This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN36481-08 to William G. Bendena.

1. Preparation of Experimental Organisms
<ol>
	<li>Pick 10 L4 staged nematodes per strain 24 h prior to commencing the assay to ensure that organisms are young adults when tested. For each mutant or control nematode tested, pick 10 L4s (10 for the control and 10 for the assay).
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		<li>Maintain L4 organisms using standard methods26,27 for 24 h on standard agar plates seeded with OP50 Escherichia coli. If organisms are lost during t...

<ol>
	<li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314 (1165), 1-340 (1986).</li><li>Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensation+in+C.+elegans+(October+25,+2006).">Chemosensation in C. elegans (October 25, 2006).</a> The C. elegans Research Community, WormBook. , (2006).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. 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This assay design modifies the food race assay24 to include a copper solution to create an aversive midline barrier and around the edge of the plate to prevent a loss of nematodes. Organisms are tested for their ability to cross the aversive barrier and reach a food patch over a 4 h period. In the context of npr-9(GF), we have utilized this assay to evaluate how starvation conditions could affect aversive responses and the detection of food. Provided that we had previously characterized <...

Caenorhabditis elegans has been used as a model for the study of neurobiology for decades due to the relative ease in analyzing the circuitry of a nervous system composed of only 302 neurons1. Provided that the organism is reliant on responding to environmental cues, much of the nervous system is dedicated to regulating the integration of environmental signals2. Despite the simplicity of its nervous system, C. elegans can detect and respond to diverse environmental signals including repellents3, attractants4, temperature5, and even humidity6. A failure to properly integrate environmental signals has been linked to a number of behavioral disorders and neurodegenerative conditions in mammalian model systems7,8,9. With a range of available neural disease models10 in C. elegans and the development of nematode pharmaceutical screens11, this organism has proven to be a useful system for the study of neurobiology. Given the availability of a mapped nematode connectome1 and mutations to almost every gene in the nematode genome12, our understanding of the nematode nervous system, and by extension our own, is partially limited by the design of creatively appropriate assays.
A number of chemotaxis assays have been developed over the past 40 years to evaluate nematode responsiveness to diverse aversive stimuli3,4,13,14,15. Initial experimentation involved the introduction of an acute environmental stimulus while a single worm roamed on an agar plate3,14,16. Immediate changes to locomotory responses were recorded. For example, the volatile odorant octanol can be applied to a hair and wafted in front of a nematode&#39;s nose to stimulate the initiation of backwards locomotion in wild type worms17. More complex assays have also been developed to incorporate multiple variables as a means of assessing behavioral choice18. A variation of this assay entails the use of a copper solution to create an aversive midline barrier4. An attractant, namely diacetyl, was placed on one side of the chemical barrier with worms transferred away from the diacetyl source. Worms defective for copper aversive responses immediately crossed the barrier to reach the diacetyl, while wild type worms were initially repulsed by the barrier. Responses were scored when worms first approached the copper barrier without long term observations.
When worms are evaluated after undergoing starvation conditions, their sensitivity to environmental stimuli is decreased19. When the aversive chemical octanol is wafted in front of the nematode nose, wild type organisms stimulate backwards movement within 3 - 5 s when on food. After these organisms have been removed from food for 10 min, they exhibit a delayed response of 8 - 10 s20. Thus, with increased starvation, nematodes display a decreased aversive response to harmful environmental signals as the search for food becomes more essential to survival. Conversely, nematodes that over-express neuropeptide receptor 9 (npr-9), do not respond to octanol on or off food and exhibit an inability to respond to a number of aversive stimuli21. These npr-9(GF) organisms also do not modulate their reversal frequency in the presence of food, but can reverse in response to harsh touch stimulations indicating that they are capable of backwards locomotion21. We have also evaluated npr-9(LF) mutants given that they exhibit an abnormally decreased reversal frequency off food yet can modulate their behavior in the presence of food21. Coupling the nutritional state of the worm with the introduction of acute external stimuli has aided in elucidating the mechanisms by which a food-related pathway can broadly modulate sensory signaling pathways22,23. The presence of food in the nematode environment has also been used to evaluate ethanol withdrawal responses24. In this experiment, worms were incubated in varying concentrations of ethanol and then were placed on an agar plate with a patch of food known as a &#34;food-race assay&#34;. The food patch was placed on one edge of the plate while the nematodes were placed away from the food source. Ethanol withdrawal was evaluated by measuring the duration of time required for worms to reach the patch of food.
This nutritional-based copper aversion assay builds upon the food-race assay to integrate additional environmental variables, namely food and copper, while assessing behavioral changes over time. This is an adaptation of a commonly use protocol throughout the C. elegans community4. This protocol has been used to evaluate aversive responses and the detection of food over a four-hour period21. Since worm&#39;s exhibit starvation behaviors after 30 min of food deprivation25, we are also able to evaluate how changes to nutritional status can influence environmental responses. The conditions of this assay measure how experimental organisms change responsiveness to aversive stimuli over time, hence this evaluates behavioral changes as organisms progress towards a starved state (and continued measurements of prolonged starvation). Since the npr-9(GF) animals do not alter their behavior in response to food or many aversive cues, we sought to identify if these behavioral deficits would persist in the context of starvation. Ultimately, this assay design has been formulated to specifically evaluate the npr-9(GF) mutants but can be further adapted to also characterize novel strains.

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			<td height="71" style="height:71px;width:332px;">M9 Solution [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Autoclave to sterilize before use.]</td>
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			<td style="width:167px;">Produced in lab</td>
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			<td height="84" style="height:84px;width:332px;">Cupric Sulfate</td>
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			<td style="width:167px;">Use water to appropriately suspend to a concentration of 0.5M</td>
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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.

We utilized wild type (N2), npr-9(tm1652), and an npr-9 overexpression strain, i.e. npr-9(GF) (IC836 -npr-9::npr-9;sur-5::gfp;odr-1::rfp), to evaluate responses to starvation and copper aversion. Wild type organisms are capable of detecting and responding to the aversive copper barrier, while npr-9(GF) mutants do not initiate an aversive response to the copper over the 4 h assay21. After 30 min of starvation, ro...

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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.

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 ...

The overall goal of this nutritional-status based copper aversion assay is to evaluate nematode responsive to an aversive chemical and a food source over a four hour period. This allows for the simultaneous evaluation of nutritional-status. This method can aid the field of behavioral neuroscience by identifying mutants that fail to modulate locomotory patterns and response to an aversive chemical despite starvation conditions.The main advantage of this technique is that it can be conducted over an extended time period, which allows for nutritional status analysis as worms become progressively more starved over four hours. To prepare experimental organisms for the assay, pick 10 L4 stage nematodes per stream, 24 hours prior to commencing the assay, to ensure that organisms are young adults when tested. For each mutant or control nematode tested, pick 10 L4s.Maintain L4 organisms, using standard methods for 24 hours on standard agar plates seeded with OP50 E.Coli. 24 hours prior to the assay, use a ruler and a thick permanent marker to draw a line on the underside of a previously prepared nematode growth media or NGM plate along the outer edge. Mark a midline barrier equidistant from each edge of the plate.Seed the plate with 50 microliters of OP50 E.Coli on only one side of the copper barrier to create a uniform lawn. Use the marked lines on the underside of the plate to ensure the bacteria will not contact the copper solution. Make sure the copper solution will line the edge of the plate and form a midline barrier and transfer the bacteria so that the copper solution will not contact the food source.Mark a second set of plates without OP50 E.Coli, to serve as the negative control. Allow the bacteria to dry, then incubate the plates at 37 degrees celsius overnight. Ensure that the bacteria patches are not disturbed when transferring the nematodes to a 37 degrees celsius incubator.Excessive disturbance could alter the location or shape of the food patch. After overnight incubation, remove plates from the incubator and using the marked underside of the plate as a guide, pipette 100 microliters of freshly prepared 0.5 molar copper(II)sulfate solution on the edge of the agar, to create an outer copper barrier. Pipette 25 microliters of the copper(II)sulfate solution to create a midline barrier.Ensure that the copper(II)sulfate solution does not contact the bacteria patch and allow copper solution to dry onto the plate. Check for dryness every five minutes after transfer, with a laboratory tissue by lightly dabbing the solution near the edge of the plate to discern. Immediately prior to the assay, transfer the experimental organisms to a bacteria free agar plate and allow the nematodes to move freely for one minute to remove excess bacteria.Next, pipette one milliliter of M9 onto the plate with young adults, that were transferred 24 hours previously in order to wash worms into a microcentrifuge tube. Centrifuge the nematodes at 3, 000 x G for one minute. Worms should form a pellet at the bottom of the tube.Aspirate M9 solution without disrupting the worm pellet. Add one milliliter of M9 solution to the worm pellet. Invert tube to mix worms with the solution.If excess bacteria were initially transferred with the worms, repeat for a total of five times. After the final wash, aspirate the supernatant until 100 microliters of M9 solution and the worm pellet remains. Immediately transfer worms from solution once the washed ups have been completed.Pipette 20 microliters of the worm pellet from the bottom of the tube onto the bacteria free half of the assay plate, ensure that 10 worms are transferred to the assay plate and that no contaminating food is present. No bacteria should be transferred to the copper food race plates. Remove excess M9 solution from the nematodes with a laboratory tissue within one minute.Ensure the M9 solution does not contact the copper(II)sulfate solution and that the worms and agar surface remain intact. Discard worms that have accidentally been removed with the laboratory tissue. Once the M9 solution has been removed, and all worms have commenced non-liquid locomotor patterns, I.e when they have stopped thrashing.Start the assay stopwatch, if extra worms were accidentally included, remove them by picking with halocarbon oil to ensure that no bacteria are added to the plate. Check assay plates every 30 minutes. For the assay plates with bacteria patches, positively score organisms if they reach the food patch over a four hour period.For the negative control plates, positively score organisms if they have crossed the barrier. We utilized the N2 and npr-9 loss of function mutant and an npr-9 overexpression strain, referred to as npr-9 gain a function, to evaluate responses to starvation and copper aversion. By the end of the assay, 100%of wild type organisms relocated to the food source, while the npr-9 over expression organisms did not modulate locomotory patterns in response to food and continued to cross the aversive barrier even after contacting the food source.On a food free plate, n2 or npr9 loss of function organisms rarely crossed the copper barrier while the npr-9 overexpression nematodes repeatedly cross the barrier back and forth. While attempting this procedure, it's important to remember to avoid disrupting the copper barrier when transferring nematodes and M9 and to also maintain consistent conditions for each strain to avoid confounding factors.

a-caenorhabditis-elegans-nutritional-status-based-copper-aversion

Research

JoVE Journal

Neuroscience

6.4K 次观看.  Queen's University. The overall goal of this nutritional-status based copper aversion assay is to evaluate nematode responsive to an aversive chemical and a food source over a four hour period. This allows for the simultaneous evaluation of nutritional-status. This method can aid the field of behavioral neuroscience by identifying mutants that fail to modulate locomotory patterns and response to an aversive chemical despite starvation conditions.The main advantage of this technique is that it can be condu...