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

Biz hipokampal nöronların düşük yoğunluklu kültürü için Shigeo Okabe ve Hitomi Matsuno teşekkür, tlcn eksikliği fareler için Masayoshi Mishina, teknik yardım için Sachiko Mitsui ve Momoko Shiozaki ve yararlı tartışmalar için Yoshihara Laboratuvarı üyeleri . Bu çalışma JSPS KAKENHI Grant NOS tarafından desteklenmektedir. JP20700307, JP22700354 ve JP24500392 ve MEXT KAKENHI Grant Nos. JP23123525 için YF ve JP20022046, JP18H04683 ve JP18H05146 YY.

Burada açıklanan tüm yöntemler RıKEN Wako kurumsal hayvan bakımı ve kullanım Komitesi tarafından onaylanmıştır.
1. hipokampal nöronların kültürü
<ol><li>Kültür ortamının hazırlanması
	<ol>
<li>200x Vitamin karışımı hazırlanması. Çözülür 100 mg D-pantothenic asit hemicalcium tuz, 100 Kolin klorür mg, 100 mg folik asit, 180 mg i-inositol, 100 mg Niacinamide, 100 mg piridoksal HCL, ve 100 mg Thiamin HCL içinde 500 ml Ultra saf su manyetik kar?...

<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>

Biz, hücre yapışma molekül TLCN ve ekstrasellüler matris protein vitronektin arasında benzeşimi kullanarak dendritik filopodia zengin fraksiyonu için bir arıtma yöntemi geliştirdi. PSD kesir ile karşılaştırıldığında, dendritik filopodia zengin fraksiyondan olgunlaşmamış sinaps üzerinde hareket sinaptik proteinlerin tanımlamak mümkün olabilir. Böylece, dendritik filopodia zengin fraksiyonu bileşenleri% 74 tarafından PSD kesir olandan farklıdır. PSD kesir farklı, biz aktif fagositik bardak o...

Dendritik filopodia, spines öncüleri olduğu düşünülmektedir. Dendritik filopodia içinde actin filamin uzatma ve retraksiyon1,2,3düzenler. Bir Axon ile temas ettikten sonra, seçilmiş dendritik filopodia, olgunlaşmasını spines içine başlar ve bir sinaps4,5oluşur. Dikenler bileşenleri postsinaptik yoğunluk kesirler6,7kapsamlı analizinden belirlendi, dendritik filopodia bileşenleri büyük ölçüde bilinmeyen kalır süre. Bu, telencephalin (tlcn), erm, syngap, RAS, pıkinaz, AKT, mTOR, Polo benzeri kinaz 2, camkii, syndecan-2, paralemin-1, ARF6 ve ephb, dendritik filopodia oluşumu5,8,9 düzenleyen gösterildi ,10,11, bir yöntem ise dendritik filopodia mevcut moleküllerin kapsamlı analizi için geliştirilmiştir değil.
Tlcn (ICAM-5) özellikle en rostral beyin segmentinde dikenli nöronlar tarafından ifade edilir, telencephalon12. TLCN, hücre dışı bölgesinde 9 IG benzeri etki alanı, bir transmembran bölgesi ve sitoplazmik kuyruk13' ü vardır. Tlcn, hücre dışı bölgesinde vitronektin (VN) ve LFA-1 integrine, transmembran bölgesinde presenilin 'e, sitoplazmik bölgesinde fosho-ERM ve α-actinin 'e bağlar5,8,14,15 ,16. Tlcn, dikenler ve dendritik şaftlar içinde dendritik filopodia ve α-actinin iplerinde phospho-ERM yoluyla akin sitoiskelet bağlar8,16.
Biz tlcn gelişmiş dendritik filopodia oluşumu ekspresyonu gösterdi ve filopodia10için dikenler reversion indüklenmiş. Teknikerleriniz 'in kurucu aktif formu tlcn sitoplazmik bölgeye bağlı ve gelişmiş dendritik filopodia oluşumu8. Böylece TLCN, akin bağlayıcı proteinlerle dendritik filopodia oluşumunu düzenler. Esselens ve ark. mikroboncuklar kültürsüz nöronlar üzerinde TLCN birikimi kaynaklı gösterdi17. Fagositik fincan yapısının, VN kaplı mikroboncuk etrafında nöronal dendritler üzerinde, TLCN-bağımlı bir biçimde15' te oluştuğunu gösterdik. Dendritik filopodia bileşenleri fagositik kupalar benzer. Dendritik filopodia toplamak zordur, ama manyetik mikroboncuk kullanarak fagositik fincan toplamak nispeten daha kolaydır. Böylece, dendritik filopodia18yerine fagositik kupayı arındırmak için bir yöntem geliştirdik. Burada, dendritik filopodia zengin fraksiyonu için arıtma yöntemi açıklanmaktadır.

<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

Dendritik filopodia akinin filament dayalı ince ve uzun çıkıntılar, ve onlar uzatmak ve bir hedef Axon ararken gibi geri çekin. Dendritik filopodia bir hedef Akon ile temas kurduğunda, bir sinaps oluşumuna yol açan, spines içine olgunlaşmak başlar. Telencephalin (TLCN) oldukça dendritik filopodia lokalize ve yavaş yavaşça spines hariç tutulur. Kültürlü hipokampal nöronlar içinde TLCN aşırı ifade dendritik filopodia oluşumu indükler. Biz telencephalin güçlü bir hücre içi matris molekül, vitronektin bağlar gösterdi. Vitronectin kaplı mikrobilyeleri nöronal dendrites üzerinde fagositik fincan oluşumu kaynaklı. Phagositik kupada, TLCN, fosforilated ezrin/Radixin/Moesin (phospho-ERM) ve F-actin gibi TLCN-bağlayıcı proteinler birikir, bu da fagositik kupanın bileşenlerinin dendritik filopodia 'ya benzer olduğunu göstermektedir. Böylece, dendritik filopodia yerine fagositik kupayı arındırmak için bir yöntem geliştirdik. Manyetik polistiren boncuklar, hipokampal nöronların kültür ortamında bulunan ve nöronal dendrites üzerinde fagositik bardak oluşumunu tetikleyen vitronektin ile kaplanmıştır. 24 saat inkübasyon yapıldıktan sonra, fagositik bardak deterjan ile hafif bir şekilde çözünmeli ve mıknatıs ayırıcı kullanılarak toplanır. Boncukları yıkadıktan sonra, bağlayıcı proteinler gümüş boyama ve Batı bloplama ile incelenmiş ve analiz edilmiştir. Bağlayıcı fraksiyonda, tlcn ve aktin bol miktarda mevcut. Buna ek olarak, kesir tespit birçok protein dendritik filopodia lokalize edildi; Bu nedenle, biz dendritik filopodia-zengin fraksiyonu olarak bağlayıcı fraksiyonu adı. Bu makalede, dendritik filopodia zengin kesir için arıtma yöntemi ile ilgili ayrıntıları açıklar.

Kültürlü hipokampal nöronlar, TLCN oldukça dendritik filopodia, şaft lokalize ve Soma ve F-actin ile kolokalize (Şekil 1a, B). Kültürlü hipokampal nöronlara polistiren mikroboncuk eklendiğinde, boncuklar otomatik olarak kültür ortamında fetal sığır serumu (FBS) elde edilen vitronektin (VN) ile kaplanmıştır; temelde dendrites bağlı ve onlar fagositik bardak oluşumu indüklenen (Şekil 1B<st...

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

Purification of the Dendritic Filopodia-rich Fraction

Bu protokolde, dendritik filopodial arasında belirli ve güçlü bir yakınlık yararlanarak kültürlü hipokampal nöronlar üzerinde fagositik fincan benzeri protrüzyon yapısı dendritik filopodia zengin fraksiyonu arındırmak için bir yöntem tanıtmak yapışma molekül, TLCN, ve bir ekstrasellüler matris molekül, vitronektin.

Dendritic Filopodia-zengin kesir arıtma

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 ...

PSD fraksiyonu ile karşılaştırıldığında, dendritik filopodia açısından zengin fraksiyonu etrafında olgunlaşmamış sinaps üzerinde hareket eden sinaptik proteini tanımlamak mümkün olabilir. Fagositik kapak oluşumunu tetiklemek için canlı nöron kullandık. Bu tekniğin en büyük avantajı, yapay yaratılıştaki aktif proteinin dendritik filopodia zengin fraksiyonundan saptanabildiğidir.Başlamak için, B5 vitamini 100 mg eriterek 200x Vitamin Mix hazırlamak, Kolin Klorür 100 mg, Folik Asit 100 mg, I-inositol 180 mg, B3 vitamini 100 mg, B6 vitamini hidroklorür 100 mg, ve 100 ultrasaf su 500 ml ultrasaf su 500 ml ultrasaf su kullanarak. Aliquot'u 50 ml'lik tüplerde dikkatlice karıştırın ve 20 santigrat derecede saklayın. Riboflavin çözeltisi hazırlamak için, manyetik bir karıştırıcı kullanarak ultrasaf su 500 mg riboflavin 100 mg çözünür.Aliquot'u 50 ml'lik tüplerde dikkatlice karıştırın ve 20 santigrat derecede saklayın. Bir azı kalsiyum diklorür hazırlamak için manyetik bir karıştırıcı kullanarak ultrasaf su 50 ml kalsiyum klorür dihidrat 7,35 gram çözünür. Minimum Esansiyel Orta hazırlık için 400 potasyum klorür mg 6, sodum klorür 800 mg, sodyum bikarbonat 200 mg, sodyum fosfat monobasic dihidrat 158 mg, 7, 000 D-glikoz mg, ve magnezyum sülfat heptahidrat 200 mg, ve ultrasaf su 950 ml, manyetik bir karıştırıcı kullanarak.Daha sonra, bir damla şekilde MEM için 1 molar kalsiyum diklorür 1.8 ml eklemek için bir manyetik karıştırıcı üzerinde sürekli ajitasyon ile 1 ml pipet kullanın. Daha sonra MEM'in pH'ını pH 7.25'e ayarlamak için hidroklorik asit ekleyin. Daha sonra MEM'e 5 ml 200x vitamin karışımı ve 200 mikrolitre riboflavin çözeltisi ekleyin.Çözeltinin hacmini ultra saf su ile 1000 ml'ye ayarlayın. Çözeltiyi 0,22 mikron filtre sistemi kullanarak filtreleyin ve 4 santigrat derecede saklayın. 10x DNase-1 stok çözeltisi hazırlamak için 100 mg Dnase-1'i 12,5 ml HBSS'de çözünür.Filtre 0.22 mikron filtre, 1.5 ml tüpler içinde aliquot, ve 20 santigrat derece tüpleri saklayın. Ara-C stok çözeltisi hazırlama için 25 mg Ara-C 8.93 ml ultrasaf suda çözünür. Filtre 0.22 mikron filtre, 1.5ml tüpler içinde aliquot ve 20 derece santigrat de saklayın.Kaplama Ortamını hazırlamak için 1 ml MEM Amino Asit Solüsyonu, 750 mikrolitre 1 molar HEPES, 1 ml B27, 125 mikrolitre 200 milimolar glutamin, 250 mikrolitre penisilin-streptomisin, 2,5 ml Fetal Sığır Serumu ve 50 ml'lik mem 44.375 ml'yi karıştırın. Durdurma ortamını hazırlamak için 1 ml MEM amino asit çözeltisi, 750 mikrolitre 1 molar HEPES, 5 ml FBS ve 50 ml'lik bir tüpte 43,25 ml MEM'i karıştırın. 15 milimolar HEPES ile desteklenen HBSS hazırlamak için 49.25 ml HBSS ve 750 mikrolitre 1 molar HEPES karıştırın.Poli-l-lizin kaplamalı yemekler in hazırlanması için, 25 santigrat derecede bir gün boyunca ml başına 0,2 mg poli-l-lizin hidrobromür ile 35 mm plastik hücre kültürü yemekleri kat. Daha sonra, bulaşıkları 2 ml ultrasaf su ile üç kez yıkayın ve kullanıma kadar 25 santigrat derecede 1,5 ml Stop Medium ile kuluçkaya yatırın. 15 milimolar HEPES, pH 7,2, 37 derece santigrat ile desteklenen HBSS 500 mikrolitre her biri 2.5% tripsin ve 10X Dnase-1 içeren bir karışım içinde diseksiyon hipokampi kuluçka 3 dakikada bir ajitasyon ile 15 dakika.Daha sonra hipokampi Stop Medium'un 10 ml'sine aktarın ve tripsinin inaktive etmek için 4 santigrat derecede 5 dakika kuluçkaya yatırın. Daha sonra, 5 dakika boyunca 4 derece santigrat taze stop orta 10 ml diseksiyon hipokampi kuluçka. Hippocampi'yi 10 ml taze stop ortasına taşıyın ve 4 santigrat derecede 5 dakika daha kuluçkaya yatırın.Daha sonra, 15 ml tüp içinde durdurmak orta 900 mikrolitre ve 10X Dnase-1 100 mikrolitre içine hipokampi taşıyın. 20 kez pipetleme tarafından izole nöronlar içine hipokampi ayrıştırmak için 1 ml pipet kullanın. Kaplama orta 9 ml ekleyin ve bir filtre 70 mikron hücreli süzgeç ile, bir 50 ml tüp içine.Hemositometre kullanan hücre sayısını ve kaplama ortamındaki mililitre başına 4 hücreye 3,5 kez 10'a ayarlayın. Daha sonra, poli-l-lisine kaplı yemeklerden stop orta aspire. Tabak 2 ml kaplamalı yemekler üzerinde hücrelerin çanak başına ve 60-64 saat boyunca 37 santigrat derece% 5 karbondioksit altında kuluçka.Kuluçkadan sonra, nöronlara 2 mikrolitre Ara-C stok çözeltisi ekleyin ve yemeği yavaşça sallayın. Kültür yemeklerini 37 santigrat derecede %5 karbondioksit altında kültür ortamını değiştirmeden nemlendirilmiş bir kutuda saklayın. İlk kültürlü nöronlar içeren 20 yemekler için çanak başına üç milyon manyetik polistiren mikroboncuklar ekleyerek 13 gün in vitro sonra dendritik filopodia zengin fraksiyonu arındırın.Bir gün sonra, orta ve bağlı olmayan mikroboncuklar kaldırmak için agiation üç kez PBS 1 ml nöronlar yıkayın. PBS çıkardıktan sonra, lysis tampon çanak başına 500 mikrolitre ile nöronları lyse. Daha sonra, bir hücre kazıyıcı ile lysate toplamak ve 10 düşük protein bağlayıcı mikrotüpler içine lysate aktarın.Bir mıknatıs ayırıcı tüpler ilerler ve bir dakika bekleyin. Supernatant toplamak ve gümüş boyama ve Batı leke analizi için sınırsız fraksiyonu olarak kullanabilirsiniz. Daha sonra, boncukları yeni bir düşük protein bağlayıcı mikrotüp e aktarın ve bir dakika lığına manyetik bir ayırıcıya ayarlayın.Tamamen supernatant çıkarın ve lysis tampon 500 mikrolitre ekleyin. 15 saniye boyunca bir Vortex karıştırıcı kullanarak boncukyı yıkayın. Boncukların yıkanması 10 kez tekrarlayın ve supernatant çıkarın.1X SDS örnek tampon 50 mikrolitre ilave edilerek boncuklar aşağı elute proteinlerve 5 dakika boyunca 98 santigrat derece tüp kaynatın. Tüpü 860xG 3000 RPM'de 10 saniye santrifüj edin ve tüpü bir dakika lığına manyetik ayırıcıya ayarlayın. Supernatant toplamak ve bağlı kesir olarak kullanabilirsiniz.Bağlı olmayan ve bağlı protein fraksiyonlarının konsantrasyonlarını BCA protein tadına göre ölçün. Protien çözeltilerini bromofenil mavisi ile görselleştirin ve SDS sayfası için konsantrasyonu mikrolitre başına 5 nanograma ayarlayın. %5-20 degrade jel kullanarak Bağlı ve bağlanmamış kesirleri SDS sayfası ile ayırın.Jelgümüş lekesi. Son olarak, batı el yazmasına göre uygun birincil ve ikincil antikorlar kullanarak leke. Kültürlü hipokampal nöronlarda, TLCN bol dendritik filopodia, şaft ve soma lokalize edildi ve F-aktin ile birlikte lokalize.Kodlanmış boncuklar esas olarak dendritlere bağlıydı ve nöronal dendritler üzerinde TLCN ve F-aktin birikimi ile fagositik kapların oluşumunu neden. Thorecense görüntüleri fagositik fincan oluşumu nun çok önemli ölçüde dendritlerde TLCN varlığına bağlı olduğunu göstermiştir. Fagositik bardaklar sadece yabani tip hipokampal nöronlarda oluşmuştur, ancak TLCN defficient hipokampal nöronlarda oluşmaz.SDS sayfa sonuçları protein bant desenleri neredeyse bağlı ve bağlı fraksiyonları için aynı olduğunu gösterdi ama yoğunlukları 50 ve 70 kilodalton, bağlı fraksiyonu bu bağlı olmayan fraksiyonu daha düşüktü. Ancak, bant yoğunluğu açıkça TLCN defficient kültür hipokampal nöronlar hazırlanan bağlanmamış ve bağlı fraksiyonları arasında farklı değildi. Bağlanmamış ve bağlı kesirlerin Batı leke analizi TLCN ve VN'nin esas olarak bağlı kesirde tespit edildiğini gösterdi.Actin, Ezrin, G alfa q, PLC beta 1, MAP-2 ve Spectrin, bağlı ve bağlı olmayan fraksiyonlarda saptanır. Belirsiz fraksiyonda Moesin, PSD-95, alfa-Aktinin ve alfa-Tubulin saptadı. Mikroboncukların kaplama proteini değişirse, bu işlem, hakem etkileşimini tanımlamak için uygulanabilir.Bu teknik yapay maddeler üzerinde çalışan proteini tanımlayabilir. Bu yüzden yapay yaratılış mekanizmasının ölçülebileceğine inanıyoruz. Biz ruhsal bozukluk ile ilişkili yeni protein dendritik filopodia açısından zengin fraksiyonu tespit edilebilir inanıyoruz.Ve terapiye kadar uzatılabilir.

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.

Kültür MSS Nöronlar Dendritik Omurga Morfoloji Analizi

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

Nörobilim

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.

Kültür sempatik nöronlar bulunan dendritik Büyüme indükleyen

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.

Organotipik Dilim Kültürleri Purkinje Hücre Dendritik Morfoloji Analizi

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.

Glutamat ve GABA hızlı Micro-iyontoforez: Synaptic Entegrasyon Araştırma yararlı bir araç

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.

Farelerde Hipokampal Bölgesi Dentate Gyrus dendritik arborization Değerlendirilmesi

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.

Fare Beyin Gemiler saflaştırılması

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.

Sütunlar ve Katmanlar dendritik Morfoloji Analizi

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.

Yaşlı Bir Farede Hipokampal Dendritik Karmaşıklığın Golgi-Cox Yöntemi ile Değerlendirilmesi

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.

Beyin Ekstraselüler Alanının Hacim Fraksiyonu ve Kıvrımını Nicelleştirmek için Tetrametilamonyum ile Gerçek Zamanlı İyontoforez

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.

Kantitatif analiz Drosophila nöronal dendritik Arborization karmaşıklığı

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