Oxidative Stress Assay for Observing Dopaminergic Neuron Loss in Caenorhabditis elegans

[Abstract] The nematode Caenorhabditis elegans is a powerful genetic model that can be used to investigate neuronal death. Research using C. elegans has been crucial to characterize cell death programmes that are conserved in mammals. Many neuronal signaling components, such as those mediating dopaminergic neurotransmission, are preserved as well. Dopaminergic neurons are progressively lost in Parkinson’s disease and an important risk factor to develop this disease appears to be oxidative stress, the increased occurrence of highly reactive oxygen species. Oxidative stressinduced dopaminergic neurodegeneration is mimicked in animal models by treatment with 6hydroxydopamine (6-OHDA), a dopamine analog, which is specifically taken up into dopaminergic neurons. After exposing C. elegans to 6-OHDA, the loss of fluorescently labeled dopaminergic neurons can be easily monitored. An organisms’ sensitivity to oxidative stress is thought to be influenced by basal levels of intrinsic oxidative stress and the ability to counteract oxidative stress and oxidative stressinduced damage. The C. elegans ‘6-OHDA model’ led to the discovery of novel genes that are required to protect dopaminergic neurons and it has helped to determine the effects of conserved cell death and cell engulfment pathways in dopaminergic neurodegeneration. Here, we describe a simple protocol that allows for the easy detection of dopaminergic neuron loss after 6-OHDA treatment in C .elegans.

elicit dopaminergic neurodegeneration in wild-type animals (Nass et al., 2002). Mutation of the dopamine transporter dat-1, which is required for neuronal 6-OHDA uptake, was shown to confer 6-OHDA resistance (Nass et al., 2002). After 6-OHDA exposure during larval stages (L3 and L4), dopaminergic neurodegeneration was scored in a low-throughput manner by mounting adult animals on cover slide (Nass et al., 2002;Tucci et al., 2011). We adapted the protocol to screen for mutants that are hypersensitive to 6-OHDA exposure by using lower 6-OHDA concentrations that do not elicit neurodegeneration in wild-type animals. We expose synchronized L1 stage larvae in 96-well plates and score adults directly on agar plates, allowing for high-throughput screening. This approach led to the characterization of the tetraspanin gene tsp-17, the neuroligin-like gene glit-1 and the transthyretin- The protocol can also be used for acute liquid exposure to other soluble compounds such as the oxidative stress-inducing drug paraquat (Offenburger et al., 2018a and2018b   1. One day before the 6-OHDA assay, pick gravid C. elegans (in adult stage and full of eggs) into a 96-well plate containing 70 μl M9 buffer per well. Only 30 μl of these 70 μl will be used for the 6-OHDA treatment, but as a substantial amount of the liquid will evaporate, it is necessary to prepare a larger volume. It is best practice to select healthy day 1 or day 2 adults for all Copyright  2. Transfer 10 adults into each well using a platinum wire, minimizing as little bacteria carryover as possible, as bacterial growth in the 96-well plates would compromise the synchronization of C. elegans L1 stage larvae.
Note: If necessary, first transfer worms to an empty NGM plate for a few seconds and then move clean animals that have crawled away from the bacteria to the 96-well plate.
3. Prepare a technical duplicate for each experiment (i.e., two wells per condition) and perform at least two biological replicates on different days. We encourage randomizing the sequence of the analyzed strains ( Figure 2). 14. Pipet the total of 200 μl of worms into oxidized 6-OHDA solution on the NGM plates opposite the bacterial stripe, directly on top of the plate label ( Figure 3B).
15. Open the lids and let the plates dry under the fume hood. When the 6-OHDA solution has soaked into the NGM, remove the adult animals and eggs with a platinum wire such that only animals that were exposed at L1 stage remain on the plate ( Figure 3C). Unless working with mutant strains that exhibit a developmental defect, there are usually no eggs or very few eggs present as all of progeny will have hatched in the 24-40 h incubation time before the assay. The L1 larvae remaining on the scoring plates will migrate towards the bacterial streak, which provides the food source ( Figure 3D).

Data analysis
We visualized scoring results with the '100% Stacked Colum' chart in Microsoft Excel. We added up numbers scored in technical duplicates and excluded data points for which the standard deviation between the technical duplicates was higher than 20% in any scoring category.
To determine statistical significance, we performed the G-Test with the 'DescTools' package of R 3. If working with a strain that exhibits a swimming defect (for example unc mutants), synchronize L1 stage animals without starvation by washing off a plate of mixed stage animals and passing them through a 5 μm nylon net filter which will retain animals of all other stages except for L1.
4. Aim to use the same lot of 6-OHDA for all experiments as efficiencies between batches can vary.
Also keep in mind that 6-OHDA might oxidize over time after opening, so older batches might become less efficient over time.
5. The ascorbic acid solution and the 6-OHDA solution must be prepared freshly immediately before treating the animals to avoid oxidization of the compounds.
6. Prepare at least 100 μl of each ascorbic acid solution and 6-OHDA solution, as otherwise the amounts will be too small to weigh them out accurately. Vortex solution thoroughly for approximately 1 min until it appears as homogeneous.