Name: methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans
Uploaded: 2014-02-04T14:00:00
Description: Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

The work was supported by an NIH Clinical Scientist Development award K08NS002083, a Shervert Frazier Research Institute Grant, and a NARSAD Young Investigator Award to Edgar A. Buttner.

1. Developmental Delay/Lethality Assay: The Wild-Type (N2) and Two Mutant (Mut1 and Mut2) Strains Are Tested in Three Clozapine Concentrations in a 12-well Plate
<ol>
	<li>On day 1, pour 2 ml NGM medium10 into each well of a 12-well plate (each well with a 2 cm diameter) and allow to harden on the bench at room temperature (RT) overnight. The same day, pick a colony of Escherichia coli OP50 bacteria, infect a bottle of 50 ml LB solution, and culture it in a 37 &#176;C shaker at 220...

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Here, we describe methods for testing the effects of APDs on the development and behavior of C. elegans. DMSO or ethanol is used to dissolve clozapine, since the drug is relatively insoluble in water. Because solvents have been reported to affect C. elegans biology12, DMSO-alone or ethanol-alone control groups are essential. The highest concentration of DMSO used in our assays is up to 3%, which does not have an obvious effect on C. elegans development. DMSO can be used for many smal...

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. C. elegans is sensitive to a wide spectrum of bioactive compounds and has therefore been used successfully to define the mechanisms of action of a variety of such compounds. For example, bioactive compounds studied using worm pharmacogenetics include acetylcholine receptor agonists (e.g.&#160;levamisole, nicotine, morantel, and pyrantel), anesthetics (e.g.&#160;halothane), caffeine, cholinesterase inhibitors (e.g.&#160;aldicarb, lannate, and trichlorfon), fluoride, GABA-related compounds (e.g.&#160;GABA and muscimol), ivermectin, paraquat, phorbol esters, and serotonin-related drugs (e.g.&#160;serotonin and imiprimine)3. Furthermore, C. elegans has been used for large-scale small molecule screens, allowing discovery of new bioactive compounds and identification of novel genetic targets4.
The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function5. Thus, C. elegans neurogenetics and neurobiology offer methods for discovering novel molecular mechanisms of action of APDs. In nematodes, APD exposure early in development produces developmental delay, and at higher concentrations, lethality2,6. APD exposure during adulthood produces behavioral phenotypes. For example, clozapine exposure inhibits locomotion and pharyngeal pumping and enhances egg laying1,2,7.
APD-induced developmental delay and lethality are powerful phenotypes for large-scale chemical genetic screens. These phenotypes are complex in so far as they likely have more than one cellular and genetic basis. Therefore, such genetic screens are expected to yield a variety of indirect drug targets. However, our laboratory has conducted candidate gene screens and a genome-wide RNAi screen for suppressors of APD-induced developmental delay and lethality and has successfully recovered genes which likely encode direct targets, including dopamine, insulin, and nicotinic acetylcholine receptors2,8. Genetic screens based on APD-induced behaviors in the adult have also led to the identification of novel APD targets, and we are now validating targets from both developmental and behavioral screens in mammals7. Thus, an invertebrate chemical genetic approach to discover novel molecular mechanisms of action of APDs appears to be feasible5,8.
The C. elegans pharynx is an organ that includes 20 neurons, 20 muscle cells, and 20 accessory cells, wrapped by a basement membrane. Similar to the mammalian heart, the pharynx is autonomous and constantly pumps food in from the external environment9. Inhibition of the pharyngeal pumping rate compromises food uptake, and thus mutations or drugs that inhibit pharyngeal pumping cause developmental delay or arrest9. APDs inhibit the pharyngeal pumping rate, accounting in part for their effects on development and viability1,2. Here, we use the atypical APD clozapine as an example to demonstrate drug assays for nematode development and pharyngeal pumping.

<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 is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

1. Developmental delay/lethality assay result
A typical result for the developmental delay/lethality assay is demonstrated in Figures&#160;1a and 1b. When wild-type animals in the control group have grown to the gravid adult stage (Figure&#160;1a), wild-type animals exposed to clozapine are still in the young larval stages or are dead (Figure&#160;1b). Figure&#160;1c shows a representative result comparing a supp...

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

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

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

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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