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.
