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 takker Shigeo Okabe og Hitomi Matsuno for den lave-density kultur af hippocampus neuroner, Masayoshi Mishina for tlcn-mangelfulde mus, Sachiko Mitsui og Momoko shiozaki for teknisk assistance, og medlemmer af yoshihara laboratorium for nyttige diskussioner . Dette arbejde blev støttet af JSPS KAKENHI Grant NOS. JP20700307, JP22700354 og JP24500392 og MEXT KAKENHI Grant NOS. JP23123525 til YF og JP20022046, JP18H04683 og JP18H05146 til åå.

Alle metoder, der er beskrevet her, er blevet godkendt af den institutionelle dyrepleje-og anvendelses Komité for RIKEN Wako.
1. kultur af hippocampus neuroner
<ol><li>Forberedelse af kulturmedium
	<ol>
<li>Fremstilling af 200x vitamin mix. Opløse 100 mg af D-pantothensyre hemicalcium salt, 100 mg cholinchlorid, 100 mg folinsyre, 180 mg af i-inositol, 100 mg af niacinamid, 100 mg af PYRIDOXAL HCl, og 100 mg thiamin HCL i 500 ml af ultrarent vand ved hjælp af en magnetisk ...

<ol>
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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. 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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. 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Vi udviklede en rensningsmetode for dendritiske filopodia-rige fraktion ved hjælp af affinitet mellem celle adhæsions molekyle TLCN og det ekstracellulære matrix protein vitronectin. Sammenlignet med PSD-fraktion kunne det være muligt at identificere de synaptiske proteiner, der optræder på den umodne synapse fra den dendritiske filopodia-rige fraktion. Bestanddelene i den dendritiske filopodia-rige fraktion adskiller sig således fra dem i PSD-fraktionen med 74%. Forskellig fra PSD fraktion, vi brugte dyrkede hipp...

Dendritiske filopodia menes at være forløbere for spines. Actin filamenter i dendritiske filopodia regulerer deres forlængelse og tilbagetrækning1,2,3. Efter kontakt med en Axon begynder udvalgte dendritiske filopodia deres modning i spines, og en synapse dannes4,5. Komponenterne i Spiner er blevet fastlagt ud fra omfattende analyse af postsynaptiske massefylde fraktioner6,7, mens komponenterne i dendritiske filopodia er stort set ukendte. Det har vist sig, at telencepin (tlcn), ERM, syngap, RAS, PI3 kinase, akt, MTOR, Polo-like kinase 2, camkii, syndecan-2, paralemin-1, ARF6 og ephb regulerer dendritiske filopodia dannelse5,8,9 ,10,11, mens en metode ikke er blevet udviklet til den omfattende analyse af molekyler, der findes i dendritiske filopodia.
TLCN (ICAM-5) udtrykkes specifikt af spiny neuroner i det mest rostrale hjerne segment, telencephalon12. Tlcn har 9 IG-lignende domæner i sin ekstracellulære region, en transmembran region, og en cytoplasmisk hale13. Tlcn binder sig til vitronectin (vn) og LFA-1 anti i dets ekstracellulære region, til presenilin i dets transmembran-region, og til phospho-ERM og α-actinin i dets cytoplasmiske region5,8,14,15 ,16. Tlcn binder sig til det cytoskelet actin gennem fosho-ERM ved spidserne af dendritiske filopodia og α-actinin i spines og dendritiske aksler8,16.
Vi viste, at overekspression af TLCN forbedret dendritiske filopodia dannelse og induceret omversion af spines til filopodia10. Den konstitutive aktive form af PMS bundet til tlcn cytoplasmiske region og forbedret dendritiske filopodia dannelse8. Således regulerer TLCN dendritiske filopodia dannelse gennem actin bindende proteiner. Esselens et al. påviste, at mikroperler inducerede TLCN-akkumulering på dyrkede neuroner17. Vi viste, at fagocytiske Cup strukturer blev dannet på neuronal dendritter omkring VN-belagte mikroperler i en TLCN-afhængige måde15. Bestanddele af dendritiske filopodia svarer til de fagocytiske bæger. Det er svært at indsamle dendritiske filopodia, men det er relativt lettere at indsamle den fagocytiske kop ved hjælp af magnetiske mikroperler. Således udviklede vi en metode til at rense den fagocytiske kop i stedet for dendritiske filopodia18. Her beskriver vi rensningsmetoden for den dendritiske filopodia-rige fraktion.

<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

Dendritiske filopodia er tynde og lange fremspring baseret på actin filament, og de udvider og trækker som om at søge efter et mål Axon. Når dendritiske filopodia etablerer kontakt med et mål Axon, begynder de at modnes i spines, hvilket fører til dannelsen af en synapse. Telencephalin (TLCN) er rigeligt lokaliseret i dendritiske filopodia og er gradvist udelukket fra spines. Overekspression af TLCN i dyrkede hippocampus neuroner inducerer dendritiske filopodia dannelse. Vi viste, at telencepin stærkt binder sig til en ekstracellulær matrix molekyle, vitronectin. Vitronectin-coatede mikroperler induceret Fagocytisk kop dannelse på neuronal dendritter. I fagocytic Cup akkumuleres tlcn-bindende proteiner såsom phosphoryleret Ezrin/Radixin/Moesin (phospho-ERM) og F-actin, hvilket tyder på, at komponenterne i det fagocytiske bæger ligner dem af dendritiske filopodia. Således udviklede vi en metode til rensning af den fagocytiske kop i stedet for dendritiske filopodia. Magnetiske polystyren perler blev belagt med vitronectin, som er rigeligt til stede i kulturen medium hippocampus neuroner og som inducerer Fagocytisk kop dannelse på neuronal dendritter. Efter 24 timers inkubation var de fagocytiske kopper mildt opløste med vaskemiddel og indsamlet ved hjælp af en magnet separator. Efter vask af perlerne blev de bindende proteiner elueret og analyseret ved sølvfarvning og vestlig blotting. I bindings brøken var TLCN og actin rigeligt til stede. Desuden blev mange proteiner identificeret fra fraktionen lokaliseret til dendritiske filopodia; således, vi navngiver bindende fraktion som dendritiske filopodia-rige fraktion. I denne artikel beskrives detaljer om rensningsmetoden for den dendritiske filopodia-rige fraktion.

I dyrkede hippocampus neuroner, TLCN var rigeligt lokaliseret til dendritiske filopodia, aksel, og Soma og colokeret med F-actin (figur 1a, B). Når polystyren mikroperler blev tilsat til dyrkede hippocampus neuroner, blev perlerne automatisk belagt med vitronectin (VN) afledt af føtal bovint serum (FBS) i dyrkningsmediet; de var hovedsageligt bundet til dendritter, og de inducerede dannelsen af fagocytiske kopper (figur...

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Purification of the Dendritic Filopodia-rich Fraction

I denne protokol introducerer vi en metode til rensning af dendritiske filopodia-rige fraktion fra den fagocytiske Cup-lignende fremspringende struktur på dyrkede hippocampus neuroner ved at drage fordel af den specifikke og stærke affinitet mellem en dendritisk filopodial adhæsions molekyle, TLCN og et ekstracellulær matrix molekyle, vitronectin.

Rensning af den dendritiske Filopodia-rige 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 ...

Sammenlignet med PSD fraktion, kan det være muligt at identificere det synaptiske protein, der handler på den umodne synapse omkring dendritiske filopodia-rige fraktion. Vi brugte levende neuron til at fremkalde fagocytisk cap dannelse. Den største fordel ved denne teknik er, at aktivt protein i kunstige tilblivelse kunne identificeres fra dendritiske filopodia rige fraktion.Til at begynde, forberede 200x Vitamin Mix ved at opløse 100 mg vitamin B5, 100 mg cholinchlorid, 100 mg folinsyre, 180 mg I-inositol, 100 mg Vitamin B3, 100 mg Vitamin B6-hydrochlorid og 100 mg thiaminhydrorid i 500 ml ultrapurevand ved hjælp af en magnetomrører. Bland forsigtigt aliquot i 50 ml rør og opbevar det ved 20 grader Celsius. For at forberede riboflavin opløsning, opløses 100 mg riboflavin i 500 mg ultrapure vand ved hjælp af en magnetisk omrører.Bland forsigtigt aliquot i 50 ml rør og opbevares ved 20 grader Celsius. Til fremstilling af en molar calciumdichlorid opløses 7,35 gram calciumchloriddihydrat i 50 ml ultrarent vand ved hjælp af et magnetisk omrører. For minimum essentielt middelpræparat opløses 400 mg kaliumchlorid 6 800 mg sodumchlorid, 2, 200 mg natriumbikarbonat, 158 mg natriumphosphatmonobasic dihydrat, 7, 000 mg D-glucose og 200 mg magnesiumsulfat heptahydrat og 950 ml ultrapurtvand ved hjælp af en magnetomrører.Dernæst skal du bruge en 1 ml pipette med en konstant omrøring på en magnetisk omrører til at tilsætte 1,8 ml 1 molar calciumdichlorid til MEM i en dråbe for dråbe måde. Derefter tilsættes saltsyre for at justere pH-koncentrationen af MEM til pH 7,25. Derefter tilsættes 5 ml 200x vitamin mix og 200 mikroliter riboflavin opløsning til MEM.Opløsningens volumen justeres til 1000 ml med ultrarent vand. Filtrere opløsningen ved hjælp af en 0,22 mikron filter system og gemme den ved 4 grader Celsius. Til fremstilling af 10x DNase-1 stock opløsning opløses 100 mg Dnase-1 i 12,5 ml HBSS.Filtrer gennem et 0,22 mikron filter, aliquot i 1,5 ml rør, og opbevar rørene ved 20 grader Celsius. Til Ara-C-beholdning opløses 25 mg Ara-C i 8,93 ml ultrarent vand. Filter gennem en 0,22 mikron filter, aliquot i 1,5 ml rør, og opbevares ved 20 grader Celsius.For at forberede Plating Medium, 1 ml MEM Aminosyreopløsning, 750 mikroliter af 1 molar HEPES, 1 ml B27, 125 mikroliter af 200 millimolarglglamin, 250 mikroliter penicillin-streptomycin, 2,5 ml føtalt kvægserum og 44,375 ml MEM i et 50 ml tube. For at klargøre stopmediet blandes 1 ml MEM-aminosyreopløsning, 750 mikroliter af 1 molar HEPES, 5 ml FBS og 43,25 ml MEM i et 50 ml rør. Til fremstilling af HBSS, suppleret med 15 millimolar HEPES, blandes 49,25 ml HBSS og 750 mikroliter af 1 molar HEPES.Til fremstilling af poly-l-lysinbelagte retter, pels 35 mm plastcellekulturretter med 0,2 mg pr. ml poly-l-lysin hydrobromid for en dag ved 25 grader Celsius. Dernæst vaskes skålene med 2 ml ultrarent vand tre gange og inkuberes dem med 1,5 ml Stop Medium ved 25 grader Celsius, indtil det bruges. Inkubere de dissekerede hippocampi i en blanding, der indeholder 500 mikroliter hver af 2,5% trypsin og 10X Dnase-1 i HBSS suppleret med 15 millimolar HEPES, pH 7,2, ved 37 grader Celsius i 15 minutter med omrøring hvert 3 minut.Derefter overføre hippocampi til 10 ml af Stop Medium og inkubere ved 4 grader Celsius i 5 minutter for at inaktivere trypsin. Dernæst inkubere de dissekeret hippocampi i 10 ml frisk stop medium ved 4 grader Celsius i 5 minutter. Flyt hippocampi i 10 ml frisk stop medium og inkubere ved 4 grader Celsius i yderligere 5 minutter.Flyt derefter hippocampi i 900 mikroliter af stopmediet og 100 mikroliter på 10X Dnase-1 i et 15 ml rør. Brug en 1 ml pipette til at adskille hippocampi i isolerede neuroner ved pipettering 20 gange. Tilsæt 9 ml plating medium, og filtreres gennem en 70 mikron celle si, i en 50 ml rør.Tæl antallet af celler ved hjælp af et hæmocytometer og en justering til 3,5 gange 10 til de fjerde celler pr. milliliter i plating medium. Dernæst aspirere stopmediet fra poly-l-lisine belagte retter. Plade 2 ml pr fad af cellerne på de belagte retter og inkuberes under 5% kuldioxid ved 37 grader Celsius i 60-64 timer.Efter inkubationen tilsættes 2 mikroliter Ara-C-lageropløsning til neuronerne og ryst skålen langsomt. Hold kulturretterne i en befugtet kasse uden at ændre kulturmediet under 5% kuldioxid ved 37 grader Celsius. Rens dendritiske filopodia rige fraktion efter 13 dage in vitro ved først at tilføje tre millioner magnetiske polystyren mikroperler pr fad til 20 retter, der indeholder de dyrkede neuroner.Efter en dag vaskes neuronerne i 1 ml PBS med agiation tre gange for at fjerne de mellemstore og ubundne mikroperler. Efter fjernelse af PBS, lyse neuroner med 500 mikroliter pr fad af lysis buffer. Dernæst indsamles lysatet med en celleskraber og overføre lysatet i 10 lavproteinbindende mikrorør.Sæt rørene på en magnet adskille og vente i et minut. Saml supernatant og bruge det som ubundet fraktion for sølv farvning og vestlige blot analyse. Dernæst overføre perlerne til en ny lav protein bindende mikrorør og sæt på en magnetisk separatr i et minut.Fjern helt supernatanten, og tilsæt 500 mikroliter af lysisbufferen. Vask perlerne ved hjælp af en Vortex mixer i 15 sekunder. Gentag vask af perlerne 10 gange og fjern supernatant.Elute proteiner ned til perlerne ved tilsætning af 50 mikroliter af 1X SDS prøve buffer og kog røret ved 98 grader Celsius i 5 minutter. Centrifuge røret ved 860xG 3000 RPM i 10 sekunder og sæt røret på en magnetisk separatr i et minut. Saml supernatant og bruge det som den bundne fraktion.Indtag koncentrationerne af de ubundne og bundne proteinfraktioner ved hjælp af BCA-proteinanalysen. Visualiser protienopløsninger med bromophenylblå og juster koncentrationen til 5 nanogram pr. mikroliter til SDS-side. Adskærg de bundne og ubundne brøker efter SDS-side ved hjælp af en 5-20%gradientgel.Sølvplette gelen. Endelig western blot ved hjælp af passende primære og sekundære antistoffer i henhold til manuskriptet. I dyrkede hippocampale neuroner, var TLCN rigeligt lokaliseret til dendritiske filopodia, aksel, og soma og co-lokaliseret med F-actin.De kodede perler var hovedsageligt bundet til dendritter og fremkalde dannelsen af fagocytiske kopper ved akkumulering af TLCN og F-actin på neuronal dendritter. Thorecense billeder viste, at fagocytiske kop dannelse var afgørende afhængig af tilstedeværelsen af TLCN i dendritter. De fagocytiske kopper blev kun dannet på vilde type hippocampal neuroner, men ikke på TLCN defficient hippocampal neuroner.SDS-sideresultaterne viste, at proteinbåndmønstrene var næsten de samme for de ubundne og bundne fraktioner, men intensiteterne ved 50 og 70 kilodalton, i den bundne fraktion, var lavere end i den ubundne fraktion. Men bandet intensitet var ikke naturligvis forskellig mellem de ubundne og bundet fraktioner udarbejdet fra TLCN defficient kultur hippocampal neuroner. Western blot-analysen af de ubundne og bundne fraktioner viste, at TLCN og VN hovedsagelig blev påvist i den bundne fraktion.Actin, Ezrin, G alpha q, PLC beta 1, MAP-2 og Spectrin blev påvist i både de bundne og ubundne fraktioner. Moesin, PSD-95, alpha-Actinin og alpha-Tubulin blev opdaget i den ubundne fraktion. Hvis belægning protein af mikroperler ændre, denne procedure kan anvendes til at identificere assay interaktion.Denne teknik kunne identificere det protein, der virker på kunstige stoffer. Så vi mener, at mekanismen kunstig tilblivelse kan kvantificeres. Vi mener, at nyt protein forbundet med psykisk lidelse kan identificeres fra dendritiske filopodia-rige fraktion.Og kan udvides til terapi.

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.

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.

Fremkalde dendritisvækst i dyrkede sympatiske neuroner

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.

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.

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge 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.

Vurdering af dendritiske arborization i gyrus dentatus i hippocampus-regionen i mus

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.

Oprensning af musehjerne Fartøjer

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.

Analyse Dendritiske Morfologi i Kolonner og Lag

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.

Vurdering af hippocampal dendritisk kompleksitet hos gamle mus ved anvendelse af 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.

Real-time iontophorese med tetramethylammonium til kvantificering af volumen fraktion og tortuosity af hjernens ekstracellulære rum

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 analyse af Neuronal dendritiske Arborization kompleksitet i Drosophila

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