A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Summary

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.

Abstract

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'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.

Introduction

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 systems^7,8,9. 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 stimuli^3,4,13,14,15. Initial experimentation involved the introduction of an acute environmental stimulus while a single worm roamed on an agar plate^3,14,16. 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'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 pathways^22,23. 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 "food-race assay". 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'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.

Protocol

1. Preparation of Experimental Organisms

1. 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).
   1. Maintain L4 organisms using standard methods^26,27 for 24 h on standard agar plates seeded with OP50 Escherichia coli. If organisms are lost during the subsequent wash steps, compensate by increasing the starting sample size (i.e. pick 20 worms rather than 10).
      Note: Behavior is an innately variable phenotype. Perform the method in triplicate for each strain on three separate days. Include additional control strains and conditions for novel strains, as highlighted in the discussion section.
2. Immediately prior to assay, transfer experimental organisms to an agar plate with no bacteria and allow nematodes to move freely for 1 min to remove excess bacteria.
   1. If experimental organisms experienced contamination conditions during the 24 h prior to the assay, discard them.
3. Pipette 1 mL of M9 onto the bacteria-free plate to wash worms into a microcentrifuge tube.
4. Centrifuge at 3,000 x g for 1 min. Worms should form a pellet at the bottom of the tube. Aspirate M9 solution without disrupting the worm pellet. Add 1 mL of M9 to the worm pellet, invert tube to mix worms with the solution.
5. Repeat steps 1.4 three more times.
   1. If excess bacteria was initially transferred with the worms, repeat for a total of 5 times. No bacteria should be transferred to the copper food race plates. If food is transferred to the assay plate, it will interfere with accurate data collection.
6. After the final wash, aspirate supernatant until 100 µL of M9 and the worm pellet remains.
   Caution: Starvation related behaviors become evident after 30 min. Consequently worms should be immediately transferred from solution once the wash steps have been completed.

2. Preparation of Assay Plates

1. Prepare standard NGM agar plates two days prior to the assay.
   1. If plates are kept in a humid environment, make agar plates 3 days prior to assay. Alternatively, remove the lid of the plate for 3 - 6 h to ensure proper dryness (if in a sterile environment).
2. With a thick permanent marker, make a line on the underside of the plate along the outer edge and another to form a mid-line barrier (Figure 1). The mid-line barrier should be equidistant from each edge of the plate. Use a ruler to ensure precise measurements. These lines will serve as a guide when transferring bacteria and the copper solution. Provided that E. coli is transferred before the copper solution, these lines will serve as indicators.
3. Seed plate with 50 µL of OP50 E. coli on only one side of the copper barrier to create a uniform lawn (Figure 2). Bacterial concentration should remain consistent across assays; however, little variability has been observed in response to mild differences in concentration.
   1. Use the marked lines on the underside of the plate to ensure the bacteria do not come into contact with the copper solution. Provided that the copper solution lines the edge of the plate and forms a mid-line barrier, transfer the bacteria so that the copper solution will not come into contact with the food source.
4. Mark a second set of plates and transfer no OP50 E. coli to them (Figure 2). These plates will serve to evaluate the negative control. These plates should also have a marking on one half of the plate to denote the initial transfer origin.
5. Allow bacteria to dry then incubate plates at 37 °C overnight. Ensure that bacterial patches are not disturbed when transferring to a 37 °C incubator or room. Excessive disturbance could alter the location or shape of the food patch.

3. Chemotaxis Assay

1. Freshly prepare a 0.5 M copper (II) sulfate solution prior to assay start time. Provided that 125 µL of the solution is used per plate, scale up this volume dependent on the number of assay plates used (e.g. 5 assay plates, 625 µL).
2. Pipette 100 µL of the copper (II) sulfate solution on the edge of the agar to create an outer copper barrier. The marked underside of the plate should serve as a guide.
3. Pipette 25 µL of the copper (II) sulfate solution to create a midline barrier.
   1. Ensure that the copper (II) sulfate solution does not come into contact with the bacterial patch. Use a spotted technique as streaking may affect locomotion due to indentations/scratches on the agar.
4. Allow the copper solution to dry onto plate. Time period can vary depending on plate and lab conditions. Visually check for dryness every 5 min after transfer.
   Note: The copper solution displays a blueish tint and is easily identifiable. Use a laboratory tissue to lightly dab the solution near the edge of the plate to discern dryness.
5. Pipette 20 µL of the worm pellet from the bottom of the tube onto the bacteria-free half of the assay plate.
   1. Ensure that 10 worms are transferred to the assay plate. If extra worms were accidentally included, remove them by picking with halocarbon oil to ensure that no bacteria are added to the plate. Each assay should have a consistent number of nematodes during the assay.
      NOTE: If too few worms are transferred during assays, increasing the initial sample size will mitigate potential losses during washes and transfers.
6. Remove excess M9 from the plate with a laboratory tissue. M9 should not come into contact with the copper (II) sulfate solution.
   Caution: Ensure that worms and the agar surface remain unaffected while performing this step. If used too harshly, the laboratory tissue can create indentations to the agar surface of the plate and can remove worms. Worms accidentally removed via KimWipe should be discarded.
7. Once the M9 solution has been removed and all worms have commenced non-liquid locomotory patterns, start the assay stopwatch.
   1. Optimally, remove the M9 solution within a min. The essential parameter is the identification of sinusoidal locomotion. Worms locomote differently, e.g. thrashing rather than sinusoidal, when in liquid. Start the assay stop watch once each of the experimental organisms stops thrashing.
8. Check assay plates every 30 min.
   1. For the assay plates with bacterial patches, positively score organisms if they reach the food patch over a 4 h period. For the negative control plates, positively score organisms if they have crossed the barrier.

Results

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...

Discussion

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 <...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.]			Produced in lab
Cupric Sulfate	Sigma	C-1297	Use water to appropriately suspend to a concentration of 0.5M