Wir möchten Donald Hutson für den Beitrag seines technischen Know-how und viel Geschick bei der Konstruktion und Herstellung von der Bühne-Top-Inkubator danken. Wir sind auch dankbar, dass Ayda Dashtaei für ihre hervorragende technische Unterstützung. Alle Arbeiten wurden von Neurosciences Research Foundation unterstützt.

<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>

Der Einsatz Lentivirus-vermittelte Expression eines fluoreszierenden Proteins gezielt an die Mitochondrien in infizierten kultivierten Neuronen und eine preiswerte Labor Baustufe-Top-Inkubator, der Bildgebung an lebenden Zellen für längere Laufzeiten ermöglicht, haben wir in der Lage, die Verbindung zwischen mitochondrialen Bewegung und neuromodulatorischen Signale zu untersuchen wie Serotonin (5-HT), Dopamin (DA) und Acetylcholin (ACh). Unsere Studien haben dazu beigetragen, einen Signalweg, der zum ersten Mal, Links mitochondrialen Menschenhandel zu Veränderungen in der Aktivität von Neuronen moduliert durch Neurotransmitter wie 5-HT-und DA-Aufklärung -, dass im Herzen der neuronalen Funktion. Wir finden, dass die Verwendung von fluoreszierenden Proteinen gezielt die Beobachtung der markierten Mitochondrien in lebenden kultivierten Neuronen über lange Zeiträume, die mehr physiologisch relevanter als die viel kürzere Laufzeiten möglich sind, mit vitalen Farbstoffe können zulässt. Darüber hinaus ermöglicht die Intensität der fluoreszierenden Proteins Signal uns zu halten Belichtungszeiten kurz während der Bildaufnahme, minimiert die Möglichkeit von Ausbleichen oder Phototoxizität. Schließlich ermöglicht eine einfache und kostengünstige Stufe-Top-Inkubator, der Umgebungstemperatur, Luftfeuchtigkeit und CO 2-Gehalt führt, bei gleichzeitiger Minimierung der Verdunstung von Medien, uns zu mitochondrialen Bewegung in lebenden Nervenzellen über Stunden oder sogar Tage zu folgen. Forscher, die auf einer Bühne-Top-Inkubator für die Langzeit-Beobachtungen von Mitochondrien Herstellung in lebenden Neuronen wollen, müssen sich nicht an die genauen Einzelheiten unserer Design, vorausgesetzt, die Eigenschaften der verwendeten Materialien (z. B. Gas-durchlässige Membran zur Verdampfung von Medien zu vermeiden ) und Grundsätze angewandt (zB Temperatur und Luftfeuchtigkeit, Pufferung des pH-Wertes, die Aufrechterhaltung der Osmolarität) sind in der Regel im Einklang mit dem, was in diesem Protokoll beschrieben.

<table border="1"><tbody><tr><th> Menge </th><th> Beschreibung (und Lokalisierung in Abbildung 1) </th></tr><tr><td> 1 </td><td> Gewebekultur-Inkubator (H) </td></tr><tr><td> 1 </td><td> Hitzebeständigem Kunststoff-Gehäuse (Delrin oder vergleichbare) (A) </td></tr><tr><td> 1 </td><td> Kunststoff-Rahmen für 35mm GBM Deckel (für die Anbringung Membran Petrischale) (M) </td></tr><tr><td> 1-2 </td><td> linear ft. Klare PFTE (Teflon) Membran-Material </td></tr><tr><td> 4 </td><td> Messing Flügelschrauben </td></tr><tr><td> 2 </td><td> Kleine 10kOhm Kühlkörper Widerstände (verwendet als Heizelemente im Inneren der Bühne-Top-Inkubator-Gehäuse) </td></tr><tr><td> 1 </td><td> Transformer (Versorgung mit 9V Strom Widerstände) </td></tr><tr><td> 1 </td><td> Terrarium Temperaturregler und Sonde (thermostatische Regulierung der Macht an Kühlkörper Widerstände über Trafo) </td></tr><tr><td> 1 </td><td> 2000ml Erlenmeyerkolben (Schalldämpfer für Schwingungsdämpfung im Schlauch vor der Stufe-Top-Inkubator) (K) </td></tr><tr><td> 1 </td><td> 1 / 8 &quot;Sorbothane Blatt (Dichtungsmaterial für die Basis der Stufe-Top-Inkubator) </td></tr><tr><td> 1 </td><td> Luftdichte ABS (oder vergleichbar) Kunststoff-Box (Gehäuse für Aquarium-Pumpe) (J) </td></tr><tr><td> 1 </td><td> Aquarium Pumpe (I) </td></tr><tr><td> 1 </td><td> Kleine Computer-Lüfter </td></tr><tr><td> 1 </td><td> Kunststoffgehäuse für Lüfter (L) </td></tr><tr><td> 1 </td><td> 9V Transformator für Computer-Lüfter </td></tr><tr><td> 20-30ft </td><td> Nalgene oder Silikonschlauch (Verbindungen zwischen Gewebekultur und Bühne-Top-Inkubatoren, Feuchtigkeit Fallen) (F, G, N) </td></tr><tr><td> 2 </td><td> Plastic Hähne (zum Öffnen und Schließen Luftstrom vor und nach der Phase-Top-Inkubator) (O) </td></tr><tr><td> 4 </td><td> Barbed Messing Schlauchverbinder (für Schlauchverbindungen zu / von der Bühne-Top-Inkubator und Aquarium Pumpe Gehäuse) </td></tr><tr><td> 2 </td><td> Messing Schnellkupplungen (für Schlauchverbindungen zu / von der Bühne-Top-Inkubator) </td></tr><tr><td> 2 </td><td> Messing Schnellkupplungen (für Schlauchverbindungen zu / von der Bühne-Top-Inkubator) </td></tr></tbody></table> Tabelle 1. Stage-top-Inkubator Teilen: <table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Poly-D-Lysin </td><td> Sigma-Aldrich </td><td> P7280-5MG </td></tr><tr><td> Laminin </td><td> Roche Applied Science </td><td> 11243217001 </td></tr><tr><td> 35 mm Glasboden-Schalen </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> Lipid-reichen BSA </td><td> Life Technologies </td><td> 11020-021 </td></tr><tr><td> L-Asparagin </td><td> Sigma-Aldrich </td><td> P0380-100G </td></tr><tr><td> L-Prolin </td><td> Sigma-Aldrich </td><td> A8381-100G </td></tr><tr><td> Vitamin 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> Dopamin </td><td> Sigma-Aldrich </td><td> H8502-5G </td></tr><tr><td> Bromocriptin </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

Um zu verstehen, die Beziehung zwischen mitochondrialen Transport und neuronale Funktion, ist es entscheidend, mitochondriale Verhalten in lebenden kultivierten Neuronen für längere Laufzeiten 1-3 beobachten. Dies ist nun durch den Einsatz von vitaler Farbstoffe und fluoreszierende Proteine, mit denen Zytoskelett-Komponenten, Organellen und anderen Strukturen in lebenden Zellen markiert und dann visualisiert werden können durch dynamische Fluoreszenz-Mikroskopie möglich. Zum Beispiel, in embryonalen Huhn sympathischen Neuronen wurde mitochondrialen Bewegung charakterisiert die vitalen Farbstoff Rhodamin 123 4. In einer anderen Studie wurden Mitochondrien visualisiert in Rattenvorderhirn Neuronen durch Transfektion von mitochondrial gezielte EYFP 5. Jedoch stellt Bildgebung von primären Neuronen über Minuten, Stunden oder sogar Tage eine Reihe von Fragen. An erster Stelle sind: 1) Pflege der Kultur, wie Temperatur, Feuchtigkeit und pH-Wert während langer Imaging-Sitzungen, 2) eine starke, stabile Fluoreszenz-Signal, um sowohl die Qualität der aufgenommenen Bilder und genaue Messung der Signalintensität während der Bildanalyse zu gewährleisten; und 3) die Begrenzung der Exposition Mal während der Bildaufnahme zu minimieren Bleichen und zur Vermeidung von Phototoxizität. Hier beschreiben wir ein Protokoll, das die Beobachtung, Visualisierung und Analyse der mitochondrialen Bewegung in kultivierten Hippocampus-Neuronen mit hoher zeitlicher Auflösung und unter optimalen Lebens unterstützen Bedingungen erlaubt. Wir haben eine erschwingliche Bühne-Top-Inkubator, dass eine gute Temperaturregelung und atmosphärischen Gasstrom bietet gebaut, und schränkt auch den Grad der Medien Verdunstung, versicherte stabilen pH-Wert und Osmolarität. Dieser Inkubator ist über Ein-und Auslass-Schläuche, um eine Standard-Gewebekultur-Inkubator, der konstante Luftfeuchtigkeit und eine Atmosphäre von 5-10% CO 2 / Luft bietet. Diese Konstruktion bietet eine kostengünstige Alternative zu wesentlich teureren Mikroskop Inkubatoren, die nicht unbedingt versichern die Lebensfähigkeit der Zellen über viele Stunden oder sogar Tage. Zur Visualisierung Mitochondrien, infizieren wir Zellen mit einem Lentivirus kodierend für ein rot fluoreszierendes Protein, das an den Mitochondrien abzielt. Dies sichert eine starke und anhaltende Signal, das in Verbindung mit dem Einsatz eines stabilen Xenon-Lichtquelle, ermöglicht es uns, Belichtungszeiten während der Bildaufnahme zu begrenzen und alle, aber schließt Bleichen und Phototoxizität. Zwei Einspritzöffnungen am oberen Rand der Bühne-Top-Inkubator ermöglicht die akute Verabreichung von Neurotransmittern und anderen Reagenzien bestimmt zu modulieren mitochondrialen Bewegung. In der Summe Lentivirus-vermittelte Expression von einer Organelle gezielte rot fluoreszierendes Protein und die Kombination unserer Bühne-Top-Inkubator, eine konventionelle invertierten Fluoreszenzmikroskop, CCD-Kamera und Xenon-Lichtquelle ermöglichen es uns, Zeitraffer-Bilder der mitochondrialen Transport in erwerben lebenden Neuronen über lange Zeiträume als möglich in Studien den Einsatz konventioneller Vitalfarbstoffen und off-the-shelf lebenserhaltenden Systemen.

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

Wir beschreiben ein Protokoll, das Bildgebung von Mitochondrien ermöglicht in lebenden Nervenzellen mittels Fluoreszenz-Mikroskopie über einen längeren Zeitraum. Imaging über längere Zeiträume durch Lentivirus-vermittelte Expression von mitochondrial gezielte fluoreszierendes Protein und Verwendung eines kostengünstigen Bühne-Top-Inkubator, entworfen und gebaut wurde in unserem Labor durchgeführt.

Neuromodulation und Mitochondriale Transport: Live Imaging in Hippocampus-Neuronen über lange Zeiträume

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 Aufrufe.  The Neurosciences Institute. Wir beschreiben ein Protokoll, das Bildgebung von Mitochondrien ermöglicht in lebenden Nervenzellen mittels Fluoreszenz-Mikroskopie über einen längeren Zeitraum. Imaging über längere Zeiträume durch Lentivirus-vermittelte Expression von mitochondrial gezielte fluoreszierendes Protein und Verwendung eines kostengünstigen Bühne-Top-Inkubator, entworfen und gebaut wurde in unserem Labor durchgeführt.

Serie: 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-Darstellung von PKC-Translokation in Sf9-Zellen und in Aplysia sensorischen Neuronen

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 ...

Forschung

Neurowissenschaften

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.

Bestimmung des mitochondrialen Membranpotentials und Reactive Oxygen Species in Live Rat kortikalen Neuronen

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.

Echtzeit-Bildgebung des axonalen Transport von Quantum Dot-markierten BDNF in primären Neuronen

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.

Live-Imaging von Nikotin induzierte Calcium Signaling und Neurotransmitterausschüttung Zusammen Ventral Hippocampus Axone

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.

Fluoreszenz- und Biolumineszenz-Bildgebung subzellulärer Ca<sup&gt; 2+</sup&gt; Im Alter von Neuronen im Hippocampus

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.

Messung der synaptischen Vesikel Endozytose in kultivierten Hippocampal Neuronen

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 von mitochondrialem Transport und Morphologie bei humaninduzierten Pluripotenten Stammzell-abgeleiteten Neuronen bei erblicher spastischer Paraplegie

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.

Bildgebung der mitochondrialen Ca2+ Aufnahme in Astrozyten und Neuronen unter Verwendung von genetisch kodierten Ca2+ Indikatoren (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.

Live-Bildgebung des mitochondrialen Systems in kultivierten Astrozyten

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.

Live-Bildgebung des mitochondrialen Glutathion-Redox-Zustands in primären Neuronen unter Verwendung eines ratiometrischen Indikators

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