal of Neurophysiology, 1996Co-Authors: Bernard G Schreurs, Matthew M Oh, Daniel L AlkonAbstract:1. Using a rabbit Cerebellar Slice preparation, we stimulated a classical conditioning procedure by stimulating parallel fiber inputs to Purkinje cells with the use of a brief, high-frequency train...
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pairing specific long term depression of purkinje cell excitatory postsynaptic potentials results from a classical conditioning procedure in the rabbit Cerebellar Slice
Journal of Neurophysiology, 1996Co-Authors: Bernard G Schreurs, Daniel L AlkonAbstract:1. Using a rabbit Cerebellar Slice preparation, we stimulated a classical conditioning procedure by stimulating parallel fiber inputs to Purkinje cells with the use of a brief, high-frequency train of eight constant-current pulses 80 ms before climbing fiber inputs to the same Purkinje cell were stimulated with the use of a brief, lower frequency train of three constant-current pulses. In all experiments, we assessed the effects of stimulation by measuring the peak amplitude of Purkinje cell excitatory postsynaptic potentials (EPSPs) to single parallel fiber test pulses. 2. Intradendritically recorded Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (30%) after paired stimulation of the parallel and climbing fibers but not after unpaired or parallel fiber alone stimulation. We call this phenomenon pairing-specific long-term depression (PSD). 3. Facilitation of the peak amplitude of a second EPSP elicited by a parallel fiber train occurred both before and after paired stimulation suggesting that the locus of depression was not presynaptic. Depression of the peak amplitude of a depolarizing response to focal application of glutamate following pairings of parallel and climbing fiber stimulation added support to a suggested postsynaptic locus of the PSD effect. 4. The application of aniracetam potentiated EPSP peak amplitude by 40%, but these values returned to baseline as a result of pairings. With the removal of aniracetam from the bath 20 min after pairings, normal levels of pairing-specific EPSP depression were observed, indicating that the effect did not result from direct desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. 5. Incubation of Slices in the protein kinase inhibitor H-7 potentiated EPSP peak amplitudes slightly (9%), but peak amplitudes returned to baseline levels after pairings. The net reduction in EPSP peak amplitude of < 10% after pairings suggested that H-7 partially blocked PSD and that, in turn, PSD involved protein kinases. 6. The means of induction and the specificity of those means suggest that the phenomenology of PSD is fundamentally different from that of long-term depression. PSD only occurs with pairings of trains of parallel fiber and climbing fiber stimulation; it occurs without the need for bicuculline; and it can overcome the blocking effects of aniracetam. 7. Nevertheless, the involvement of protein kinases and the potential role of calcium suggest that the mechanisms involved in the induction of PSD and long-term depression have a number of features in common. 8. Because of the pairing-specific nature of the long-term synaptic depression observed in these experiments, PSD provides a mechanism that may contribute to the role of the Cerebellar cortex in classical conditioning.
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Incorporation of fluorescent lipids into living rabbit hippocampal and Cerebellar Slices.
NeuroImage, 1994Co-Authors: David S. Lester, Bernard G Schreurs, James L. Olds, Donna L. Mcphie, Clive R. Braham, Daniel L AlkonAbstract:Incorporation of exogenously applied fluorescent lipids into living cells was exploited to probe cellular structure and function in living hippocampal and Cerebellar Slices as assessed by fluorescent imaging techniques and intracellular recording. Nitrobenzoxadiole-phosphatidylcholine (NBD-PC) and BODIPY phorbol ester, in vitro substrates of phospholipase activity and protein kinase C, respectively, were incorporated and distributed into specific cell populations. In the hippocampal Slice, both probes labeled the somata and proximal dendrites of pyramidal and granule cells but were hetrogeneously distributed across the different hippocampal fields. Changes in fluorescent properties of NBD-PC in individual pyramidal cell and granule cell somata were quantified upon challenge with a muscarinic agonist known to modulate phospholipase A2 activity. In the Cerebellar Slice, both probes labeled Purkinje cell bodies and dendrites but only NBD-PC labeled stellate and granule cells. The cellular and functional specificity of these fluorescent lipid probes shows great promise for monitoring biochemical events in complex neuronal systems with significant spatial and temporal resolution.
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Rabbit Cerebellar Slice analysis of long-term depression and its role in classical conditioning.
Brain research, 1993Co-Authors: Bernard G Schreurs, Daniel L AlkonAbstract:Abstract Cerebellar long-term depression (LTD) has been proposed as a mechanism underlying classical conditioning of the rabbit nictitating membrane/eyelid response (NMR). However, LTD has only been obtained reliably when (1) Cerebellar Slices are bathed in GABA antagonists which abolish disynaptic inhibitory post synaptic potentials, and (2) the temporal sequence of stimulation used in Slice or intact preparations is the opposite of that used in classical conditioning. Based on intradendritic Purkinje cell recordings obtained from rabbit Cerebellar Slices, we report that stimulation of climbing fibers and then parallel fibers in the presence of the GABA antagonist, bicuculline, produced significant depression of parallel fiber excitatory post synaptic potential (epsp) amplitude that continued to increase for at least 20 min after stimulation. However, application of the same stimulation protocol without GABA antagonists produced a brief depression of parallel fiber epsps that disappeared within minutes. Activation of parallel fibers and then climbing fibers in an order opposite to the LTD-producing sequence (i.e. a classical conditioning-like order) produced a brief depression that dissipated quickly. Stimulation of parallel fibers alone produced a small, slowly developing potentiation, but stimulation of parallel fibers during depolarization-induced local dendritic calcium spikes produced significant depression almost immediately which then declined slowly to more modest levels. Finally, stimulation of parallel fibers at frequencies used in in vivo parallel fiber-climbing fiber stimulation experiments (e.g. 100 Hz) produced an immediate and profound long-lasting epsp depression. The depression occurred, however, whether parallel and climbing fibers were stimulated separately (unpaired) or in a classical conditioning-like protocol (paired) where parallel fiber stimulation coterminated with climbing fiber stimulation (10 Hz). The depression observed in both cases was reminiscent of transmitter depletion. Thus, despite obvious differences between in vitro and in vivo preparations, the present intradendritic Purkinje cell recordings in a rabbit Cerebellar Slice do caste some doubt on the hypothesis that LTD functions as a mechanism underlying classical conditioning of the rabbit NMR.
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GABA-induced responses in Purkinje cell dendrites of the rabbit Cerebellar Slice.
Brain research, 1992Co-Authors: Bernard G Schreurs, J.‐v. Sanchez‐andres, Daniel L AlkonAbstract:Abstract Pressure applications of GABA localized to Purkinje cell somas in a rabbit Cerebellar Slice produced uniphasic hyperpolarizing responses, whereas applications of GABA that were directed at the Purkinje cell dendrites produced complex, triphasic responses with hyperpolarizing and depolarizing components. Both somatic and dendritic application of GABA elicited fast hyperpolarization (GABAhf), but dendritic application also elicited a slower depolarization (GABAd) and a later, long-lasting hyperpolarization (GABAhf). All three types of responses were accompanied by increased conductance. Use of either GABA antagonist, bicuculline or picrotoxin, eliminated the GABAhf and GABAd responses but left the GABAhl response intact. Pressure delivery of the GABA agonist, baclofen, to the dendrites but not the soma elicited a GABAhl response. Application of baclofen paired with membrane depolarization sufficient to elicit local, calcium-dependent dendritic spiking produced a persistent reduction in the GABAhl response, whereas alternating presentations of baclofen and membrane depolarization or presentations of baclofen alone could not. The fact that GABA and baclofen inhibited Purkinje cell activity in the rabbit Cerebellar Slice and that picrotoxin and bicuculline eliminated some, but not all of the components of the GABA response suggests the presence of both GABAA and GABAB receptors. The ability of baclofen to inhibit Purkinje cells if it was applied to the applied to the soma suggests that GABAB receptors are located predominantly on Purkinje cell dendrites. The pairing-specific change in the baclofen response suggests the existence of GABAB-mediated modifiability of Purkinje cell dendrites. Taken together, the present data provide the first electrophysiological evidence that (1) GABAA receptors are located predominantly at the Purkinje cell soma, (2) GABAB receptors are located predominantly on Purkinje cell dendrites, and (3) these GABAB receptors may be susceptible to modification.
Jean-louis Bossu - One of the best experts on this subject based on the ideXlab platform.
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Participation of low-threshold Ca2+ spike in the Purkinje cells complex spike.
NeuroReport, 2008Co-Authors: Pauline Cavelier, Huguette Beekenkamp, Ann M Lohof, Etienne Lonchamp, Jean Mariani, Jean-louis BossuAbstract:In Purkinje cells from Cerebellar Slice cultures, low-threshold Ca spike (LTS) gives rise to complex bursts in the soma that resemble the complex spike induced by climbing fibers stimulation. We show that LTS is reduced by T-type and R-type Ca channel blockers (SNX-482, nickel, or mibefradil). We propose that LTS is generated by openings of T-type Ca channels (alpha-1G and/or alpha-1I subunits) and R-type Ca channels (alpha-1E subunit isoforms with a weak sensitivity to SNX-482 and to nickel). Using mibefradil we show that climbing fiber stimulation activates LTS, which contributes to the shape of the response. This Ca entry may be involved in Ca-dependent synaptic plasticity of the parallel fiber input induced by climbing fiber activation.
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Cerebellar Slice cultures from mice lacking the P/Q calcium channel: electroresponsiveness of Purkinje cells
Neuroscience letters, 2002Co-Authors: Pauline Cavelier, Huguette Beekenkamp, Hee-sup Shin, Kisun Jun, Jean-louis BossuAbstract:Abstract To investigate the role of P/Q type Ca 2+ channels in determining the firing pattern of Purkinje cells (PCs) we compared the somatically evoked discharge of action potentials (APs) in PCs from 3 to 4 week old Cerebellar Slice cultures obtained with ataxic mice lacking α 1A -subunit (α −/− ) and with normal mice (non-ataxic α +/− or α +/+ ) using the whole-cell configuration of the patch-clamp recording method. Whereas evoked responses of PCs in normal mice were mainly fast APs, those of PCs from ataxic mice were mainly low-threshold Ca 2+ spikes (LTS). Furthermore, a sustained plateau potential due to the activation of cadmium sensitive Ca 2+ conductances was not observed in PCs from ataxic mice by blocking K + channels. These results confirm that P/Q Ca 2+ channels elicit Ca 2+ -dependent plateau potentials and control the propagation of the dendritic LTS to the soma.
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control of the propagation of dendritic low threshold ca2 spikes in purkinje cells from rat Cerebellar Slice cultures
The Journal of Physiology, 2002Co-Authors: Pauline Cavelier, Huguette Beekenkamp, Frederic Pouille, Thomas Desplantez, Jean-louis BossuAbstract:To investigate the ionic mechanisms controlling the dendrosomatic propagation of low-threshold Ca2+ spikes (LTS) in Purkinje cells (PCs), somatically evoked discharges of action potentials (APs) were recorded under current-clamp conditions. The whole-cell configuration of the patch-clamp method was used in PCs from rat Cerebellar Slice cultures. Full blockade of the P/Q-type Ca2+ current revealed slow but transient depolarizations associated with bursts of fast Na+ APs. These can occur as a single isolated event at the onset of current injection, or repetitively (i.e. a slow complex burst). The initial transient depolarization was identified as an LTS Blockade of P/Q-type Ca2+ channels increased the likelihood of recording Ca2+ spikes at the soma by promoting dendrosomatic propagation. Slow rhythmic depolarizations shared several properties with the LTS (kinetics, activation/inactivation, calcium dependency and dendritic origin), suggesting that they correspond to repetitively activated dendritic LTS, which reach the soma when P/Q channels are blocked. Somatic LTS and slow complex burst activity were also induced by K+ channel blockers such as TEA (2.5 × 10−4m) charybdotoxin (CTX, 10−5m), rIberiotoxin (10−7m), and 4-aminopyridine (4-AP, 10−3m), but not by apamin (10−4m). In the presence of 4-AP, slow complex burst activity occurred even at hyperpolarized potentials (−80 mV). In conclusion, we suggest that the propagation of dendritic LTS is controlled directly by 4-AP-sensitive K+ channels, and indirectly modulated by activation of calcium-activated K+ (BK) channels via P/Q-mediated Ca2+ entry. The slow complex burst resembles strikingly the complex spike elicited by climbing fibre stimulation, and we therefore propose, as a hypothesis, that dendrosomatic propagation of the LTS could underlie the complex spike.
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dendro somatic distribution of calcium mediated electrogenesis in purkinje cells from rat Cerebellar Slice cultures
The Journal of Physiology, 2000Co-Authors: Frederic Pouille, Pauline Cavelier, Huguette Beekenkamp, Thomas Desplantez, Peter J Craig, Ruth E Beattie, S G Volsen, Jean-louis BossuAbstract:The role of Ca2+ entry in determining the electrical properties of Cerebellar Purkinje cell (PC) dendrites and somata was investigated in Cerebellar Slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the α1A subunit (P/Q type Ca2+ channel), the α1G subunit (T type) and the α1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 ± 15 ms) triggering one or two fast (∼1 ms) action potentials (APs). The slow depolarization was identified as a low-threshold non-P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane-permeable Ca2+ chelator abolished repetitive firing. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to ω-conotoxin MVII C or ω-agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel-specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. In summary, we demonstrate that Ca2+ entry through low-voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+-dependent hyperpolarization. Synaptic activity in response to transmitter release on dendritic and somatic post-synaptic sites is generally transduced into trains of action potentials (APs) at the axon hillock. The firing pattern coding the input-output relationship in a given neuronal population is determined mainly by the intrinsic properties of each neuron, based on sets of voltage-dependent channels with specific membrane distributions. Furthermore, the axo-somatic compartment communicates with the dendrites via back-propagating Na+ APs, which modulate the impact of synaptic inputs on the dendritic membrane potential (see Magee et al. 1998). Whether or not the dendrites communicate actively with the axo-somatic compartment by propagating APs, such as Ca2+ APs probably initiated in the dendrites (see Yuste & Tank, 1996) is not yet clear, nor is the nature of the channels involved. Purkinje cells (PCs) of the Cerebellar cortex represent a unique model to study the role of ionic channels in determining the firing pattern generated by various synaptic inputs as well as, owing to their large dendritic arborization, the electrophysiology of the dendro-somatic interactions. These neurons occupy a central position in the Cerebellar circuitry: they integrate excitatory post-synaptic potentials from climbing and parallel fibres into, respectively, ‘complex’ or ‘simple’ spikes (Eccles et al. 1967). Furthermore, PCs display spontaneous and rhythmic activity characterized by prolonged periods of bursting followed by periods of electrical quiescence (Llinas & Sugimori, 1980a). These neurons express a large number of voltage-gated ionic channels and based on the results of voltage-clamp studies of PCs ionic conductances, a computational model of PCs has been developed. This reproduces many features of the discharge pattern of PCs in response to current injections (De Schutter & Bower, 1994a) and generates appropriate responses to climbing and parallel fibre activation (De Schutter & Bower, 1994b). A compartmental model of PCs with Na+ APs restricted to the soma and Ca2+ APs to the dendrites had been proposed as early as 1980 (Llinas & Sugimori, 1980a, b). Na+ APs have been shown to be initiated in the PC axon close to the soma and to spread passively into the dendrites where the density of Na+ channels decreases with the distance from the soma (Stuart & Hausser, 1994). Ca2+ APs are generated in dendrites (Llinas & Sugimori, 1980b). Among voltage-gated neuronal Ca2+ channels, PCs in organotypic Slice cultures as well as PCs in acute Slices (Usowicz et al. 1992) expressed P/Q type Ca2+ channels and a high density of low-threshold transient type Ca2+ channels mainly in the dendrites (Mouginot et al. 1997). We describe here the role of these Ca2+ conductances in determining the dendritic and somatic firing behaviour of PCs in this culture model. This preparation appears to be an advantageous model to characterize the interactions between ionic currents in relation to the establishment of somatic and dendritic firing patterns. Somatic and dendritic compartments are accessible for patch-clamp recordings to determine the localization and the biophysical properties of various channels types. The relation between the firing pattern and a given channel type could be easily determined by using specific pharmacological and/or molecular tools. Some of the present results have been published in a preliminary communication (Bossu et al. 1998).
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low threshold ca2 currents in dendritic recordings from purkinje cells in rat Cerebellar Slice cultures
The Journal of Neuroscience, 1997Co-Authors: Didier Mouginot, Jean-louis Bossu, Beat H. GähwilerAbstract:Voltage-dependent Ca2+ conductances were investigated in Purkinje cells in rat Cerebellar Slice cultures using the whole-cell and cell-attached configurations of the patch-clamp technique. In the presence of 0.5 mm Ca2+ in the extracellular solution, the inward current activated with a threshold of −55 ± 1.5 mV and reached a maximal amplitude of 2.3 ± 0.4 nA at −31 ± 2 mV. Decay kinetics revealed three distinct components: a fast (24.6 ± 2 msec time constant), a slow (304 ± 46 msec time constant), and a nondecaying component. Rundown of the slow and sustained components of the current, or application of antagonists for the P/Q-type Ca2+ channels, allowed isolation of the fast-inactivating Ca2+ current, which had a threshold for activation of −60 mV and reached a maximal amplitude of 0.7 nA at a membrane potential of −33 mV. Both activation and steady-state inactivation of this fast-inactivating Ca2+ current were described with Boltzmann equations, with half-activation and inactivation at −51 mV and −86 mV, respectively. This Ca2+ current was nifedipine-insensitive, but its amplitude was reduced reversibly by bath-application of NiCl2 and amiloride, thus allowing its identification as a T-type Ca2+ current. Channels with a conductance of 7 pS giving rise to a fast T-type ensemble current (insensitive to ω-Aga-IVA) were localized with a high density on the dendritic membrane. Channel activity responsible for the ensemble current sensitive to ω-Aga-IVA was detected with 10 mm Ba2+ as the charge carrier. These channels were distributed with a high density on dendritic membranes and in rare cases were also seen in somatic membrane patches.
John E. Greenlee - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological o
2016Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, L. Clardy, Ikuo Tsunoda, Noel G. CarlsonAbstract:Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Pur-kinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic Cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat Cerebellar Slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in dis-ease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo anti-body to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody bind-ing; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, in-cubation of rat Cerebellar Slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microgli
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anti yo antibody uptake and interaction with its intracellular target antigen causes purkinje cell death in rat Cerebellar Slice cultures a possible mechanism for paraneoplastic Cerebellar degeneration in humans with gynecological or breast cancers
PLOS ONE, 2015Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Stacey L Clardy, Noel G. CarlsonAbstract:Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic Cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat Cerebellar Slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat Cerebellar Slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.
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Comparison of Purkinje cell cytotoxicity produced by anti-Yo antibodies with cytotoxicity produced by commercial and patient antibodies reactive with other Purkinje cell cytoplasmic proteins.
2015Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Stacey L Clardy, Noel G. CarlsonAbstract:Rat Cerebellar Slice cultures were incubated for 72 hours with either 1) sera from patients with anti-Yo antibodies; 2) commercial antibodies to calbindin, calmodulin, or PCP-2/L7; 3) anti-Purkinje cells antibodies from the three neurologically normal patients studied, whose sera labeled Purkinje cell cytoplasm but did not react with Yo antigens; or 4) normal human IgG. Cultures were quantified for cell death as indicated by uptake of SYTOX dyes. Extensive Purkinje cell death was seen in cultures incubated with all 4 anti-Yo sera (data for 2 anti-Yo sera not shown). In contrast, cell death was not observed in cultures incubated with any of the three commercially obtained antibodies reactive with intracellular anti-Purkinje cell proteins nor with sera from Patients 1–3, despite extensive Purkinje cell uptake. Cultures incubated with normal human IgG exhibited only faint antibody accumulation by Purkinje cells and no detectable Purkinje cell death. Statistical significance between groups was determined by non-parametric Mann-Whitney ANOVA. Death in cultures incubated with anti-Yo antisera was statistically significantly greater than that seen in cultures incubated with commercial antisera, sera from patients 1–3, or normal human IgG.
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comparative uptake and cytotoxicity of anti hu and anti ri antibodies in rat Cerebellar Slice cultures p02 161
Neurology, 2013Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Noel G. CarlsonAbstract:OBJECTIVE: To evaluate neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in organotypic cultures of rat cerebellum. BACKGROUND: The paraneoplastic autoantibodies, anti-Hu and anti-Ri, recognize intracellular antigens present in essentially all neurons. At autopsy, brains of patients with anti-Hu antibody show neuronal destruction. In contrast, anti-Ri antibody is less clearly associated with neuronal death, and patients with anti-Ri antibodies may respond to treatment. The role of antibodies in causing paraneoplastic neurological injury has not been defined. However, we have previously demonstrated that anti-Yo antibodies, associated with paraneoplastic Cerebellar degeneration, accumulate in Purkinje cells in Slice cultures of rat cerebellum and that antibody accumulation was followed by cell death. The present study was conducted to determine 1) whether anti-Hu and anti-Ri antibodies are also taken up by neurons in this culture system; 2) whether uptake is restricted to Purkinje cells, and 3) whether uptake of either antibody is followed by cell death. DESIGN/METHODS: Rat Cerebellar Slice cultures were incubated with either anti-Hu or anti-Ri antibodies and were followed over time. TUNEL and FLICA methods were used to detect apoptosis. The dead cell stain, SYTOX green, was used to detect non-apoptotic cell death. RESULTS: Anti-Hu and anti-Ri antibodies accumulated not only in Purkinje cells but also in multiple other neuronal populations. Accumulation of anti-Hu antibody was followed by apoptosis involving multiple neuronal populations: this became detectable within 72-96 hours. In contrast, as compared to controls, cells accumulating anti-Ri antibodies showed no evidence of apoptosis or necrotic cell death over a period of 200 hours. CONCLUSIONS: Uptake of anti-Hu and anti-Ri antibodies was observed in viable neurons throughout the cerebellum and was not restricted to Purkinje cells. While anti-Hu antibody appears to be specifically cytotoxic, anti-Ri antibody did not affect neuronal viability and may predominantly cause neuronal dysfunction rather than death. Supported by: United States Department of Veterans Affairs. Disclosure: Dr. Greenlee has received personal compensation for activities with American Academy of Neurology and Oliver Maner LLP. Dr. Greenlee has received personal compensation in an editorial capacity for Medlink. Dr. Clawson has nothing to disclose. Dr. Wood has nothing to disclose. Dr. Hill has nothing to disclose. Dr. Tsunoda has nothing to disclose. Dr. Carlson has nothing to disclose.
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purkinje cell death after uptake of anti yo antibodies in Cerebellar Slice cultures
Journal of Neuropathology and Experimental Neurology, 2010Co-Authors: John E. Greenlee, Susan A. Clawson, Kenneth E. Hill, Blair Wood, Ikuo Tsunoda, Noel G. CarlsonAbstract:Paraneoplastic Cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of Cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in Slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic Cerebellar degeneration.
Josef P. Kapfhammer - One of the best experts on this subject based on the ideXlab platform.
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PKCγ-Mediated Phosphorylation of CRMP2 Regulates Dendritic Outgrowth in Cerebellar Purkinje Cells
Molecular Neurobiology, 2020Co-Authors: Sabine C. Winkler, Etsuko Shimobayashi, Josef P. KapfhammerAbstract:The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of Cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinoCerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in Cerebellar Slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in Cerebellar Slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated Cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.
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Chronic pharmacological blockade of the Na+/Ca2+ exchanger modulates the growth and development of the Purkinje cell dendritic arbor in mouse Cerebellar Slice cultures
The European journal of neuroscience, 2017Co-Authors: Pradeep Sherkhane, Josef P. KapfhammerAbstract:The Na+ /Ca2+ exchanger (NCX) is a bidirectional plasma membrane antiporter involved in Ca2+ homeostasis in eukaryotes. NCX has three isoforms, NCX1-3, and all of them are expressed in the cerebellum. Immunostaining on Cerebellar Slice cultures indicates that NCX is widely expressed in the cerebellum, including expression in Purkinje cells. The pharmacological blockade of the forward mode of NCX (Ca2+ efflux mode) by bepridil moderately inhibited growth and development of Purkinje cell dendritic arbor in Cerebellar Slice cultures. However, the blockade of the reverse mode (Ca2+ influx mode) by KB-R7943 severely reduced the dendritic arbor and induced a morphological change with thickened distal dendrites. The effect of KB-R7943 on dendritic growth was unrelated to the activity of voltage-gated calcium channels and was also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. We have used additional NCX inhibitors including CB-DMB, ORM-10103, SEA0400, YM-244769, and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse, or both modes. These inhibitors caused a strong dendritic reduction similar to that seen with KB-R7943, but did not elicit thickening of distal dendrites. Our findings indicate that disturbance of the NCX-dependent calcium transport in Purkinje cells induces a reduction of dendritic arbor, which is presumably caused by changes in the calcium handling, and underline the importance of the calcium equilibrium for the dendritic development in Cerebellar Purkinje cells.
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the plasma membrane ca2 atpase2 pmca2 is involved in the regulation of purkinje cell dendritic growth in Cerebellar organotypic Slice cultures
Neural Plasticity, 2013Co-Authors: Pradeep Sherkhane, Josef P. KapfhammerAbstract:Purkinje cells are the principal neurons of the Cerebellar cortex and have an extensive and elaborate dendritic tree. Chronic activation of type I metabotropic glutamate receptors inhibits Purkinje cell dendritic growth in organotypic Cerebellar Slice cultures. This effect is mediated by calcium influx through P/Q-type and T-type Ca(2+) channels. We have now studied the role of the plasma membrane Ca(2+)-ATPase2 (PMCA2), a major calcium extrusion pump, for Purkinje cell dendritic development. We found that PMCA2 is strongly expressed in the plasma membrane and dendritic spines of Purkinje cells in organotypic Slice cultures compatible with a role for controlling the local dendritic calcium equilibrium. Inhibition of PMCA2 activity by carboxyeosin resulted in a moderate reduction of Purkinje cell dendritic tree size indicating that the extrusion of calcium by PMCA2 is important for maintaining the dendritic calcium concentration and controlling dendritic growth. When inhibition of PMCA2 was combined with stimulation of type I metabotropic glutamate receptors, it partially rescued dendritic morphology. This protection can be explained by a compensatory inactivation of voltage-gated calcium channels in Purkinje cells after PMCA2 inhibition. Our results demonstrate that PMCA2 activity is an important regulator of the dendritic calcium equilibrium controlling Purkinje cell dendritic growth.
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The analysis of purkinje cell dendritic morphology in organotypic Slice cultures.
Journal of Visualized Experiments, 2012Co-Authors: Josef P. Kapfhammer, Olivia S. GuggerAbstract:Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents (3). Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells (11) are available through Jackson labs. Organotypic Cerebellar Slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period (4). We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic Cerebellar Slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained Cerebellar Slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic Slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.
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different regulation of purkinje cell dendritic development in Cerebellar Slice cultures by protein kinase calpha and beta
Journal of Neurobiology, 2003Co-Authors: Anja Gundlfinger, Josef P. Kapfhammer, Friederike Kruse, Michael Leitges, Friedrich MetzgerAbstract:Activity of protein kinase C (PKC), and in particular the PKCgamma-isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity-dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca(2+)-dependent PKC isoforms, PKCalpha and -beta, in Purkinje cell survival and dendritic morphology in Slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild-type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCbeta, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild-type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCalpha-deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild-type cells. Together with our previous work on the role of PKCgamma, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCgamma-isoform, which is highly activated by developmental processes. The PKCalpha isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCbeta isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in Cerebellar Slice cultures.