Nous tenons à remercier Donald Hutson pour contribuer son expertise technique et une grande habileté lors de la conception et la fabrication de l&#39;incubateur en scène haut. Nous sommes également reconnaissants à Ayda Dashtaei pour son excellente assistance technique. Tout le travail a été soutenu par Neurosciences Research Foundation.

<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>

Employant lentivirus médiée par l&#39;expression d&#39;une protéine fluorescente ciblé à la mitochondrie dans les neurones infectés cultivé et un peu coûteux en laboratoire construit étape-top incubateur qui permet l&#39;imagerie de cellules vivantes pour des durées prolongées, nous avons été en mesure d&#39;étudier le lien entre le mouvement des mitochondries et des signaux neuromodulateurs , tels que la sérotonine (5-HT), la dopamine (DA), et l&#39;acétylcholine (ACh). Nos études ont permis d&#39;élucider une voie de signalisation qui, pour la première fois, le trafic mitochondrial liens à des changements dans l&#39;activité des neurones modulée par des neurotransmetteurs tels que la 5-HT et DA - qui sont au cœur de la fonction neuronale. Nous constatons que l&#39;utilisation de protéines fluorescentes ciblées permet l&#39;observation des mitochondries étiquetés dans la vie des neurones cultivés sur de longues périodes qui peuvent être plus physiologiquement pertinents que les durées beaucoup plus courtes qui sont possibles en utilisant des colorants vitaux. Par ailleurs, l&#39;intensité du signal fluorescent protein nous permet de garder le temps d&#39;exposition courts lors de l&#39;acquisition, en minimisant la possibilité de photoblanchiment ou de phototoxicité. Enfin, une étape-top simple et peu coûteux incubateur qui maintient la température ambiante, l&#39;humidité et de CO 2 niveaux, tout en minimisant l&#39;évaporation de médias, nous permet de suivre le mouvement des mitochondries dans la vie des neurones pendant des heures voire des jours. Les chercheurs qui souhaitent fabriquer un incubateur étape-top pour les observations à long terme des mitochondries dans des neurones vivants ne doivent pas suivre les détails précis de notre conception, à condition que les propriétés des matériaux utilisés (par exemple, le gaz membrane perméable pour éviter l&#39;évaporation des médias ) et les principes appliqués (par exemple, contrôle de la température et l&#39;humidité, du pH tampon, le maintien de l&#39;osmolarité) sont généralement compatibles avec ce qui est décrit dans ce protocole.

<table border="1"><tbody><tr><th> Quantité </th><th> Description (et l&#39;emplacement dans la Figure 1) </th></tr><tr><td> 1 </td><td> Incubateur de culture tissulaire (H) </td></tr><tr><td> 1 </td><td> Résistant à la chaleur boîtier en plastique (delrin ou comparables) (A) </td></tr><tr><td> 1 </td><td> Cadre en plastique pour le 35mm GBM couvercle (pour l&#39;apposition membrane pour boîte de Petri) (M) </td></tr><tr><td> 1-2 </td><td> linéaires pi matériels Effacer PTFE membrane (téflon) </td></tr><tr><td> 4 </td><td> Vis à oreilles en laiton </td></tr><tr><td> 2 </td><td> Petites résistances radiateur 10kOhm (utilisés comme éléments de chauffage à l&#39;intérieur en scène haut de l&#39;enceinte incubateur) </td></tr><tr><td> 1 </td><td> Transformateur (fourniture de courant 9V à résistances) </td></tr><tr><td> 1 </td><td> Régulateur de température et sonde terrarium (régulation thermostatique de la puissance aux résistances du radiateur via transformateur) </td></tr><tr><td> 1 </td><td> 2000ml Erlenmeyer (silencieux pour la suppression des vibrations dans les tuyaux avant l&#39;étape-dessus incubateur) (K) </td></tr><tr><td> 1 </td><td> 1 / 8 feuilles sorbothane &quot;(matériau de joint pour la base de l&#39;étape-dessus incubateur) </td></tr><tr><td> 1 </td><td> Hermétique ABS (ou comparable) boîte en plastique (boîtier de la pompe d&#39;aquarium) (J) </td></tr><tr><td> 1 </td><td> Pompe d&#39;aquarium (I) </td></tr><tr><td> 1 </td><td> Petit ventilateur d&#39;ordinateur </td></tr><tr><td> 1 </td><td> Boîtier en plastique pour ventilateur de refroidissement (L) </td></tr><tr><td> 1 </td><td> 9V transformateur pour ventilateur d&#39;ordinateur </td></tr><tr><td> 20-30ft </td><td> Tuyau Nalgene ou de silicone (liens entre la culture de tissus et le stade-top incubateurs; pièges à humidité) (F, G, N) </td></tr><tr><td> 2 </td><td> Robinets en plastique (pour ouvrir et fermer le débit d&#39;air avant et après l&#39;étape-dessus incubateur) (O) </td></tr><tr><td> 4 </td><td> Raccords de tuyaux en laiton barbelé (pour les raccords de tuyau de / vers l&#39;étape-top incubateur et enceinte de la pompe d&#39;aquarium) </td></tr><tr><td> 2 </td><td> Coupleurs rapides en laiton (pour les raccords de tuyau de / vers l&#39;étape-top incubateur) </td></tr><tr><td> 2 </td><td> Coupleurs rapides en laiton (pour les raccords de tuyau de / vers l&#39;étape-top incubateur) </td></tr></tbody></table> Tableau 1. Stage-dessus des pièces incubateur: <table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> Poly-D-lysine </td><td> Sigma-Aldrich </td><td> P7280-5MG </td></tr><tr><td> laminine </td><td> Roche Applied Science </td><td> 11243217001 </td></tr><tr><td> 35 mm à fond de verre plats </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> Riche en lipides BSA </td><td> Life Technologies </td><td> 11020-021 </td></tr><tr><td> L-asparagine </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> La vitamine 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> Dopamine </td><td> Sigma-Aldrich </td><td> H8502-5G </td></tr><tr><td> Bromocriptine </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

Pour comprendre la relation entre le transport mitochondrial et la fonction neuronale, il est essentiel d&#39;observer le comportement des mitochondries en direct neurones cultivés pour des durées étendu 1-3. C&#39;est maintenant possible grâce à l&#39;utilisation de colorants vitaux et des protéines fluorescentes avec laquelle les composants du cytosquelette, les organites, et d&#39;autres structures dans les cellules vivantes peuvent être étiquetées et ensuite visualisé par microscopie à fluorescence dynamique. Par exemple, dans les neurones embryonnaires de poulet sympathique, le mouvement mitochondrial a été caractérisée en utilisant la rhodamine colorant vital 123 4. Dans une autre étude, les mitochondries ont été visualisées dans les neurones du cerveau antérieur chez le rat par transfection de mitochondrially ciblées EYFP 5. Cependant, l&#39;imagerie des neurones primaires en quelques minutes, heures, jours ou même présente un certain nombre de questions. Ce sont notamment les suivants: 1) le maintien de conditions de culture telles que la température, l&#39;humidité et du pH au cours des séances d&#39;imagerie longue; 2) un signal fort et stable, fluorescentes pour assurer la qualité des images acquises et la mesure précise de l&#39;intensité du signal pendant l&#39;analyse d&#39;image; et 3) la limitation des temps d&#39;exposition lors de l&#39;acquisition d&#39;images pour minimiser photoblanchiment et d&#39;éviter la phototoxicité. Ici, nous décrivons un protocole qui permet l&#39;observation, la visualisation et l&#39;analyse du mouvement des mitochondries dans des cultures de neurones de l&#39;hippocampe avec une résolution temporelle élevée et dans des conditions optimales de survie. Nous avons construit un prix abordable étape-top incubateur qui permet une régulation de température et de débit de gaz bonne atmosphère, et limite également le degré d&#39;évaporation des médias, en assurant un pH stable et l&#39;osmolarité. Cet incubateur est connecté, via l&#39;entrée et la sortie de tuyaux, d&#39;une norme incubateur de culture tissulaire, qui fournit des niveaux d&#39;humidité constante et une atmosphère de 5-10% de CO 2 / air. Cette conception offre une alternative rentable aux incubateurs microscope sensiblement plus cher que ne garantit pas nécessairement la viabilité des cellules pendant plusieurs heures voire plusieurs jours. Pour visualiser la mitochondrie, nous infecter les cellules avec un lentivirus codant pour une protéine fluorescente rouge qui est ciblé vers la mitochondrie. Ceci assure un signal fort et persistant, qui, en conjonction avec l&#39;utilisation d&#39;une source de lumière au xénon stable, nous permet de limiter les temps d&#39;exposition lors de l&#39;acquisition d&#39;images et de tous, mais exclut photoblanchiment et de phototoxicité. Deux ports d&#39;injection sur le dessus de l&#39;incubateur en scène haut permettent l&#39;administration aiguë de neurotransmetteurs et autres réactifs destinés à moduler le mouvement des mitochondries. En somme, lentivirus médiée par l&#39;expression d&#39;un organite ciblées protéine fluorescente rouge et la combinaison de notre stade-dessus incubateur, une conventionnelles microscope inversé à fluorescence, caméra CCD, et la source lumineuse au xénon nous permettent d&#39;acquérir des images en accéléré du transport mitochondrial neurones vivants sur de longues durées que celles possibles dans les études conventionnelles de déployer des colorants vitaux et off-the-shelf systèmes de survie.

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

Nous décrivons un protocole qui permet l&#39;imagerie de la mitochondrie dans la vie des neurones par l&#39;intermédiaire de microscopie par fluorescence sur des durées longues. Imagerie sur de longues périodes est accompli par des lentivirus médiée par l&#39;expression d&#39;une protéine fluorescente mitochondrially ciblées et l&#39;utilisation d&#39;un moyen peu coûteux étape-top incubateur qui a été conçu et construit dans notre laboratoire.

Neuromodulation et mitochondriale de transport: Live in Imaging neurones de l&#39;hippocampe sur des durées longues

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

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Neuroscience

15.8K vues.  The Neurosciences Institute. Nous décrivons un protocole qui permet l'imagerie de la mitochondrie dans la vie des neurones par l'intermédiaire de microscopie par fluorescence sur des durées longues. Imagerie sur de longues périodes est accompli par des lentivirus médiée par l'expression d'une protéine fluorescente mitochondrially ciblées et l'utilisation d'un moyen peu coûteux étape-top incubateur qui a été conçu et construit dans notre laboratoire.

Vidéo: 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-imagerie de la translocation de la PKC dans les cellules Sf9 et dans les neurones sensoriels aplysie

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 ...

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Neurosciences

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.

Détermination des espèces d&#39;oxygène Potentiel membrane mitochondriale et réactive dans les neurones corticaux de rat en direct

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.

Imagerie en temps réel des axonale Transport de Quantum Dot marqué BDNF dans les neurones primaires

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.

Imagerie en direct de la nicotine induit la signalisation du calcium et de la libération de neurotransmetteurs long ventrales hippocampe 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.

Fluorescence et bioluminescence imagerie des Subcellular Ca<sup&gt; 2+</sup&gt; In Aged neurones de l&#39;hippocampe

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.

Mesurer l’endocytose de la vésicule synaptique dans des neurones Hippocampal cultivés

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.

Analyse du transport mitochondrial et de la morphologie dans les neurones dérivés de cellules souches pluripotentes induites par l’homme dans la paraplégie spastique héréditaire

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.

Imagerie de l’absorption mitochondriale de Ca2+ dans les astrocytes et les neurones à l’aide d’indicateurs ca2+ codés génétiquement (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.

Imagerie en direct du système mitochondrial dans les astrocytes en culture

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.

Imagerie en direct de l’état redox du glutathion mitochondrial dans les neurones primaires à l’aide d’un indicateur ratiométrique

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