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

हम हिप्पोकैम्पस न्यूरॉन्स की कम घनत्व संस्कृति के लिए Shigeo Okabe और Hitomi Matsuno धन्यवाद, TLCN कमी चूहों के लिए Masayoshi Mishina, Sachiko Mitsui और Momoko Shiozaki तकनीकी सहायता के लिए, और सहायक विचार विमर्श के लिए Yoshihara प्रयोगशाला के सदस्यों . इस कार्य को जेएसपीएस काकेनही ग्रांट नं. JP20700307, JP22700354, और JP24500392 और MEXT KAKENHI अनुदान Nos. JP23123525 YF और JP20022046, JP18H04683, और JP18H05146 YY करने के लिए.

यहां वर्णित सभी विधियों को रिकेन वाको की संस्थागत पशु देखभाल और उपयोग समिति द्वारा अनुमोदित किया गया है।
1. हिप्पोकैम्पस न्यूरोंस की संस्कृति
<ol><li>संस्कृति माध्यम की तैयारी
	<ol>
<li>200x विटा...

<ol>
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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>

हमने सेल आसंजन अणु TLCN और extracellular मैट्रिक्स प्रोटीन vitronectin के बीच संबंध का उपयोग कर के डेन्ड्रिटिक filopodia समृद्ध अंश के लिए एक शुद्धि विधि विकसित की है. PSD अंश की तुलना में, यह डेन्ड्रिटिक filopodia समृद्ध अंश से अपरिपक्...

डेन्ड्रिटिक फिलोपोडिया को रीढ़ की हड्डी का अग्रदूत माना जाता है। डेन्ड्रिटिक फिलोपोडिया में एक्टिन फिलामेंट उनके विस्तार और वापसी1,2,3को विनियमित करते हैं . एक एक्सॉन के साथ संपर्क करने के बाद, चयनित डेन्ड्रिटिक फिलोपोडिया रीढ़ की हड्डी में अपनी परिपक्वता शुरू करते हैं, और एक सिनेप्स का गठन4,5होता है। रीढ़ के घटक पोस्टीनैप्टिक घनत्व के व्यापक विश्लेषण से निर्धारित किए गए हैं6,7, जबकि डेन्ड्रिटिक फिलोपोडिया के घटक काफी हद तक अज्ञात रहते हैं। यह दिखाया गया है कि टेलीनसेफेलिन (TLCN), ERM, SynGAP, रास, PI3 kinase, Akt, MTOR, पोलो की तरह kinase 2, CaMKII, syndecan-2, paralemin-1, ARF6, और EphB को विनियमित डेन्ड्रिटिक filopodia गठन5,8,9 10,11, जबकि डेन्ड्रिटिक फिलोपोडिया में मौजूद अणुओं के व्यापक विश्लेषण के लिए एक विधि विकसित नहीं की गई है।
TLCN (ICAM-5) विशेष रूप से सबसे रोस्ट्रल मस्तिष्क खंड में spiny न्यूरॉन्स द्वारा व्यक्त की है, telencephalon12. TLCN अपने extracellular क्षेत्र में 9 आईजी की तरह डोमेन है, एक transmembrane क्षेत्र, और एक साइटोप्लाज्मिक पूंछ13. TLCN अपने extracellular क्षेत्र में vitronectin (VN) और LFA-1 integrin के लिए बांधता है, अपने transmembrane क्षेत्र में presenilin करने के लिए, और फॉस्फो-ईआरएम और अपने साइटोप्लाज्मिक क्षेत्र में जेड-actinin5,8,14,15 ,16. TLCN रीढ़ की हड्डी और डेन्ड्रिटिक शाफ्ट8,16में डेन्ड्रिटिक फिलोपोडिया और जेड-एक्टिनिन के सुझावों पर फॉस्फो-ईआरएम के माध्यम से actin cytoskeleton को बांधता है।
हमने दिखाया कि टीएलसीएन ने डेन्ड्रिटिक फिलोपोडिया के गठन को बढ़ाया और रीढ़ की हड्डी को फिर से शुरू करने के लिए फिलोपोडिया10को प्रेरित किया . TLCN कोशिकाद्रव्यी क्षेत्र और उन्नत डेन्ड्रिटिक फिलोपोडिया गठन8के लिए बाध्य ezrin के गठन सक्रिय रूप . इस प्रकार, TLCN actin-बाध्यकारी प्रोटीन के माध्यम से डेन्ड्रिटिक filopodia गठन को नियंत्रित करता है. Esselens एट अल. प्रदर्शन किया है कि microbeads सुसंस्कृत न्यूरॉन्स17पर TLCN संचय प्रेरित . हमने दिखाया कि वीएन लेपित माइक्रोबीड्स के चारों ओर न्यूरॉनल डेन्ट्रेट्स पर टीएलसीएन-निर्भर तरीके से15के बारे में फेगोसाइटिक कप संरचनाओं का गठन किया गया था। डेन्ड्रिटिक फिलोपोडिया के घटक फैगोसाइटिक कप के समान होते हैं। डेन्ड्रिटिक फिलोपोडिया को एकत्र करना मुश्किल है, लेकिन चुंबकीय माइक्रोबीड्स का उपयोग करके फैगोसाइटिक कप एकत्र करना अपेक्षाकृत आसान है। इस प्रकार हमने डेन्ड्रिटिक फिलोपोडिया18के स्थान पर फागोसाइटिक कप को शुद्ध करने की एक विधि विकसित की। यहाँ, हम डेन्ड्रिटिक फिलोपोडिया समृद्ध अंश के लिए शुद्धि विधि का वर्णन करते हैं।

<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

डेन्ड्रिटिक फिलोपोडिया एक्टिन फिलामेंट के आधार पर पतले और लंबे समय तक बाहर निकालते हैं, और वे एक लक्ष्य एक्सॉन के लिए खोज के रूप में विस्तार और वापस ले लेते हैं। जब डेन्ड्रिटिक फिलोपोडिया एक लक्ष्य एक्सॉन के साथ संपर्क स्थापित करता है, तो वे रीढ़ की हड्डी में परिपक्व होने लगते हैं, जिससे एक synapse के गठन के लिए अग्रणी होता है। टेलीनसेफेलिन (टीएलसीएन) डेन्ड्रिटिक फिलोपोडिया में प्रचुर मात्रा में स्थानीयकृत है और धीरे-धीरे रीढ़ की हड्डी से बाहर रखा गया है। सुसंस्कृत हिप्पोकैम्पस न्यूरॉन्स में टीएलसीएन का अतिव्यंजक डेन्ड्रिटिक फिलोपोडिया गठन को प्रेरित करता है। हमने दिखाया कि टेलिनेसेफेलिन एक अतिरिक्त कोशिकीय मैट्रिक्स अणु, विट्रोनेक्टिन से दृढ़ता से बांधता है। विट्रोनेक्टिन लेपित माइक्रोबीड्स न्यूरोनल डेन्ड्राइट पर फेगोसाइटिक कप गठन को प्रेरित करता है। फागोसाइटिक कप में, टीएलसीएन, टीएलसीएन-बाइंडिंग प्रोटीन जैसे फॉस्फोरिलेटिड एज़रिन/रेडिक्सिन/मोसिन (फॉस्फो-ईआरएम) और एफ-एक्टिन जमा होते हैं, जो बताते हैं कि फागोसाइटिक कप के घटक डेन्ड्रिटिक फिलोपोडिया के समान होते हैं। इस प्रकार, हमने डेन्ड्रिटिक फिलोपोडिया के स्थान पर फागोसाइटिक कप को शुद्ध करने के लिए एक विधि विकसित की। चुंबकीय पॉलीस्टाइरीन मोती विट्रोनेक्टिन के साथ लेपित थे, जो हिप्पोकैम्पस न्यूरॉन्स की संस्कृति माध्यम में प्रचुर मात्रा में मौजूद है और जो न्यूरोनल डेन्ड्राइट पर फेगोसाइटिक कप गठन को प्रेरित करता है। 24 ज ऊष्मायन के बाद, phagocytic कप डिटर्जेंट के साथ हल्के solubilized थे और एक चुंबक विभाजक का उपयोग कर एकत्र. मोती धोने के बाद, बाध्यकारी प्रोटीन eluted और चांदी धुंधला और पश्चिमी सोख्ता द्वारा विश्लेषण किया गया. बाध्यकारी अंश में, TLCN और actin प्रचुर मात्रा में मौजूद थे. इसके अलावा, अंश से पहचाने गए कई प्रोटीन को डेन्ड्रिटिक फिलोपोडिया में स्थानीयकृत किया गया; इस प्रकार, हमने बंधनीय अंश को डेन्ड्रिटिक फिलोपोडिया समृद्ध अंश के रूप में नामित किया। यह लेख डेन्ड्रिटिक फिलोपोडिया समृद्ध अंश के लिए शुद्धि विधि के बारे में विवरण का वर्णन करता है।

सुसंस्कृत हिप्पोकैम्पस न्यूरॉन्स में, TLCN प्रचुर मात्रा में डेन्ड्रिटिक filopodia, शाफ्ट, और सोमा के लिए स्थानीयकृत किया गया था और एफ-एक्टिन के साथ colocalized (चित्र 1A, बी)। जब polystyrene microbeads स?...

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

Purification of the Dendritic Filopodia-rich Fraction

इस प्रोटोकॉल में, हम एक डेन्ड्रिटिक फिलोपोडायल के बीच विशिष्ट और मजबूत संबंध का लाभ लेने के द्वारा सुसंस्कृत हिप्पोकैम्पस न्यूरॉन्स पर phagocytic कप की तरह बहिर्वेश संरचना से डेन्ड्रिटिक filopodia समृद्ध अंश को शुद्ध करने के लिए एक विधि परिचय आसंजन अणु, TLCN, और एक extracellular मैट्रिक्स अणु, vitronectin.

डेन्ड्रिटिक फिलोपोडिया समृद्ध अंश का शुद्धिकरण

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 ...

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.

संवर्धित सीएनएस न्यूरॉन्स में वृक्ष के समान रीढ़ आकृति विज्ञान के विश्लेषण

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.

संवर्धित सहानुभूति न्यूरॉन्स में वृक्ष के समान विकास उत्प्रेरण

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.

Organotypic टुकड़ा संस्कृतियों में Purkinje सेल वृक्ष के समान आकृति विज्ञान के विश्लेषण

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.

ग्लूटामेट और गाबा की फास्ट माइक्रो योणोगिनेसिस: Synaptic एकता की जांच के लिए एक उपयोगी उपकरण

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.

चूहे में hippocampal क्षेत्र के दांतेदार गाइरस में वृक्ष के समान द्रुमायण का आकलन

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.

माउस ब्रेन वेसल्स की शुद्धि

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.

कॉलम और परतों में वृक्ष के समान आकृति विज्ञान का विश्लेषण

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.

गोल्गी-कॉक्स विधि का उपयोग करते हुए वृद्ध चूहों में हिप्पोकैम्पल वृक्ष वृत्तीय जटिलता का आकलन

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.

रीयल-टाइम Iontophoresis Tetramethylammonium के साथ मात्रा मात्रा और ब्रेन एक्स्ट्रासेल्यूलर स्पेस के Tortuosity मात्रा

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.

Drosophila में न्यूरॉनी वृक्ष Arborization जटिलता का मात्रात्मक विश्लेषण

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