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1.
Sci Rep ; 8(1): 3417, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467426

RESUMO

Neuronal avalanches have become an ubiquitous tool to describe the activity of large neuronal assemblies. The emergence of scale-free statistics with well-defined exponents has led to the belief that the brain might operate near a critical point. Yet not much is known in terms of how the different exponents arise or how robust they are. Using calcium imaging recordings of dissociated neuronal cultures we show that the exponents are not universal, and that significantly different exponents arise with different culture preparations, leading to the existence of different universality classes. Naturally developing cultures show avalanche statistics consistent with those of a mean-field branching process, however, cultures grown in the presence of folic acid metabolites appear to be in a distinct universality class with significantly different critical exponents. Given the increased synaptic density and number of feedback loops in folate reared cultures, our results suggest that network topology plays a leading role in shaping the avalanche dynamics. We also show that for both types of cultures pronounced correlations exist in the sizes of neuronal avalanches indicating size clustering, being much stronger in folate reared cultures.


Assuntos
Neurônios/fisiologia , Animais , Avalanches , Encéfalo/fisiologia , Técnicas de Cultura de Células/métodos , Pareamento Cromossômico/fisiologia , Rede Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley
2.
Genet Med ; 20(5): 503-512, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28933792

RESUMO

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.


Assuntos
Canais de Cálcio Tipo T/genética , Variações do Número de Cópias de DNA , Frequência do Gene , Haplótipos , Padrões de Herança , Triptases/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular , Duplicação Gênica , Estudos de Associação Genética , Loci Gênicos , Técnicas de Genotipagem , Humanos , Desequilíbrio de Ligação , Mutação , Fenótipo , Análise de Sequência de DNA , Triptases/metabolismo
3.
Nat Neurosci ; 19(3): 432-42, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26854804

RESUMO

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.


Assuntos
Conexinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Quinases da Família src/fisiologia , Animais , Cálcio/metabolismo , Morte Celular/fisiologia , Conexinas/metabolismo , Maleato de Dizocilpina/farmacologia , Magnésio/farmacologia , Potenciais da Membrana/fisiologia , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia
4.
Glia ; 63(11): 2023-2039, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26119281

RESUMO

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo-glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non-neuronal cells exogenously expressing NL3's binding partners NX1α or NX1ß. Conversely, blocking axo-glial NX-NL3 signaling by saturating NX with exogenous soluble NL protein (NL-ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX-NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL-axon signaling occur normally as this peptide does not disrupt NX-NL3 axo-glial interactions. Our findings provide evidence not only for a new pathway in axo-glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023-2039.

5.
Cell Mol Biol Lett ; 19(3): 381-92, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25034033

RESUMO

The large microtubule-associated/Ca(2+)-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca(2+) dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca(2+).


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Dendritos/metabolismo , Hipocampo/citologia , Luz , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Neurônios/efeitos da radiação , Interferência de RNA , Ratos , Análise de Célula Única/métodos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Mol Brain ; 7: 42, 2014 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-24886461

RESUMO

BACKGROUND: Inorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels. RESULTS: Using patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride. CONCLUSIONS: We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.


Assuntos
Potenciais de Ação/fisiologia , Ativação do Canal Iônico/fisiologia , Neurônios/fisiologia , Polifosfatos/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Técnicas de Cocultura , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Indóis/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
7.
Sci Rep ; 3: 1465, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23492951

RESUMO

Maternal folic acid supplementation is essential to reduce the risk of neural tube defects. We hypothesize that high levels of folic acid throughout gestation may produce neural networks more susceptible to seizure in offspring. We hence administered large doses of folic acid to rats before and during gestation and found their offspring had a 42% decrease in their seizure threshold. In vitro, acute application of folic acid or its metabolite 4Hfolate to neurons induced hyper-excitability and bursting. Cultured neuronal networks which develop in the presence of a low concentration (50 nM) of 4Hfolate had reduced capacity to stabilize their network dynamics after a burst of high-frequency activity, and an increase in the frequency of mEPSCs. Networks reared in the presence of the folic acid metabolite 5M4Hfolate developed a spontaneous, distinctive bursting pattern, and both metabolites produced an increase in synaptic density.


Assuntos
Suplementos Nutricionais , Ácido Fólico/farmacologia , Convulsões/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Ácido Fólico/administração & dosagem , Idade Gestacional , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Gravidez , Ratos , Ratos Long-Evans , Sinaptofisina/metabolismo
8.
PLoS One ; 7(7): e41185, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815963

RESUMO

BACKGROUND: Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners. RESULTS: We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca(2+) influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca(2+)/CaM-dependent protein kinase IIα (CaMKII) a in Ca(2+)/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells. CONCLUSIONS: Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , DNA/metabolismo , Epitopos/química , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Plasticidade Neuronal , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo
9.
PLoS One ; 7(5): e37755, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22666389

RESUMO

Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aß(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aß(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aß(42) toxicity. In this study we show that neither extracellular Aß(42) nor Aß(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aß(42), Western analysis of Aß(42) was performed following external Aß(42) application. Five hours after a conditioning heat shock, Aß(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aß(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aß(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aß(42) indicating that Hsp40 modulation of Aß(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aß(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aß(42). Our data reveal a biochemical link between Hsp40 expression and Aß(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Bovinos , Linhagem Celular Tumoral , Demência/metabolismo , Demência/patologia , Regulação da Expressão Gênica , Hipocampo/citologia , Humanos , Cinética , Camundongos , Príons/metabolismo , Ratos
10.
Cell Calcium ; 47(5): 441-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20434769

RESUMO

Loss of the ability to regulate calcium is a central event leading to neuronal cell death during a wide range of pathological conditions including stroke and seizure. Here we present a new dissociated hippocampal cell culture model of acute electrical activity which incorporates the photoconductive stimulation of neuronal networks grown on silicon wafers. This technology allows precise modeling of user defined neuronal activity patterns, and the study of their effect on neuronal physiology. Here, seizure-like conditions were created by continuous stimulation, causing hundreds of neurons to fire synchronously at 50Hz for 4min. This stimulation protocol induced cell death as monitored by propidium iodide staining. The number of dead cells per stimulation region increased from 3.6+/-2.1 preceding stimulation to 81+/-21 30min following stimulation. Excitotoxicity primarily affected excitatory rather than inhibitory neurons, and was preceded by an increase in intracellular calcium as well as changes in the mitochondrial membrane potential, as measured by a tetramethylrhodamine methyl ester (TMRM) assay. Cyclosporin A (CsA), a mitochondrial permeability transition pore (PTP) blocker, was effective in preventing cell death. We propose that photoconductive stimulation is a useful tool for investigating the pathogenesis of excitotoxicity in vitro.


Assuntos
Cálcio/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Animais , Apoptose , Células Cultivadas , Ciclosporina/farmacologia , Luz , Neurônios/fisiologia , Neurônios/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismo , Fatores de Tempo
11.
Brain Behav Immun ; 24(1): 31-40, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19559784

RESUMO

Cognition and behavior primarily arise from the communication that occurs between brain cells. By using photoconductive stimulation to trigger localized regions of neuronal action potentials and astrocyte Ca(2+) waves in dissociated rat hippocampal cultures, we can directly study microglia behavior in response to physiological and pathological levels of activity. Connections between neurons can be modified by microglia, which regulate gap junctions and synapses through secretion of proteins such as cytokines, proteases and neurotrophic factors. Activated microglia participate in bidirectional communication with the excitable tissues that they support. Through feedback from the many ion channels and surface receptors they express, microglia are informed of neuronal and astrocytic activity that may indicate disruption in the homeostasis of the CNS. Such disturbances alert microglia to locations of such activity and promote their transformation into a reactive state, in which they perform adaptive functions that can be either neuroprotective, neurotoxic, or neuromodulatory. Under physiological conditions, normal brain activity has the effect of suppressing microglia inflammatory responses. This report summarizes available data about the interaction of microglia and brain activity and presents a new in vitro paradigm to study the mechanisms involved. We propose that photoconductive stimulation is a powerful tool for studying the cellular and molecular mechanisms underlying the dynamic interactions between neurons, astrocytes and microglia.


Assuntos
Microcomputadores , Microglia/fisiologia , Neurônios/fisiologia , Silício/química , Animais , Animais Recém-Nascidos , Astrócitos/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Quimiotaxia de Leucócito/fisiologia , Corantes , Imuno-Histoquímica , Estimulação Luminosa , Propídio , Ratos , Ratos Sprague-Dawley
12.
Eur J Neurosci ; 30(11): 2042-52, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-20128843

RESUMO

Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post-synaptic components. Here, we investigate the activity-driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post-synaptic proteins PSD-95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high-frequency activity altered the trajectory, but not velocity, of PSD-95-GFP and Shank-YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin-CFP reorganized into puncta following activity and approximately 50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1-R473C. These results demonstrate activity-dependent changes that may result in the formation of new post-synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity-dependent remodeling of excitatory synapses.


Assuntos
Actinas/metabolismo , Molécula de Adesão de Leucócito Ativado/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Actinas/genética , Molécula de Adesão de Leucócito Ativado/genética , Análise de Variância , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso , Estimulação Luminosa/métodos , Transporte Proteico/fisiologia , Transporte Proteico/efeitos da radiação , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção/métodos
13.
PLoS One ; 3(11): e3692, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19002247

RESUMO

Activity plays a critical role in network formation during developmental, experience-dependent, and injury related remodeling. Here we report a mechanism by which axon trajectory can be altered in response to remote neuronal activity. Using photoconductive stimulation to trigger high frequency action potentials in rat hippocampal neurons in vitro, we find that activity functions as an attractive cue for growth cones in the local environment. The underlying guidance mechanism involves astrocyte Ca(2+) waves, as the connexin-43 antagonist carbenoxolone abolishes the attraction when activity is initiated at a distance greater than 120 microm. The asymmetric growth cone filopodia extension that precedes turning can be blocked with CNQX (10 microM), but not with the ATP and adenosine receptor antagonists suramin (100 microM) and alloxazine (4 microM), suggesting non-NMDA glutamate receptors on the growth cone mediate the interaction with astrocytes. These results define a potential long-range signalling pathway for activity-dependent axon guidance in which growth cones turn towards directional, temporally coordinated astrocyte Ca(2+) waves that are triggered by neuronal activity. To assess the viability of the guidance effect in an injury paradigm, we performed the assay in the presence of conditioned media from lipopolysaccharide (LPS) activated purified microglial cultures, as well as directly activating the glia present in our co-cultures. Growth cone attraction was not inhibited under these conditions, suggesting this mechanism could be used to guide regeneration following axonal injury.


Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/citologia , Axônios/metabolismo , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/metabolismo , Hipocampo/citologia , Microglia/metabolismo , Estimulação Luminosa , Pseudópodes/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
14.
Neuron ; 58(5): 749-62, 2008 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-18549786

RESUMO

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.


Assuntos
Integrina beta3/fisiologia , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endocitose/efeitos dos fármacos , Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Integrina beta3/genética , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios , Técnicas de Patch-Clamp/métodos , Peptídeos/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Sinapses/efeitos dos fármacos , Tiazolidinas/farmacologia , Fatores de Tempo , Transfecção , Proteínas rap1 de Ligação ao GTP
15.
Mol Biol Cell ; 19(5): 2026-38, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18287537

RESUMO

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.


Assuntos
Espinhas Dendríticas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Pseudópodes/metabolismo , Processamento Alternativo/genética , Animais , Células COS , Membrana Celular/metabolismo , Lipoilação , Camundongos , Transporte Proteico , Ratos , Receptores de AMPA/metabolismo , Fatores de Tempo
16.
Proc Natl Acad Sci U S A ; 104(46): 18091-6, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17986607

RESUMO

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by beta-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Cálcio/metabolismo , Morte Celular , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Metabolismo Energético , Proteínas de Fluorescência Verde/metabolismo , Humanos , Transporte de Íons , Potenciais da Membrana , Mitocôndrias/enzimologia , NAD/metabolismo
17.
J Neurosci ; 26(49): 12807-15, 2006 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-17151284

RESUMO

Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retina/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/análise , Ativação Enzimática/fisiologia , Transferência Ressonante de Energia de Fluorescência , Neurônios/química , Neurônios/metabolismo , Ratos , Retina/química
18.
J Exp Biol ; 209(Pt 12): 2312-9, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16731807

RESUMO

Information processing in the central nervous system is primarily mediated through synaptic connections between neurons. This connectivity in turn defines how large ensembles of neurons may coordinate network output to execute complex sensory and motor functions including learning and memory. The synaptic connectivity between any given pair of neurons is not hard-wired; rather it exhibits a high degree of plasticity, which in turn forms the basis for learning and memory. While there has been extensive research to define the cellular and molecular basis of synaptic plasticity, at the level of either pairs of neurons or smaller networks, analysis of larger neuronal ensembles has proved technically challenging. The ability to monitor the activities of larger neuronal networks simultaneously and non-invasively is a necessary prerequisite to understanding how neuronal networks function at the systems level. Here we describe recent breakthroughs in the area of various bionic hybrids whereby neuronal networks have been successfully interfaced with silicon devices to monitor the output of synaptically connected neurons. These technologies hold tremendous potential for future research not only in the area of synaptic plasticity but also for the development of strategies that will enable implantation of electronic devices in live animals during various memory tasks.


Assuntos
Rede Nervosa/fisiologia , Plasticidade Neuronal , Neurônios/citologia , Neurônios/fisiologia , Silício/química , Sinapses/fisiologia , Animais , Humanos , Tecnologia
19.
Neuron ; 49(4): 547-62, 2006 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-16476664

RESUMO

Nonsynaptic clusters of postsynaptic proteins have been documented; however, their role remains elusive. We monitored the trafficking of several candidate proteins implicated in synaptogenesis, when nonsynaptic clusters of scaffold proteins are most abundant. We find a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. A proportion of mobile clusters that exhibits slow movement and travels short distances contains neuroligin-1. The second group consists of stationary nonsynaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Fosfoproteínas Fosfatases/fisiologia , Sinapses/fisiologia , Actinas/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Células Cultivadas , Diagnóstico por Imagem/métodos , Proteína 4 Homóloga a Disks-Large , Fosfatases de Especificidade Dupla , Embrião de Mamíferos , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Imuno-Histoquímica/métodos , Proteínas de Membrana/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Nocodazol/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Transporte Proteico/efeitos dos fármacos , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Fatores de Tempo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo
20.
Nat Protoc ; 1(1): 461-7, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17406269

RESUMO

Photoconductive stimulation allows the noninvasive depolarization of neurons cultured on a silicon wafer. This technique relies on a beam of light to target a cell of interest while applying a voltage bias across the silicon wafer. The targeted cell is excited with minimal physiological manipulation, and, therefore, long-term modulation of activity patterns and investigations of biochemical mechanisms sensitive to physiological perturbations are possible. Ideologically similar to transistor-based neuronal interfaces, the photoconductive-stimulation method has the advantage of being able to extracellularly excite any neuron in a network regardless of its spatial position on the silicon substrate. This protocol can be easily implemented on a conventional reflected-light fluorescence microscope using materials and resources that are readily available. Time requirements are comparable to standard cell-culture and electrophysiology techniques. When combined with fluorescence imaging of various molecular probes, activity-dependent cellular processes can be dynamically monitored.


Assuntos
Técnicas Citológicas/métodos , Estimulação Elétrica/métodos , Luz , Neurônios/fisiologia , Silício/química , Células Cultivadas , Condutividade Elétrica
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