Gostaríamos de agradecer Donald Hutson para contribuir seus conhecimentos técnicos e muita habilidade durante o projeto e fabricação da incubadora em estágio superior. Estamos também gratos a Ayda Dashtaei por sua excelente assistência técnica. Todo o trabalho foi apoiado pela Fundação de Pesquisa Neurociências.

<ol>
	<li>Ligon, L. A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+mitochondria+in+the+axons+and+dendrites+of+cultured+hippocampal+neurons.">Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.</a> J Comp Neurol. 427, 340-350 (2000).</li><li>Miller, K. E., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+coherent+low+velocity+axonal+transport+of+mitochondria.">Direct evidence for coherent low velocity axonal transport of mitochondria.</a> J Cell Biol. 173, 373-381 (2006).</li><li>Macaskill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miro1+is+a+calcium+sensor+for+glutamate+receptor-dependent+localization+of+mitochondria+at+synapses.">Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.</a> Neuron. 61, 541-555 (2009).</li><li>Morris, R. L., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+mitochondria+along+microtubules+and+F-actin+in+living+vertebrate+neurons.">Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.</a> J Cell Biol. 131, 1315-1326 (1995).</li><li>Rintoul, G. L., Filiano, A. J., Brocard, J. B., Kress, G. J., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+decreases+mitochondrial+size+and+movement+in+primary+forebrain+neurons.">Glutamate decreases mitochondrial size and movement in primary forebrain neurons.</a> J Neurosci. 23, 7881-7888 (2003).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in neuroscience. , (1999).</li><li>Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for neural cell culture. , (2001).</li><li>Chen, S., Owens, G. C., Crossin, K. L., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+stimulates+mitochondrial+transport+in+hippocampal+neurons.">Serotonin stimulates mitochondrial transport in hippocampal neurons.</a> Mol Cell Neurosci. 36, 472-483 (2007).</li><li>Chen, S., Owens, G. C., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+inhibits+mitochondrial+motility+in+hippocampal+neurons.">Dopamine inhibits mitochondrial motility in hippocampal neurons.</a> PLoS One. 3, e2804-e2804 (2008).</li><li>Chen, S., Owens, G. C., Makarenkova, H., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+regulates+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 regulates mitochondrial transport in hippocampal neurons.</a> PLoS One. , e10848-e10848 (2010).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol Ther. 1, 31-38 (2000).</li></ol>

Não há conflitos de interesse declarados.

Empregando lentivírus mediada expressão de uma proteína fluorescente alvejado a mitocôndria em neurônios cultivados infectados e uma incubadora barata laboratório construído estágio-top que permite imagens de células vivas por longos períodos, temos sido capazes de investigar a ligação entre o movimento mitocondrial e os sinais de neuromoduladores , como a serotonina (5-HT), dopamina (DA) e acetilcolina (ACh). Nossos estudos têm ajudado a elucidar uma via de sinalização que, pela primeira vez, links tráfico mitocondrial para mudanças na atividade dos neurônios-modulado por neurotransmissores como a 5-HT e DA - que são o cerne da função neural. Nós achamos que o uso de proteínas fluorescentes alvo permite a observação de mitocôndrias rotulados na vida neurônios cultivados por longos períodos que podem ser mais fisiologicamente relevante do que a duração muito mais curta que são possíveis usando corantes vitais. Além disso, a intensidade do sinal de uma proteína fluorescente nos permite manter os tempos de exposição curtos durante a aquisição de imagem, minimizando a possibilidade de fotodegradação ou fototoxicidade. Finalmente, um estágio-top simples e barato incubadora que mantém a temperatura ambiente, umidade e níveis de CO 2, enquanto minimiza a evaporação da mídia, nos permite acompanhar o movimento mitocondrial em neurônios vivos por horas ou mesmo dias. Pesquisadores que desejam fabricar uma incubadora de estágio-top para as observações a longo prazo de mitocôndrias nos neurônios viver não precisa seguir os detalhes precisos do nosso projeto, desde que as propriedades dos materiais utilizados (por exemplo, o gás membrana permeável para evitar a evaporação da mídia ) e os princípios aplicados (por exemplo, controle de temperatura e umidade, o buffer de manutenção do pH, da osmolaridade) são geralmente consistentes com o que é descrito neste protocolo.

<table border="1"><tbody><tr><th> Quantidade </th><th> Descrição (e localização na Figura 1) </th></tr><tr><td> 1 </td><td> Cultura de tecidos incubadora (H) </td></tr><tr><td> 1 </td><td> Resistente ao calor gabinete plástico (delrin ou comparáveis) (A) </td></tr><tr><td> 1 </td><td> Armação de plástico para 35mm tampa GBM (para a aposição de membrana para placa de Petri) (M) </td></tr><tr><td> 02/01 </td><td> linear pé Limpar PFTE material da membrana (Teflon) </td></tr><tr><td> 4 </td><td> Polegar parafusos de latão </td></tr><tr><td> 2 </td><td> Pequena 10kOhm resistores dissipador de calor (usados ​​como elementos de aquecimento no interior estágio-top gabinete incubadora) </td></tr><tr><td> 1 </td><td> Transformador (fonte de corrente 9V para resistores) </td></tr><tr><td> 1 </td><td> Terrário controlador de temperatura e sonda (regulação termostática de poder para resistores dissipador de calor através de transformador) </td></tr><tr><td> 1 </td><td> 2000ml Erlenmeyer (silenciador para a supressão de vibração na mangueira antes da fase-top incubadora) (K) </td></tr><tr><td> 1 </td><td> 1 / 8 &quot;folha Sorbothane (junta material para base de estágio-top incubadora) </td></tr><tr><td> 1 </td><td> Hermético ABS (ou comparável) caixa de plástico (carcaça para a bomba de aquário) (J) </td></tr><tr><td> 1 </td><td> Aquário da bomba (I) </td></tr><tr><td> 1 </td><td> Ventilador pequeno computador </td></tr><tr><td> 1 </td><td> Invólucro de plástico para ventilador de arrefecimento (L) </td></tr><tr><td> 1 </td><td> 9V transformador para computador ventoinha de refrigeração </td></tr><tr><td> 20-30 pés </td><td> Nalgene ou silicone mangueira (conexões entre cultura de tecidos e-top estágio incubadoras; armadilhas umidade) (F, G, N) </td></tr><tr><td> 2 </td><td> Torneiras de plástico (para abrir e fechar o fluxo de ar antes e depois do estágio-top incubadora) (O) </td></tr><tr><td> 4 </td><td> Conectores de bronze farpado mangueira (mangueira para conexões de / para o estágio-top incubadora e gabinete de bomba de aquário) </td></tr><tr><td> 2 </td><td> Acopladores de bronze rápida (para conexões de mangueira de / para o estágio-top incubadora) </td></tr><tr><td> 2 </td><td> Acopladores de bronze rápida (para conexões de mangueira de / para o estágio-top incubadora) </td></tr></tbody></table> Tabela 1. Stage-top incubadora feita com partes: <table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Poli-D-lisina </td><td> Sigma-Aldrich </td><td> P7280-5mg </td></tr><tr><td> laminina </td><td> Roche Applied Science </td><td> 11243217001 </td></tr><tr><td> 35 milímetros com fundo de vidro pratos </td><td> Mattek </td><td> P35GC-0-14-C </td></tr><tr><td> DMEM </td><td> Life Technologies </td><td> 10567 </td></tr><tr><td> B27 </td><td> Life Technologies </td><td> 17504-044 </td></tr><tr><td> Glutamax </td><td> Life Technologies </td><td> 35050 </td></tr><tr><td> Rica em lipídios BSA </td><td> Life Technologies </td><td> 11020-021 </td></tr><tr><td> L-Asparagina </td><td> Sigma-Aldrich </td><td> P0380-100G </td></tr><tr><td> L-Proline </td><td> Sigma-Aldrich </td><td> A8381-100G </td></tr><tr><td> A vitamina B-12 </td><td> Sigma-Aldrich </td><td> V2876-100mg </td></tr><tr><td> 5-HT </td><td> Sigma-Aldrich </td><td> H9523-25mg </td></tr><tr><td> 8-OH-DPAT </td><td> Sigma-Aldrich </td><td> H8520-25mg </td></tr><tr><td> Dopamina </td><td> Sigma-Aldrich </td><td> H8502-5G </td></tr><tr><td> Bromocriptina </td><td> Sigma-Aldrich </td><td> B2134-25mg </td></tr><tr><td> SKF38393 </td><td> Sigma-Aldrich </td><td> D047-100mg </td></tr></tbody></table>

neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations

Para entender a relação entre o transporte mitocondrial e função neuronal, é fundamental observar o comportamento mitocondrial em viver neurônios cultivados por longos períodos 1-3. Isto é agora possível através do uso de corantes vitais e proteínas fluorescentes com que os componentes do citoesqueleto, organelas, e outras estruturas em células vivas podem ser rotulados e então visualizados através de microscopia de fluorescência dinâmica. Por exemplo, nos neurônios de frango embrionárias Simpático, movimento mitocondrial foi caracterizada utilizando o corante vital rodamina 123 4. Em outro estudo, as mitocôndrias foram visualizados nos neurônios do prosencéfalo rato por transfecção de eYFP mitochondrially alvo 5. No entanto, imaging de neurônios primários ao longo de minutos, horas ou mesmo dias apresenta uma série de questões. Lugar entre estes estão: 1) manutenção das condições de cultivo, como temperatura, umidade e pH durante as sessões de imagem longa, 2) um forte sinal estável fluorescentes para garantir a qualidade das imagens adquiridas e medição precisa da intensidade do sinal durante a análise de imagem; e 3) limitar tempos de exposição durante a aquisição de imagens para minimizar e evitar fotobranqueamento fototoxicidade. Aqui, nós descrevemos um protocolo que permite a observação, visualização e análise do movimento mitocondrial em culturas de neurônios do hipocampo com alta resolução temporal e sob condições de apoio ótima vida. Temos construído um preço acessível estágio-top incubadora que fornece boa regulação da temperatura e fluxo de gás na atmosfera, e também limita o grau de evaporação media, assegurando pH estável e osmolaridade. Esta incubadora é conectado, via de entrada e saída de mangueiras, com um padrão de cultura de tecidos da incubadora, que oferece níveis de umidade constante e uma atmosfera de CO% 05-10 fevereiro de ar /. Este projeto oferece uma alternativa de baixo custo para incubadoras microscópio significativamente mais caros que não necessariamente garantem a viabilidade das células durante muitas horas ou mesmo dias. Para visualizar as mitocôndrias, que infectar as células com um lentivírus que codifica uma proteína fluorescente vermelha que é direcionado para a mitocôndria. Isso garante um sinal forte e persistente, que, em conjunto com o uso de uma fonte estável de luz xenon, que nos permite limitar tempos de exposição durante a aquisição de imagem e tudo, mas impede fotobranqueamento e fototoxicidade. Duas portas de injeção na parte superior da incubadora estágio-top permitir que a administração aguda de neurotransmissores e outros reagentes destinados para modular o movimento mitocondrial. Na expressão de soma, mediada por lentivírus de uma organela-alvo a proteína fluorescente vermelha ea combinação de nosso palco-top incubadora, um microscópio invertido de fluorescência convencional, câmera CCD, e fonte de luz xenon nos permitem adquirir imagens de lapso de tempo de transporte mitocondrial em neurônios vivos mais durações mais longas do que as possíveis em estudos a implantação convencional corantes vitais e off-the-shelf sistemas de suporte de vida.

Watch this Scientific Journal Video about Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations at JoVE.com

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

Nós descrevemos um protocolo que permite imagens de mitocôndrias na vida neurônios através de microscopia de fluorescência durante muito tempo. Imagem por longos períodos é realizado através de lentivírus mediada expressão de uma proteína fluorescente mitochondrially alvo e uso de uma infra-top estágio incubadora que foi projetada e construída em nosso laboratório.

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

neuromodulation-mitochondrial-transport-live-imaging-hippocampal

Research

JoVE Journal

Neuroscience

15.8K visualizações.  The Neurosciences Institute. Nós descrevemos um protocolo que permite imagens de mitocôndrias na vida neurônios através de microscopia de fluorescência durante muito tempo. Imagem por longos períodos é realizado através de lentivírus mediada expressão de uma proteína fluorescente mitochondrially alvo e uso de uma infra-top estágio incubadora que foi projetada e construída em nosso laboratório.

Vídeo: Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imagem de translocação da PKC em células Sf9 e em neurônios sensoriais Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Neurociência

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Determinação de Membrana Mitocondrial Espécies de Oxigênio Reativas Potencial e em neurônios de rato vivo Cortical

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Imagens em tempo real de axonal Transporte de Quantum Dot marcado BDNF na Primária Neurônios

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Imagens ao vivo de nicotina Induzida Sinalização de Cálcio e Neurotransmitter lançamento Junto ventrais do hipocampo Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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.

Medindo a endocitose da vesícula sináptica nos neurônios Hippocampal culturas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analisando o Transporte Mitocondrial e a Morfologia em neurônios derivados de células-tronco pluripotentes induzidos por humanos em Paraplegia Espástica Hereditária

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imagem Mitocondrial Ca2+ Absorção em Astrócitos e Neurônios usando Indicadores Ca2+ Codificados Geneticamente (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

Imagem ao vivo do Sistema Mitocondrial em Astrócitos Cultivados

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Imagem ao vivo do Estado De Glutathione Mitocondrial Redox em Neurônios Primários usando um Indicador Ratiométrico

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...