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

Agradecemos a Shigeo Okabe y Hitomi Matsuno por la cultura de baja densidad de las neuronas hipocampales, Masayoshi Mishina por ratones con deficiencia de TLCN, Sachiko Mitsui y Momoko Shiozaki por la asistencia técnica, y miembros del laboratorio Yoshihara para discusiones útiles . Este trabajo fue apoyado por JSPS KAKENHI Grant Nos. JP20700307, JP22700354, y JP24500392 y MEXT KAKENHI Grant Nos. JP23123525 a YF y JP20022046, JP18H04683 y JP18H05146 a YY.

Todos los métodos descritos aquí han sido aprobados por el Comité Institucional de Cuidado y Uso de Animales de RIKEN Wako.
1. Cultura de las neuronas del hipocampo
<ol><li>Preparación del medio de cultivo
	<ol>
<li>Preparación de la mezcla de vitamina 200x. Disolver 100 mg de sal hemicalcio ácido D-pantoténico, 100 mg de cloruro de colina, 100 mg de ácido fólico, 180 mg de i-inositol, 100 mg de niacinamida, 100 mg de pyridoxal HCl, y 100 mg de tiamina HCl en 500 ml...

<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. 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Desarrollamos un método de purificación para la fracción dendrítica rica en filopodia utilizando la afinidad entre la molécula de adhesión celular TLCN y la matriz extracelular profisitecina. En comparación con la fracción de PSD, podría ser posible identificar las proteínas sinápticas que actúan sobre la sinapsis inmadura de la fracción dendritica rica en filopodia. Por lo tanto, los componentes de la fracción dendrítica rica en filopodia son diferentes de los de la fracción PSD por 74%. A diferencia de ...

Filopodia dendrítica se cree que son precursores de las espinas. Los filamentos de actina en la filopodia dendrítica regulan su extensión y retracción1,2,3. Después de entrar en contacto con un axón, la filopodia dendrítica seleccionada comienza su maduración en espinas, y se forma una sinapsis4,5. Los componentes de las espinas se han determinado a partir de un análisis exhaustivo de las fracciones de densidad postsináptica6,7, mientras que los componentes de la filopodia dendrítica siguen siendo en gran medida desconocidos. Se ha demostrado que la telencefalina (TLCN), ERM, SynGAP, Ras, PI3 quinasa, Akt, mTOR, polo-como quinasa 2, CaMKII, syndecan-2, paralemin-1, ARF6, y EphB regulan la formación de filopodia dendrítica5,8,9 ,10,11, mientras que no se ha desarrollado un método para el análisis integral de moléculas presentes en la filopodia dendrítica.
TLCN (ICAM-5) se expresa específicamente por las neuronas espinosas en el segmento cerebral más rostral, el telencéfalo12. TLCN tiene 9 dominios similares a Ig en su región extracelular, una región transmembrana y una cola citoplasmática13. EL TLCN se une a vitronectina (VN) y a la integrina LFA-1 en su región extracelular, a la presenilinicina ensu región transmembrana, y a la fosfo-ERM y a la actina en su región citoplasmática 5,8,14,15 ,16. El TLCN se une al citoesqueleto de actina a través de fosfo-ERM en las puntas de filopodia dendrítica y de actinina en espinas y ejes dendríticos8,16.
Mostramos que la sobreexpresión de TLCN mejoró la formación de filopodia dendrítica e indujo la reversión de las espinas a la filopodia10. La forma activa constitutiva de ezrin ligada a la región citoplasmática TLCN y la formación de filopodia dendrítica mejorada8. Por lo tanto, TLCN regula la formación de filopodia dendrítica a través de proteínas de unión a la actina. Esselens y otros demostraron que las microperlas indujeron la acumulación de TLCN en las neuronas cultivadas17. Mostramos que las estructuras de copa shagocíticas se formaron sobre dendritas neuronales alrededor de microperlas recubiertas de VN de una manera dependiente del TLCN15. Los componentes de la filopodia dendrítica son similares a los de la copa fagocítica. Es difícil recoger filopodia dendrítica, pero es relativamente más fácil recoger la copa fagocítica usando microperlas magnéticas. Así, desarrollamos un método para purificar la copa fagocítica en lugar de la filopodia dendrítica18. Aquí, describimos el método de purificación para la fracción dendrítica rica en filopodia.

<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

La filopodia dendrítica son protuberancias delgadas y largas basadas en el filamento de actina, y se extienden y se retraen como si buscaran un axón objetivo. Cuando la filopodia dendrítica establece contacto con un axón objetivo, comienzan a madurar en espinas, lo que conduce a la formación de una sinapsis. La telencefalina (TLCN) se localiza abundantemente en filopodia dendrítica y se excluye gradualmente de las espinas. La sobreexpresión de TLCN en neuronas hipocampales cultivadas induce la formación de filopodia dendrítica. Mostramos que la telencefalia se une fuertemente a una molécula de matriz extracelular, vitronectina. Microperlas de Vitronectina inducida por la formación de copas fagocíticas en dendritas neuronales. En la copa fagocítica, se acumulan TLCN, proteínas de unión a TLCN como Ezrin/Radixin/Moesin(phospho-ERM) foso-ERM, y F-actina, lo que sugiere que los componentes de la copa fagocítica son similares a los de la filopodia dendrítica. Por lo tanto, desarrollamos un método para purificar la copa fagocítica en lugar de filopodia dendrítica. Las perlas de poliestireno magnético fueron recubiertas con vitronectina, que está abundantemente presente en el medio de cultivo de las neuronas del hipocampo y que induce la formación de copas fagocíticas en dendritas neuronales. Después de 24 horas de incubación, las copas fagocíticas fueron ligeramente solubilizadas con detergente y recogidas con un separador de imán. Después de lavar las cuentas, las proteínas de unión fueron eluidas y analizadas por la tinción de plata y la hincha occidental. En la fracción de unión, TLCN y actina estaban abundantemente presentes. Además, muchas proteínas identificadas a partir de la fracción fueron localizadas a la filopodia dendrítica; por lo tanto, nombramos la fracción de unión como la fracción dendrítica rica en filopodia. Este artículo describe los detalles sobre el método de purificación para la fracción dendrítica rica en filopodia.

En las neuronas del hipocampo cultivadas, el TLCN fue localizado abundantemente a la filopodia dendrítica, el eje y el soma y colocalizó con F-actina (Figura1A,B). Cuando se añadieron microperlas de poliestireno a las neuronas del hipocampo cultivadas, las cuentas fueron recubiertas automáticamente con vitronectina (VN) derivada del suero bovino fetal (FBS) en el medio de cultivo; estaban destinados principalmente a dendritas, e indujeron...

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Purification of the Dendritic Filopodia-rich Fraction

En este protocolo, introducimos un método para purificar la fracción dendrítica rica en filopodia a partir de la estructura de protrusión phagocítica similar a la copa en las neuronas del hipocampo cultivadas aprovechando la afinidad específica y fuerte entre un filopodial dendrítico molécula de adhesión, TLCN, y una molécula de matriz extracelular, vitroctina.

Purificación de la fracción defípoda dendrítica

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 ...

En comparación con la fracción PSD, puede ser posible identificar la proteína sináptica que actúa sobre la sinapsis inmadura alrededor de la fracción rica en filopodia dendrítica. Usamos neuronas vivas para inducir la formación de gorra fagocítica. La principal ventaja de esta técnica es que la proteína activa en la génesis artificial podría identificarse a partir de la fracción rica en filopodia dendrítica.Para empezar, preparar 200x mezcla de vitaminas disolviendo 100 mg de vitamina B5, 100 mg de cloruro de colina, 100 mg de ácido fólico, 180 mg de I-inositol, 100 mg de vitamina B3, 100 mg de clorhidrato de vitamina B6, y 100 mg de clorhidrato de tiamina en 500 ml de agua ultrapura utilizando un agitador magnético. Mezcle cuidadosamente la alícuota en tubos de 50 ml y guárdelo a 20 grados centígrados. Para preparar la solución de riboflavina, disolver 100 mg de riboflavina en 500 mg de agua ultrapura utilizando un agitador magnético.Mezcle cuidadosamente la alícuota en tubos de 50 ml y guárdelo a 20 grados centígrados. Para preparar un dicloruro de calcio molar disolver 7,35 gramos de cloruro de calcio dihidrato en 50 ml de agua ultrapura utilizando un agitador magnético. Para la preparación de Minimum Essential Medium disolver 400 mg de cloruro de potasio 6, 800 mg de cloruro de sodum, 2, 200 mg de bicarbonato de sodio, 158 mg de fosfato sódico monobásico dihidrato, 7, 000 mg de D-glucosa, y 200 mg de heptahidrato de sulfato de magnesio, y 950 ml de agua ultrapura, utilizando un agitador magnético.A continuación, utilice una pipeta de 1 ml con una agitación constante en un agitador magnético para añadir 1,8 ml de 1 dicloruro de calcio molar al MEM de una manera gota por gota. A continuación, agregue ácido clorhídrico para ajustar el pH del MEM a pH 7.25. A continuación, añadir 5 ml de mezcla de vitaminas 200x y 200 microlitros de solución de riboflavina al MEM.Ajustar el volumen de la solución a 1000 ml con agua ultrapura. Filtre la solución utilizando un sistema de filtro de 0,22 micras y guárdela a 4 grados centígrados. Para preparar 10x DNase-1 solución en stock disolver 100 mg de Dnase-1 en 12,5 ml de HBSS.Filtrar a través de un filtro de 0,22 micras, alícuota en tubos de 1,5 ml, y almacenar los tubos a 20 grados centígrados. Para la preparación de la solución en stock de Ara-C disolver 25 mg de Ara-C en 8,93 ml de agua ultrapura. Filtrar a través de un filtro de 0,22 micras, alícuota en tubos de 1,5 ml y almacenar a 20 grados centígrados.Para preparar el medio de chapado, mezclar 1 ml de mem amino acid Solution, 750 microlitros de 1 HEPES molar, 1 ml de B27, 125 microlitros de 200 milivolares glutamina, 250 microlitros de penicilina-estreptomicina, 2,5 ml de suero bovino fetal y 44,375 ml de MEM en un tubo de 50 ml. Para preparar el medio de parada, mezclar 1 ml de solución de aminoácidos MEM, 750 microlitros de 1 HEPES molar, 5 ml de FBS y 43,25 ml de MEM en un tubo de 50 ml. Para preparar HBSS, complementado con 15 HEPES mililolares, mezclar 49,25 ml de HBSS, y 750 microlitros de 1 HEPES molar.Para la preparación de platos recubiertos de poli-l-lisina, cubra los platos de cultivo celular de plástico de 35 mm con 0,2 mg por ml de hidrobromuro de poli-l-lisina durante un día a 25 grados centígrados. A continuación, lavar los platos con 2 ml de agua ultrapura tres veces e incubarlos con 1,5 ml de Stop Medium a 25 grados centígrados hasta su uso. Incubar el hipocampo diseccionado en una mezcla que contenga 500 microlitros cada uno de 2.5%trypsin y 10X Dnase-1 en HBSS complementado con 15 HEPES mililolares, pH 7.2, a 37 grados Celsius durante 15 minutos con agitación cada 3 minutos.Luego, transfiera el hipocampo a 10 ml del Medio Stop e incubar a 4 grados Celsius durante 5 minutos para inactivar la trippsina. A continuación, incubar el hipocampo diseccionado en 10 ml de medio de parada fresco a 4 grados centígrados durante 5 minutos. Mueva el hipocampo a 10 ml de medio de parada fresco e incubar a 4 grados centígrados durante otros 5 minutos.A continuación, mueva el hipocampo a 900 microlitros del medio de parada y 100 microlitros de 10X Dnase-1 en un tubo de 15 ml. Utilice una pipeta de 1 ml para disociar el hipocampo en neuronas aisladas pipeteando 20 veces. Añadir 9 ml de medio de chapado, y filtrar a través de un colador celular de 70 micras, en un tubo de 50 ml.Cuente el número de células usando un hemocitoómetro y ajuste a 3.5 veces 10 a las cuartas células por mililitro en medio de chapado. A continuación, aspirar el medio de parada de los platos recubiertos de poli-l-lisina. Plato 2 ml por plato de las células en los platos recubiertos e incubar menos de 5% dióxido de carbono a 37 grados Celsius durante 60-64 horas.Después de la incubación, añadir 2 microlitros de la solución de ara-C a las neuronas y agitar el plato lentamente. Mantenga los platos de cultivo en una caja humidificada sin cambiar el medio de cultivo por debajo del 5% de dióxido de carbono a 37 grados centígrados. Purificar la fracción rica en filopodia dendrítica después de 13 días in vitro añadiendo primero tres millones de microbeads de poliestireno magnético por plato a 20 platos que contienen las neuronas cultivadas.Después de un día, lavar las neuronas en 1 ml de PBS con agición tres veces para eliminar las micropersas medianas y no unidas. Después de eliminar PBS, ane las neuronas con 500 microlitros por plato del tampón de lisis. A continuación, recoger el izado con un rascador celular y transferir el izado a 10 microtubos de baja unión a proteínas.Ponga los tubos en un separador de imán y espere un minuto. Recoger el sobrenadante y utilizarlo como la fracción sin ataduras para la tinción de plata y el análisis de manchas occidentales. A continuación, transfiera las perlas a un nuevo microtubo de unión a proteínas baja y establezca un separado magnético durante un minuto.Retire completamente el sobrenadante y agregue 500 microlitros del búfer de lelisis. Lave las cuentas con un mezclador Vortex durante 15 segundos. Repita el lavado de las perlas 10 veces y retire el sobrenadante.Eluir las proteínas hasta las cuentas por la adición de 50 microlitros de tampón de muestra 1X SDS y hervir el tubo a 98 grados Celsius durante 5 minutos. Centrifugar el tubo a 860xG 3000 RPM durante 10 segundos y ajuste el tubo en un separado magnético durante un minuto. Recoja el sobrenadante y utilícelo como fracción enlazada.Mida las concentraciones de las fracciones de proteínas no unidas y unidas por el ensayo de proteína BCA. Visualice las soluciones protien con bromofenilo azul y ajuste la concentración a 5 nanogramos por microlitro para la página SDS. Separe las fracciones enlazadas y no enlazadas por la página SDS usando un gel de 5-20%gradiente.La plata mancha el gel. Finalmente, Western blot utilizando anticuerpos primarios y secundarios apropiados de acuerdo con el manuscrito. En las neuronas del hipocampo cultivadas, TLCN fue abundantemente localizado a la filopodia dendrítica, eje, y soma y co-localizado con F-actina.Las cuentas codificadas se unieron principalmente a dendritas e inducen la formación de copas fagocíticas por acumulación de TLCN y F-actin en dendritas neuronales. Las imágenes de Thorecense mostraron que la formación de la copa fagocítica dependía crucialmente de la presencia de TLCN en las dendritas. Las copas fagocíticas sólo se formaron en neuronas hipocampales de tipo salvaje, pero no en las neuronas hipocampales deficientes de TLCN.Los resultados de la página SDS mostraron que los patrones de banda de proteína eran casi los mismos para las fracciones no enlazadas y enlazadas, pero las intensidades a 50 y 70 kilodalton, en la fracción enlazada eran inferiores a las de la fracción sin enlazar. Sin embargo, la intensidad de la banda no era obviamente diferente entre las fracciones no enlazadas y enlazadas preparadas a partir de las neuronas hipocampales de cultivo deficiente de TLCN. El análisis de la mancha occidental de las fracciones no enlazadas y enlazadas mostró que TLCN y VN se detectaron principalmente en la fracción enlazada.Actin, Ezrin, G alfa q, PLC beta 1, MAP-2 y Spectrin, se detectaron tanto en las fracciones enlazadas como no enlazadas. Moesin, PSD-95, alfa-Actinin y alfa-Tubulina se detectaron en la fracción sin enlazar. Si la proteína de recubrimiento de las micropersas cambia, este procedimiento se puede aplicar para identificar la interacción del ensayo.Esta técnica podría identificar la proteína que trabaja en sustancias artificiales. Así que creemos que la génesis artificial del mecanismo se puede cuantificar. Creemos que la nueva proteína asociada con el trastorno mental se puede identificar a partir de la fracción rica en filopodia dendrítica.Y se puede extender a la terapia.

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.

Análisis de la morfología espina dendrítica en las neuronas del SNC cultivadas

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

Investigación

Neurociencias

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.

Inducir el crecimiento dendríticas en las neuronas simpáticas cultivadas

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.

El análisis de la morfología de células dendríticas Purkinje en cultivos de corte organotípicos

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-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

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.

Evaluación de la arborización dendrítica en el giro dentado de la región del hipocampo en ratones

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.

La purificación de los buques de cerebro de ratón

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.

Analizando dendríticas morfología en las columnas y capas

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.

Evaluación de la complejidad dendrítica del hipocampo en ratones envejecidos utilizando el método de Golgi-Cox

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.

Iontoforesis en tiempo real con tetrametilamonio para cuantificar la fracción de volumen y la tortuosidad del espacio extracelular cerebral

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.

Análisis cuantitativo de la complejidad de la arborización dendrítica Neuronal en Drosophila

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