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 lav tetthet kultur hippocampus neurons, Masayoshi Mishina for TLCN-mangelfull mus, Sachiko Mitsui og Momoko Shiozaki for teknisk assistanse, og medlemmer av Yoshihara laboratoriet for nyttige diskusjoner . Dette arbeidet ble støttet av JSP KAKENHI Grant NOS. JP20700307, JP22700354 og JP24500392 og MEXT KAKENHI Grant NOS. JP23123525 til YF og JP20022046, JP18H04683 og JP18H05146 til åå.

Alle metoder beskrevet her over ha blitt anerkjent av det institusjonell dyr bekymre og bruk komité av RIKEN Wako.
1. kultur hippocampus neurons
<ol><li>Utarbeidelse av kultur medium
	<ol>
<li>Utarbeidelse av 200x vitamin mix. Oppløse 100 mg av D-Pantotensyre acid hemicalcium salt, 100 mg av kolin klorid, 100 mg folsyre, 180 mg av i-Inositol, 100 mg av Niacinamide, 100 mg pyridoxal HCl, og 100 mg av Tiamin HCl i 500 mL av ultrarent vann ved hjelp av en magnetisk rører. L?...

<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. 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 utviklet en rensing metode for dendrittiske filopodia-rike brøkdel ved hjelp av affinitet mellom celle vedheft molekylet TLCN og ekstracellulære Matrix protein vitronectin. Sammenlignet med PSD brøkdel, kan det være mulig å identifisere Synaptic proteiner opptrer på umodne synapse fra dendrittiske filopodia-rik brøk. Dermed er bestanddelene av dendrittiske filopodia-rik brøk er forskjellig fra de av PSD brøkdel av 74%. Forskjellig fra PSD brøkdel, brukte vi kultivert hippocampus neurons å aktivt danne phago...

Dendrittiske filopodia antas å være forløpere for pigger. Utgangen filamenter i dendrittiske filopodia regulere sin forlengelse og uttrekk1,2,3. Etter å ha kontaktet en axon, valgte dendrittiske filopodia begynne sin modning i pigger, og en synapse er dannet4,5. Komponenter av pigger har blitt bestemt fra omfattende analyse av postsynaptic tetthet fraksjoner6,7, mens komponenter av dendrittiske filopodia fortsatt i stor grad ukjent. Det har blitt vist at telencephalin (TLCN), erm, SynGAP, RAS, PI3 kinase, akt, mTOR, Polo-like kinase 2, CaMKII, syndecan-2, paralemin-1, ARF6, og EphB regulere dendrittiske filopodia formasjon5,8,9 ,10,11, mens en metode ikke er utviklet for den omfattende analyse av molekyler til stede i dendrittiske filopodia.
TLCN (ICAM-5) er spesielt uttrykt ved spiny neurons i de mest rostral hjernen segmentet, Telencephalon12. TLCN har 9 IG-lignende domener i sin ekstracellulære region, en transmembrane region, og en cytoplasmatiske hale13. TLCN binder seg til vitronectin (vn) og LFA-1 integrin i sin ekstracellulære region, til presenilin i sin transmembrane region, og til fosfat-erm og α-actinin i sin cytoplasmatiske region5,8,14,15 ,16. TLCN binder seg til utgangen cytoskeleton gjennom fosfat-erm på tuppen av dendrittiske filopodia og α-actinin i pigger og dendrittiske aksler8,16.
Vi viste at overuttrykte av TLCN forbedret dendrittiske filopodia formasjon og indusert reversion av pigger til filopodia10. Den konstituerende aktive formen av Ezrin bundet til TLCN cytoplasmatiske regionen og forbedret dendrittiske filopodia formasjon8. Således regulerer TLCN dendrittiske filopodia formasjon gjennom utgangen-bindende proteiner. Esselens et al. demonstrerte at mikroperler indusert TLCN akkumulering på kultivert neurons17. Vi viste at phagocytic Cup strukturer ble dannet på neuronal dendrites rundt VN-belagt mikroperler i en TLCN-avhengig måte15. Bestanddeler av dendrittiske filopodia er lik de av phagocytic koppen. Det er vanskelig å samle dendrittiske filopodia, men det er relativt lettere å samle phagocytic koppen ved hjelp av magnetisk mikroperler. Dermed har vi utviklet en metode for å rense phagocytic koppen i stedet for dendrittiske filopodia18. Her beskriver vi rensing metoden for dendrittiske filopodia-rik brøk.

<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

Dendrittiske filopodia er tynne og lange utstikkende basert på utgangen filament, og de strekker og trekker seg som om du søker etter et mål axon. Når dendrittiske filopodia etablere kontakt med en mål axon, begynner de å modnes i pigger, noe som fører til dannelsen av en synapse. Telencephalin (TLCN) er rikelig lokalisert i dendrittiske filopodia og blir gradvis ekskludert fra pigger. Overuttrykte av TLCN i kultivert hippocampus neurons induserer dendrittiske filopodia formasjon. Vi viste at telencephalin sterkt binder seg til en ekstracellulære matrise molekyl, vitronectin. Vitronectin-belagt mikroperler indusert phagocytic kopp formasjon på neuronal dendrites. I phagocytic Cup, TLCN, TLCN-bindende proteiner som fosforylert Ezrin/Radixin/Moesin (fosfat-ERM), og F-utgangen er akkumulert, noe som tyder på at komponentene i phagocytic koppen er lik de av dendrittiske filopodia. Derfor utviklet vi en metode for rensing av phagocytic koppen i stedet for dendrittiske filopodia. Magnetiske polystyren perler var belagt med vitronectin, som er rikelig til stede i kulturen medium for hippocampus neurons og som induserer phagocytic kopp formasjon på neuronal dendrites. Etter 24 h av inkubasjons, phagocytic koppene var mildt solubilized med vaskemiddel og samlet inn ved hjelp av en magnet separator. Etter vask av perlene, ble de bindende proteinene eluert og analysert av sølv flekker og vestlig blotting. I bindingen brøk, TLCN og utgangen var rikelig til stede. I tillegg ble mange proteiner identifisert fra fraksjonen lokalisert til dendrittiske filopodia; dermed kalte vi bindingen brøk som dendrittiske filopodia-rik brøk. Denne artikkelen beskriver detaljer om rensing metoden for dendrittiske filopodia-rik brøk.

I kulturperler hippocampus neurons, var TLCN rikelig lokalisert til dendrittiske filopodia, aksel, og Soma og colocalized med F-utgangen (figur 1a, B). Når polystyren mikroperler ble lagt til kultivert hippocampus neurons, ble perlene automatisk belagt med vitronectin (VN) avledet fra fosterets storfe serum (FBS) i kulturen medium; de var hovedsakelig bundet til dendrites, og de induserte dannelsen av phagocytic kopper (...

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Purification of the Dendritic Filopodia-rich Fraction

I denne protokollen introduserer vi en metode for rensing av dendrittiske filopodia fraksjon fra phagocytic Cup-lignende protrusion struktur på kultivert hippocampus neurons ved å utnytte den spesifikke og sterke affinitet mellom en dendrittiske filopodial vedheft molekyl, TLCN, og en ekstracellulære matrise molekyl, vitronectin.

Rensing av dendrittiske Filopodia-rik brøk

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-fraksjonen kan det være mulig å identifisere det synaptiske proteinet som virker på den umodne synapsen rundt dendrittiske filopodiarike fraksjonen. Vi brukte levende nevron for å indusere fagocytisk hettedannelse. Den største fordelen med denne teknikken er at aktivt protein i kunstig genese kan identifiseres fra dendrittiske filopodia rik fraksjon.For å begynne, forberede 200x vitamin mix ved å oppløse 100 mg vitamin B5, 100 mg kolin klorid, 100 mg folsyre, 180 mg I-inositol, 100 mg vitamin B3, 100 mg vitamin B6 hydroklorid, og 100 mg tiaminhydroklorid i 500 ml ultrapure vann ved hjelp av en magnetisk røre. Bland aliquot forsiktig i 50 ml rør og oppbevar den ved 20 grader Celsius. For å forberede riboflavin oppløsning, oppløs 100 mg riboflavin i 500 mg ultrarent vann ved hjelp av en magnetisk rører.Bland aliquot forsiktig i 50 ml rør og oppbevar ved 20 grader Celsius. For å forberede en molar kalsiumdiklorid oppløse 7,35 gram kalsiumkloriddihydrat i 50 ml ultrapure vann ved hjelp av en magnetisk rører. For minimum essensiell medium preparat oppløses 400 mg kaliumklorid 6, 800 mg sodumklorid, 2, 200 mg natriumbikarbonat, 158 mg natriumfosfatmonobasisk dihydrat, 7, 000 mg D-glukose og 200 mg magnesiumsulfatheptahydrat og 950 ml ultrarent vann, ved hjelp av magnetisk rører.Deretter bruker du en 1 ml pipette med en konstant agitasjon på en magnetisk rører for å tilsette 1,8 ml 1 molar kalsiumdiklorid til MEM i en dråpe for dråpe. Tilsett deretter saltsyre for å justere pH-en til pH 7,25. Deretter legger du til 5 ml 200x vitaminblanding og 200 mikroliter riboflavin-oppløsning til MEM.Juster volumet volumet av oppløsningen til 1000 ml med ultrarent vann. Filtrer løsningen ved hjelp av et 0,22 mikron filtersystem og lagre den ved 4 grader Celsius. For å forberede 10x DNase-1 lagerløsning oppløses 100 mg Dnase-1 i 12,5 ml HBSS.Filtrer gjennom et 0,22 mikronfilter, aliquot i 1,5 ml rør, og lagre rørene ved 20 grader Celsius. For Ara-C lager oppløsning forberedelse oppløse 25 mg Ara-C i 8,93 ml ultrapure vann. Filtrer gjennom et 0,22 mikron filter, aliquot i 1,5 ml rør, og lagre på 20 grader Celsius.For å forberede platingmediet, bland 1 ml MEM aminosyreoppløsning, 750 mikroliter med 1 molar HEPES, 1 ml B27, 125 mikroliter på 200 millimolar glutamin, 250 mikroliter med penicillin-streptomycin, 2,5 ml fetal storfe serum og 44,375 ml MEM i et 50 ml rør. For å forberede stoppmediet, bland 1 ml MEM aminosyreoppløsning, 750 mikroliter med 1 molar HEPES, 5 ml FBS og 43,25 ml MEM i et 50 ml rør. For å forberede HBSS, supplert med 15 millimolar HEPES, bland 49,25 ml HBSS og 750 mikroliter med 1 molar HEPES.For poly-l-lysinbelagte retter forberedelse, coat 35 mm plast celle kultur retter med 0,2 mg per ml poly-l-lysin hydrobromid for en dag på 25 grader Celsius. Deretter vasker du oppvasken med 2 ml ultrarent vann tre ganger og inkuberer dem med 1,5 ml Stop Medium ved 25 grader Celsius til bruk. Inkuber den dissekerte hippocampi i en blanding som inneholder 500 mikroliter hver av 2,5% trypsin og 10X Dnase-1 i HBSS supplert med 15 millimolar HEPES, pH 7,2, ved 37 grader Celsius i 15 minutter med agitasjon hvert tredje minutt.Deretter overfører hippocampi til 10 ml av Stop Medium og inkuberer ved 4 grader Celsius i 5 minutter for å inaktivere trypsin. Deretter inkuberer du den dissekerte hippocampi i 10 ml friskt stoppmedium ved 4 grader Celsius i 5 minutter. Flytt hippocampi i 10 ml friskt stoppmedium og inkuber ved 4 grader Celsius i ytterligere 5 minutter.Deretter flytter du hippocampi inn i 900 mikroliter av stoppmediet og 100 mikroliter med 10X Dnase-1 i et 15 ml rør. Bruk en 1 ml pipette til å ta avstand fra hippocampi i isolerte nevroner ved å pipettering 20 ganger. Tilsett 9 ml plating medium, og filtrer gjennom en 70 mikron celle sil, i en 50 ml rør.Telle antall celler ved hjelp av et hemocytometer og en justering til 3,5 ganger 10 til de fjerde cellene per milliliter i plating medium. Deretter aspirerer du stoppmediet fra poly-l-lisinebelagte retter. Plate 2 ml per tallerken av cellene på belagte retter og inkubere under 5% karbondioksid ved 37 grader Celsius i 60-64 timer.Etter inkubasjonen, tilsett 2 mikroliter Ara-C lagerløsning til nevronene og rist parabolen sakte. Hold kulturrettene i en fuktet boks uten å endre kulturmediet under 5% karbondioksid ved 37 grader Celsius. Rens den dendrittiske filopodiarike fraksjonen etter 13 dager in vitro ved først å legge til tre millioner magnetiske polystyrenmikroperler per tallerken til 20 retter som inneholder de kultiverte nevronene.Etter en dag vask nevronene i 1 ml PBS med agiation tre ganger for å fjerne middels og ubundet mikroperler. Etter å ha fjernet PBS, lyse nevronene med 500 mikroliter per tallerken av lysis buffer. Deretter samler du lysatet med en celleskraper og overfører lysatet til 10 lave proteinbindende mikrorør.Sett rørene på en magnetseparer og vent i ett minutt. Samle supernatant og bruke den som den ubundne fraksjonen for sølvfarging og vestlig flekkanalyse. Deretter overfører du perlene til et nytt lavt proteinbindende mikrorør og setter på en magnetisk separatør i ett minutt.Fjern det supernatante fullstendig, og tilsett 500 mikroliter av lysisbufferen. Vask perlene med en Vortex-mikser i 15 sekunder. Gjenta vaskingen av perlene 10 ganger og fjern det overnaturlige.Elute proteiner ned til perlene ved tilsetning av 50 mikroliter av 1X SDS prøvebuffer og kok røret ved 98 grader Celsius i 5 minutter. Sentrifuger røret ved 860xG 3000 RPM i 10 sekunder og sett røret på en magnetisk separatør i ett minutt. Samle supernatant og bruke den som den bundne brøken.Mål konsentrasjonene av de ubundet og bundne proteinfraksjonene ved BCA-proteinanalysen. Visualiser protienløsninger med bromofenyblå og juster konsentrasjonen til 5 nanogram per mikroliter for SDS-siden. Skill de bundne og ubundne brøkene etter SDS-siden ved hjelp av en 5-20 % graderingsgel.Sølv flekk gelen. Til slutt, Western blot ved hjelp av passende primære og sekundære antistoffer i henhold til manuskriptet. I kultiverte hippocampale nevroner ble TLCN rikelig lokalisert til dendrittiske filopodia, aksel og soma og sam-lokalisert med F-actin.De kodede perlene var hovedsakelig bundet til dendritter og indusere dannelsen av fagocytiske kopper ved akkumulering av TLCN og F-actin på nevronale dendritter. Thorecense bilder viste at fagocytisk koppdannelse var avgjørende avhengig av tilstedeværelsen av TLCN i dendritter. De fagocytiske koppene ble bare dannet på ville hippocampale nevroner, men ikke på TLCN mangelfulle hippocampal nevroner.SDS-sideresultater viste at proteinbåndmønstrene var nesten de samme for de ubundet og bundne fraksjonene, men intensitetene på 50 og 70 kilodalton, i den bundne fraksjonen var lavere enn de i den ubundne fraksjonen. Imidlertid var båndintensiteten ikke åpenbart forskjellig mellom de ubundne og bundne fraksjonene utarbeidet fra TLCN-mangelfulle kultur hippocampal nevroner. Western blot analyse av de ubundet og bundne brøkene viste at TLCN og VN hovedsakelig ble oppdaget i den bundne fraksjonen.Actin, Ezrin, G alpha q, PLC beta 1, MAP-2 og Spectrin, ble oppdaget i både bundne og ubundne fraksjoner. Moesin, PSD-95, alfa-Actinin og alfa-Tubulin ble oppdaget i den ubundne fraksjonen. Hvis beleggproteinet i mikroperlene endres, kan denne prosedyren brukes til å identifisere analyseinteraksjon.Denne teknikken kan identifisere proteinet som arbeider med kunstige stoffer. Så vi tror at mekanismen kunstig genese kan kvantifiseres. Vi tror at nytt protein forbundet med psykisk lidelse kan identifiseres fra den dendrittiske filopodia-rike fraksjonen.Og kan utvides til terapien.

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.

Inducing dendrittiske Veksten i dyrkede sympatiske 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 ...

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.

Fast Micro-iontoforese av glutamat og GABA: Et nyttig verktøy for å etterforske 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 av Dendritic arborization i Dentate Gyrus av 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.

Rensing av musehjerne 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 ...

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.

Analysere Dendritic Morfologi i kolonner og 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 ...

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 av hippocampal dendritisk kompleksitet hos gamle mus ved bruk 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.

Real-time Iontophoresis med tetrametylammonium for å kvantifisere volumfraksjon og tortuositet i hjernens ekstracellulære rom

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 av Neuronal dendrittiske 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. ...