Die Arbeit wurde durch ein NIH Clinical Scientist Award K08NS002083 Entwicklung, einer Shervert Frazier Research Institute Grant, und ein NARSAD Young Investigator Award an Edgar A. Büttner unterstützt.

1. Entwicklungsverzögerung / Letalität Assay: Die Wildtyp-(N2) und Zwei Mutant (Mut1 und Mut2) Die Stämme werden in drei Clozapin Konzentrationen in einer 12-Well-Platte getestet <ol><li> (Jeweils auch mit einem 2 cm Durchmesser) Am Tag 1 pour 2 ml NGM Medium 10 in jede Vertiefung einer 12-Well-Platte und lassen auf der Bank bei Raumtemperatur (RT) über Nacht aushärten. Am selben Tag, wählen Sie eine Kolonie von Escherichia coli OP50 Bakterien infizieren, eine Flasche von 50 ml LB-Lösung und Kultur, die sie in einem 37 ° C-Schüttler bei 220 rpm Dreh Übernachtung. </li><li> Am Tag 2 Transfer 20 ul OP50 Bakterienkultur auf die Mitte jeder NGM auch. Die Inkubation in einem 37 ° C-Inkubator oder lassen Sie sie über Nacht bei RT, damit der Bakterienrasen dicker zu wachsen. </li><li> Machen Sie eine 80 mM Stammlösung Clozapin durch Auflösen von Clozapin in DMSO (Dimethylsulfoxid) Lösungsmittel. Dann machen Sie eine 80-ul-Arbeitslösung mit der Stammlösung in 1,7 mM Essigsäure verdünnt bin Tabelle 1 ased. Hinweis: Die Arbeitslösung für ein bestimmtes Medikament von Interesse sind, indem Sie eine Reihe von vorläufigen Konzentrationstests, um die am besten geeignete Konzentration für die Unterscheidung der Wildtyp von den Mutanten zu finden bestimmt werden. </li><li> Über 80 ul Clozapin Arbeitslösung auf die Platte ausgesät NGM gut und wirbeln die Platte, damit die Lösung gleichmäßig auf der Oberfläche verteilen. Warten Sie, bis die Platte zu trocknen. Verwenden Sie die Platte für den Test am gleichen Tag oder legen Sie sie in einem 20 ° C-Inkubator, am nächsten Tag verwendet werden. Hinweis: Die Arbeitslösung ist eine Wirkstoffsuspension aufgrund der geringen Löslichkeit von Clozapin, insbesondere bei höheren Konzentration. Daher vortexen Rohr vor der Lösung in der Medikamentenplatte, um zu gewährleisten, dass die Wirkstoffkonzentration genau ist. </li><li> Waschen Würmer aus einem 3,5 cm-Platte mit vielen schwangeren Erwachsenen mit M9-Puffer und dann drehen sie in einem 15-ml-Tubebei 2000 Upm für 1 min. </li><li> Saugen Sie den Überstand. 5 ml Bleichlösung (NaOH: Hypochlorit: ddH 2 O bei 4.01.05) und stören die Nematoden durch vorsichtiges Umdrehen der Rohre. </li><li> Beachten Sie die Tiere, bis die Hälfte von ihnen sind mit rund 5 min gelöst. Spin down die Eier bei 2.000 rpm für 1 min. </li><li> Saugen Sie den Überstand und fügen Sie 14 ml M9-Puffer schnell. </li><li> Spin down die Eier bei 2.000 rpm für 1 min und saugen Sie den Überstand, so dass etwa 100 ul Lösung. Vortex, um die Eier zu suspendieren. </li><li> Transfer 2 ul Eier in M9 aufgehängt auf einer regulären NGM Platte zu testen, wie viele Eier übertragen werden. Stellen Sie die Lautstärke der suspendierten Eier, um sicherzustellen, dass es etwa 30 bis 35 Eier in jeder Übertragung. Übertragen 30-35 Eier in jede Vertiefung der Testplatte, und dann in einem 20 ° C-Inkubator inkubiert für 24 Stunden. </li><li> Am ersten Tag des Tests, Note die Anzahl der Eier geschlüpft. Stellen Sie die Gesamtzahl der Tiere, die von schraffierten Picki 25/wellng von den zusätzlichen Würmer, wenn nötig. </li><li> An den folgenden Tagen, die Tiere zu beobachten und Gäste sie alle 24 Stunden. Entwicklungsstufe wird basierend auf der Größe des Tieres und der Form der Vulva 11 erzielt. Der Versuch dauert 5-6 Tage mit den robusten Effekt in der Regel am dritten oder vierten Tag gesehen. </li><li> Eingabe der Daten in eine Excel-Datei und berechnen Sie den Prozentsatz der Tiere in jeder Entwicklungsstufe für jeden Tag des Experiments. Erzeugung von Diagrammen mit der Funktion &#39;100% Gestapelte Säule &quot;in der Spalte &#39;2-D&quot;-Menü. </li></ol> 2. Rachen-Pumping Assay: Junger Erwachsener Tiere für den Wildtyp-und Mutanten-Stämme sind auf Clozapin Assay-Platten Partitur <ol><li> Die Testplatte wird nach dem gleichen Protokoll für die Entwicklungsverzögerung / Letalität Assay verwendet werden. </li><li> Wählen L4 50 Tieren für jeden Stamm auf NGM-Platten ausgesät 24 Stunden vor dem Test und bebrüten die Würmer bei 20 ° C </li><li> Wählen Sie 10 Tiere zu einem assay Well-Platte. Dann wählen Sie 10 Tiere auf den nächsten gut, alle 15-20 min. </li><li> Nachdem die Tiere in der ersten und haben den Drogen für 30 Minuten ausgesetzt wurde, starten Sie den Test durch Beobachtung Rachenpump unter einem Dissektionsmikroskop und Gäste die Pumpen für 20 sec 9. Sobald Rachenpump erzielt wird, wählen Sie den Wurm von der Platte. </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. R., Hodgkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+glycosyltransferase+BUS-8+has+two+distinct+and+essential+roles+in+epidermal+morphogenesis.">The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis.</a> Dev. Biol. 317, 549-559 (2008).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> J. Vis. Exp. (51), (2011).</li></ol>

Die Autoren erklären, dass es keine Interessenkonflikt beteiligt.

Hier Verfahren zum Testen der Wirkung von APDs auf die Entwicklung und das Verhalten von C beschreiben wir elegans. DMSO oder Ethanol eingesetzt wird Clozapin lösen, da der Wirkstoff in Wasser relativ unlöslich ist. Da Lösungsmittel wurde berichtet, daß C. beeinflussen elegans Biologie 12, DMSO-alone-oder Ethanol-alone-Kontrollgruppen sind unerlässlich. Die höchste Konzentration an DMSO in unseren Tests verwendet wird bis zu 3%, was eine offensichtliche Wirkung auf <e...

Caenorhabditis elegans ist eine einfache genetische Organismus zugänglich Groß Vorwärts-und Rückwärts genetischen Screens und chemisch-genetischen Bildschirme. C elegans ist gegenüber einem breiten Spektrum von biologisch aktiven Verbindungen und ist daher nicht erfolgreich verwendet worden, um die Mechanismen der Wirkung von einer Vielzahl von solchen Verbindungen zu definieren. Zum Beispiel mit Schnecken Pharmakogenetik gehören Acetylcholin-Rezeptor-Agonisten (zB Levamisol, Nikotin, Morantel und Pyrantel), Anästhetika (zB Halothan), Koffein, Cholinesterase-Inhibitoren (z. B. Aldicarb, Lannate und Trichlorfon), Fluorid, GABA-verwandten studierte bioaktiven Verbindungen Verbindungen (z. B. GABA und Muscimol), Ivermectin, Paraquat, Phorbolester und Serotonin-bezogene Medikamente (z. B. Serotonin und imiprimine) 3. Außerdem C. elegans hat für große Bildschirme kleines Molekül verwendet worden, so dass Entdeckung von neuen bioaktiven Verbindungs und Identifizierung neuer genetischer Ziele 4. Die C. elegans Genom umfasst mögliche Antipsychotikum (APD) Ziele im Menschen konserviert, einschließlich der Gene, die Proteine ​​für die Neurotransmitter-Synthese und für die synaptische Struktur und Funktion 5 erforderlich kodiert. So, C. elegans Neurogenetik und Neurobiologie bieten Methoden für die Entdeckung neuer molekularer Wirkmechanismen von APDs. In Nematoden, APD Exposition früh in der Entwicklung produziert Entwicklungsverzögerung, und bei höheren Konzentrationen, Letalität 2,6. APD Exposition im Erwachsenenalter produziert Verhaltens Phänotypen. Zum Beispiel hemmt Clozapin Exposition Fortbewegung und Rachenpump und verbessert Eiablage 1,2,7. APD-induzierten Entwicklungsverzögerung und Letalität sind starke Phänotypen für Groß chemisch-genetischen Bildschirme. Diese Phänotypen sind komplex, soweit sie wahrscheinlich mehr als einen zellulären und genetischen basis. Daher werden solche genetischen Bildschirme erwartet, dass eine Vielzahl von indirekten Wirkstoffziele ergeben. Allerdings hat unser Labor Kandidaten-Gen-Bildschirme und eine genomweite RNAi-Bildschirm für die Unterdrücker der APD-induzierten Entwicklungsverzögerung und Letalität durchgeführt und erfolgreich wiederhergestellt Gene, die wahrscheinlich zu kodieren direkte Ziele, einschließlich Dopamin, Insulin und Nikotinsäure Acetylcholin-Rezeptoren 2,8. Genetische Screens auf der Grundlage APD-induzierte Verhalten beim Erwachsenen haben auch die Identifizierung neuer APD Ziele führte, und wir jetzt Validierung von Targets sowohl Entwicklungs-und Verhaltens Bildschirme in Säugetieren 7. So scheint, ein wirbel chemisch-genetischen Ansatz für neuartige molekulare Wirkmechanismen von APDs entdecken machbar 5,8 sein. Die C. elegans Rachen ist ein Organ, 20 Neuronen, Muskelzellen, 20, 20 und akzessorischen Zellen durch eine Basalmembran gewickelt enthält. Ähnlich wie bei der Säugetier-Herz, ist der Rachen eine autonomend Pumpen ständig Essen in von der äußeren Umgebung 9. Die Hemmung der Rachenpumprate beeinträchtigt Nahrungsaufnahme und damit Mutationen oder Medikamente, die Rachenpump Ursache Entwicklungsverzögerung hemmen oder zu verhaften 9. APDs hemmen die Rachenpumprate Anteil zum Teil auf ihre Auswirkungen auf die Entwicklung und Lebensfähigkeit 1,2. Hier verwenden wir die atypische APD Clozapin als Beispiel für Drogentests zur Nematodenentwicklung und Rachenpump demonstrieren.

<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 ist eine einfache genetische Organismus zugänglich Groß Vorwärts-und Rückwärts genetischen Screens und chemisch-genetischen Bildschirme. Die C. elegans Genom umfasst mögliche Antipsychotikum (APD) Ziele im Menschen konserviert, einschließlich der Gene, die Proteine ​​für die Neurotransmitter-Synthese und für die synaptische Struktur und Funktion erforderlich kodiert. APD Exposition produziert Entwicklungsverzögerung und / oder Letalität bei Nematoden in einer konzentrationsabhängigen Weise. Diese Phänotypen verursacht, teilweise durch die APD-induzierte Hemmung der Rachenpump 1,2. Somit hat die Entwicklungs Phänotyp eine neuromuskuläre Basis, so dass es nützlich pharmakogenetische Studien von Neuroleptika. Hier zeigen wir detaillierte Verfahren für die Prüfung APD Auswirkungen auf die Nematodenentwicklung und Rachen Pumpen. Für die Entwicklungs-Assay werden synchronisiert Embryonen auf Nematoden-Wachstumsmedium (NGM)-Platten, die APD, und die Stadien der Entwicklung ani platziertmals werden dann täglich erzielt. Für die Rachenpumprate-Test, inszenierte junge Tiere werden auf NGM-Platten mit APDs getestet. Die Anzahl der Pharynx Pumpen pro Zeiteinheit aufgezeichnet werden, und die Pumpgeschwindigkeit wird berechnet. Diese Assays können zur Untersuchung von vielen anderen Arten von kleinen Molekülen oder sogar großen Molekülen verwendet werden.

1. Entwicklungsverzögerung / Letalität Testergebnis Ein typisches Ergebnis für die Entwicklungsverzögerung / Letalität Test wird in den 1a 1b gezeigt und. Bei Wildtyp-Tiere in der Kontrollgruppe wurden dem schwangeren Erwachsenen-Stadium (Abbildung 1a) gewachsen ist, sind Wildtyp-Tiere auf Clozapin ausgesetzt noch in den jungen Larvenstadien oder tot sind (Abbildung 1b). 1c zeigt ein repräsentatives Ergebnis zu vergleichen ein Suppressor-...

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

Ansätze zum Testen der Wirkung von Neuroleptika (APD) in Caenorhabditis elegans gezeigt werden. Die Tests werden für die Prüfung Arzneimittelwirkungen auf die Entwicklung und Lebensfähigkeit und Rachenpumprate beschrieben. Diese Methoden sind auch für pharmakogenetische Versuche mit anderen als APDs Medikamentenklassen.

Methoden zur Untersuchung der Wirkmechanismen von Psychopharmaka in<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.  ...

methods-for-studying-mechanisms-action-antipsychotic-drugs

Research

JoVE Journal

Neuroscience

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

11.4K Aufrufe.  Harvard Medical School.  Ansätze zum Testen der Wirkung von Neuroleptika (APD) in Caenorhabditis elegans gezeigt werden. Die Tests werden für die Prüfung Arzneimittelwirkungen auf die Entwicklung und Lebensfähigkeit und Rachenpumprate beschrieben. Diese Methoden sind auch für pharmakogenetische Versuche mit anderen als APDs Medikamentenklassen. 

Serie: 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.

Maus in Utero Elektroporation: Kontrollierte Spatiotemporal Gene Transefection

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

Forschung

Neurowissenschaften

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.

Direkt Intraventrikuläre Abgabe von Arzneimitteln an die Nager 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 ...

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.

A<em&gt; Caenorhabditis elegans</em&gt; Modell-System für 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 ...

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.

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

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.

Ein Test zur Messung der Wirkung von Ethanol auf die Fortbewegungsgeschwindigkeit der<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.

Agarose Mikrokammern für Langzeit Calcium Imaging von<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.

Novel Metrics zur Charakterisierung von Embryonic Dehnung des Nematode<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.

EIN<em&gt; Caenorhabditis elegans</em&gt; Nährwert-basierter Kupfer-Aversion-Assay

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;

Ratiometrischen Kalzium Imaging einzelner Neuronen im Verhalten Caenorhabditis Elegans

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.

Bildgebung dendritischer Dornen bei Caenorhabditis elegans

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