Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.393
Filtrar
Filtros adicionais











País/Região como assunto
Intervalo de ano
1.
Nihon Yakurigaku Zasshi ; 154(3): 133-137, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527363

RESUMO

Hydrogen sulfide (H2S) has been focused as a biological mediator, which modulates signal transduction and protects cells and tissues from oxidative stress. H2S is also expected as a neuroprotectant because it has a neuroprotective activity. Endogenous H2S is mainly generated from L-cysteine. However, it is difficult to use L-cysteine as a neuroprotectant because of its neurotoxicity. In 2013, a novel biogenesis pathway of H2S from D-cysteine has been identified. In this pathway, D-amino acid oxidase (DAO) converts D-cysteine to 3-mercaptopyruvate (3MP), followed by the generation of H2S from 3MP by 3-mercaptopyrvate sulfurtransferase. DAO is especially abundant in cerebellum among various brain regions and mediates efficient generation of H2S from D-cysteine in the cerebellar tissues. In addition, D-cysteine has more potent neuroprotective activity in cerebellar primary neurons than L-cysteine. Cerebella Purkinje cells (PCs) are characterized by the highly-branched dendrites and are important for cerebellar functions. The dendritic shrinkage and degeneration of PCs are frequently observed in patients and model mice of cerebellar ataxias. We revealed that D-cysteine enhanced dendritic development of primary cultured PCs, but L-cysteine impaired the dendritic development. This effect of D-cysteine was inhibited by DAO inhibitors and reproduced by 3MP and a H2S donor, suggesting that this enhancement of dendritic development is caused by the production of H2S from D-cysteine. Taken together, D-cysteine would be available as a neuroprotectant against cerebellar ataxias, which are accompanied with dendritic shrinkage of cerebellar PCs.


Assuntos
Cisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Animais , Células Cultivadas , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Humanos , Camundongos , Neurogênese , Estresse Oxidativo , Células de Purkinje/citologia
2.
Genes Cells ; 24(7): 464-472, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31095815

RESUMO

Dendrites of neurons receive and process synaptic or sensory inputs. The Drosophila class IV dendritic arborization (da) neuron is an established model system to explore molecular mechanisms of dendrite morphogenesis. The total number of dendritic branch terminals is one of the frequently employed parameters to characterize dendritic arborization complexity of class IV neurons. This parameter gives a useful phenotypic readout of arborization during neurogenesis, and it is typically determined by laborious manual analyses of numerous images. Ideally, an automated analysis would greatly reduce the workload; however, it is challenging to automatically discriminate dendritic branch terminals from signals of surrounding tissues in whole-mount live larvae. Here, we describe our newly developed software, called DeTerm, which automatically recognizes and quantifies dendrite branch terminals via an artificial neural network. Once we input an image file of a neuronal dendritic arbor and its region of interest information, DeTerm is capable of labeling terminals of larval class IV neurons with high precision, and it also provides positional data of individual terminals. We further show that DeTerm is applicable to other types of neurons, including mouse cerebellar Purkinje cells. DeTerm is freely available on the web and was successfully tested on Mac, Windows and Linux.


Assuntos
Cerebelo/fisiologia , Dendritos/fisiologia , Redes Neurais (Computação) , Neurogênese , Neurônios/fisiologia , Células de Purkinje/fisiologia , Software , Animais , Cerebelo/citologia , Drosophila , Proteínas de Drosophila/metabolismo , Larva , Camundongos , Neurônios/citologia , Células de Purkinje/citologia
3.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979012

RESUMO

Staufen2 (Stau2) is an RNA-binding protein that is involved in dendritic spine morphogenesis and function. Several studies have recently investigated the role of Stau2 in the regulation of its neuronal target mRNAs, with particular focus on the hippocampus. Here, we provide evidence for Stau2 expression and function in cerebellar Purkinje cells. We show that Stau2 downregulation (Stau2GT) led to an increase of glutamate receptor ionotropic delta subunit 2 (GluD2) in Purkinje cells when animals performed physical activity by voluntary wheel running compared with the age-matched wildtype (WT) mice (C57Bl/6J). Furthermore, Stau2GT mice showed lower performance in motor coordination assays but enhanced motor learning abilities than did WT mice, concomitantly with an increase in dendritic GluD2 expression. Together, our results suggest the novel role of Stau2 in Purkinje cell synaptogenesis in the mouse cerebellum.


Assuntos
Envelhecimento , Encéfalo/fisiologia , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Células de Purkinje/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de Glutamato/genética , Animais , Cerebelo/citologia , Cerebelo/fisiologia , Feminino , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora , Células de Purkinje/citologia , RNA Mensageiro/genética , Receptores de Glutamato/análise
4.
Neurosci Bull ; 35(3): 378-388, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30888608

RESUMO

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.


Assuntos
Vetores Genéticos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Vírus da Floresta de Semliki/genética , Animais , Células Cultivadas , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Células de Purkinje/citologia , Células de Purkinje/metabolismo
5.
Int J Mol Sci ; 20(2)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642045

RESUMO

Endogenous γ-aminobutyric acid (GABA)-dependent activity induces death of developing Purkinje neurons in mouse organotypic cerebellar cultures and the synthetic steroid mifepristone blocks this effect. Here, using brain-derived neurotrophic factor (BDNF) heterozygous mice, we show that BDNF plays no role in immature Purkinje cell death. However, interestingly, BDNF haploinsufficiency impairs neuronal survival induced by mifepristone and GABAA-receptors antagonist (bicuculline) treatments, indicating that the underlying neuroprotective mechanism requires the neurotrophin full expression.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Mifepristona/farmacologia , Fármacos Neuroprotetores/farmacologia , Células de Purkinje/citologia , Ácido gama-Aminobutírico/efeitos adversos , Animais , Apoptose , Bicuculina/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sobrevivência Celular , Células Cultivadas , Haploinsuficiência , Camundongos , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo
6.
Cell Mol Neurobiol ; 38(7): 1399-1412, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066224

RESUMO

Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.


Assuntos
Cerebelo/citologia , Cultura Primária de Células/métodos , Células de Purkinje/citologia , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Camundongos , Células de Purkinje/fisiologia , Ratos , Ratos Wistar
7.
Cell Mol Life Sci ; 75(19): 3495-3505, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29982847

RESUMO

Purkinje neurons, the sole output of the cerebellar cortex, deliver GABA-mediated inhibition to the deep cerebellar nuclei. To subserve this critical function, Purkinje neurons fire repetitively, and at high frequencies, features that have been linked to the unique properties of the voltage-gated sodium (Nav) channels expressed. In addition to the rapidly activating and inactivating, or transient, component of the Nav current (INaT) present in many types of central and peripheral neurons, Purkinje neurons, also expresses persistent (INaP) and resurgent (INaR) Nav currents. Considerable progress has been made in detailing the biophysical properties and identifying the molecular determinants of these discrete Nav current components, as well as defining their roles in the regulation of Purkinje neuron excitability. Here, we review this important work and highlight the remaining questions about the molecular mechanisms controlling the expression and the functioning of Nav currents in Purkinje neurons. We also discuss the impact of the dynamic regulation of Nav currents on the functioning of individual Purkinje neurons and cerebellar circuits.


Assuntos
Potenciais de Ação/fisiologia , Cerebelo/citologia , Células de Purkinje/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologia , Potenciais de Ação/genética , Animais , Cerebelo/fisiologia , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Células de Purkinje/citologia , Canais de Sódio Disparados por Voltagem/classificação , Canais de Sódio Disparados por Voltagem/genética
8.
Neurosci Res ; 135: 13-20, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29614249

RESUMO

Alternative splicing of RNAs diversifies the functionalities of proteins, and it is optimized for each cell type and each developmental stage. nElavl (composed of Elavl2, Elavl3, and Elavl4) proteins are the RNA-binding proteins that is specifically expressed in neurons, regulate the alternative splicing of target RNAs, and promote neuronal differentiation and maturation. Recent studies revealed that Elavl3 knockout (Elavl3-/-) mice completely lost the expression of nElavl proteins in the Purkinje cells and exhibited cerebellar dysfunction. Here, we found that the alternative splicing of AnkyrinG exon 34 was misregulated in the cerebella of Elavl3-/- mice. AnkyrinG is an essential factor for the formation of neuronal polarity and is required for normal neuronal functions. We revealed that exon 34 of AnkyrinG was normally included in immature neurons and was mostly excluded in mature neurons; however, it was included in the cerebella of Elavl3-/- mice even in adulthood. In the Purkinje cells of adult Elavl3-/- mice, the length of the AnkyrinG-positive region shortened and somatic organelles leaked into the axons. These results suggested that exon 34 of AnkyrinG is an embryonic-stage-preferential exon that should be excluded from mature neurons and that Elavl3 regulates neuronal polarity through alternative splicing of this exon.


Assuntos
Anquirinas/genética , Proteína Semelhante a ELAV 3/genética , Éxons , Células de Purkinje/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Anquirinas/metabolismo , Polaridade Celular/genética , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Doenças Cerebelares/patologia , Cerebelo/patologia , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 3/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Homologia de Sequência de Aminoácidos
9.
Proc Natl Acad Sci U S A ; 115(19): 5004-5009, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29691318

RESUMO

Tonic inhibition in the brain is mediated through an activation of extrasynaptic GABAA receptors by the tonically released GABA, resulting in a persistent GABAergic inhibitory action. It is one of the key regulators for neuronal excitability, exerting a powerful action on excitation/inhibition balance. We have previously reported that astrocytic GABA, synthesized by monoamine oxidase B (MAOB), mediates tonic inhibition via GABA-permeable bestrophin 1 (Best1) channel in the cerebellum. However, the role of astrocytic GABA in regulating neuronal excitability, synaptic transmission, and cerebellar brain function has remained elusive. Here, we report that a reduction of tonic GABA release by genetic removal or pharmacological inhibition of Best1 or MAOB caused an enhanced neuronal excitability in cerebellar granule cells (GCs), synaptic transmission at the parallel fiber-Purkinje cell (PF-PC) synapses, and motor performance on the rotarod test, whereas an augmentation of tonic GABA release by astrocyte-specific overexpression of MAOB resulted in a reduced neuronal excitability, synaptic transmission, and motor performance. The bidirectional modulation of astrocytic GABA by genetic alteration of Best1 or MAOB was confirmed by immunostaining and in vivo microdialysis. These findings indicate that astrocytes are the key player in motor coordination through tonic GABA release by modulating neuronal excitability and could be a good therapeutic target for various movement and psychiatric disorders, which show a disturbed excitation/inhibition balance.


Assuntos
Astrócitos/metabolismo , Cerebelo/metabolismo , Desempenho Psicomotor/fisiologia , Células de Purkinje/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Astrócitos/citologia , Bestrofinas/genética , Bestrofinas/metabolismo , Cerebelo/citologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Células de Purkinje/citologia , Ácido gama-Aminobutírico/genética
10.
Dis Markers ; 2018: 5046372, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29651324

RESUMO

Background: After perinatal asphyxia, the cerebellum presents more damage than previously suggested. Objectives: To explore if the antioxidant N-acetylcysteine amide (NACA) could reduce cerebellar injury after hypoxia-reoxygenation in a neonatal pig model. Methods: Twenty-four newborn pigs in two intervention groups were exposed to 8% oxygen and hypercapnia, until base excess fell to -20 mmol/l or the mean arterial blood pressure declined to <20 mmHg. After hypoxia, they received either NACA (NACA group, n = 12) or saline (vehicle-treated group, n = 12). One sham-operated group (n = 5) served as a control and was not subjected to hypoxia. Observation time after the end of hypoxia was 9.5 hours. Results: The intranuclear proteolytic activity in Purkinje cells of asphyxiated vehicle-treated pigs was significantly higher than that in sham controls (p = 0.03). Treatment with NACA was associated with a trend to decreased intranuclear proteolytic activity (p = 0.08), There were significantly less mutations in the mtDNA of the NACA group compared with the vehicle-treated group, 2.0 × 10-4 (±2.0 × 10-4) versus 4.8 × 10-5(±3.6 × 10-4, p < 0.05). Conclusion: We found a trend to lower proteolytic activity in the core of Purkinje cells and significantly reduced mutation rate of mtDNA in the NACA group, which may indicate a positive effect of NACA after neonatal hypoxia. Measuring the proteolytic activity in the nucleus of Purkinje cells could be used to assess the effect of different neuroprotective substances after perinatal asphyxia.


Assuntos
Acetilcisteína/análogos & derivados , Asfixia Neonatal/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Células de Purkinje/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Animais , Asfixia Neonatal/genética , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , Modelos Animais de Doenças , Humanos , Recém-Nascido , Taxa de Mutação , Fármacos Neuroprotetores/farmacologia , Proteólise , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Suínos
11.
Curr Neuropharmacol ; 16(2): 151-159, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-28554312

RESUMO

BACKGROUND: Spinocerebellar ataxias (SCAs) are a group of cerebellar diseases characterized by progressive ataxia and cerebellar atrophy. Several forms of SCAs are caused by missense mutations or deletions in genes related to calcium signaling in Purkinje cells. Among them, spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations in PRKCG gene which encodes protein kinase C gamma (PKCγ). It is remarkable that in several cases in which SCA is caused by point mutations in an individual gene, the affected genes are involved in the PKCγ signaling pathway and calcium signaling which is not only crucial for proper Purkinje cell function but is also involved in the control of Purkinje cell dendritic development. In this review, we will focus on the PKCγ signaling related genes and calcium signaling related genes then discuss their role for both Purkinje cell dendritic development and cerebellar ataxia. METHODS: Research related to SCAs and Purkinje cell dendritic development is reviewed. RESULTS: PKCγ dysregulation causes abnormal Purkinje cell dendritic development and SCA14. Carbonic anhydrase related protein 8 (Car8) encoding CAR8 and Itpr1 encoding IP3R1were identified as upregulated genes in one of SCA14 mouse model. IP3R1, CAR8 and PKCγ proteins are strongly and specifically expressed in Purkinje cells. The common function among them is that they are involved in the regulation of calcium homeostasis in Purkinje cells and their dysfunction causes ataxia in mouse and human. Furthermore, disruption of intracellular calcium homeostasis caused by mutations in some calcium channels in Purkinje cells links to abnormal Purkinje cell dendritic development and the pathogenesis of several SCAs. CONCLUSION: Once PKCγ signaling related genes and calcium signaling related genes are disturbed, the normal dendritic development of Purkinje cells is impaired as well as the integration of signals from other neurons, resulting in abnormal development, cerebellar dysfunction and eventually Purkinje cell loss.


Assuntos
Sinalização do Cálcio/genética , Dendritos/fisiologia , Proteína Quinase C/genética , Células de Purkinje/citologia , Ataxias Espinocerebelares , Animais , Biomarcadores Tumorais/genética , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteína Quinase C/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
12.
Heart Rhythm ; 15(2): 258-264, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28987458

RESUMO

BACKGROUND: Pulmonary vein (PV) myocardium is a known source of atrial fibrillation. A debated question is whether myocardial extensions into caval veins and coronary sinus (CS) have similar properties. No studies have documented specific pacemaker and/or conducting properties of human extracardiac myocardium. OBJECTIVE: The purpose of this study was to characterize the histology and immunohistochemical features of myocardial sleeves in the wall of cardiac veins. METHODS: Sections of 32 human hearts were investigated. Specimens of PVs, superior caval vein (SVC), CS, sinoatrial and atrioventricular nodes, and left ventricle were stained with Best's Carmine for selective staining of intracellular glycogen. Anti-connexin45 (Cx45)- and Cx43-specific antibodies were used to determine the conduction properties of extracardiac myocardium. RESULTS: Myocardial sleeve was found in the wall of PVs of 15 of 16 hearts, 21 of 22 SVCs, and 8 of 8 CSs. Bundles of glycogen-positive cardiomyocytes exhibiting pale cytoplasm and peripheral myofibrils were observed in the venous sleeves. Strong Cx45 and weak Cx43 labeling was detected in the extracardiac myocardium. Similar staining pattern was observed for the pacemaker and conduction system, whereas ventricular myocardium exhibited prominent Cx43 and no Cx45 immunoreactivity. CONCLUSION: Myocardial fibers of PVs, SVC, and CS exhibit morphology similar to that of Purkinje fibers and are enriched in glycogen. We provide data for the first time on prominent positive staining for Cx45 in the extracardiac myocardium, indicating its potential pacemaker and/or conducting nature.


Assuntos
Conexinas/análise , Vasos Coronários/metabolismo , Miócitos Cardíacos/citologia , Células de Purkinje/citologia , Idoso , Idoso de 80 Anos ou mais , Cadáver , Seio Coronário/citologia , Seio Coronário/metabolismo , Vasos Coronários/citologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Células de Purkinje/metabolismo
13.
J Mol Neurosci ; 64(2): 300-311, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29285738

RESUMO

Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.


Assuntos
Neurogênese , Células de Purkinje/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Potenciais da Membrana , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células de Purkinje/citologia , Células de Purkinje/fisiologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/genética , Sinapses/fisiologia
14.
Cell Rep ; 21(8): 2066-2073, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29166599

RESUMO

Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor δ2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity.


Assuntos
Cálcio/metabolismo , Cerebelo/metabolismo , Rede Nervosa/citologia , Sinapses/metabolismo , Animais , Axônios/metabolismo , Dendritos/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos Knockout , Células de Purkinje/citologia , Receptores de Glutamato/genética
15.
Dev Neurosci ; 39(6): 487-497, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28972955

RESUMO

Cerebellar growth is impeded following very preterm birth in human infants and the observed reduction in cerebellar volume is associated with neurodevelopmental impairment. Decreased levels of circulating insulin-like growth factor 1 (IGF-1) are associated with decreased cerebellar volume. The relationship between preterm birth, circulating IGF-1, and key cell populations supporting cerebellar proliferation is unknown. The aim of this study was to evaluate the effect of preterm birth on postnatal growth, circulating IGF-1, and cerebellar maturation in a preterm rabbit pup model. Preterm rabbit pups (PT) were delivered by cesarean section at day 29 of gestation, cared for in closed incubators with humidified air, and gavage fed with formula. Control term pups (T) delivered by spontaneous vaginal delivery at day 32 of gestation were housed and fed by their lactating doe. In vivo perfusion-fixation for immunohistochemical evaluation of cerebellar proliferation, cell maturation, and apoptosis was performed at repeated time points in PT and T pups. Results show that the mean weight of the pups and circulating IGF-1 protein levels were lower in the PT group at all time points (p < 0.05) than in the T group. Postnatal weight development correlated with circulating IGF-1 (r2 = 0.89) independently of gestational age at birth and postnatal age. The proliferative (Ki-67-positive) portion of the external granular layer (EGL) was decreased in the PT group at postnatal day 2 (P2) compared to in the T group (p = 0.01). Purkinje cells exhibited decreased calbindin staining at P0 (p = 0.003), P2 (p = 0.004), and P5 (p = 0.04) in the PT group compared to in the T group. Staining for sonic hedgehog was positive in neuronal EGL progenitors and Purkinje cells at early time points but was restricted to a well-defined Purkinje cell monolayer at later time points. Preterm birth in rabbit pups is associated with lower circulating levels of IGF-1, decreased postnatal growth, and decreased cerebellar EGL proliferation and Purkinje cell maturation. The preterm rabbit pup model exhibits important characteristics of human preterm birth, and may thus be suitable for the evaluation of interventions aiming to modify growth and cerebellar development in the preterm population.


Assuntos
Cerebelo/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like I/biossíntese , Tamanho do Órgão/fisiologia , Células de Purkinje/citologia , Animais , Animais Recém-Nascidos , Feminino , Idade Gestacional , Lactação/fisiologia , Neurogênese/fisiologia , Gravidez , Coelhos
16.
J Neurochem ; 143(6): 660-670, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29049849

RESUMO

Cerebellar Purkinje cells (PCs) express two members of the classical protein kinase C (cPKC) subfamily, namely, PKCα and PKCγ. Previous studies on PKCγ knockout (KO) mice have revealed a critical role of PKCγ in the pruning of climbing fibers (CFs) from PCs during development. The question remains as to why only PKCγ and not PKCα is involved in CF synapse elimination from PCs. To address this question, we assessed the expression levels of PKCγ and PKCα in wild-type (WT) and PKCγ KO PCs using PC-specific quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis. The results revealed that the vast majority of cPKCs in PCs were PKCγ, whereas PKCα accounted for the remaining minimal fraction. The amount of PKCα was not up-regulated in PKCγ KO PCs. Lentiviral expression of PKCα in PKCγ KO PCs resulted in a 10-times increase in the amount of PKCα mRNA in the PKCγ KO PCs, compared to that in WT PCs. Our quantification showed that the expression levels of cPKC mRNA in PKCγ KO PCs increased roughly from 1% to 22% of that in WT PCs solely through PKCα expression. The up-regulation of PKCα in PKCγ KO PCs significantly rescued the impaired CF synapse elimination. Although both PKCα and PKCγ are capable of pruning supernumerary CF synapses from developing PCs, these results suggest that the expression levels of cPKCs in PKCγ KO PCs are too low for CF pruning.


Assuntos
Cerebelo/enzimologia , Cerebelo/crescimento & desenvolvimento , Proteína Quinase C/biossíntese , Células de Purkinje/citologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas/metabolismo , Isoformas de Proteínas , Transcriptoma
17.
Development ; 144(20): 3686-3697, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28893945

RESUMO

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.


Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Células de Purkinje/citologia , Fatores de Transcrição/fisiologia , Anormalidades Múltiplas/genética , Alelos , Animais , Diferenciação Celular , Divisão Celular , Proliferação de Células , Cerebelo/anormalidades , Dano ao DNA , Anormalidades do Olho/genética , Deleção de Genes , Doenças Renais Císticas/genética , Camundongos , Mutação , Domínios Proteicos , Retina/anormalidades , Fuso Acromático/metabolismo , Dedos de Zinco
18.
Dev Biol ; 430(1): 18-31, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28802829

RESUMO

Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.


Assuntos
Embrião não Mamífero/citologia , Imagem Tridimensional , Cultura Primária de Células/métodos , Transfecção/métodos , Peixe-Zebra/embriologia , Animais , Forma Celular , Células Cultivadas , Eletroporação , Embrião não Mamífero/metabolismo , Citometria de Fluxo , Macrófagos/citologia , Neurônios Motores/citologia , Neuroglia/citologia , Células de Purkinje/citologia , Coloração e Rotulagem , Frações Subcelulares/metabolismo , Transgenes
19.
Neuron ; 95(1): 51-62.e4, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28648497

RESUMO

Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively-a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV-L7-ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.


Assuntos
Potenciais de Ação/fisiologia , Vermis Cerebelar/fisiologia , Optogenética/métodos , Células de Purkinje/fisiologia , Movimentos Sacádicos/fisiologia , Animais , Córtex Cerebelar/citologia , Córtex Cerebelar/fisiologia , Vermis Cerebelar/citologia , Cerebelo/citologia , Cerebelo/fisiologia , Dependovirus/genética , Medições dos Movimentos Oculares , Imuno-Histoquímica , Macaca mulatta , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
20.
Cell Rep ; 19(10): 1977-1986, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28591570

RESUMO

The role of neurotrophic factors as endogenous survival proteins for brain neurons remains contentious. In the cerebellum, the signals controlling survival of molecular layer interneurons (MLIs) are unknown, and direct evidence for the requirement of a full complement of MLIs for normal cerebellar function and motor learning has been lacking. Here, we show that Purkinje cells (PCs), the target of MLIs, express the neurotrophic factor GDNF during MLI development and survival of MLIs depends on GDNF receptors GFRα1 and RET. Conditional mutant mice lacking either receptor lose a quarter of their MLIs, resulting in compromised synaptic inhibition of PCs, increased PC firing frequency, and abnormal acquisition of eyeblink conditioning and vestibulo-ocular reflex performance, but not overall motor activity or coordination. These results identify an endogenous survival mechanism for MLIs and reveal the unexpected vulnerability and selective requirement of MLIs in the control of cerebellar-dependent motor learning.


Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Aprendizagem/fisiologia , Atividade Motora/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Células de Purkinje/metabolismo , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-ret/genética , Células de Purkinje/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA