Este trabalho foi financiado pelo Instituto Nacional de Surdez e Outros Distúrbios de Comunicação (NIDCD) Programa intramuros.

1. Transfecção neurônio <ol><li> Cultura embrionária neurônios dia 18 (E18) de ratos do hipocampo em poli-d-lisina revestido Mattek de 35 mm com fundo de vidro pratos 1. Em 16-18 dias in vitro (DIV), os neurônios transfecção usando o Clontech CalPhos Kit Transfection mamíferos. Primeiro, substituir o meio de cultura com 1,5 ml <a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/Classical_Media/dmem.html">Modified Dulbecco Eagle Medium</a> (DMEM) por 35 mm prato 0,5 horas antes da transfecção. Salvar o meio de cultura original em um tubo estéril 15 ml para uso posterior (passo 1.6). </li><li> Misturar 10 mg DNA pEGFP-N1 do plasmídeo com estéril H 2 O (Clontech) e 12,4 mL solução de cálcio 2 M (Clontech) para um volume total de 100 l. </li><li> Adicione a mistura a partir do passo 1.2) para 100 l 2 × HBS gota a gota enquanto vórtex 2 × HBS em velocidade média. </li><li> Deixe a mistura descansar em temperatura ambiente por 20 minutos e depois adicione a mistura final a partir do passo 1.3) em DMEM-incubadas neurônios. </li><li> Coloque os neurônios de volta para a 37 ° C incubadora de 1-1,5 horas. </li><li> Remover o fosfato de cálcio contendo meio, em seguida, lavar as células com DMEM três vezes. Antes de devolver o prato de cultura para a incubadora, troca o meio DMEM com o meio de cultura original. </li></ol> 2. FRAP experimento em uma coluna <ol><li> Neurônios são usados ​​para o experimento FRAP 2-4 dias após transfecção. </li><li> Substituir o meio de cultura a partir do 35-mm prato fundo de vidro, por acrescentando imediatamente pré-aquecido solução Tyrode, que contém (em mM) NaCl 145, KCl 5, HEPES 10, Glucose 10, Glycine 0,005, CaCl 2 2,6, e MgCl 2 1.3 (pH ajustado para 7,4 com NaOH). </li><li> A Zeiss LSM 710 microscópio confocal é usado para o experimento FRAP. O sistema TempModule Zeiss é usado para controlar a temperatura (37 ° C), a umidade e as emissões de CO 2 (5%) do sistema de trabalho. Certifique-se que o CO 2 tanque está conectado ea garrafa de água, que é usado para balanceamento de umidade, é preenchido com água. </li><li> Encontrar uma dendrite transfectadas maduros com o objetivo × 100 (αplan Apochromat-100 × / 1,46 óleo). Se as células transfectadas no prato são pesquisa, escasso para uma célula desejada com o 40 × objetivo (plano-Neofluar 40 × / 1.3 de petróleo), e depois mudar para o 100 × objetivo de capturar imagens. </li><li> Use 5 × zoom óptico e uma resolução de 256 × 256 pixels para a imagem de um pequeno pedaço de dendrite com espinhas várias. Para capturar as imagens, use a velocidade nominal 9 (pixel tempo de permanência 3,15 ms), que leva 0,5 segundo para terminar um scan. O pinhole é definida como 2μm para a obtenção de fluorescência forte. Ao tirar as imagens, tente usar laser de baixa transmissão, por exemplo 1-5%, para evitar a fotodegradação toda a imagem. </li><li> Selecione a coluna de interesse. Em nosso experimento, escolhemos espinhas cogumelo com seus diâmetros coluna cabeça de ~ 1 mícron. </li><li> Para fazer um experimento FRAP, tomar 5 imagens de controle antes de branqueamento, em seguida, água sanitária a espinha de juros de 10 vezes na transmissão do laser nominal de 100% [A potência do laser de argônio é de 30 mW, com 50% (15 mW) na linha de laser de 488, e mW aproximadamente 3-4 chega ao microscópio através da linha de laser de 488], e então capturar uma série de imagens imediatamente após o clareamento. Para este experimento, as imagens são capturadas a cada 1 segundo durante 15 segundos após o clareamento. O intervalo de tempo deve ser ajustado de acordo com diferentes proteínas alvo e diferentes desenhos experimentais (Fig. 1). </li><li> Guardar as imagens. </li></ol> 3. Análise de dados <ol><li> Abrir imagens com ImageJ software. </li><li> Alinhe a pilha de imagens utilizando a ferramenta align (&quot;pilhas - embaralhando &#39;plugins&#39; → →&quot; align fatias na pilha de &#39;corpo rígido &quot;&quot; tradução &quot;e / ou →) no ImageJ, de modo que a coluna de interesse não flutua , em outras palavras - ele permanece na mesma posição na imagem. </li><li> Medir a intensidade de fluorescência relativa da coluna de interesse (F s), uma região transfectadas mas crus (controle), e uma região de fundo (F b) em imagens de lapso de tempo, usando a ferramenta &#39;Intensidade v Time Monitor &quot;do ImageJ (&quot; plugins &#39;→&#39; stacks - embaralhando &#39;→&#39; Intensidade Tempo v Monitor &#39;). A região de controle poderia ser um pedaço de bem focada dendrite distal. A região de fundo é uma região não-fluorescente. </li><li> Calcular a taxa de fotodegradação (r), comparando a fluorescência da região controle antes (F c0) e depois (F c) fotobranqueamento. r = F c / F c0. </li><li> Normalizar a intensidade de fluorescência da coluna de destino (F) como segue: F = (F s - F b) / r. </li><li> A curva é adequada a intensidade de fluorescência da coluna de destino com uma equação de uma fase exponencial de Graphpad software Prism (Fig. 2) ou outrr software similar. </li><li> Calcular a fração móvel (f m) e da fração de imóvel (f i) pelas seguintes equações: f m = ∞ F / F 0, onde F ∞ é a intensidade de fluorescência após a recuperação completa, e F 0 é a intensidade de fluorescência antes de fotodegradação. f i = 1 - f m. </li></ol> 4. Resultados representativos: Neste estudo, realizamos um experimento sobre FRAP madura neurônios do hipocampo. Em 18-22 DIV, espinhas de cogumelos já estão formados. Utilizando nosso método, as mudanças dinâmicas da intensidade de fluorescência em uma região pequena, como uma coluna, pode ser gravado. Para analisar o processo de recuperação de fluorescência de EGFP, tomamos 5 imagens como controles antes de branqueamento e, em seguida, uma imagem a cada 1 segundo imediatamente após o clareamento por 15 segundos. A resolução da imagem é suficiente para a análise quantitativa. Os perfis de recuperação de fluorescência de proteínas tag fluorescência são altamente reprodutíveis. Também mostrarei brevemente como para definir as frações móvel e imóvel de uma proteína de fluorescência, usando ImageJ e Graphpad software Prism. O método FRAP e análise mostramos aqui pode ser amplamente utilizado em neurociência, biologia celular e outros estudos. <img src="/files/ftp_upload/2568/2568fig1.jpg" alt="Figura 1" /> Figura 1. Medições FRAP de EGFP fluorescência em uma coluna de um neurônio culta do hipocampo. Das setas vermelhas indicam o tempo de fotodegradação. Fotografias representam a mesma área antes (Pré) e em 0, 1, 5, 10, 15 segundos após a fotodegradação. A região da coluna, controle e de fundo são marcadas com as letras S, C e B, respectivamente. Neurônios foram mantidos a 37 ° C durante o experimento. Escala de bar, 1 mícron. <img src="/files/ftp_upload/2568/2568fig2.jpg" alt="Figura 2" /> Figura 2. Curvas FRAP de EGFP de fluorescência ao longo de um período de 15 segundos. A linha verde mostra a curva original; a linha vermelha mostra a curva normalizada. Os pontos em curvas mostram a FRAP a cada 1 segundo. As curvas foram ajustadas por uma fase equações exponenciais. A fluorescência média antes fotobranqueamento foi contado como 100%. Neste experimento, a fração móvel (MF) é de 94% e da fração de imóvel (IF) é de 6%.

<ol>
	<li>Zheng, C. Y., Petralia, R. S., Wang, Y. X., Kachar, B., Wenthold, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAP102+is+a+highly+mobile+MAGUK+in+spines.">SAP102 is a highly mobile MAGUK in spines.</a> J Neurosci. 30, 4757-4766 (2010).</li><li>Grati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+turnover+of+stereocilia+membrane+proteins:+evidence+from+the+trafficking+and+mobility+of+plasma+membrane+Ca(2+)-ATPase+2.">Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.</a> J Neurosci. 26, 6386-6395 (2006).</li><li>Liu, L. N., Aartsma, T. J., Thomas, J. C., Zhou, B. C., Zhang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+on+Red+Alga+Reveals+the+Fluorescence+Recovery+Is+Ascribed+to+Intrinsic+Photoprocesses+of+Phycobilisomes+than+Large-Scale+Diffusion.">FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion.</a> PLoS ONE. 4, e5295-e5295 (2009).</li><li>Wiedenmann, J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+with+EosFP+and+other+photoactivatable+marker+proteins+of+the+GFP+family.">Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.</a> Expert Rev Proteomics. 3, 361-374 (2006).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Sprague, B. L., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+binding:+proper+and+fitting.">FRAP analysis of binding: proper and fitting.</a> Trends Cell Biol. 15, 84-91 (2005).</li></ol>

Não há conflitos de interesse declarados.

FRAP análise tem sido amplamente utilizada in vivo e in vitro estudos 1-2. Esta técnica utiliza comumente GFP <a href="http://en.wikipedia.org/wiki/Fusion_protein" title="Proteína de fusão">proteínas de fusão</a> , embora possa também usar as proteínas de fusão alga vermelha 3. Esta análise é sensível e pode ser usado para caracterizar a mobilidade das proteínas GFP. Para produzir análises FRAP significativa, é importante para evitar a fotodegradação desnecessários antes e durante o experimento FRAP. Há duas maneiras de conseguir isso. Primeiro, o processo para pesquisar e observar o neurônio experimental deve ser rápido. Especialmente, a observação de neurônios com um objetivo 100X por um longo tempo significativamente alvejantes a fluorescência. Segundo, potência do laser e digitalização de alta freqüência, muitas vezes aumentar a possibilidade de fotodegradação. Assim, será necessário calcular a taxa de fotodegradação em uma região de controle e depois normalizar a curva FRAP na região experimental. O método de normalização tem sido descrita no protocolo (veja o passo 3,3-3,5 para detalhes). O passo fotobranqueamento também é crítico para assegurar resultados FRAP bom. Neste experimento, lixívia que a espinha de juros de 10 a 100 vezes a transmissão a laser%. Esta condição é suficiente para branquear a fluorescência de uma coluna para o nível de fundo em uma preparação fixo. Assim, vamos definir a intensidade de fluorescência para o mesmo número como pano de fundo em 0 segundos após a fotodegradação. Dependendo da velocidade da primeira varredura, uma quantidade significativa de recuperação de fluorescência já podem ser detectados quando a proteína de interesse é altamente móvel. Muitas sondas proteína fluorescente têm sido desenvolvidos para estudar a dinâmica de proteínas com FRAP. Uma abordagem complementar é, por exemplo, para usar photoactivatable GFP (PA-GFP), ou variantes photoconvertible. Juntamente com a técnica FRAP, essas ferramentas estão se tornando indispensável para estudo de células vivas de imagem 4-6.

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<td>35 mm glass-bottom dishes </td>
<td>&nbsp;</td>
<td>MatTek</td>
<td>P35GC-1.0-14-C</td>
<td>&nbsp;</td>
<tr>
<td>CalPhos Mammalian Transfection Kit</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>631312</td>
<td>&nbsp;</td>
<tr>
<td>pEGFP-N1 plasmid</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>6085-1</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss confocal microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>LSM 710</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss TempModule system</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>N.A.</td>
<td>&nbsp;</td>
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<td>Graphpad Prism software</td>
<td>&nbsp;</td>
<td>Graphpad software</td>
<td>N.A.</td>
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fluorescence recovery after photobleaching (frap) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons

FRAP tem sido utilizada para quantificar a mobilidade das proteínas GFP. Usando um laser de excitação forte, a fluorescência de uma proteína GFP-tagged é branqueada na região de interesse. A fluorescência da região se recupera quando a proteína GFP-tagged crus de fora da região difunde para a região de interesse. A mobilidade da proteína é então analisada através da medição da taxa de recuperação de fluorescência. Esta técnica poderia ser usada para caracterizar a mobilidade de proteína e taxa de rotatividade. Neste estudo, nós expressamos o (proteína verde fluorescente melhorada) vector EGFP em culturas de neurônios do hipocampo. Usando o microscópio confocal Zeiss 710, nós photobleach o sinal de fluorescência da proteína GFP em uma coluna única, e depois tirar fotos lapso de tempo para registrar a recuperação de fluorescência após a fotodegradação. Finalmente, estimamos a porcentagem de frações móvel e imóvel da GFP em espinhas, analisando os dados de imagem usando ImageJ e softwares Graphpad. Este protocolo FRAP mostra como executar um experimento FRAP básica, bem como a forma de analisar os dados.

Watch this Scientific Journal Video about Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons at JoVE.com

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP tem sido utilizada para quantificar a mobilidade de proteína de fluorescência verde (GFP) marcadas proteínas em células em cultura. Examinamos as frações móveis / imóveis da GFP, analisando o percentual de recuperação de fluorescência após a fotodegradação. Neste estudo, foi realizada na FRAP espinhas de neurônios do hipocampo.

Recuperação de fluorescência Após Fotodegradação (FRAP) de fluorescência Proteínas Tagged em espinhas dendríticas dos neurônios do hipocampo Cultivadas

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

fluorescence-recovery-after-photobleaching-frap-fluorescence-tagged

Research

JoVE Journal

Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

54.1K visualizações.  National Institutes of Health, Bethesda. FRAP tem sido utilizada para quantificar a mobilidade de proteína de fluorescência verde (GFP) marcadas proteínas em células em cultura. Examinamos as frações móveis / imóveis da GFP, analisando o percentual de recuperação de fluorescência após a fotodegradação. Neste estudo, foi realizada na FRAP espinhas de neurônios do hipocampo.

Vídeo: Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Análise de Morfologia Spine dendríticas em neurônios cultivados CNS

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Neurociência

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induzir o crescimento dendrítico no cultivadas neurônios simpáticos

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Tensão sensível Gravação Dye de axônios, dendritos e espinhas dendríticas de neurônios individuais em fatias de cérebro

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Detecção de palmitoylation proteína em neurônios cultivados por imunoprecipitação e Acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Rotulagem diferencial de superfície celular e internalizado Proteínas após Anticorpo Alimentação da Vivo Cultivadas neurônios

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagiologia espinhas dendríticas dos neurônios do hipocampo de rato primária usando Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescência e imagem de bioluminescência subcelular Ca<sup&gt; 2+</sup&gt; Aged em neurônios do hipocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.