Background

The gradual loss of dopaminergic neurons can be recapitulated in animal models following exposure to the oxidative stress-inducing drug 6-hydroxydopamine (6-OHDA) (for review Schober, 2004). In contrast to other neurodegenerative drugs such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), 6-OHDA is safer to handle as it does not pass the blood-brain-barrier. 6-OHDA is a hydroxylated dopamine analog, which is specifically taken up into dopaminergic neurons by the dopamine transporter and blocks complex I of the respiratory chain (Schober, 2004). The resulting formation of reactive oxygen species is thought to trigger 6-OHDA-induced neurodegeneration (Schober, 2004).

6-OHDA exposure of the nematode Caenorhabditis elegans leads to the selective loss of dopaminergic neurons (Nass et al., 2002) and components of dopaminergic neurotransmission are highly conserved compared to mammals (for review Nass et al., 2001). C. elegans hermaphrodites possess eight dopaminergic neurons: four CEP (cephalic sensilla) and two ADE (anterior deirids) dopaminergic neurons in the head, and two PDE (posterior deirids) dopaminergic neurons in the midbody (Sulston et al., 1975). These neurons can be specifically labeled by expression of a fluorescent protein driven by the promoter of the dat-1 dopamine transporter (Nass et al., 2002). The C. elegans 6-OHDA model can be used to understand how dopaminergic neurons maintain their integrity when subjected to oxidative stress.

The first published 6-OHDA intoxication protocol for C. elegans used high concentrations of 6-OHDA 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-related gene ttr-33, all of which protect C. elegans dopaminergic neurons from 6-OHDA-induced neurodegeneration (Masoudi et al., 2014; Offenburger et al., 2018a and 2018b). The C. elegans 6-OHDA assay was further used to describe the roles of known stress response and cell death pathways in oxidative stress-induced dopaminergic neurodegeneration (Nass et al., 2002; Tóth et al., 2007; Offenburger et al., 2018a and 2018b).

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 and 2018b). The procedures we describe here are generally useful to test if compounds influence dopaminergic neuron death.