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

Ringraziamo Shigeo Okabe e Hitomi Matsuno per la cultura a bassa densità dei neuroni ippocampali, Masayoshi Mishina per topi carenti di TLCN, Sachiko Mitsui e Momoko Shiozaki per l'assistenza tecnica, e membri del laboratorio Yoshihara per discussioni utili . Questo lavoro è stato supportato da JSPS KAKENHI Grant Nos. JP20700307, JP22700354 e JP24500392 e MEXT KAKENHI Grant Nos. da JP23123525 a YF e JP20022046, JP18H04683 e da JP18H05146 a YY.

Tutti i metodi qui descritti sono stati approvati dal Comitato istituzionale per la cura e l'uso degli animali di RIKEN Wako.
1. Cultura dei neuroni ippocampali
<ol><li>Preparazione del mezzo culturale
	<ol>
<li>Preparazione del mix di vitamine 200x. Sciogliere 100 mg di sale di emicalcio acido Pantotenico, 100 mg di cloruro di colina, 100 mg di acido folico, 180 mg di i-inositolo, 100 mg di niacinamide, 100 mg di HCl piruridossiale e 100 mg di tiamina HCl in 500 mL di acqua...

<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>

Abbiamo sviluppato un metodo di purificazione per la frazione dendritica ricca di filopodia utilizzando affinità tra la molecola di adesione cellulare TLCN e la proteina matrice extracellulare vitronectina. Rispetto alla frazione PSD, potrebbe essere possibile identificare le proteine sinaptiche che agiscono sulla sinapsi immatura dalla frazione dendritica ricca di filopodia. Così, i costituenti della frazione dendritica ricca di filopodia sono diversi da quelli della frazione PSD del 74%. A differenza della frazione P...

Si pensa che la filopodi dendritica sia precursore delle spine. I filamenti di Actin nella filopodi adendriticane regolano l'estensione e la retrazione 1,2,3. Dopo aver contattato un assone, la filopodia dendritica selezionata inizia la loro maturazione in spine, e si forma una sinapsi4,5. I componenti delle spine sono stati determinati dall'analisi completa delle frazioni di densità postsinaptica6,7, mentre i componenti della filopodia dendritica rimangono in gran parte sconosciuti. È stato dimostrato che telencephalin (TLCN), ERM, SynGAP, Ras, PI3 kinase, Akt, mTOR, polo-like kinase 2, CaMKII, syndecan-2, paralemin-1, ARF6, e EphB regolano la formazione di filopodi dendritici5,8,9 ,10,11, mentre un metodo non è stato sviluppato per l'analisi completa delle molecole presenti nella filopodia dendritica.
TLCN (ICAM-5) è specificamente espresso dai neuroni spinosi nel segmento cerebrale più rostrale, il telencephalon12. TLCN ha 9 domini simili a Ig nella sua regione extracellulare, una regione transmembrana e una coda citoplasmica13. TLCN si lega alla vitronectina (VN) e all'integrina LFA-1 nella sua regione extracellulare, alla presenilina nella sua regione transmembrana, e alla fosfora-ERM e alla z-actinina nella sua regione citoplasmica5,8,14,15 ,16. TLCN si lega al citoscheletro dell'actina attraverso il fosforo-ERM su punta di filopodia dendritica e di z-actininina nelle spine e negli alberi dendritici8,16.
Abbiamo dimostrato che la sovraespressione del TLCN ha migliorato la formazione di filopodi a dendritici e ha indotto la reversione delle spine alla filopodia10. La forma attiva costituiscista di ezrin legata alla regione citoplasmica TLCN e la formazione di filopodia dendritica migliorata8. Così, TLCN regola la formazione di filopodi dendritici attraverso proteine che legano l'atto. Esselens et al. ha dimostrato che le microsfere hanno indotto l'accumulo di TLCN sui neuroni coltivati17. Abbiamo dimostrato che le strutture della coppa fagocitica si sono formate sui dendriti neuronali intorno alle microsfere rivestite di VN in modo dipendente dalla TLCN15. Costituenti di filopodi dendritici sono simili a quelli della coppa fagocitica. È difficile raccogliere la filopodi dendritica, ma è relativamente più facile raccogliere la coppa fagocitica utilizzando microsfere magnetiche. Così, abbiamo sviluppato un metodo per purificare la coppa fagocitica invece di filopodia dendritica18. Qui, descriviamo il metodo di purificazione per la frazione dendritica ricca di 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

I filodidi dendrici sono sporche sottili e lunghe basate sul filamento di actin, e si estendono e si ritraggono come se cercassero un assone bersaglio. Quando la filopodia dendritica stabilisce il contatto con un assone bersaglio, iniziano a maturare in spine, portando alla formazione di una sinapsi. La telencefine (TLCN) è abbondantemente localizzata nella filopodia dendritica ed è gradualmente esclusa dalle spine. La sovraespressione del TLCN nei neuroni ippocampali coltivati induce la formazione di filopodia dendritica. Abbiamo dimostrato che la telencefine si lega fortemente a una molecola di matrice extracellulare, la vitronectina. Le microsfere rivestite di cellavanguardia in vitronectine hanno indotto la formazione di coppe fagocitiche sui dendriti neuronali. Nella tazza fagocitica, si accumulano proteine che legano il TLCN, come Ezrin/Radixin/Moesin (phospho-ERM), e F-actin, il che suggerisce che i componenti della coppa fagocitica sono simili a quelli della filodia dendritica. Così, abbiamo sviluppato un metodo per purificare la coppa fagocitica invece di filopodi a dendritica. Le perle di polistirene magnetico sono state rivestite con vitronectina, che è abbondantemente presente nel mezzo di coltura dei neuroni ippocampali e che induce la formazione di coppe fagocitiche sui dendriti neuronali. Dopo 24 h di incubazione, le tazze fagocitiche erano leggermente solubili con detergente e raccolte utilizzando un separatore magnete. Dopo aver lavato le perline, le proteine leganti sono state eluite e analizzate dalla colorazione d'argento e dalla gonfiore occidentale. Nella frazione vincolante, TLCN e actin erano abbondantemente presenti. Inoltre, molte proteine identificate dalla frazione sono state localizzate alla filopodica dendritica; così, abbiamo chiamato la frazione di legame come la frazione dendritica ricca di filopodia. Questo articolo descrive i dettagli relativi al metodo di purificazione per la frazione dendritica ricca di filopodia.

Nei neuroni ippocampali coltivati, TLCN è stato abbondantemente localizzato alla filopodia dendritica, albero, e soma e colocalizzato con F-actin (Figura 1A, B). Quando le microsfere di polistirolo sono state aggiunte ai neuroni ippocampali coltivati, le perline sono state automaticamente rivestite con vitronectina (VN) derivata dal siero bovino fetale (FBS) nel mezzo di coltura; erano principalmente legati ai dendriti, e indotto la formazio...

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

Purification of the Dendritic Filopodia-rich Fraction

In questo protocollo, introduciamo un metodo per purificare la frazione dendritica ricca di filopodia dalla struttura di protrusione simile a una coppa fagocitica sui neuroni ippocampali coltivati sfruttando l'affinità specifica e forte tra un filopopodiale dendritico molecola di adesione, TLCN, e una molecola di matrice extracellulare, vitronectina.

Purificazione della Frazione ricca di Filopodia Dendritica

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 ...

Rispetto alla frazione PSD, può essere possibile identificare la proteina sinaptica che agisce sulla sinapsi immatura attorno alla frazione ricca di filopodi dendritici. Abbiamo usato il neurone vivo per indurre la formazione di tappi fagocitici. Il principale vantaggio di questa tecnica è che la proteina attiva nella genesi artificiale potrebbe essere identificata dalla frazione ricca di filopodi dendritici.Per iniziare, preparare 200x Vitamin Mix sciogliendo 100 mg di vitamina B5, 100 mg di cloruro di colina, 100 mg di acido folico, 180 mg di I-inositolo, 100 mg di vitamina B3, 100 mg di cloridrato di vitamina B6 e 100 mg di cloridrato di tiammina in 500 ml di acqua ultrapura utilizzando un agitatore magnetico. Mescolare con cura l'aliquota in tubi da 50 ml e conservarla a 20 gradi Celsius. Per preparare la soluzione di riboflavina, sciogliere 100 mg di riboflavina in 500 mg di acqua ultrapura utilizzando un agitatore magnetico.Mescolare con cura l'aliquota in tubi da 50 ml e conservare a 20 gradi Celsius. Per preparare un cloruro di calcio molare sciogliere 7,35 grammi di diidrato di cloruro di calcio in 50 ml di acqua ultrapura utilizzando un agitatore magnetico. Per la preparazione minima del mezzo essenziale sciogliere 400 mg di cloruro di potassio 6, 800 mg di cloruro di sodo, 2,200 mg di bicarbonato di sodio, 158 mg di diidrato monobasico di fosfato di sodio, 7.000 mg di D-glucosio e 200 mg di eptaidrato di solfato di magnesio e 950 ml di acqua ultrapura, utilizzando un agitatore magnetico.Successivamente, utilizzare una pipetta da 1 ml con agitazione costante su un agitatore magnetico per aggiungere 1,8 ml di 1 cloruro di calcio molare al MEM in modo drop by drop. Quindi aggiungere acido cloridrico per regolare il pH del MEM a pH 7,25. Quindi, aggiungere 5 ml di miscela di vitamine 200x e 200 microlitri di soluzione di riboflavina al MEM.Regolare il volume della soluzione a 1000 ml con acqua ultrapura. Filtrare la soluzione utilizzando un sistema di filtri da 0,22 micron e conservarla a 4 gradi Celsius. Per preparare 10x DNasi-1 soluzione stock sciogliere 100 mg di Dnase-1 in 12,5 ml di HBSS.Filtrare attraverso un filtro da 0,22 micron, aliquota in tubi da 1,5 ml e conservare i tubi a 20 gradi Celsius. Per la preparazione della soluzione di approvvigionamento Ara-C sciogliere 25 mg di Ara-C in 8,93 ml di acqua ultrapura. Filtrare attraverso un filtro da 0,22 micron, aliquota in tubi da 1,5 ml e conservare a 20 gradi Celsius.Per preparare il mezzo di placcatura, mescolare 1 ml di soluzione di amminoacido MEM, 750 microlitri di 1 HEPES molare, 1 ml di B27, 125 microlitri di glutammina millimolare, 250 microlitri di penicillina-streptomicina, 2,5 ml di siero bovino fetale e 44,375 ml di MEM in un tubo da 50 ml. Per preparare il mezzo di arresto, mescolare 1 ml di soluzione amminoacido MEM, 750 microlitri di 1 HEPES molare, 5 ml di FBS e 43,25 ml di MEM in un tubo da 50 ml. Per preparare l'HBSS, integrato con 15 HEPES millimolare, mescolare 49,25 ml di HBSS e 750 microlitri di 1 HEPES molare.Per la preparazione di piatti rivestiti in poli-l-lisina, rivestire 35 mm di stoviglie di coltura cellulare in plastica con 0,2 mg per ml di poli-l-lisina idrobromide per un giorno a 25 gradi Celsius. Successivamente, lavare i piatti con 2 ml di acqua ultrapura tre volte e incubarli con 1,5 ml di Stop Medium a 25 gradi Celsius fino all'uso. Incubare gli ippocampi sezionati in una miscela contenente 500 microlitri ciascuno del 2,5% di tripside e 10X Dnase-1 in HBSS integrati con HEPES millimolare, pH 7,2, a 37 gradi Celsius per 15 minuti con agitazione ogni 3 minuti.Quindi, trasferire gli ippocampi a 10 ml del mezzo di arresto e incubare a 4 gradi Celsius per 5 minuti per inattivare la tripside. Successivamente, incubare gli ippocampi sezionati in 10 ml di mezzo di arresto fresco a 4 gradi Celsius per 5 minuti. Spostare gli ippocampi in 10 ml di mezzo di arresto fresco e incubare a 4 gradi Celsius per altri 5 minuti.Quindi, spostare gli ippocampi in 900 microlitri del mezzo di arresto e 100 microlitri di Dnase-1 10X in un tubo da 15 ml. Utilizzare una pipetta da 1 ml per dissociare gli ippocampi in neuroni isolati pipettando 20 volte. Aggiungere 9 ml di mezzo di placcatura e filtrare attraverso un colino a celle da 70 micron in un tubo da 50 ml.Contare il numero di cellule che usano un emocitometro e una regolazione a 3,5 per 10 fino alle quattro cellule per millilitro nel mezzo di placcatura. Successivamente, aspirare il mezzo di arresto dalle stoviglie rivestite in poli-l-lisina. Piatto 2 ml per piatto delle cellule sui piatti rivestiti e incubare sotto il 5% di anidride carbonica a 37 gradi Celsius per 60-64 ore.Dopo l'incubazione, aggiungere 2 microlitri di soluzione di calcio Ara-C ai neuroni e scuotere lentamente il piatto. Conservare i piatti di coltura in una scatola umidificata senza cambiare il mezzo di coltura sotto il 5% di anidride carbonica a 37 gradi Celsius. Purificare la frazione ricca di filopodi dendritici dopo 13 giorni in vitro aggiungendo prima tre milioni di microperline magnetiche in polistirolo per piatto a 20 piatti contenenti i neuroni coltivati.Dopo un giorno, lavare i neuroni in 1 ml di PBS con agitazione tre volte per rimuovere le microperline medie e non unite. Dopo aver rimosso pbs, lyse i neuroni con 500 microlitri per piatto del tampone dilisi. Successivamente, raccogliere il lisato con un raschietto cellulare e trasferire il lisato in 10 microtubi leganti a basso contenuto proteico.Impostare i tubi su un separatore di magneti e attendere un minuto. Raccogli il supernatante e usalo come frazione non vincolata per la colorazione dell'argento e l'analisi western blot. Successivamente, trasferire le perline su un nuovo microtubo legante a basso contenuto proteico e impostare su un separatore magnetico per un minuto.Rimuovere completamente il supernatante e aggiungere 500 microlitri del tampone dilisi. Lavare le perline utilizzando un miscelatore Vortex per 15 secondi. Ripetere il lavaggio delle perline 10 volte e rimuovere il supernatante.Elute proteine fino alle perline con l'aggiunta di 50 microlitri di 1X SDS tampone campione e far bollire il tubo a 98 gradi Celsius per 5 minuti. Centrifugare il tubo a 860xG 3000 RPM per 10 secondi e impostare il tubo su un separatore magnetico per un minuto. Raccogliere il supernatante e utilizzarlo come frazione associata.Misurare le concentrazioni delle frazioni proteiche non legate e legate dal saggio proteico BCA. Visualizza le soluzioni di protien con blu bromofile e regola la concentrazione a 5 nanogrammi per microlitro per la pagina SDS. Separare le frazioni associate e non associate per pagina SDS utilizzando un gel gradiente 5-20%..Macchia d'argento nel gel. Infine, western blot usando anticorpi primari e secondari appropriati secondo il manoscritto. Nei neuroni ippocampali coltivati, il TLCN era abbondantemente localizzato nei filopodi dendritici, nell'albero e nel soma e co-localizzato con F-actin.Le perline codificate erano principalmente legate a dendriti e inducevano la formazione di tazze fagocitiche per accumulo di TLCN e F-actina su dendriti neuronali. Le immagini di Thorecense mostravano che la formazione di coppe fagocitiche dipendeva in modo cruciale dalla presenza di TLCN nei dendriti. Le tazze fagocitiche si sono formate solo su neuroni ippocampali di tipo selvaggio, ma non su neuroni ippocampali defficienti TLCN.I risultati della pagina SDS hanno mostrato che i modelli di banda proteica erano quasi gli stessi per le frazioni non legate e legate, ma le intensità a 50 e 70 kilodalton, nella frazione legata erano inferiori a quelle della frazione non legata. Tuttavia, l'intensità della banda non era ovviamente diversa tra le frazioni non legate e legate preparate dai neuroni ippocampali di coltura defficiente TLCN. L'analisi western delle frazioni non legate e legate ha mostrato che TLCN e VN sono stati rilevati principalmente nella frazione associata.Actin, Ezrin, G alpha q, PLC beta 1, MAP-2 e Spectrin, sono stati rilevati sia nelle frazioni legate che in uscita. Moesin, PSD-95, alfa-actinina e alfa-tubulina sono stati rilevati nella frazione non vincolata. Se la proteina di rivestimento delle microperline cambia, questa procedura può essere applicata per identificare l'interazione del saggio.Questa tecnica potrebbe identificare la proteina che lavora su sostanze artificiali. Quindi crediamo che il meccanismo della genesi artificiale possa essere quantificato. Crediamo che nuove proteine associate al disturbo mentale possano essere identificate dalla frazione ricca di filopodi dendritici.E può essere esteso alla 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.

Analisi di Morfologia spine dendritiche in colture di neuroni del SNC

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

Ricerca

Neuroscienze

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.

Indurre crescita dendritica in colture di neuroni simpatici

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.

L&#39;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

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.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione 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.

Valutazione della dendritiche arborizzazione nel giro dentato della Regione ippocampale nei topi

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.

Purificazione delle navi cervello di topo

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.

Analizzando dendritiche Morfologia in colonne e strati

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.

Valutazione della complessità dendritica ippocampale nei topi invecchiati utilizzando il metodo 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.

Iontoforesi in tempo reale con tetrametilammonio per quantificare la frazione del volume e la tortuosità dello spazio extracellulare del cervello

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.

Analisi quantitativa di un neurone Arborization dentritico complessità in Drosophila

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