Name: preparation of acute subventricular zone slices for calcium imaging
Uploaded: 2012-09-19T11:00:00
Description: Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Dette arbejde blev støttet af tilskud fra NIH (DC007681, AB), CT Stem Cell tilskud (AB), Pardee fundament (AB), Predoctoral Ruth L. Kirschstein National Research Service Awards (NRSA) (SZY), og en NSF Graduate Research Fellowship (BL). Vi takker Bordey lab medlemmer for personer kommentarer til manuskriptet. Det foreliggende materiale er baseret på arbejde delvis støttet af staten Connecticut under Connecticut Stem Cell Research Grants Program. Dens indhold er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter i staten Connecticut, Institut for Folkesundhed i staten Connecticut eller CT Innovations, Incorporated.

1. Udarbejdelse af løsninger, dissektion og Vibratome <ol><li> Opløsninger skal fremstilles ved den korrekte osmolaritet og pH (tabel 1). Sammenlignet med kunstig cerebrospinalvæske (ACSF), der dissektion opløsning fremstillet med lavere koncentrationer af natrium og calcium, og højere koncentrationer af magnesium. Derved mindskes excitotoksicitet virkninger under udskæring. </li><li> Både dissektion og ACSF løsninger skal være mættet med 95% O2 / 5% CO2 ved gennembobli...

<ol>
	<li>Peretto, P., Giachino, C., Aimar, P., Fasolo, A., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+formation+and+glial+tube+assembly+in+the+shift+from+neonatal+to+adult+subventricular+zone+of+the+rodent+forebrain.">Chain formation and glial tube assembly in the shift from neonatal to adult subventricular zone of the rodent forebrain.</a> J. Comp Neurol. 487, 407-427 (2005).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice.">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Front. Neurosci. 4, 10-3389 (2010).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction-mediated+calcium+waves+define+communication+networks+among+murine+postnatal+neural+progenitor+cells.">Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.</a> Eur. J. Neurosci. , (2011).</li><li>Platel, J. C., Dave, K. A., Gordon, V., Lacar, B., Rubio, M. E., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+activated+by+subventricular+zone+astrocytic+glutamate+are+critical+for+neuroblast+survival+prior+to+entering+a+synaptic+network.">NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network.</a> Neuron. 65, 859-872 (2010).</li><li>Platel, J. C., Dupuis, A., Boisseau, S., Villaz, M., Albrieux, M., Brocard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrony+of+spontaneous+calcium+activity+in+mouse+neocortex+before+synaptogenesis.">Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis.</a> Eur. J. Neurosci. 25, 920-928 (2007).</li><li>Young, S. Z., Platel, J. C., Nielsen, J. V., Jensen, N. A., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA+increases+calcium+in+subventricular+zone+astrocyte-like+cells+through+L-+and+T-type+voltage-gated+calcium+channels.">GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels.</a> Front. Cell. Neurosci. 4, 8 (2010).</li><li>Bolteus, A. J., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+Release+and+Uptake+Regulate+Neuronal+Precursor+Migration+in+the+Postnatal+Subventricular+Zone.">GABA Release and Uptake Regulate Neuronal Precursor Migration in the Postnatal Subventricular Zone.</a> J. Neurosci. 24, 7623-7631 (2004).</li><li>Platel, J., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+a+whole+mount+preparation+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in a whole mount preparation of the subventricular zone.</a> J. Physiol. (Lond). 586, 3783-3793 (2008).</li><li>Nam, S. C., Kim, Y., Dryanovski, D., Walker, A., Goings, G., Woolfrey, K., Kang, S. S., Chu, C., Chenn, A., Erdelyi, F., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Lacar, B. L., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+controls+Ca2++activity-dependent+network+synchrony+in+the+adult+neurogenic+forebrain.">GABA controls Ca2+ activity-dependent network synchrony in the adult neurogenic forebrain.</a> , (2007).</li><li>Liu, X., Bolteus, A. J., Balkin, D. M., Henschel, O., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+cells+in+the+postnatal+subventricular+zone+display+a+unique+glial+phenotype+intermediate+between+radial+glia+and+astrocytes.">GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes.</a> Glia. 54, 394-410 (2006).</li><li>Wang, D. D., Krueger, D. D., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+ionic+signature+of+neuronal+progenitors+of+the+postnatal+subventricular+zone+in+situ.">Biophysical properties and ionic signature of neuronal progenitors of the postnatal subventricular zone in situ.</a> J. Neurophysiol. 90, 2291-2302 (2003).</li><li>Tian, L., Hires, S. A., Mao, T., Huber, D., Chiappe, M. E., Chalasani, S. H., Petreanu, L., Akerboom, J., McKinney, S. A., Schreiter, E. R., Bargmann, C. I., Jayaraman, V., Svoboda, K., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li><li>Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca(2)+indicators.">An expanded palette of genetically encoded Ca(2) indicators.</a> Science. 333, 1888-1891 (2011).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat. Neurosci. 13, 759-766 (2010).</li></ol>

Calcium billeddannelse af SVZ-celler er blevet anvendt til at studere mønstrene på spontan aktivitet i neuroblaster 10, receptorkanalen ekspression i både neuroblaster og astrocytter 4,6,8 og astrocytiske calcium bølger 3. Da celler i SVZ er enten umodne eller har gliale egenskaber, de ikke fyre handling potentialer 11,12, hvilket betyder, at millisekund ændringer i spændingspotentiale vejledende aktivitet i modne netværk ikke finder anvendelse i denne region. Derfor op...

<table border="1"><tbody><tr><td> Solut </td><td> Firma </td><td> Catalog Number </td><td> Dissektion (mM) </td></tr><tr><td> Saccharose </td><td> Sigma </td><td> S0389 </td><td> Dissektion: 147 mm ACSF: 0 mM </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S9888 </td><td> Dissektion: 42 mM ACSF: 126 mM </td></tr><tr><td> KCl </td><td> Sigma </td><td> P3911 </td><td> Dissektion: 2,5 mM ACSF: 2,5 mM </td></tr><tr><td> MgCl2 0,6 H2O </td><td> Sigma </td><td> M9272 </td><td> Dissektion: 4,33 mM ACSF: 1 mM </td></tr><tr><td> NaH 2 PO4. H2O </td><td> Sigma </td><td> S8282 </td><td> Dissektion: 1,25 mM ACSF: 1,25 mM </td></tr><tr><td> Glucose </td><td> Sigma </td><td> G8270 </td><td> Thesektion: 10 mM ACSF: 10 mM </td></tr><tr><td> NaHCO3 </td><td> Sigma </td><td> S6014 </td><td> Dissektion: 26 mM ACSF: 26 mM </td></tr><tr><td> CaCl2 0,2 H2O </td><td> Sigma </td><td> C3306 </td><td> Dissektion: 1,33 mM ACSF: 2 mM </td></tr></tbody></table> Tabel 1. Kemisk liste og opskrifter på dissektion løsning og ACSF. <table border="1"><tbody><tr><td></td><td></td><td></td><td> [Header] </td></tr><tr><td> Navn </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Vibratome </td><td> Leica </td><td> VT 1000S </td><td></td></tr><tr><td> Super Glue </td><td> Surehold eller 3M </td><td> Surehold 3G Super Glue eller 3M Vet-Bond </td><td></td></tr><tr><td> Dissektionsværktøj </td><td> Roboz eller Ted Pella </td><td></td><td></td></tr><tr><td> Fluo-4:00 calcium-sensitive farvestof </td><td> Invitrogen </td><td> F14201 </td><td></td></tr><tr><td> Oregon Green BAPTA-1:00 calcium-sensitive farvestof </td><td> Invitrogen </td><td> O6807 </td><td></td></tr><tr><td> Pluronic F-127 opløst i 20% DMSO </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> Opretstående konfokalt mikroskop </td><td> Olympus </td><td> FV300 eller FV1000 </td><td></td></tr><tr><td> Vand-nedsænkning mål </td><td> Olympus </td><td> LUMPlanFl 40 x W / IR (NA 0,80), LUMPlanFl 60 x W / I (NA 0,90) </td><td></td></tr><tr><td> Mikromanipulatorer </td><td> Sutter </td><td> MPC-200/MPC-325/MPC-385 </td><td></td></tr><tr><td> Pressure controller &lt;/ Td&gt; <td> Parker Hannifin </td><td> Picospritzer </td><td> &lt;3 PSI under anvendelse </td></tr><tr><td> Pipette aftrækker </td><td> Sutter eller Narshige </td><td> Sutter P-97 eller Narshige PP-830 </td><td></td></tr><tr><td> Glaspipetter </td><td> Sutter </td><td> BF 150-110-10 </td><td> ID: 1,10, OD: 1,50 </td></tr><tr><td> Peristaltisk pumpe </td><td> Harvard Apparatus </td><td> Model 720 </td><td> strømningshastighed: 1 ml / min </td></tr><tr><td> Chamber bad </td><td> Warner Instruments </td><td> RC-26 GLP </td><td> Lavprofil giver mulighed for objektiv clearance </td></tr><tr><td> Tubing </td><td> Tygon </td><td></td><td></td></tr><tr><td> Temperatur Controller </td><td> Warner Instruments </td><td> TC-324B/344B </td><td></td></tr></tbody></table> Tabel 2. Materialer / udstyrsliste.

preparation of acute subventricular zone slices for calcium imaging

The subventricular zone (SVZ) er en af ​​de to neurogene områder i den postnatale hjernen. The SVZ indeholder tætpakkede celler, herunder neurale progenitorceller med astrocytiske features (kaldet SVZ astrocytter), neuroblaster og mellemliggende progenitorceller. Neuroblaster født i SVZ tangentialt migrere en stor afstand til lugtekolben, hvor de differentierer til interneuroner. Intercellulære signalering gennem adhæsionsmolekyler og diffunderbare signaler spiller vigtige roller i kontrollerende neurogenese. Mange af disse signaler udløser intercellulær calcium aktivitet, der transmitterer information i og mellem celler. Calcium aktivitet er således afspejler aktiviteten af ​​ekstracellulære signaler og er en optimal måde at forstå funktionel intercellulær signalering blandt SVZ celler. Calcium-aktivitet er blevet undersøgt i mange andre regioner og celletyper, herunder modne astrocytter og neuroner. Men den traditionelle metode til load celler med calcium indikatorfarvestof (dvs. bad loading) var ikke effektiv ved pålæsning alle SVZ celletyper. Faktisk den cellulære tæthed i SVZ udelukker farvestoffet diffusion inde i vævet. Desuden vil fremstilling sagittale udsnit bedre bevarer den tredimensionelle arrangement af SVZ celler, især strømmen af ​​neuroblast migration på den rostrale-caudale akse. Her beskriver vi metoder til at forberede sagittale sektioner og indeholder SVZ, at lastningen af ​​SVZ celler med calcium indikatorfarvestof, og købet af calcium aktivitet med time-lapse film. Vi anvendte Fluo-4:00 farvestof til lastning SVZ astrocytter hjælp trykpåføring inde i vævet. Calcium-aktivitet blev registreret ved hjælp af en konfokal mikroskop giver en præcis løsning for skelne de enkelte celler. Vores tilgang er anvendelig til andre neurogene zoner, herunder den voksne hippocampus subgranular zone og embryonale neurogene zoner. Endvidere kan andre typer af farvestofferpåføres ved anvendelse af den beskrevne fremgangsmåde.

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

En fremgangsmåde til at indlæse subventricular zone (SVZ) celler med calcium indikatorfarvestoffer til optagelse calcium aktivitet er beskrevet. Den postnatale SVZ indeholder tætpakkede celler, herunder neurale progenitorceller og neuroblaster. Snarere end at bruge bad loading vi injicerede farvestoffet ved tryk inde i vævet giver bedre farve diffusion.

Fremstilling af akutte Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Research

JoVE Journal

Neuroscience

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Time-lapse Imaging af neuroblast Migration i Akutte Skiver af den voksne Mouse forhjerne

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Imaging Calcium respons i GFP-mærkede neuroner i hypothalamus musehjerne Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Neonatal Subventricular Zone Electroporation

Neonatal Subventricular Zone Elektroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Fødselsdepression Elektroporation og Time-lapse Imaging af neuroblast Migration i Mouse Akut hjerneskiver

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Celle Sortering af neurale stam- og progenitorceller fra voksne mus subventrikulære Zone og Live-billeddannelse af deres cellecyklus Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Forberedelse af akut hjernen skiver bruger en optimeret N-Methyl-D-glucamine beskyttende genoprettelsesmetode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Langvarig Inkubation af akut neuronalt væv for Elektrofysiologi og Calcium-billeddannelse

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

To-foton Calcium Imaging i Neuronal dendritter i hjernen skiver

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...