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 Shigeo Okabe e Hitomi Matsuno pela cultura de baixa densidade de neurônios hipocampais, Masayoshi Mishina para camundongos deficientes em TLCN, Sachiko Mitsui e Momoko Shiozaki para assistência técnica, e membros do laboratório Yoshihara para discussões úteis . Este trabalho foi apoiado por JSPS KAKENHI Grant nos. JP20700307, JP22700354, e JP24500392 e MEXT KAKENHI Grant nos. JP23123525 para YF e JP20022046, JP18H04683 e JP18H05146 para YY.

Todos os métodos aqui descritos foram aprovados pelo Comitê institucional de cuidado e uso de animais da RIKEN Wako.
1. cultura dos neurônios hipocampais
<ol><li>Preparação do meio de cultura
	<ol>
<li>Preparação da mistura da vitamina 200x. Dissolver 100 mg de D-ácido pantotênico hemicalcium sal, 100 mg de cloreto de colina, 100 mg de ácido fólico, 180 mg de i-inositol, 100 mg de niacinamida, 100 mg de HCl piridoxal, e 100 mg de tiamina HCl em 500 mL de água ult...

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

Nós desenvolvemos um método da purificação para a fração filopodia-rica dendríticas usando a afinidade entre a molécula tlcn da adesão da pilha e a vitronectina extracelular da proteína da matriz. Comparado à fração PSD, pode ser possível identificar as proteínas sinápticas atuando na sinapse imatura da fração dendrítica rica em filopodia. Assim, os constituintes da fração dendrítica filopodia-rica são diferentes dos da fração PSD em 74%. Diferente da fração PSD, usamos neurônios hipocampais c...

O filopódia dendrítico é pensado para ser precursores dos espinhas. Os filamentos de actina no filopódia dendrítico regulam sua extensão e retração1,2,3. Depois de entrar em contato com um axão, o filopódia dendrítico selecionado começa sua maturação em espinhos, e uma sinapse é formada em4,5. Componentes de espinhos foram determinados a partir da análise abrangente das frações de densidade pós-sináptica6,7, enquanto os componentes de filopódia dendrítico permanecem em grande parte desconhecidos. Demonstrou-se que a telencérola (tlcn), erm, syngap, Ras, ARF6 quinase, Akt, mTOR, Polo-como a quinase 2, camkii, syndecan-2, paralemin-1, e ephb regulam a formação dendrítica defilopódia5,8,9 ,10,11, enquanto um método não foi desenvolvido para a análise abrangente de moléculas presentes no filopódio dendrítico.
Tlcn (ICAM-5) é expressado especificamente por neurônios spiny no segmento o mais rostral do cérebro, o telencéfalo12. Tlcn tem 9 domínios do IG-like em sua região extracelular, uma região do transmembrana, e uma cauda cytoplasmic13. A tlcn liga-se à vitronectina (vn) e à integrina LFA-1 em sua região extracelular, à presenilina em sua região transmembranar e ao fosho-ERM e α-actinina em sua região citoplasmática5,8,14,15 ,16. Tlcn liga-se ao citoesqueleto de actina através de fosho-ERM nas pontas de filopódia dendrítico e α-actinina em espinhos e eixos dendríticos8,16.
Nós mostramos que o superexpressão de tlcn aumentou a formação dendríticas do filopódia e induziu a reversão dos espinhos ao filopódia10. A forma ativa constitutiva de Ezrin ligada à região citoplasmática do TLCN e a formação de filopótico dendrítica reforçada8. Assim, a TLCN regula a formação de filopódios dendríticos através de proteínas de ligação actina. Esselens et al. demonstraram que microgrânulos induziu acúmulo de TLCN em neurônios cultivados17. Nós mostramos que as estruturas fagocitária do copo foram dadas forma em dendritos neuronal em torno dos microesferas vn-revestidos em uma maneira tlcn-dependente15. Constituintes de filopódios dendríticos são semelhantes aos do copo fagocitítico. É difícil coletar filopódios dendríticos, mas é relativamente mais fácil coletar o copo fagocitítico usando microgrânulos magnéticos. Assim, desenvolvemos um método para purificar o copo fagocitítico em vez de filopódia dendrítico18. Aqui, nós descrevemos o método da purificação para a fração filopodia-rica dendríticas.

<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

Os filopódios dendríticos são protrusões finas e longas com base no filamento de actina, e eles se estendem e se retraem como se estivessem buscando um AXON alvo. Quando o filopódia dendrítico estabelecer contato com um AXON alvo, eles começam a amadurecer em espinhos, levando à formação de uma sinapse. A telencífica (tlcn) está abundantemente localizada em filopódia dendrítico e é gradualmente excluída de espinhas. O overexpression de tlcn em neurônios hippocampal cultivados induz a formação dendríticas do filopódia. Nós mostramos que o Telencephalin liga fortemente a uma molécula extracelular da matriz, Vitronectin. Microesferas Vitronectin-revestidos induziu a formação fagocitária do copo em dendrites neuronal. No copo fagocitítico, TLCN, proteínas de ligação TLCN tais como Ezrin fosforilado/Radixina/Moesina (fosho-ERM), e F-Actina são acumuladas, o que sugere que os componentes do copo fagocítico são semelhantes aos de filopodia dendrítico. Assim, desenvolvemos um método para purificação do copo fagocítico em vez de filopópodes dendrítico. Os grânulos magnéticos do poliestireno foram revestidos com o Vitronectin, que é abundantemente atual no meio de cultura de neurônios hippocampal e que induz a formação fagocitária do copo em dendrites neuronal. Após 24 h de incubação, os copos fagocíticos foram levemente solubilizados com detergente e coletados por meio de um separador magnético. Após a lavagem dos grânulos, as proteínas de ligação foram eluidas e analisadas por coloração de prata e western blotting. Na fração de ligação, TLCN e actina estavam abundantemente presentes. Além, muitas proteínas identificadas da fração foram localizadas ao filopodia dendríticas; assim, denominamos a fração de ligação como a fração rica em filopóticos dendrítico. Este artigo descreve detalhes a respeito do método da purificação para a fração filopodia-rica dendríticas.

Nos neurônios hipocampais cultivados, a TLCN foi localizada abundantemente ao filopótico dendrítica, ao eixo e ao soma e colocalizada com F-Actina (Figura 1a, B). Quando os microgrânulos de poliestireno foram adicionados aos neurônios do hipocampal cultivado, os grânulos foram revestidos automaticamente com o vitronectina (vn) derivado do soro bovino fetal (FBS) no meio de cultura; Eles foram principalmente vinculados a dendritos, e ind...

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

Purification of the Dendritic Filopodia-rich Fraction

Neste protocolo, nós introduzimos um método para purificante a fração filopodia-rica dendríticas do fagocitária copo-como a estrutura do protrusão em neurônios hippocampal cultivados aproveitando-se da afinidade específica e forte entre um filopodial dendríticas molécula de adesão, TLCN, e uma molécula de matriz extracelular, vitronectina.

Purificação da fração rica de Filopodia 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 ...

Em comparação com a fração PSD, pode ser possível identificar a proteína sináptica agindo na sinapse imatura em torno da fração dendrítica rica em filopodia. Usamos neurônio vivo para induzir a formação de tampas fagocíticas. A principal vantagem dessa técnica é que a proteína ativa na gênese artificial poderia ser identificada a partir da fração rica em filopodia dendrítica.Para começar, prepare o 200x Vitamin Mix dissolvendo 100 mg de Vitamina B5, 100 mg de Cloreto de Colina, 100 mg de Ácido Fólico, 180 mg de I-inositol, 100 mg de Vitamina B3, 100 mg de cloridrato de vitamina B6, e 100 mg de cloridrato de tiamina em 500 ml de água ultrapura usando um agitador magnético. Misture cuidadosamente a alíquota em tubos de 50 ml e armazene-a a 20 graus Celsius. Para preparar a solução riboflavina, dissolva 100 mg de riboflavina em 500 mg de água ultrapura usando um agitador magnético.Misture cuidadosamente a alíquota em tubos de 50 ml e armazene a 20 graus Celsius. Para preparar um diclorito de cálcio molar dissolva 7,35 gramas de dihidrato de cloreto de cálcio em 50 ml de água ultrauso usando um agitador magnético. Para o mínimo essencial preparação média dissolver 400 mg de cloreto de potássio 6, 800 mg de cloreto de sodum, 2.200 mg de bicarbonato de sódio, 158 mg de dihidrato monobásico de fosfato de sódio, 7.000 mg de D-glicose, e 200 mg de heptahydrate de sulfato de magnésio, e 950 ml de água ultrapura, usando um agitador magnético.Em seguida, use uma pipeta de 1 ml com uma agitação constante em um agitador magnético para adicionar 1,8 ml de 1 dicloreto de cálcio molar ao MEM de forma gota a gota. Em seguida, adicione ácido clorídrico para ajustar o pH do MEM ao pH 7.25. Em seguida, adicione 5 ml de mistura de vitaminas de 200x e 200 microliters de solução de riboflavina ao MEM.Ajuste o volume do volume da solução para 1000 ml com água ultrapura. Filtre a solução usando um sistema de filtro de 0,22 míccro e armazene-a a 4 graus Celsius. Para preparar 10x solução de estoque DNase-1 dissolva 100 mg de Dnase-1 em 12,5 ml de HBSS.Filtre através de um filtro de 0,22 mícrons, aliquot em tubos de 1,5 ml e armazene os tubos a 20 graus Celsius. Para a preparação da solução de estoque Ara-C dissolver 25 mg de Ara-C em 8,93 ml de água ultrapura. Filtre através de um filtro de 0,22 míccro, alíquota em tubos de 1,5ml e armazene a 20 graus Celsius.Para preparar o Meio de Revestimento, misture 1 ml de SOLUÇÃO DE Aminoácido MEM, 750 microliters de 1 HEPES molar, 1 ml de B27, 125 microliters de 200 milimilous de glutamina, 250 microliters de penicilina-estreptomicina, 2,5 ml de Soro Bovino Fetal e 44,375 ml de MEM em um tubo de 50 ml. Para preparar o meio de parada, misture 1 ml de solução de aminoácido MEM, 750 microliters de 1 molar HEPES, 5 ml de FBS e 43,25 ml de MEM em um tubo de 50 ml. Para preparar o HBSS, suplementado com HEPES de 15 mililitros, misture 49,25 ml de HBSS e 750 microlitres de 1 molar HEPES.Para a preparação de pratos revestidos de poli-l-lysina, cubra pratos de cultura de células plásticas de 35 mm com 0,2 mg por ml de hidrobromida poli-l-lysina por um dia a 25 graus Celsius. Em seguida, lave os pratos com 2 ml de água ultrauso três vezes e incuba-os com 1,5 ml de Stop Medium a 25 graus Celsius até usar. Incubar o hipocampi dissecado em uma mistura contendo 500 microliters cada um de 2,5% de trippsina e 10X Dnase-1 no HBSS suplementado com HEPES de 15 milimolares, pH 7.2, a 37 graus Celsius por 15 minutos com agitação a cada 3 minutos.Em seguida, transfira o hipocampi para 10 ml do Stop Medium e incubar a 4 graus Celsius por 5 minutos para inativar a trippsina. Em seguida, incubar o hipocampi dissecado em 10 ml de meio de parada fresco a 4 graus Celsius por 5 minutos. Mova o hipocampi para 10 ml de stop médio fresco e incubar a 4 graus Celsius por mais 5 minutos.Em seguida, mova o hipocampi para 900 microliters do meio stop e 100 microliters de 10X Dnase-1 em um tubo de 15 ml. Use uma pipeta de 1 ml para dissociar o hipocampi em neurônios isolados por pipetar 20 vezes. Adicione 9 ml de meio de revestimento e filtre através de um coador de células de 70 mn, em um tubo de 50 ml.Conte o número de células usando um hemócito e ajuste para 3,5 vezes 10 para a quarta célula por mililitro em meio de revestimento. Em seguida, aspire o meio de parada dos pratos revestidos de poli-l-lisina. Placa 2 ml por prato das células nos pratos revestidos e incubar sob 5% de dióxido de carbono a 37 graus Celsius por 60-64 horas.Após a incubação, adicione 2 microliters de solução de estoque Ara-C aos neurônios e agite o prato lentamente. Mantenha os pratos da cultura em uma caixa umidificada sem alterar o meio cultural abaixo de 5% de dióxido de carbono a 37 graus Celsius. Purifique a fração rica de filopodia dendrítica após 13 dias in vitro adicionando primeiro três milhões de microesferas de poliestireno magnético por prato a 20 pratos contendo os neurônios cultivados.Depois de um dia, lave os neurônios em 1 ml de PBS com agiação três vezes para remover as microesferas médias e desvinculados. Depois de remover a PBS, lise os neurônios com 500 microliters por prato do tampão de lise. Em seguida, colete o lysate com um raspador de células e transfira o lysate em 10 microtubos de ligação de proteínas baixas.Coloque os tubos em um separador de ímãs e espere por um minuto. Colete o supernante e use-o como a fração desvinculada para coloração de prata e análise de manchas ocidentais. Em seguida, transfira as contas para um novo microtubo de ligação de baixa proteína e coloque em um separador magnético por um minuto.Remova completamente o supernascer e adicione 500 microliters do tampão de lise. Lave as contas usando um misturador de vórtice por 15 segundos. Repita a lavagem das contas 10 vezes e remova o sobrenatante.Proteínas elutos até as contas pela adição de 50 microliters de 1X SDS tampão de amostra e ferver o tubo a 98 graus Celsius por 5 minutos. Centrifugar o tubo a 860xG 3000 RPM por 10 segundos e definir o tubo em um separador magnético por um minuto. Colete o supernante e use-o como fração vinculada.Medir concentrações das frações proteicas ilimitadas e vinculadas pelo ensaio proteico BCA. Visualize soluções de protien com azul bromofenil e ajuste a concentração para 5 nanogramas por microliter para página SDS. Separe as frações vinculadas e ilimitadas pela página SDS usando um gel de gradiente de 5-20%.Mancha de prata o gel. Finalmente, a mancha ocidental usando anticorpos primários e secundários apropriados de acordo com o manuscrito. Em neurônios hipocampais cultivados, o TLCN foi abundantemente localizado para a filopodia dendrítica, eixo e soma e co-localizado com F-actin.As contas codificadas eram principalmente ligadas aos dendritos e induzir a formação de copos fagocíticos por acúmulo de TLCN e F-actin em dendritos neuronais. Imagens thorecenses mostraram que a formação de copos fagocíticos dependia crucialmente da presença de TLCN em dendritos. Os copos fagocíticos só eram formados em neurônios hipocampais do tipo selvagem, mas não em neurônios hipocampais defucientes TLCN.Os resultados das páginas da SDS mostraram que os padrões de banda de proteína eram quase os mesmos para as frações ilimitadas e vinculadas, mas as intensidades a 50 e 70 kilodalton, na fração vinculada eram menores do que as da fração não vinculada. No entanto, a intensidade da banda não era obviamente diferente entre as frações desvinculadas e ligadas preparadas a partir de neurônios hipocampais de cultura defucientes TLCN. A análise das manchas ocidentais das frações desvinculadas e vinculadas mostrou que tlcn e VN foram detectados principalmente na fração vinculada.Actin, Ezrin, G alfa q, PLC beta 1, MAP-2 e Spectrin, foram detectados tanto nas frações vinculadas quanto não ligadas. Moesin, PSD-95, alfa-Actinin e alfa-Tubulin foram detectados na fração desvinculada. Se a proteína de revestimento das microesferas mudar, este procedimento pode ser aplicado para identificar a interação do ensaio.Essa técnica poderia identificar a proteína que trabalha em substâncias artificiais. Então acreditamos que o mecanismo da gênese artificial pode ser quantificado. Acreditamos que novas proteínas associadas ao transtorno mental podem ser identificadas a partir da fração rica em filopodia dendrítica.E pode ser estendido à 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.

Análise de Morfologia Spine dendríticas em neurônios cultivados CNS

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

Neurociência

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.

Induzir o crescimento dendrítico no cultivadas neurônios simpáticos

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.

A Análise de Purkinje Morfologia de células dendríticas em Culturas fatia organotípicas

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.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

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.

Avaliação de dendríticas Arborização no giro dentado do hipocampo em Ratos Região

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.

Purificação de embarcações do cérebro do rato

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.

Analisando dendríticas Morfologia em Colunas e Camadas

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.

Avaliação da complexidade dendrítica do hipocampo em ratos idosos usando o 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.

Itoforese em tempo real com tetrametilamônio para quantificar fração de volume e tortuosidade do espaço 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.

Análise quantitativa da arborização dendrítica Neuronal complexidade em Drosophila

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