Il lavoro è stato supportato da un NIH premio Sviluppo Scienziato Clinico K08NS002083, un Shervert Frazier Research Institute Grant, e un NARSAD Young Investigator Award di Edgar A. Buttner.

1. Developmental Delay / letalità saggio: The Wild-Type (N2) e due Mutant (Mut1 e Mut2) ceppi sono stati testati in tre Clozapina concentrazioni in un 12-pozzetti <ol><li> Il giorno 1, versare 2 ml NGM medio 10 in ciascun pozzetto di una piastra 12 pozzetti (ogni pozzetto con un diametro di 2 cm) e lasciare indurire sul banco a temperatura ambiente (RT) per una notte. Lo stesso giorno, scegliere una colonia di batteri Escherichia coli OP50, infettare un flacone di 50 ml di soluzione LB, e la cultura in uno shaker 37 ° C a 220 giri al minuto di rotazione durante la notte. </li><li> Il giorno 2, trasferire 20 microlitri coltura batterica OP50 sul centro di ogni NGM bene. Incubare le piastre in un incubatore a 37 ° C o lasciarli a temperatura ambiente nella notte per consentire il prato batterica di crescere più spessa. </li><li> Fare un clozapina soluzione stock 80 mM sciogliendo clozapina in DMSO (dimetilsolfossido) solvente. Poi fare una soluzione operativa 80 microlitri di soluzione madre diluita in 1.7 mM acido acetico based nella Tabella 1. Nota: La soluzione di lavoro per un particolare farmaco di interesse deve essere determinato effettuando una serie di prove preliminari di concentrazione per determinare la concentrazione più adeguato per distinguere il tipo selvaggio dai mutanti. </li><li> Trasferimento 80 microlitri clozapina soluzione di lavoro sulla piastra NGM seminato bene e agitare la piastra per consentire alla soluzione per distribuire uniformemente sulla superficie. Attendere che la piastra si asciughi. Utilizzare la piastra per il saggio lo stesso giorno o metterla in un incubatore a 20 ° C da utilizzare il giorno successivo. Nota: La soluzione di lavoro è una sospensione farmaco a causa della bassa solubilità della clozapina, soprattutto a maggiore concentrazione. Pertanto, agitare la provetta prima di trasferire la soluzione alla piastra farmaco per garantire che la concentrazione di farmaco è accurato. </li><li> Lavare i vermi fuori un piatto 3,5 centimetri contenente molti adulti gravide con tampone M9 e poi girare giù in una provetta da 15 mla 2.000 rpm per 1 min. </li><li> Aspirare il surnatante. Aggiungere 5 ml di soluzione di candeggina (NaOH: ipoclorito: DDH 2 O a 4:01:05) e disturbare i nematodi invertendo delicatamente i tubi. </li><li> Osservare gli animali fino a metà di essi vengono sciolti a circa 5 min. Spin giù le uova a 2.000 rpm per 1 min. </li><li> Aspirare il surnatante e aggiungere rapidamente 14 ml di tampone M9. </li><li> Spin giù le uova a 2.000 rpm per 1 min e aspirare il surnatante, lasciando circa 100 microlitri soluzione. Vortex per sospendere le uova. </li><li> Transfer 2 uova microlitri sospesi in M9 su una piastra NGM regolare per testare quante uova sono trasferiti. Regolare il volume delle uova sospese per garantire che ci sono circa 30-35 uova per ogni trasferimento. Trasferire 30-35 uova in ciascun pozzetto della piastra di dosaggio e quindi incubare in un incubatore a 20 ° C per 24 ore. </li><li> Il primo giorno del test, segnerà il numero di uova schiuse. Regolare il numero totale di animali nati per 25/well da picking fuori i vermi supplementari, se necessario. </li><li> Nei giorni successivi, osservare gli animali e il punteggio loro ogni 24 ore. Stadio di sviluppo è ottenuto in base alla dimensione dell&#39;animale e la forma della vulva 11. L&#39;esperimento dura 5-6 giorni con l&#39;effetto più robusto genere visto il terzo o quarto giorno. </li><li> Inserire i dati in un file di Excel e calcolare la percentuale di animali in ogni fase di sviluppo per ogni giorno dell&#39;esperimento. Generare grafici con la funzione di &#39;100% Stacked Colonna &#39;in &#39;2-D colonna&#39; menu. </li></ol> 2. Faringeo pompaggio saggio: Giovane adulto Animals per il wild-type e mutanti ceppi sono segnati a tavola Clozapina Assay <ol><li> La piastra di saggio viene effettuata seguendo lo stesso protocollo usato per il saggio ritardo dello sviluppo / letalità. </li><li> Accompagnamento 50 L4 animali per ogni ceppo di piastre NGM seminati 24 ore prima del dosaggio e incubare i vermi a 20 ° C. </li><li> Scegli 10 animali ad un assay pozzetti. Poi scegliere 10 animali al prossimo bene ogni 15-20 min. </li><li> Dopo gli animali del primo pozzo sono stati esposti al farmaco per 30 minuti, iniziare l&#39;analisi osservando faringeo pompaggio sotto un microscopio dissezione e segnare le pompe per 20 secondi 9. Una volta faringea pompaggio è segnato, scegliere il verme dalla piastra. </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. 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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. 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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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Gli autori dichiarano che non vi sia conflitto di interesse coinvolti.

Qui, descriviamo metodi per testare gli effetti della APD sullo sviluppo e comportamento di C. elegans. DMSO o etanolo è usato per sciogliere clozapina, in quanto il farmaco è relativamente insolubile in acqua. Dato che i solventi sono stati segnalati per influenzare C. elegans biologia 12, DMSO-alone o gruppi di controllo etanolo-alone sono essenziali. La massima concentrazione di DMSO utilizzata nei nostri test è fino al 3%, che non ha un effetto evidente sul C. elegans svilupp...

Caenorhabditis elegans è un organismo semplice genetica suscettibili di grandi schermi genetiche in avanti e all&#39;indietro e schermi genetici chimici. C. elegans è sensibile ad un ampio spettro di composti bioattivi e pertanto è stato usato con successo per definire i meccanismi di azione di una varietà di tali composti. Ad esempio, i composti bioattivi studiate usando farmacogenetica vermi sono agonisti del recettore dell&#39;acetilcolina (ad esempio Levamisolo, nicotina, morantel, e Pyrantel), anestetici (es. alotano), la caffeina, gli inibitori della colinesterasi (ad esempio aldicarb, Lannate, e triclorfon), fluoruro, GABA-correlate composti (ad esempio GABA e muscimolo), ivermectina, paraquat, esteri del forbolo, e farmaci serotonina legati (es. serotonina e imiprimine) 3. Inoltre, C. elegans è stato usato per grandi schermi piccole molecole, permettendo scoperta di nuovi composti bioattivis e identificazione di nuovi bersagli genetici 4. La C. elegans genoma include potenziale farmaco antipsicotico (APD) obiettivi conservati negli esseri umani, compresi i geni che codificano le proteine ​​necessarie per la sintesi dei neurotrasmettitori e per la struttura e la funzione sinaptica 5. Così, C. elegans neurogenetica e offrono neurobiologia metodi per la scoperta di nuovi meccanismi molecolari d&#39;azione di APD. In nematodi, l&#39;esposizione APD precoce dello sviluppo produce ritardo dello sviluppo, e a concentrazioni più elevate, letalità 2,6. L&#39;esposizione APD durante l&#39;età adulta produce fenotipi comportamentali. Ad esempio, l&#39;esposizione clozapina inibisce la locomozione e faringea pompaggio e migliora la deposizione delle uova 1,2,7. APD-indotta ritardo e letalità dello sviluppo sono fenotipi potenti per grandi schermi genetici chimici. Questi fenotipi sono complesse nella misura in cui probabilmente hanno più di un BASI cellulari e geneticis. Pertanto, tali schermi genetici dovrebbero produrre una varietà di bersagli indiretti droga. Tuttavia, il nostro laboratorio ha condotto candidati schermi di geni e uno schermo RNAi genome-wide per soppressori di APD indotta ritardi e letalità dello sviluppo e ha recuperato con successo geni che codificano probabili bersagli diretti, tra cui la dopamina, l&#39;insulina, e recettori nicotinici 2,8. Schermi genetici basati sui comportamenti APD-indotte nel adulti hanno portato anche all&#39;identificazione di nuovi bersagli APD, e ora stiamo convalida bersagli dagli schermi sia sviluppo e comportamentali nei mammiferi 7. Così, un approccio invertebrato chimica genetica per scoprire i meccanismi molecolari d&#39;azione delle APD sembra essere fattibile 5,8. La C. elegans faringe è un organo che include 20 neuroni, cellule muscolari, 20 e 20 cellule accessorie, avvolto da una membrana basale. Simile al cuore dei mammiferi, la faringe è un autonomond pompe costantemente cibo dall&#39;ambiente esterno 9. L&#39;inibizione del tasso di pompaggio faringeo compromette l&#39;assorbimento di cibo, e quindi le mutazioni o farmaci che inibiscono faringeo pompaggio causa ritardo nello sviluppo o arresto 9. APD inibiscono il tasso faringeo di pompaggio, che rappresentano in parte per i loro effetti sullo sviluppo e la redditività 1,2. Qui, usiamo il atipico APD clozapina come esempio per dimostrare saggi di droga per lo sviluppo nematode e della faringe pompaggio.

<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 è un organismo semplice genetica suscettibili di larga scala in avanti e retromarcia schermi genetici e schermi genetici chimici. La C. elegans genoma include potenziale farmaco antipsicotico (APD) obiettivi conservati negli esseri umani, compresi i geni che codificano le proteine ​​necessarie per la sintesi dei neurotrasmettitori e per la struttura e la funzione sinaptica. L&#39;esposizione APD produce ritardo di sviluppo e / o letalità in nematodi in modo concentrazione-dipendente. Questi fenotipi sono causati, in parte, mediante inibizione APD-indotta faringeo pompaggio 1,2. Così, il fenotipo di sviluppo ha una base neuromuscolare, rendendolo utile per studi di farmacogenetica dei neurolettici. Qui mostriamo le procedure dettagliate per testare gli effetti APD sullo sviluppo nematode e della faringe pompaggio. Per il saggio di sviluppo, gli embrioni sincronizzati sono immessi sul (NGM) piastre contenenti APD nematode mezzo di crescita, e le fasi di sviluppo di animali vengono quindi segnati giornalmente. Per il saggio faringeo tasso di pompaggio, in scena giovani animali adulti sono testati su piastre NGM contenenti APD. Il numero di pompe faringei per unità di tempo viene registrato, e la velocità di pompaggio è calcolato. Questi saggi possono essere utilizzati per studiare molti altri tipi di piccole molecole o anche grandi molecole.

1. Il ritardo dello sviluppo / letalità risultato del test Un risultato tipico per il dosaggio ritardo dello sviluppo / letalità è dimostrato nelle figure 1a e 1b. Quando wild-type animali del gruppo di controllo sono cresciuti allo stadio adulto gravida (Figura 1a), wild-type animali esposti a clozapina sono ancora nelle giovani stadi larvali o sono morti (Figura 1b). Figura 1c mostra un risultato rappresentativo confronto ...

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

Approcci per testare gli effetti dei farmaci antipsicotici (APD) in Caenorhabditis elegans sono dimostrati. I dosaggi sono descritti per testare gli effetti della droga sullo sviluppo e la redditività e sulla faringe velocità di pompaggio. Questi metodi sono applicabili per esperimenti di farmacogenetica con classi di farmaci diversi da APD anche.

Metodi per studiare i meccanismi d&#39;azione di farmaci antipsicotici 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 Views.  Harvard Medical School.  Approcci per testare gli effetti dei farmaci antipsicotici (APD) in Caenorhabditis elegans sono dimostrati. I dosaggi sono descritti per testare gli effetti della droga sullo sviluppo e la redditività e sulla faringe velocità di pompaggio. Questi metodi sono applicabili per esperimenti di farmacogenetica con classi di farmaci diversi da APD anche. 

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.

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

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

Ricerca

Neuroscienze

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.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

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; Sistema modello per Amylopathy Studio

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.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

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.

Un test per misurare gli effetti di etanolo sulla velocità di Locomotion<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.

Microchambers Agarose per lungo termine Imaging di calcio<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.

Nuovi parametri per caratterizzare embrionale Allungamento del 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.

UN<em&gt; Caenorhabditis elegans</em&gt; Analisi di Aversione di rame basata sullo stato nutrizionale

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;

Raziometrici calcio Imaging di singoli neuroni nel comportarsi 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.

Imaging delle spine dendritiche in Caenorhabditis elegans

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