Name: purification of the dendritic filopodia-rich fraction
Uploaded: 2019-05-02T13:30:00
Description: Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

Vi tackar Shigeo Okabe och Hitomi Matsuno för låg densitet kulturen i hippocampus nervceller, Masayoshi Mishina för TLCN-brist möss, Sachiko Mitsui och Momoko Shiozaki för tekniskt stöd, och medlemmar av Yoshihara laboratorium för hjälpsamma diskussioner . Detta arbete stöddes av JSPS KAKENHI Grant nos. JP20700307, JP22700354, och JP24500392 och MEXT KAKENHI Grant nos. JP23123525 till YF och JP20022046, JP18H04683 och JP18H05146 till YY.

Alla metoder som beskrivs här har godkänts av den institutionella djuromsorg och användning kommitté RIKEN Wako.
1. kultur av hippocampus nervceller
<ol><li>Beredning av odlingssubstrat
	<ol>
<li>Beredning av 200x vitamin mix. Lös 100 mg D-pantotensyra hemikalciumsalt, 100 mg kolinklorid, 100 mg folsyra, 180 mg i-inositol, 100 mg niacinamide, 100 mg pyridoxal HCl, och 100 mg tiamin HCL i 500 ml av ultrarent vatten med hjälp av en magnetisk omrörare. Lösningen är int...

<ol>
	<li>Fiala, J. C., Feinberg, M., Popov, V., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptogenesis+via+dendritic+filopodia+in+developing+hippocampal+area+CA1.">Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.</a> Journal of Neuroscience. 18 (21), 8900-8911 (1998).</li><li>Portera-Cailliau, C., Pan, D. T., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-regulated+dynamic+behavior+of+early+dendritic+protrusions:+evidence+for+different+types+of+dendritic+filopodia.">Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia.</a> Journal of Neuroscience. 23 (18), 7129-7142 (2003).</li><li>Ziv, N. E., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+role+of+dendritic+filopodia+in+synaptogenesis+and+spine+formation.">Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.</a> Neuron. 17 (1), 91-102 (1996).</li><li>Lohmann, C., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+local+calcium+signaling+in+rapid+synaptic+partner+selection+by+dendritic+filopodia.">A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia.</a> Neuron. 59 (2), 253-260 (2008).</li><li>Yoshihara, Y., De Roo, M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+formation+and+stabilization.">Dendritic spine formation and stabilization.</a> Current Opinion in Neurobiology. 19 (2), 146-153 (2009).</li><li>Bayes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+human+and+mouse+postsynaptic+proteomes+finds+high+compositional+conservation+and+abundance+differences+for+key+synaptic+proteins.">Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins.</a> PLoS One. 7 (10), e46683 (2012).</li><li>Bayes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+proteome,+diseases+and+evolution+of+the+human+postsynaptic+density.">Characterization of the proteome, diseases and evolution of the human postsynaptic density.</a> Nature Neuroscience. 14 (1), 19-21 (2011).</li><li>Furutani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+telencephalin+and+ERM+family+proteins+mediates+dendritic+filopodia+formation.">Interaction between telencephalin and ERM family proteins mediates dendritic filopodia formation.</a> Journal of Neuroscience. 27 (33), 8866-8876 (2007).</li><li>Mao, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+Conduct+Target+Selection+in+Cortical+Neurons+Using+Differences+in+Signal+Kinetics+of+a+Single+Kinase.">Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.</a> Neuron. 98 (4), 767-782 (2018).</li><li>Matsuno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telencephalin+slows+spine+maturation.">Telencephalin slows spine maturation.</a> Journal of Neuroscience. 26 (6), 1776-1786 (2006).</li><li>Raemaekers, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF6-mediated+endosomal+transport+of+Telencephalin+affects+dendritic+filopodia-to-spine+maturation.">ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation.</a> The EMBO Journal. 31 (15), 3252-3269 (2012).</li><li>Mori, K., Fujita, S. C., Watanabe, Y., Obata, K., Hayaishi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telencephalon-specific+antigen+identified+by+monoclonal+antibody.">Telencephalon-specific antigen identified by monoclonal antibody.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (11), 3921-3925 (1987).</li><li>Yoshihara, Y., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telencephalin:+a+neuronal+area+code+molecule?.">Telencephalin: a neuronal area code molecule?.</a> Neuroscience Research. 21 (2), 119-124 (1994).</li><li>Annaert, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+telencephalin+and+the+amyloid+precursor+protein+predicts+a+ring+structure+for+presenilins.">Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins.</a> Neuron. 32 (4), 579-589 (2001).</li><li>Furutani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitronectin+induces+phosphorylation+of+ezrin/radixin/moesin+actin-binding+proteins+through+binding+to+its+novel+neuronal+receptor+telencephalin.">Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.</a> Journal of Biological Chemistry. 287 (46), 39041-39049 (2012).</li><li>Nyman-Huttunen, H., Tian, L., Ning, L., Gahmberg, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Actinin-dependent+cytoskeletal+anchorage+is+important+for+ICAM-5-mediated+neuritic+outgrowth.">alpha-Actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth.</a> Journal of Cell Biology. 119 (Pt 15), 3057-3066 (2006).</li><li>Esselens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+1+mediates+the+turnover+of+telencephalin+in+hippocampal+neurons+via+an+autophagic+degradative+pathway.">Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway.</a> Journal of Cell Biology. 166 (7), 1041-1054 (2004).</li><li>Furutani, Y., Yoshihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+Dendritic+Filopodia-Rich+Fraction+Isolated+by+Telencephalin+and+Vitronectin+Interaction.">Proteomic Analysis of Dendritic Filopodia-Rich Fraction Isolated by Telencephalin and Vitronectin Interaction.</a> Frontiers in Synaptic Neuroscience. 10, 27 (2018).</li><li>Lu, Z., Piechowicz, M., Qiu, 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+Simplified+Method+for+Ultra-Low+Density,+Long-Term+Primary+Hippocampal+Neuron+Culture.">A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (109), (2016).</li><li>Okabe, S., Miwa, A., Okado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+the+C-terminal+domain+regulates+cell+surface+expression+of+the+NMDA+receptor+NR1+subunit.">Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit.</a> The Journal of Neuroscience. 19 (18), 7781-7792 (1999).</li><li>Okabe, S., Vicario-Abejon, C., Segal, M., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+synaptogenesis+of+hippocampal+neurons+without+NMDA+receptor+function+in+culture.">Survival and synaptogenesis of hippocampal neurons without NMDA receptor function in culture.</a> European Journal of Neuroscience. 10 (6), 2192-2198 (1998).</li></ol>

Vi utvecklade en reningsmetod för den dendritiska filopodia-rika fraktionen med hjälp av affinitet mellan celladhesionmolekylen TLCN och det extracellulära matrix proteinet vitronectin. Jämfört med PSD fraktion, det kan vara möjligt att identifiera de synaptiska proteiner som verkar på omogen synapsen från dendritiska filopodia-rika fraktionen. Sålunda, beståndsdelarna i dendritiska filopodia-rika fraktionen skiljer sig från de av PSD fraktion av 74%. Skiljer sig från PSD fraktion, vi använde odlade Hippocam...

Dendritic filopodia tros vara föregångare till SPINES. Actin glödtrådar i dendritiska filopodia reglera deras förlängning och indragning1,2,3. Efter kontakt med en Axon, valda dendritiska filopodia börjar sin mognad i taggar, och en synaps bildas4,5. Komponenterna i taggar har fastställts från omfattande analys av postsynaptiska densitet fraktioner6,7, medan komponenterna i dendritiska filopodia fortfarande i stort sett okända. Det har visats att telencephalin (tlcn), ERM, syngap, ras, PI3 kinas, akt, mTOR, Polo-liknande Kinas 2, camkii, syndecan-2, paralemin-1, ARF6, och EphB reglera dendritiska filopodia formation5,8,9 ,10,11, medan en metod inte har utvecklats för en omfattande analys av molekyler som finns i dendritiska filopodia.
Tlcn (ICAM-5) är uttryckligen uttrycks av taggig nervceller i de mest rostralt hjärn segmentet, den telencephalon12. TLCN har 9 ig-liknande domäner i sin extracellulära regionen, en transmembranregion, och en cytoplasmisk svans13. Tlcn binder till aktie (VN) och LFA-1 integrin i dess extracellulära regionen, till presenilin i dess transmembranregionen, och till Phospho-ERM och α-actinin i dess cytoplasmiska region5,8,14,15 ,16. Tlcn binder till aktin cytoskelettet genom Phospho-ERM på spetsarna av dendritiska filopodia och α-actinin i taggar och dendritiska axlar8,16.
Vi visade att överuttryck av tlcn förstärkt dendritiska filopodia formation och inducerade återgång av taggar till filopodia10. Den konstitutiva aktiva formen av Ezrin bunden till TLCN cytoplasmiska regionen och förstärkt dendritiska filopodia formation8. Sålunda reglerar TLCN dendritiska filopodia formation genom Actin-bindande proteiner. Esselens et al. visade att mikrokulor inducerad tlcn ackumulering på odlade nervceller17. Vi visade att fagocytiska Cup strukturer bildades på neuronala dendriter runt VN-belagda mikrokulor i en tlcn-beroende sätt15. Beståndsdelar i dendritiska filopodia liknar de i fagocytos Cup. Det är svårt att samla dendritiska filopodia, men det är relativt lättare att samla fagocytos kopp med magnetiska mikropärlor. Således utvecklade vi en metod för att rena fagocytos Cup istället för dendritiska filopodia18. Här beskriver vi reningsmetoden för den dendritiska filopodia-rika fraktionen.

<table>
	<tbody>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml Low Binding MCT</td>
			<td>Sorenson BioScience</td>
			<td>39640T</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>2530149</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic cell culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-actin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-Actinin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T-9026</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ezrin</td>
			<td>Sigma-Aldrich</td>
			<td>clone3C12, SAB4200806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Galphaq</td>
			<td>Santacruz</td>
			<td>sc-393</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>Chemicon</td>
			<td>clone AP20, MAB3418</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Moesin</td>
			<td>Sigma-Aldrich</td>
			<td>clone 38/87, M7060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PLCbeta1</td>
			<td>Santacuz</td>
			<td>sc-5291</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PSD95</td>
			<td>MA2</td>
			<td>ABR</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Spectrin beta</td>
			<td>Chemicon</td>
			<td>MAB1622</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Merck</td>
			<td>1.08122.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chrolide, hydrous</td>
			<td>Wako</td>
			<td>038-19735</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine beta-D-arabinofuranoside</td>
			<td>Sigma-Aldrich</td>
			<td>C-6645</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase-I</td>
			<td>Sigma-Aldrich</td>
			<td>DN-25</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P5155</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Thermo</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Prime Western Blotting Detection Reagent</td>
			<td>GE</td>
			<td>RPN2232</td>
			<td></td>
		</tr>
		<tr>
			<td>e-PAGEL 5-20% SDS-PAGE gradient gel</td>
			<td>ATTO</td>
			<td>E-T520L</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8758</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr>
			<td>i-Inositol</td>
			<td>Sigma-Aldrich</td>
			<td>I7508</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-1000 mini</td>
			<td>Fuji Film</td>
			<td>LAS-1000 mini</td>
			<td>For detection of luminescence from WB membrane</td>
		</tr>
		<tr>
			<td>Magnetic polystyrene microbeads</td>
			<td>Sperotech</td>
			<td>PM-20-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acid solution</td>
			<td>Gibco</td>
			<td>11130-051</td>
			<td>30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H2O, 20 mM L-Isoleucine, 20 mM L-Leucine, 19.8 mM L-Lysine hydrochloride, 5.1 mM L-Methionine, 10 mM L-Phenylalanine, 20 mM L-Threonine, 2.5 mM L-Tryptophan, 10 mM L-Tyrosine, and 20 mM L-Valine</td>
		</tr>
		<tr>
			<td>Mini-slab size electrophoresis system</td>
			<td>ATTO</td>
			<td>AE-6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Niacinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin / Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP phosphatase inhibitor cocktail</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Nacali</td>
			<td>28360-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P6155</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Sigma-Aldrich</td>
			<td>R9504</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain 2 Kit wako</td>
			<td>Wako</td>
			<td>291-5031</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-rad</td>
			<td>1703940JA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water</td>
			<td>MilliQ</td>
			<td></td>
			<td>For production of ultra pure water</td>
		</tr>
	</tbody>
</table>

purification of the dendritic filopodia-rich fraction

Dendritic filopodia är tunna och långa utskjutande bygger på aktin glödtråden, och de sträcker sig och dra som om du söker efter ett mål Axon. När dendritiska filopodia etablera kontakt med ett mål Axon, börjar de mognar i taggar, vilket leder till bildandet av en Synapse. Telencephalin (tlcn) är rikligt lokaliserad i dendritiska filopodia och gradvis utesluts från taggar. Överuttryck av TLCN i odlade Hippocampus nervceller inducerar dendritiska filopodia formation. Vi visade att telencephalin starkt binder till en extracellulär matrix molekyl, vitronectin. Vitronectin-belagda mikropärlor inducerad fagocytos kopp bildas på neuronala dendriter. I fagocytos Cup, tlcn, tlcn-bindande proteiner såsom fosforylerade Ezrin/radixin/moesin (Phospho-ERM), och F-Actin ackumuleras, vilket tyder på att komponenterna i fagocytos Cup liknar dendritiska filopodia. Således utvecklade vi en metod för rening av fagocytos Cup istället för dendritiska filopodia. Magnetisk polystyren pärlor var belagda med vitronectin, som är rikligt närvarande i kulturen medium Hippocampus nervceller och som inducerar fagocytos Cup formation på neuronala dendriter. Efter 24 h av inkubering, de fagocytos kopparna var milt solubilized med rengöringsmedel och samlas med hjälp av en magnet separator. Efter tvättning av pärlor, var bindande proteiner elueras och analyseras med silver färgning och Western blotting. I bindnings delen var tlcn och aktin rikligt närvarande. Dessutom var många proteiner som identifierats från fraktionen lokaliserade till dendritiska filopodia; Sålunda, vi kallade den bindande fraktionen som dendritiska filopodia-rika fraktion. Den här artikeln beskriver detaljer om reningsmetoden för den dendritiska filopodia-rika fraktionen.

I odlade Hippocampus nervceller, TLCN var rikligt lokaliserad till dendritiska filopodia, skaft, och Soma och samlokaliserade med F-Actin (figur 1a, B). När polystyren mikropärlor lades till odlade Hippocampus nervceller, var pärlorna automatiskt belagda med aktie (VN) härstammar från foster bovint serum (FBS) i odlingsmediet; de var främst bundna till dendriter, och de inducerade bildandet av fagocytos koppar (figu...

Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

Purification of the Dendritic Filopodia-rich Fraction

I detta protokoll introducerar vi en metod för rening av dendritiska filopodia-rika fraktionen från fagocytiska Cup-liknande utskjutande struktur på odlade Hippocampus nervceller genom att dra nytta av den specifika och starka samhörighet mellan en dendritiska filopodial adhesion molekyl, TLCN, och en extracellulär matris molekyl, vitronectin.

Rening av dendritiska Filopodia-rik fraktion

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Jämfört med PSD-fraktion kan det vara möjligt att identifiera det synaptiska protein som verkar på den omogna synapsen runt dendritiska filopodia-rika fraktionen. Vi använde levande neuron för att inducera fagocytisk kapsylbildning. Den största fördelen med denna teknik är att aktivt protein i artificiella uppkomsten skulle kunna identifieras från dendritiska filopodia rika fraktion.Till att börja med, förbereda 200x Vitamin Mix genom att lösa upp 100 mg vitamin B5, 100 mg kolinklorid, 100 mg folsyra, 180 mg I-inositol, 100 mg vitamin B3, 100 mg B6-vitaminhydroklorid, och 100 mg tiaminhydroklorid i 500 ml ultrarent vatten med hjälp av en magnetisk omrörare. Blanda försiktigt alikvot i 50 ml-rör och förvara den i 20 grader Celsius. För att förbereda riboflavinlösning, lös 100 mg riboflavin i 500 mg ultrarent vatten med hjälp av en magnetisk omrörare.Blanda försiktigt alikvot i 50 ml rör och förvara i 20 grader Celsius. För att förbereda en molar kalciumdiklorid lös 7,35 gram kalciumklorid dihydrat i 50 ml ultrapure vatten med hjälp av en magnetisk omrörare. För Minimum Essential Medium förberedelse lös 400 mg kaliumklorid 6, 800 mg sodumklorid, 2, 200 mg natriumbikarbonat, 158 mg natriumfosfatmonbasatet, 7, 000 mg D-glukos, och 200 mg magnesiumsulfatheptahydrat, och 950 ml ultrarent vatten, med hjälp av en magnetisk omrörare.Därefter använder en 1 ml-pipett med en konstant agitation på en magnetisk omrörare för att lägga till 1,8 ml 1 molar kalciumdiklorid till MEM i en droppe för droppe sätt. Tillsätt sedan saltsyra för att justera pH-värdet i MEM till pH 7.25. Tillsätt sedan 5 ml 200x vitaminblandning och 200 mikroliter riboflavinlösning till MEM.Justera volymen av lösningen till 1000 ml med ultrarent vatten. Filtrera lösningen med hjälp av ett 0,22 mikronfiltersystem och förvara den i 4 grader Celsius. För att förbereda 10x DNase-1 stamlösning lös 100 mg Dnase-1 i 12,5 ml HBSS.Filtrera genom ett 0,22 mikronfilter, alikvot i 1,5 ml-rör, och förvara rören i 20 grader Celsius. För Ara-C stamlösning beredning lösa 25 mg Ara-C i 8,93 ml ultrarent vatten. Filtrera genom ett 0,22 mikronfilter, alikvot i 1,5 ml-rör och förvara vid 20 grader Celsius.För att förbereda Plating Medium, blanda 1 ml MEM Amino Acid Solution, 750 mikroliter av 1 molar HEPES, 1 ml B27, 125 mikroliter på 200 millimolar glutamin, 250 mikroliter av penicillin-streptomycin, 2,5 ml av Fetalt bovin serum, och 44,375 ml MEM i ett 50 ml rör. För att förbereda stoppmediet, blanda 1 ml MEM aminosyralösning, 750 mikroliter av 1 molar HEPES, 5 ml FBS, och 43,25 ml MEM i ett 50 ml rör. För att förbereda HBSS, kompletterat med 15 millimolar HEPES, blanda 49,25 ml HBSS, och 750 mikroliter av 1 molar HEPES.För poly-l-lysinbelagda rätter beredning, coat 35 mm plast cell kultur rätter med 0,2 mg per ml poly-l-lysin hydrobromid för en dag vid 25 grader Celsius. Därefter diska med 2 ml ultrarent vatten tre gånger och inkubera dem med 1,5 ml Stop Medium vid 25 grader Celsius tills användning. Inkubera den dissekerade hippocampi i en blandning som innehåller 500 mikroliter vardera av 2,5%trypsin och 10X Dnase-1 i HBSS kompletterad med 15 millimolar HEPES, pH 7.2, vid 37 grader Celsius i 15 minuter med agitation var 3:e minut.Överför sedan hippocampi till 10 ml av Stop Medium och inkubera vid 4 grader Celsius i 5 minuter för att inaktivera trypsin. Därefter inkubera dissekerade hippocampi i 10 ml färskt stopp medium vid 4 grader Celsius i 5 minuter. Flytta hippocampi i 10 ml färskt stoppmedium och inkubera vid 4 grader Celsius i ytterligare 5 minuter.Flytta sedan hippocampi till 900 mikroliter av stoppmediet och 100 mikroliter av 10X Dnase-1 i ett 15 ml-rör. Använd en 1 ml pipet för att separera hippocampi i isolerade nervceller genom pipettering 20 gånger. Tillsätt 9 ml plätering medium, och filtrera genom en 70 mikron cell sil, i en 50 ml rör.Räkna antalet celler med hjälp av en hemocytometer och en justera till 3,5 gånger 10 till fjärde celler per milliliter i plätering medium. Därefter aspirera stoppmediet från poly-l-lisinbelagda rätter. Plate 2 ml per maträtt av cellerna på de belagda rätterna och inkubera under 5%koldioxid vid 37 grader Celsius i 60-64 timmar.Efter inkubationen, tillsätt 2 mikroliter av Ara-C stamlösning till nervcellerna och skaka skålen långsamt. Förvara kulturrätterna i en fuktad låda utan att ändra odlingsmediet under 5 %koldioxid vid 37 grader Celsius. Rena den dendritiska filopodia rik fraktion efter 13 dagar in vitro genom att först lägga till tre miljoner magnetiska polystyren mikropärlor per maträtt till 20 rätter som innehåller odlade nervceller.Efter en dag, tvätta nervcellerna i 1 ml PBS med agiation tre gånger för att ta bort medium och obundna microbeads. Efter avlägsnande PBS, lysa nervceller med 500 mikroliter per maträtt av lysbuffert. Därefter samla lysate med en cell skrapa och överföra lysate till 10 låg proteinbindande mikrorör.Ställ in rören på en magnetavskiljare och vänta i en minut. Samla supernatanten och använd den som den obundna fraktionen för silverfärgning och Western blot-analys. Därefter överför pärlorna till en ny lågproteinbindande mikrorör och ställ på en magnetisk separater i en minut.Ta helt bort supernatanten, och tillsätt 500 mikroliter av lysbufferten. Tvätta pärlorna med hjälp av en Vortex-mixer i 15 sekunder. Upprepa tvättningen av pärlorna 10 gånger och ta bort supernatanten.Elute proteiner ner till pärlorna genom tillsats av 50 mikroliter av 1X SDS provbuffert och koka röret vid 98 grader Celsius i 5 minuter. Centrifugera röret vid 860xG 3000 RPM i 10 sekunder och ställ in röret på en magnetisk separater i en minut. Samla upp supernatanten och använd den som bunden fraktion.Mät koncentrationer av de obundna och bundna proteinfraktionerna av BCA-proteinanalysen. Visualisera protienlösningar med bromophenylblått och justera koncentrationen till 5 nanogram per mikroliter för SDS-sida. Separera de bundna och obundna bråken efter SDS-sida med hjälp av en 5-20%toningsgel.Silverfläck gelen. Slutligen, Western blot med hjälp av lämpliga primära och sekundära antikroppar enligt manuskriptet. I odlade hippocampus nervceller, TLCN var rikligt lokaliserade till dendritic filopodia, skaft och soma och co-lokaliserade med F-aktin.De kodade pärlor var huvudsakligen bundna till dendrites och inducera bildandet av fagocytic koppar genom ackumulering av TLCN och F-aktin på neuronal dendrites. Thorecense bilder visade att phagocytic kopp bildandet var avgörande beroende av förekomsten av TLCN i dendrites. De fagocytic koppar bildades endast på vilda typ hippocampus nervceller, men inte på TLCN defficient hippocampal nervceller.SDS-sidans resultat visade att proteinbandsmönstren var nästan desamma för de obundna och bundna fraktionerna men intensiteterna vid 50 och 70 kilodalton, i den bundna fraktionen var lägre än de i den obundna fraktionen. Bandet intensitet var dock inte uppenbart annorlunda mellan obundna och bundna fraktioner som utarbetats från TLCN defficient kultur hippocampus nervceller. Western blot analys av de obundna och bundna fraktioner visade att TLCN och VN främst upptäcktes i den bundna fraktionen.Actin, Ezrin, G alpha q, PLC beta 1, MAP-2 och Spectrin, upptäcktes i både de bundna och obundna fraktionerna. Moesin, PSD-95, alpha-Actinin och alpha-Tubulin upptäcktes i obundna fraktion. Om beläggning protein av mikropärlorna förändras, kan denna procedur tillämpas för att identifiera analys interaktion.Denna teknik skulle kunna identifiera proteinet som arbetar med konstgjorda ämnen. Så vi tror att mekanismen konstgjorda uppkomsten kan kvantifieras. Vi tror att nya protein som är associerade med psykisk störning kan identifieras från dendritiska filopodia-rika fraktionen.Och kan utvidgas till terapin.

purification-of-the-dendritic-filopodia-rich-fraction

Research

JoVE Journal

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Analys av Dendritiska Spine Morfologi i odlade CNS Neuroner

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induktion Dendritic Tillväxten i Odlade sympatiska nervceller

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Analysen av Purkinje Cell Dendritic Morfologi i Organotypiska slice kulturer

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Snabb Micro-iontophoresis av glutamat och GABA: Ett användbart verktyg för att undersöka Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Bedömning av dendritiska arborization i gyrus dentatus i hippocampus regionen hos möss

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Rening av Mouse Brain Fartyg

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analysera Dendritic morfologi i kolumner och lager

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Bedömning av hippocampal dendritisk komplexitet hos åldrade möss med användning av Golgi-Cox-metoden

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

Realtidsanalytik med tetrametylammonium för att kvantifiera volymfraktioner och tortuositet i hjärnens extracellulära utrymme

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Kvantitativ analys av neuronala dendritiska Arborization komplexitet i Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...