Çalışma NIH Klinik Bilim Geliştirme ödül K08NS002083, bir Shervert Frazier Araştırma Enstitüsü Grant ve Edgar A. Büttner bir NARSAD Genç Araştırmacı Ödülü ile desteklenmiştir.

1.. Gelişim Delay / Lethality Deneyi: Vahşi tip (N2) ve iki Mutant (Mut1 ve Mut2) Suşlar, 12 oyuklu bir plaka içerisinde üç Klozapin Konsantrasyonlar test edilir <ol><li> 1. günde, (2 cm çaplı her oyuğa), 12 oyuklu plakanın her oyuğuna 2 mi NGM ortamı 10 dökün ve gece boyunca oda sıcaklığında (RT) de tezgah üzerinde sertleşmesine olanak sağlar. Aynı gün, bir gece boyunca 220 rpm devirle, Escherichia coli OP50 bir bakteri kolonisi seçmek 50 ml LB solüsyonu bir şişe enfekte eder ve 37 ° C bir çalkalayıcı içinde kültür o. </li><li> 2. günde, her bir oyuğa NGM merkezi üzerine 20 ul OP50 bakteri kültürü aktarın. 37 ° C inkübatör içinde inkübe edilir ya da bakteri çim kalın büyümesine izin vermek için gece boyunca oda sıcaklığında bekletin. </li><li> DMSO (dimetil sülfoksit) solvent içinde eritilmesi ile klozapin bir 80 mM stok solüsyonu klozapin olun. Daha sonra 1.7 mM asetik asit B içinde seyreltilmiş stok çözeltisi ile bir 80 ul çalışma çözeltisi yapmakTablo 1 ased. Not: ilgi belirli bir ilaç için hazır çözelti mutantlardan alınan yabani tür ayırt için en uygun konsantrasyonunu bulmak için, ön konsantrasyon bir dizi test yaparak tespit edilmelidir. </li><li> Aktarım 80 ul klozapin de tohumlanmış NGM plaka üzerine çalışma çözeltisi ve çözelti yüzeyi üzerinde eşit olarak dağıtmak için izin vermek için plaka girdap. Plaka kurumasını bekleyin. Aynı gün deneyi için plaka kullanarak ya da bir sonraki gün kullanılmak üzere 20 ° C inkübatör içine yerleştirin. Not: çalışma çözeltisi, özellikle yüksek konsantrasyonda, Klozapin düşük çözünürlüğe sahip bir ilaç bir süspansiyondur. Bu nedenle, ilaç konsantrasyonu doğru olduğunu garanti etmek için ilaç plakaya çözüm aktarmadan önce tüp vorteks. </li><li> M9 tampon ile birçok gebe yetişkin içeren 3,5 cm plaka solucanlar yıkayın ve daha sonra bir 15 ml tüp onları aşağı spin1 dakika boyunca 2000 rpm&#39;de. </li><li> Süpernatant aspire. 5 ml çamaşır suyu (: hipoklorit: NaOH 04:01:05 de GKD 2 O) ekleyin ve hafifçe tersini tüpleri nematodları bozabilir. </li><li> Yarısı yaklaşık 5 dakika ile eritilir kadar hayvanlar dikkat edin. 1 dakika 2.000 rpm&#39;de yumurta aşağı spin. </li><li> Süpernatant aspirat ve hızla 14 ml M9 tampon ekleyin. </li><li> 1 dakika 2.000 rpm&#39;de yumurta aşağı Spin ve yaklaşık 100 ul çözüm bırakarak, süpernatant aspire. Yumurta askıya Vortex. </li><li> Transferi 2 ul yumurta transfer kaç yumurta test etmek için düzenli bir NGM plaka üzerine M9 asılı. Yaklaşık 30-35 yumurta her transferi olmadığından emin olmak için askıya yumurta seviyesini ayarlayın. Deney plakasının her oyuğuna 30-35 yumurta transferi ve daha sonra 24 saat için bir 20 ° C kuluçka makinesi içinde inkübe edilir. </li><li> Tahlilin ilk gününde, yumurtadan yumurta sayısını skor. Picki tarafından 25ul/çukur için taranmış hayvanların toplam sayısını ayarlayınGerekirse ekstra kapalı solucanlar ng. </li><li> Daha sonraki günlerde, hayvanları gözlemlemek ve onları her 24 saatte skor. Gelişim aşama hayvanın boyutu ve vulva 11 şekline göre puanlanmıştır. Deney tipik olarak, üçüncü ya da dördüncü gününde görülen en güçlü etkisi ile 5-6 gün sürer. </li><li> Bir Excel dosyası içine girdi veri ve deney her gün için her gelişimsel aşamada hayvanların yüzdesi hesaplanır. &#39;100% Menüsünden &#39;&#39;2-B sütun içinde&#39; sütun Yığılmış fonksiyonu ile grafikler oluşturun. </li></ol> 2. Pharyngeal Kaldırma Deneyi: Yabani-tür ve mutant suşu için genç yetişkin Hayvan Deneyi Klozapin tabakalarına atacak <ol><li> Analiz plakası gelişimsel gecikme / letalite deneyinde kullanılan aynı protokol takip edilerek yapılır. </li><li> 24 saat analizden önce ekim yapılmış olan NGM plakalarına her bir suş için 50 L4 hayvanlar almak ve 20 ° C de inkübe solucanlar </li><li> Bir assa 10 hayvan almaky iyi plaka. Ardından bir sonraki de her 15-20 dk 10 hayvan almak. </li><li> İlk kuyudaki hayvanlar 30 dakika için bir ilaca maruz bırakıldıktan sonra, bir diseksiyon mikroskobu altında yutak pompalama gözlemleyerek deneyi başlatın ve 20 saniye için pompalar 9 puan. Faringeal pompalama attı sonra, plaka kapalı solucan almak. </li></ol>

<ol>
	<li>Donohoe, D. R., Jarvis, R. A., Weeks, K., Aamodt, E. J., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+adaptation+in+C.+elegans+produced+by+antipsychotic+drugs+requires+serotonin+and+is+associated+with+calcium+signaling+and+calcineurin+inhibition.">Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.</a> Neurosci. Res. 64, 280-289 (2009).</li><li>Karmacharya, R., 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=Clozapine+interaction+with+phosphatidyl+inositol+3-kinase+(PI3K)/insulin-signaling+pathway+in+Caenorhabditis+elegans.">Clozapine interaction with phosphatidyl inositol 3-kinase (PI3K)/insulin-signaling pathway in Caenorhabditis elegans.</a> Neuropsychopharmacology. 34, 1968-1978 (2009).</li><li>Rand, J. B., Johnson, C. D., Estein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Carnorhabditis elegans Modern Biological Analysis of an Organism. 48, 187-204 (1995).</li><li>Kwok, T. 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=A+small-molecule+screen+in+C.+elegans+yields+a+new+calcium+channel+antagonist.">A small-molecule screen in C. elegans yields a new calcium channel antagonist.</a> Nature. 441, 91-95 (2006).</li><li>Wang, X., Sliwoski, G. R., Buttner, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+Caenorhabditis+elegans+genetics+for+understanding+human+psychiatric+disease.">The relevance of Caenorhabditis elegans genetics for understanding human psychiatric disease.</a> Harv. Rev. Psychiatry. 19, 210-218 (2011).</li><li>Donohoe, D. R., Aamodt, E. J., Osborn, E., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antipsychotic+drugs+disrupt+normal+development+in+Caenorhabditis+elegans+via+additional+mechanisms+besides+dopamine+and+serotonin+receptors.">Antipsychotic drugs disrupt normal development in Caenorhabditis elegans via additional mechanisms besides dopamine and serotonin receptors.</a> Pharmacol. Res. 54, 361-372 (2006).</li><li>Karmacharya, R., 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=Behavioral+effects+of+clozapine:+involvement+of+trace+amine+pathways+in+C.+elegans+and+M.+musculus.">Behavioral effects of clozapine: involvement of trace amine pathways in C. elegans and M. musculus.</a> Brain Res. 1393, 91-99 (2011).</li><li>Saur, 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=A+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans+Identifies+the+Nicotinic+Acetylcholine+Receptor+Subunit+ACR-7+as+an+Antipsychotic+Drug+Target.">A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target.</a> PLoS Genet. 9, (2013).</li><li>Avery, L., You, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans feeding. WormBook, ed. The C. elegans Research Community, doi:10.1895/wormbook.1.150.1. , (2012).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. 47, (2011).</li><li>Lewis, J. A., Fleming, J. T., Estein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Carnorhabditis elegans Modern Biological Analysis of an Organism. 48, 3-29 (1995).</li><li>Davis, J. R., Li, Y., Rankin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+developmental+exposure+to+ethanol+on+Caenorhabditis+elegans.">Effects of developmental exposure to ethanol on Caenorhabditis elegans.</a> Alcohol Clin. Exp. Res. 32, 853-867 (2008).</li><li>Kage-Nakadai, E., 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=Two+very+long+chain+fatty+acid+acyl-CoA+synthetase+genes,+acs-20+and+acs-22,+have+roles+in+the+cuticle+surface+barrier+in+Caenorhabditis+elegans.">Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.</a> PLoS One. 5, (2010).</li><li>Partridge, F. A., Tearle, A. W., Gravato-Nobre, M. J., Schafer, W. 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Yazarlar dahil faiz çatışma olmadığını beyan ederim.

Burada, C ve davranış ile ilgili APDS etkilerini test etmek için yöntemleri tarif eder elegans. İlaç suda çözünmediği DMSO ya da etanol, klozapin çözmek için kullanılır. Çözücüler C etkilediği bildirilmiştir Çünkü elegans biyoloji 12, DMSO-tek başına veya etanol başına bir kontrol grubu gereklidir. Analizlerde kullanılan en yüksek DMSO konsantrasyonu, C üzerinde belirgin bir etkisi yoktur% 3 e kadar olduğu elegans gelişimi....

Caenorhabditis elegans büyük ölçekli ileri ve geri genetik ekranları ve kimyasal genetik ekranlarında mükellef basit bir genetik organizmadır. C. elegans biyolojik olarak aktif bileşikler, geniş bir spektrum duyarlıdır ve bu nedenle bu bileşiklerin, çeşitli etki mekanizmalarını tanımlamak için başarılı bir şekilde kullanılmıştır. Örneğin, biyolojik olarak aktif bileşikler, solucan farmakogenetik asetilkolin reseptörü agonistleri (örneğin, levamisol, nikotin, morantel ve pirantel), anestezikler (ör., halotan), kafein, kolinesteraz inhibitörleri (örneğin, aldikarb, lannate ve triklorfon), florür, GABA ile ilişkili bulunmaktadır kullanılarak incelendi bileşikleri (örneğin, GABA ve Muscimol), ivermektin, parakuat, forbol esterler ve serotonin ile ilgili ilaçlar (örneğin, serotonin ve imiprimine) 3. Ayrıca, C. elegans sağlayan, büyük ölçekli küçük molekül ekranlar için kullanılmış olan, yeni biyolojik olarak aktif bileşiğin keşfis ve yeni genetik hedefler 4. tanımlanması. C. elegans genomu nörotransmitter sentezi ve sinaptik yapısı ve fonksiyonu için gerekli olan 5 proteinleri kodlayan genler dahil olmak üzere potansiyel antipsikotik ilaç (APD), insanlarda muhafaza hedefleri içerir. Bu nedenle, C. elegans nörogenetik ve APDS etki yeni moleküler mekanizmalarını keşfetmek için nörobiyoloji teklif yöntemleri. Nematod olarak, APD maruz erken gelişim gelişme gecikmesi ve daha yüksek konsantrasyonlarda, ölümcül 2,6 üretir. Yetişkinlik döneminde APD poz davranışsal fenotipleri üretir. Örneğin, klozapin pozlama lokomosyonu ve faringeal pompalama engeller ve 1,2,7 döşeme yumurta geliştirir. APD kaynaklı gelişimsel gecikme ve öldürücülüğü büyük ölçekli kimyasal genetik ekranlar için güçlü fenotipleri vardır. Büyük olasılıkla birden fazla hücresel ve genetik Basi gibi bu fenotipleri kadar karmaşıktırs. Bu nedenle, genetik ekranlar dolaylı ilaç hedefleri bir dizi elde etmek için beklenir. Ancak, bizim laboratuvar aday gen ekranları ve APD kaynaklı gelişimsel gecikme ve letalitesinin bastırıcılarının için bir genom RNAi ekran gerçekleştirilen ve başarıyla olasılıkla dopamin, insülin ve nikotinik asetilkolin reseptörleri 2,8 dahil olmak üzere doğrudan hedefleri, kodlayan genleri kurtarıldı. Yetişkin APD kaynaklı davranışları dayalı genetik ekranlar da yeni APD hedeflerinin tanımlanmasına yol açmıştır, ve biz şimdi memelilerde 7 gelişimsel ve davranışsal hem de ekranlardan hedefleri doğrulayarak edilir. Bu nedenle, APDS aksiyon yeni moleküler mekanizmaları keşfetmek için bir omurgasız kimyasal bir genetik yaklaşım 5,8 mümkün görünmektedir. C. elegans yutak 20 nöronlar, 20 kas hücreleri ve Bir taban zan ile sarılmış 20 aksesuar hücreleri içeren bir organdır. Memeli kalp benzer şekilde, yutak özerk a,nd sürekli dış ortamdan 9 gıda pompaları. Faringeal pompalama oranı engellenmesi gıda alımını uzlaşma ve böylece mutasyonlar veya uyuşturucu faringeal pompalama nedeni gelişimsel gecikme inhibe veya 9 tutuklama söyledi. APDS gelişimi ve canlılığı 1,2 üzerindeki etkileri kısmen muhasebe, yutak pompalama hızını inhibe. Burada, nematod gelişimi ve yutak pompalama için ilaç deneyleri göstermek için bir örnek olarak atipik AKB klozapini kullanın.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

Caenorhabditis elegans ileri büyük ölçekli mükellef ve genetik ekranları ve kimyasal genetik ekranları ters basit bir genetik organizmadır. C. elegans genomu nörotransmitter sentezi ve sinaptik yapısı ve fonksiyonu için gerekli olan proteinleri kodlayan genler dahil olmak üzere potansiyel antipsikotik ilaç (APD), insanlarda muhafaza hedefleri içerir. APD maruz kalması, konsantrasyona bağlı bir şekilde gelişme gecikmesi ve / veya kurt öldürücü olarak üretir. Bu fenotipler 1,2 kaldırma farenks ve APD kaynaklı inhibisyonu ile, kısmen, kaynaklanır. Böylece, gelişimsel fenotip nöroleptiklerin farmakogenetik çalışmalar için yararlıdır, bir nöromüsküler temeli vardır. Burada nematod gelişimi ve yutak pompalamayı APD etkilerini test için ayrıntılı prosedürleri göstermektedir. Gelişimsel testi için, senkronize embriyolar nematod büyüme ortamı APDS içeren (NGM) levhalar, ve ani gelişen aşamalarında yerleştirilirmals sonra günlük puanlanır. Faringeal pompalama hızı testi için, genç yetişkin hayvanlar APDS içeren NGM plakalar üzerinde test edilir düzenledi. Birim zaman başına yutak pompaların sayısı kaydedilir ve pompalama hızı hesaplanır. Bu deneyler, küçük moleküller, büyük moleküller ya da birçok diğer türleri çalışmak için de kullanılabilir.

1.. Gelişimsel gecikme / letalite tahlil sonucu Gelişimsel gecikme / öldürücülüğü deneyi için tipik bir sonucu Şekil 1a gösterdi ve 1b edilir. Kontrol grubunda, vahşi tip hayvanlara gebe yetişkin aşamasında (Şekil 1a) büyümüştür zaman, klozapin maruz vahşi tip hayvanların ölmüş (Şekil 1b). Şekil 1c karşılaştırma temsili bir sonuç göstermektedir Genç larva aşamalarında hala ya da vardır bas...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans antipsikotik ilaçlar (APDS) etkilerini test etmek için yaklaşımlar gösterilmektedir. Deneyler gelişimi ve canlılığı üzerinde ve yutak pompalama hızı üzerindeki, ilaç etkilerini test etmek için tarif edilmiştir. Bu yöntemler aynı zamanda APDS başka ilaç sınıfları ile farmakogenetik deneyler için geçerlidir.

Antipsikotik İlaçların Etki Mekanizmaları incelenmesi için Yöntemleri<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

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Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

11.4K Görüntüleme.  Harvard Medical School.  Caenorhabditis elegans antipsikotik ilaçlar (APDS) etkilerini test etmek için yaklaşımlar gösterilmektedir. Deneyler gelişimi ve canlılığı üzerinde ve yutak pompalama hızı üzerindeki, ilaç etkilerini test etmek için tarif edilmiştir. Bu yöntemler aynı zamanda APDS başka ilaç sınıfları ile farmakogenetik deneyler için geçerlidir. 

Video: Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Nörobilim

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Kemirgen Merkezi Sinir Sistemi İlaçların doğrudan Intraventriküler teslim

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Bir<em&gt; Caenorhabditis elegans</emAmylopathy Çalışmaları&gt; Model Sistemi

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Locomotion Speed ​​Etanol Etkilerinin Ölçülmesi için bir Testi<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Uzun süreli Kalsiyum Görüntüleme için Agaroz microchambers<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Roman Metrik Nematodu Embriyonik Uzama karakterize etmek<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

bir<emCaenorhabditis elegans</emBeslenme Durumuna Dayalı Bakır Kaçınma Deneyi

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Ratiometric kalsiyum görüntüleme Caenorhabditis Elegans davranışlar içinde bireysel nöronların

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Caenorhabditis elegans'ta Dendritik Dikenleri Görüntüleme

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...