Nos gustaría dar las gracias a Donald Hutson por contribuir con su experiencia técnica y una gran habilidad en el diseño y la fabricación de la incubadora de etapa superior. También estamos agradecidos a Ayda Dashtaei por su excelente asistencia técnica. Todo el trabajo fue apoyado por la Fundación de Investigación de Neurociencias.

<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>

El empleo de lentivirus mediada por la expresión de una proteína fluorescente dirigidos a la mitocondria en cultivos de neuronas infectadas y un bajo costo de laboratorio construido etapa superior incubador que permite imágenes de células vivas para la concesión de prórrogas, que han sido capaces de investigar la relación entre el movimiento mitocondrial y señales de neuromoduladores , como la serotonina (5-HT), dopamina (DA), y la acetilcolina (ACh). Nuestros estudios han contribuido a esclarecer una vía de señalización que, por primera vez, los enlaces tráfico mitocondrial de los cambios en la actividad de las neuronas-modulado por neurotransmisores como la 5-HT y DA - que están en el corazón de la función neural. Nos encontramos con que el uso de proteínas fluorescentes dirigidas permite la observación de las mitocondrias en la etiqueta que viven las neuronas cultivadas durante largos períodos que pueden ser fisiológicamente más relevante que la duración mucho más corta que son posibles con colorantes vitales. Además, la intensidad de la señal de la proteína fluorescente que nos permite mantener los tiempos de exposición a corto durante la adquisición de la imagen, reduciendo al mínimo la posibilidad de photobleaching o fototoxicidad. Por último, una etapa superior sencillo y barato incubadora que mantiene la temperatura ambiente, humedad, y los niveles de CO 2, mientras se minimiza la evaporación de los medios de comunicación, nos permite seguir el movimiento de las mitocondrias en el que viven las neuronas durante horas o incluso días. Los investigadores que deseen fabricar una incubadora de etapa superior de las observaciones a largo plazo de las mitocondrias en neuronas vivas no necesita seguir los detalles precisos de nuestro diseño, siempre que las propiedades de los materiales utilizados (por ejemplo, la membrana permeable a los gases para evitar la evaporación de los medios de comunicación ) y los principios aplicados (por ejemplo, control de temperatura y humedad, de amortiguación del pH, el mantenimiento de la osmolaridad) son generalmente consistentes con lo que se describe en este protocolo.

<table border="1"><tbody><tr><th> Cantidad </th><th> Descripción (y ubicación en la Figura 1) </th></tr><tr><td> 1 </td><td> Incubadora de cultivo de tejidos (H) </td></tr><tr><td> 1 </td><td> Resistente al calor, caja de plástico (delrin o similar) (A) </td></tr><tr><td> 1 </td><td> Marco de plástico de 35 mm la tapa GBM (para la colocación de membrana a una caja de petri) (M) </td></tr><tr><td> 2.1 </td><td> pies lineales claras PTFE (Teflón) de la membrana de material </td></tr><tr><td> 4 </td><td> Tornillos de bronce del pulgar </td></tr><tr><td> 2 </td><td> Pequeñas resistencias disipador 10kOhm (utilizados como elementos de calefacción dentro de la etapa superior de la caja-incubadora) </td></tr><tr><td> 1 </td><td> Transformador (fuente de corriente de 9V a las resistencias) </td></tr><tr><td> 1 </td><td> Terrario controlador de temperatura y de la sonda (regulación termostática de la energía a las resistencias del disipador de calor a través de transformador) </td></tr><tr><td> 1 </td><td> Erlenmeyer de 2000 ml (silenciador para la supresión de vibraciones en la manguera antes de la etapa superior-incubadora) (K) </td></tr><tr><td> 1 </td><td> 1 / 8 &quot;hoja de Sorbothane (material de la junta para la base de la etapa superior-incubadora) </td></tr><tr><td> 1 </td><td> Hermético ABS (o similar) caja de plástico (de vivienda para la bomba de acuario) (J) </td></tr><tr><td> 1 </td><td> Bomba de acuario (I) </td></tr><tr><td> 1 </td><td> Equipo pequeño ventilador de refrigeración </td></tr><tr><td> 1 </td><td> Carcasa de plástico para ventilador de refrigeración (L) </td></tr><tr><td> 1 </td><td> 9V transformador para el ventilador de refrigeración del ordenador </td></tr><tr><td> 20-30ft </td><td> Manguera de Nalgene o de silicona (las conexiones entre el cultivo de tejidos y las incubadoras de etapa superior, atrapa la humedad) (F, G, N) </td></tr><tr><td> 2 </td><td> Llaves de plástico (para abrir y cerrar el flujo de aire antes y después de la etapa superior-incubadora) (O) </td></tr><tr><td> 4 </td><td> De púas de latón conectores de manguera (para las conexiones de la manguera hacia / desde la etapa-top incubadora y el recinto de la bomba de acuario) </td></tr><tr><td> 2 </td><td> Enchufes de bronce (para las conexiones de la manguera a / desde el escenario-top incubadora) </td></tr><tr><td> 2 </td><td> Enchufes de bronce (para las conexiones de la manguera a / desde el escenario-top incubadora) </td></tr></tbody></table> Tabla 1. Etapa superior incubadora partes: <table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </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 mm con fondo de cristal platos </td><td> MatTek </td><td> P35GC-0-14-C </td></tr><tr><td> DMEM </td><td> Tecnologías de la Vida </td><td> 10567 </td></tr><tr><td> B27 </td><td> Tecnologías de la Vida </td><td> 17504-044 </td></tr><tr><td> Glutamax </td><td> Tecnologías de la Vida </td><td> 35050 </td></tr><tr><td> Ricas en lípidos BSA </td><td> Tecnologías de la Vida </td><td> 11020-021 </td></tr><tr><td> L-asparagina </td><td> Sigma-Aldrich </td><td> P0380-100G </td></tr><tr><td> L-prolina </td><td> Sigma-Aldrich </td><td> A8381-100G </td></tr><tr><td> La 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> La 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 la relación entre el transporte mitocondrial y la función neuronal, es fundamental para observar el comportamiento de las mitocondrias en vivo las neuronas cultivadas de duración extendida 1-3. Esto es ahora posible mediante el uso de colorantes vitales y las proteínas fluorescentes con los componentes del citoesqueleto, orgánulos y otras estructuras en las células vivas pueden ser etiquetados y luego visualizados a través de microscopía de fluorescencia dinámica. Por ejemplo, en las neuronas simpáticas embrionarias de pollo, el movimiento se caracterizó mitocondrial utilizando el colorante vital importancia rodamina 123 4. En otro estudio, las mitocondrias se visualizan en las neuronas del cerebro anterior de rata mediante la transfección de mitocondrias objetivo EYFP 5. Sin embargo, las imágenes de las neuronas primarias en cuestión de minutos, horas o incluso días, presenta una serie de cuestiones. La primera de ellas son: 1) el mantenimiento de las condiciones de cultivo, tales como temperatura, humedad y pH durante las sesiones de formación de imágenes de largo, 2) una señal fuerte y estable fluorescentes para asegurar tanto la calidad de las imágenes obtenidas y la medición exacta de la intensidad de la señal durante el análisis de la imagen; y 3) la limitación de los tiempos de exposición durante la adquisición de la imagen para reducir al mínimo y evitar photobleaching fototoxicidad. Aquí se describe un protocolo que permite la observación, la visualización y análisis del movimiento mitocondrial en cultivos de neuronas del hipocampo de alta resolución temporal y bajo condiciones óptimas de mantenimiento de vida. Hemos construido un escenario económico-top incubadora que proporciona una buena regulación de la temperatura y el flujo de gas atmosférico, y también limita el grado de evaporación de medios, asegurando la estabilidad del pH y osmolaridad. Esta incubadora está conectado, a través de mangueras de entrada y de salida, a un nivel de cultivo de tejidos incubadora, que ofrece un nivel constante de humedad y una atmósfera de 5-10% CO 2 / aire. Este diseño ofrece una alternativa rentable a las incubadoras de microscopio mucho más caros que no necesariamente asegurar la viabilidad de las células durante muchas horas o incluso días. Para visualizar las mitocondrias, que infectan a las células con un lentivirus que codifica una proteína fluorescente roja que se dirige a la mitocondria. Esto asegura una señal fuerte y persistente, que, junto con el uso de una fuente de luz de xenón estable, nos permite limitar el tiempo de exposición durante la adquisición de imágenes y todo, pero se opone a photobleaching y fototoxicidad. Dos puertos de inyección en la parte superior de la incubadora de etapa superior permiten la administración aguda de los neurotransmisores y otros reactivos destinados a modular el movimiento mitocondrial. En suma, lentivirus mediada por la expresión de un orgánulo orientada proteína fluorescente de color rojo y la combinación de nuestra etapa-top incubadora, un microscopio de fluorescencia convencional invertido, cámara CCD, y la fuente de luz de xenón permiten obtener imágenes con lapso de tiempo de transporte mitocondrial en neuronas vivas más prolongados que los que sea posible en los estudios convencionales despliegue colorantes vitales dentro y fuera de la plataforma de sistemas de soporte vital.

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

Se describe un protocolo que permite imágenes de las mitocondrias en la vida neuronas a través de microscopía de fluorescencia durante largos períodos de tiempo. Imágenes durante períodos prolongados se lleva a cabo a través de lentivirus mediada por la expresión de una proteína mitocondrial, dirigida fluorescentes y el uso de una etapa de bajo costo-top incubadora que fue diseñado y construido en nuestro laboratorio.

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

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 vistas.  The Neurosciences Institute. Se describe un protocolo que permite imágenes de las mitocondrias en la vida neuronas a través de microscopía de fluorescencia durante largos períodos de tiempo. Imágenes durante períodos prolongados se lleva a cabo a través de lentivirus mediada por la expresión de una proteína mitocondrial, dirigida fluorescentes y el uso de una etapa de bajo costo-top incubadora que fue diseñado y construido en nuestro laboratorio.

Video: 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.

En vivo de imágenes de la translocación de PKC en células Sf9 y en las neuronas sensoriales 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 ...

Investigación

Neurociencias

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.

Determinación de Especies de la membrana mitocondrial de oxígeno potencial y reactiva en las neuronas corticales de rata en vivo

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.

Imágenes en tiempo real de Axonal Transporte de Quantum Dot-etiquetado BDNF en las neuronas primarias

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.

Imágenes en vivo de nicotina señalización de calcio inducido y neurotransmisor lanzamiento Junto ventrales hipocampo axones

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.

Fluorescencia y bioluminiscencia de imágenes de subcelular Ca<sup&gt; 2 +</sup&gt; Envejecido en las neuronas del 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.

Medición de endocitosis vesícula sináptica en neuronas Hippocampal cultivadas

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.

Análisis del transporte mitocondrial y la morfología en neuronas derivadas de células madre pluripotentes inducidas por el hombre en paraplejia espástica hereditaria

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.

Obtención de imágenes de ca2+ mitocondrial en astrocitos y neuronas utilizando indicadores de Ca2+ codificados genéticamente (GECI)

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.

Imágenes en vivo del sistema mitocondrial en astrocitos 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.

Imágenes en vivo del estado redox de glutatión mitocondrial en neuronas primarias utilizando un indicador ratiométrico

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