RESUMO
BACKGROUND INFORMATION: During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. RESULTS: We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1-4 (day in vitro 1-4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14-23). CONCLUSION: These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Munc18/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/fisiologia , Animais , Cones de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Transgênicos , Proteínas Munc18/deficiência , Proteínas do Tecido Nervoso/deficiência , Pseudópodes/metabolismoRESUMO
Dendritic spines are major sites of morphological plasticity in the CNS, but the molecular mechanisms that regulate their dynamics remain poorly understood. Here we show that the association of drebrin with actin filaments plays a major role in regulating dendritic spine stability and plasticity. Overexpressing drebrin or the internal actin-binding site of drebrin in rat hippocampal neurons destabilized mature dendritic spines so that they lost synaptic contacts and came to resemble immature dendritic filopodia. Drebrin-induced spine destabilization was dependent on Ras activation: expression of constitutively active Ras destabilized spine morphology whereas drebrin-induced spine destabilization was rescued by co-expressing dominant negative Ras. Conversely, RNAi-mediated drebrin knockdown prevented Ras-induced destabilization and promoted spine maturation in developing neurons. Together these data demonstrate a novel mechanism in which the balance between stability and plasticity in dendritic spines depends on binding of drebrin to actin filaments in a manner that is regulated by Ras.
Assuntos
Dendritos/metabolismo , Hipocampo/embriologia , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Neuropeptídeos/metabolismo , Proteínas ras/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Diferenciação Celular/genética , Dendritos/ultraestrutura , Hipocampo/ultraestrutura , Neuropeptídeos/genética , Ligação Proteica/fisiologia , Interferência de RNA , Ratos , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
INTRODUCTIONThis protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. One to two percent transfected cells is a typical result. A disadvantage of the calcium phosphate method is that hippocampal neurons become "fragile" after treatment.
RESUMO
The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.
Assuntos
Núcleo Celular/metabolismo , Neurônios/metabolismo , Profilinas/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Cálcio/deficiência , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/metabolismo , Espinhas Dendríticas/metabolismo , Glicina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Magnésio/farmacologia , Camundongos , Camundongos Transgênicos , Mutação/genética , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Profilinas/genética , Ligação Proteica , Transporte Proteico , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.
Assuntos
Espinhas Dendríticas , Microscopia Confocal/métodos , Técnicas de Cultura de Tecidos , Espinhas Dendríticas/ultraestrutura , Eletrofisiologia , Hipocampo/ultraestrutura , Fatores de TempoRESUMO
Synapses in the brain must maintain a balance between learning-related plasticity and the stability necessary for reliable function. In this issue of Neuron, Calabrese and Halpain describe cell-transfection experiments implicating MARCKS, a protein that binds to both the cell surface and actin cytoskeleton, in the maintenance of dendritic spines.
Assuntos
Espinhas Dendríticas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Animais , Tamanho Celular , Proteínas de Fluorescência Verde/biossíntese , Substrato Quinase C Rico em Alanina Miristoilada , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologiaRESUMO
Brain hypothermia is at present the most effective neuroprotective treatment against brain ischemia in man. Ischemia induces a redistribution of proteins involved in synaptic functions, which is markedly diminished by therapeutic hypothermia (33 degrees C). Dendritic spines at excitatory synapses are motile and show both shape changes and rearrangement of synaptic proteins as a consequence of neuronal activity. We investigated the effect of reduced temperature (33 degrees C and 27 degrees C compared with 37 degrees C), on spine motility, length and morphology by studying the distribution of GFP-actin before, during and after induction of in vitro ischemia. Because high-concentration actin filaments are located inside spines, dissociated hippocampal neurons (7-11 DIV) from transgenic mice expressing GFP-actin were used in this study. The movement of the spines and the distribution of GFP-actin were recorded using time-lapse fluorescence microscopy. Under normal conditions rapid rearrangement of GFP-actin was seen in dendritic spines, indicating highly motile spines at 37 degrees C. Decreasing the incubation temperature to 33 degrees C or 27 degrees C, dramatically reduces actin dynamics (spine motility) by approximately 50% and 70%, respectively. In addition, the length of the spine shaft was reduced by 20%. We propose that decreasing the temperature from 37 degrees C to 33 degrees C during ischemia decreases the neuronal actin polymerization rate, which reduces spine calcium kinetics, disrupts detrimental cell signaling and protects neurons against damage.
Assuntos
Actinas/metabolismo , Isquemia Encefálica/patologia , Espinhas Dendríticas/patologia , Hipotermia , Animais , Sobrevivência Celular , Células Cultivadas , Proteínas de Fluorescência Verde , Hipocampo/citologia , Cinética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , TemperaturaRESUMO
Most excitatory synapses in the brain are made on spines, small protrusions from dendrites that exist in many different shapes and sizes. Spines are highly motile, a process that reflects rapid rearrangements of the actin cytoskeleton inside the spine, and can also change shape and size over longer timescales. These different forms of morphological plasticity are regulated in an activity-dependent way, involving calcium influx through glutamate receptors and voltage-gated calcium channels. Many proteins regulating the turnover of filamentous actin (F-actin) are calcium-dependent and might transduce intracellular calcium levels into spine shape changes. On the other hand, the morphology of a spine might affect the function of the synapse residing on it. In particular, the induction of synaptic plasticity is known to require large elevations in the postsynaptic calcium concentration, which depend on the ability of the spine to compartmentalize calcium. Since the actin cytoskeleton is also known to anchor postsynaptic glutamate receptors, changes in the actin polymerization state have the potential to influence synaptic function in a number of ways. Here we review the most prominent types of changes in spine morphology in hippocampal pyramidal cells with regard to their calcium-dependence and discuss their potential impact on synaptic function.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Espinhas Dendríticas/fisiologia , Animais , Canais de Cálcio/metabolismo , HumanosRESUMO
Dendritic spines, which are present at the vast majority of excitatory synapses in the central nervous system, have a specialized cytoskeleton of dynamic actin filaments that makes them capable of rapid morphological plasticity. During development, structural remodeling of nascent spines is an important factor in experience-dependent shaping of neuronal circuits, whereas in the adult brain spines maintain a balance between morphological stability and plasticity.
Assuntos
Diferenciação Celular/fisiologia , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Animais , HumanosRESUMO
Dendritic spines contain a specialized cytoskeleton composed of dynamic actin filaments capable of producing rapid changes in their motility and morphology. Transient changes in Ca2+ levels in the spine cytoplasm have been associated with the modulation of these effects in a variety of ways. To characterize the contribution of Ca2+ fluxes originating through different pathways to these phenomena, we used time-lapse imaging of cultured hippocampal neurons expressing GFP-actin to follow the influence of postsynaptic neurotransmitter receptors, voltage-activated Ca2+ channels and release from internal Ca2+ stores on spine actin dynamics. Stimulation of AMPA receptors produced a rapid blockade of actin-dependent spine motility that was immediately reversible when AMPA was removed. Stimulation of NMDA receptors also blocked spine motility but in this case suppression of actin dynamics was delayed by up to 30 min depending on NMDA concentration and motility was never seen to recover when NMDA was removed. These effects could be mimicked by depolarizing neurons under appropriate circumstances demonstrating the involvement of voltage-activated Ca2+ channels in AMPA receptor-mediated effects and the receptor associated Ca2+ channel in the effects of NMDA. Caffeine, an agent that releases Ca2+ from internal stores, had no immediate effect on spine actin, a result compatible with the lack of caffeine-releasable Ca2+ in cultured hippocampal neurons under resting conditions. Blocking internal store function by thapsigargin led to a delayed suppression of spine actin dynamics that was dependent on extracellular Ca2+. Together these results indicate the common involvement of changes in Ca2+ levels in modulating actin-dependent effects on dendritic spine motility and morphology through several modes of electrophysiological activation.
Assuntos
Actinas/fisiologia , Cálcio/farmacologia , Dendritos/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/fisiologia , Animais , Cafeína/farmacologia , Células Cultivadas , Dendritos/efeitos dos fármacos , N-Metilaspartato/farmacologia , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Mechanisms of synaptic plasticity in CNS circuits are commonly investigated using in vitro preparations such as brain slices or slice culture. During their preparation, slices are exposed to low temperatures, and electrophysiological measurements are sometimes made below physiological temperature. Because dendritic spines, which occur at the majority of excitatory synapses, are morphologically plastic, we investigated the influence of reduced temperature on their morphology and plasticity using live cell imaging of hippocampal slices from transgenic mice expressing a green fluorescent protein-based neuronal surface marker and electron microscopy of adult brain slices. Our data show that dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed at longer times by reversible loss of the entire spine structure. Thus, reduced temperature significantly affects synaptic morphology, which is in turn known to influence several key aspects of synaptic transmission. Evidence that hypothermia potentiates anesthesia and is associated with spine loss in hibernating animals further suggests that spine morphology may have a widespread influence on brain function.
Assuntos
Artefatos , Temperatura Baixa , Dendritos/ultraestrutura , Hipocampo/ultraestrutura , Manejo de Espécimes/métodos , Vesículas Sinápticas/ultraestrutura , Animais , Genes Reporter , Proteínas de Fluorescência Verde/análise , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Proteínas do Tecido Nervoso/análise , Técnicas de Cultura de Órgãos , Fatores de TempoRESUMO
The stability of neuronal networks is thought to depend on synaptic transmission which provides activity-dependent maintenance signals for both synapses and neurons. Here, we tested the relationship between presynaptic secretion and neuronal maintenance using munc18-1-null mutant mice as a model. These mutants have a specific defect in secretion from synaptic and large dense-cored vesicles [Verhage et al. (2000), Science, 287, 864-869; Voets et al. (2001), Neuron, 31, 581-591]. Neuronal networks in these mutants develop normally up to synapse formation but eventually degenerate. The proposed relationship between secretion and neuronal maintenance was tested in low-density and organotypic cultures and, in vivo, by conditional cell-specific inactivation of the munc18-1 gene. Dissociated munc18-1-deficient neurons died within 4 days in vitro (DIV). Application of trophic factors, insulin or BDNF delayed degeneration up to 7 DIV. In organotypic cultures, munc18-1-deficient neurons survived until 9 DIV. On glial feeders, these neurons survived up to 10 DIV and 14 DIV when insulin was applied. Co-culturing dissociated mutant neurons with wild-type neurons did not prolong survival beyond 4 DIV, but coculturing mutant slices with wild-type slices prolonged survival up to 19 DIV. Cell-specific deletion of munc18-1 expression in cerebellar Purkinje cells in vivo resulted in the specific loss of these neurons without affecting connected or surrounding neurons. Together, these data allow three conclusions. First, the lack of synaptic activity cannot explain the degeneration in munc18-1-null mutants. Second, trophic support delays but cannot prevent degeneration. Third, a cell-intrinsic yet unknown function of munc18-1 is essential for prolonged survival.
Assuntos
Hipocampo/patologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , Sinapses/metabolismo , Proteínas de Transporte Vesicular/deficiência , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Calbindinas , Sobrevivência Celular/genética , Células Cultivadas , Técnicas de Cocultura/métodos , Estimulação Elétrica/métodos , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Hipocampo/fisiopatologia , Imuno-Histoquímica/métodos , Insulina/uso terapêutico , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Munc18 , Degeneração Neural/tratamento farmacológico , Degeneração Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurotransmissores/genética , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp/métodos , Fenotiazinas , Proteínas Qa-SNARE , Proteína G de Ligação ao Cálcio S100/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/genética , Fatores de Tempo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiologiaRESUMO
Morphological changes in dendritic spines have been implicated in connective plasticity in brain circuitry, but the underlying pathway leading from synaptic transmission to structural change is unknown. Using primary neurons expressing GFP-tagged proteins, we found that profilin, a regulator of actin polymerization, is targeted to spine heads when postsynaptic NMDA receptors are activated and that actin-based changes in spine shape are concomitantly blocked. Profilin targeting was triggered by electrical stimulation patterns known to induce the long-term changes in synaptic responsiveness associated with memory formation. These results suggest that, in addition to electrophysiological changes, NMDA receptor activation initiates changes in the actin cytoskeleton of dendritic spines that stabilize synaptic structure.
Assuntos
Proteínas Contráteis , Dendritos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios/citologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Toxinas Botulínicas Tipo A/farmacologia , Cálcio/metabolismo , Contagem de Células , Células Cultivadas , Galinhas , Dendritos/fisiologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Luminescentes/metabolismo , Magnésio/farmacologia , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Fármacos Neuromusculares/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Cloreto de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Profilinas , Receptores de Glutamato/classificação , Receptores de Glutamato/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The adenomatous polyposis coli (APC) tumour suppressor protein is a component of the Wnt signalling pathway in which it plays a major role in controlling nuclear accumulation of beta-catenin and hence in the modulation of beta-catenin-regulated gene transcription. APC also associates with microtubules at the ends of cytoplasmic extensions in epithelial cells, a distribution that can be reproduced in COS cells ectopically expressing APC. To examine the effect of APC on microtubule properties, we monitored directly the behaviour of APC and of APC-decorated microtubules by time-lapse imaging of cytoplasmic extensions in live COS cells expressing APC tagged with a green fluorescent protein. On the proximal part of microtubules, APC was visualised as particulate material moving unidirectionally towards the plus end of microtubules. The distal parts of microtubules were uniformly decorated by APC and were animated by a motile behaviour in the form of aperiodic bending. This behaviour is likely to be the consequence of compression forces acting on microtubules encountering obstacles while elongating. The majority of APC-decorated microtubules in transfected COS cells was sensitive to depolymerisation by nocodazole, but they contained detyrosinated and acetylated alpha-tubulin, suggesting a reduction in the rate of subunit exchange at their growing end. Taken together, these results demonstrate that microtubule domains uniformly decorated by APC display dynamic and motile properties that may be significant for the postulated role of APC in targeting microtubules to specialised membrane sites.