Dopamine (DA) signaling depends upon the coordination of multiple presynaptic processes, including those supporting DA biosynthesis, packaging, release and reuptake. The presynaptic dopamine transporter (DAT-1) is of particular importance in regulating both the exposure of pre- and postsynaptic DA receptors as well as supporting recycling of DA for further rounds of release. Using Caenorhabditis elegans, our lab demonstrated that loss of DAT (dat-1) elicits a DA-dependent, locomotory phenotype termed Swimming Induced Paralysis (SWIP) (Mcdonald et al., 2007). Whereas wild-type (N2) L4 stage worms swim at a continuous rate for ~20 min., dat-1 worms initiate normal swimming, but then paralyze over the next six minutes. SWIP can be rescued by pretreatment with the vesicular DA transporter (CAT-1) inhibitor reserpine, loss of DA biosynthesis (cat-2) or loss of the DA receptor DOP-3 (dop-3). In our initial studies, dat-1 SWIP was thought to be evident in either water or M9 buffer. Recently, however, we tested N2 and dat-1 worms for SWIP in both media, as well as three sucrose-supplemented water solutions. As shown in Fig. 1, as the osmolarity of the aqueous media increases, dat-1 SWIP penetrance decreases significantly. In contrast, swimming of wild-type worms is unaffected by media osmolarity. Although the effects of osmolarity on neural activity in worms have been well documented, here we show that media composition modulates a DA-modulated C. elegans motor behavior by DA and possibly other biogenic amines like serotonin. Candidate, reverse or forward genetic manipulations of SWIP in water vs. M9 may provide an opportunity to further dissect DA signaling in the nematode in vivo.

C. elegans dwells in humus where it feeds on microorganisms. Movement in the worm is the result of environmental and internal signals creating stimulatory and inhibitory output from sensory neurons. Mutants deficient in neurexin (nrx-1) and/or neuroligin (nlg-1) genes exhibit impairment in excitatory and inhibitory neuronal synapses. It has been shown that nlg-1 deficient mutants are defective in detecting osmotic strength (Calahorro et al., 2009) and other sensory behavior (Hunter et al., 2010). Here we illustrate a simple experiment that allows quantifying the roaming behaviour phenotype in neuroligin and neurexin defective mutants of C. elegans. The results of a representative experiment are shown in Figure 1.

The experiment was carried out as follow: 1) Worms were grown synchronously at 20°C on NGM agar plates to L4 stage. Animals were washed three times with CTX buffer (5 mM K₂HPO₄, KH₂PO₄ buffer pH 6.6; 1 mM CaCl₂; 1 mM MgSO₄). 2) One central circle, 1 cm of diameter, was drawn on the back of two types of assay plates: NGM plates without OP50 bacteria, and NGM plates seeding with 100 µl of OP50 outlining a ring (0.5 cm of thickness) in the perimeter, maintaining free of bacteria the centre of the plate. The plates were incubated for 24 hours at 37°C before used. 3) Around 20-30 washed worms were pipetted in the middle of the agar, coinciding with the centre of the circle drawn on the back of the plate. The excess of liquid was gently removed with a small sterile filter paper. 4) The animals, out of the ring, were counted every 15 minutes.

In toxicity and pharmacological studies C. elegans are exposed to different compounds and the effects analyzed by observing worm viability or behavior. An alternative approach is to use locomotor activity as a readout, which has been instrumental for assessing compound toxicity (Dhawan et al., 2000). Data acquisition usually requires microscope observation and manual counting, which is time consuming and inconvenient for extensive compound screenings. We have previously developed an automated method to detect C. elegans movement in which swimming worms are detected as they cross through infrared microbeams (Simonetta et al., 2007).

We are currently adapting this methodology for high throughput analyses. We have developed a 384 channel apparatus and successfully recorded the behavioral changes produced by toxic compounds (Figure 1). The effect increases with compound concentration and is dependent on exposure time. Our “Worm Microtracker” might be useful for the community to develop easier and faster toxicity and paralysis assays, opening the possibility of performing high throughput studies in C. elegans.

Dichlorofluorescin diacetate (DCFDA) is a popular fluorescence-based probe for reactive oxygen species (ROS) detection in vitro and in vivo, and has been used for this purpose in C. elegans. DCFDA is first deacetylated by endogenous esterases to dichlorofluorescein (DCFH), which can react with several ROS to form the fluorophore DCF (reviewed by Halliwell and Gutteridge, 2007). The DCFDA assay in C. elegans is sometimes performed using lysed worms, following high intensity sonication. This process disrupts the outer cuticle and internal membranes, causing intracellular, as well as intraorganelle, contents to be released. Worm lysis will cause the release of transition metal ions such as iron. Free iron may participate in redox cycling to generate ROS, for example, by Fenton chemistry (Halliwell and Gutteridge, 2007). Therefore, apparent ROS levels detected in lysed worms may be un-physiological. However, DCFDA readily diffuses into cells where it undergoes deacetylation, and this allows ROS measurement in whole worms. If whole animals are utilized, cells are not disrupted and iron remains sequestered.

Using DCFDA and a synchronized worm cohort, we measured the amount of ROS in equivalent numbers of lysed and whole worms. The gradient of the linear regression curve (expressed as ΔRFU/Δmin) (Figure 1) is the rate of fluorescent DCF production. This gradient can be, very approximately, converted to the rate of H2O2-equivalent ROS production (nmol/min) by spiking known amounts of H₂O₂. It should, however, be noted that H₂O₂ itself does not oxidize DCFH, and hence, the free radical reactions resulting in increased DCF signal following H₂O₂ spiking are likely complex. Therefore, the calibration ratio can only be considered to provide an approximate estimation of ROS produced. Nevertheless, we found that the rate of H₂O₂-equivalent production was more than an order of magnitude faster in lysed (4.1×10-3nmol/min/worm) compared to whole animals (3.6×10-4 nmol/min/worm). Using the O₂ consumption rate measured in the same worm cohort (0.018 nmol/min/worm), the % of O₂ consumed leading to ROS generation can be estimated by dividing the rate of H₂O₂-equivalent production by the rate of O₂ consumption. Using this approach, we found apparent ROS production to be around 23% for lysed worms, but only 2% for whole worms. Current estimates of in vivo mitochondrial ROS production range from 0.2 to 2% (Balaban et al., 2005). Assuming all ROS measured using the DCFDA assay to be of mitochondrial origin, ROS production is clearly overestimated by a factor of at least 10-fold, and may be as much as 100-fold, in lysed worms compared to whole worms. Therefore, when using the DCFDA assay, whole worms should be utilized instead.

C. elegans is greatly suitable in suppressor screens and has strong potential to screen for the effects of modifiers at the subcellular level as it is transparent at all stages of lifespan. Here, we describe a 96-well plate assay that illustrates how high resolution imaging may be used in day-to-day research as well as RNAi and drug screens. We used the Plate Runner HD® (Trophos, France), a 96/384-well plate motorized device able to collect fluorescence at resolutions ranging from 1024×1024 px (1 px is 7.4 µm) to 8192×8192 px (1 px is 1 µm). This device has high depth of field (40 µm at resolution of 7.4 µm; 8 µm at resolution of 1 µm), thus allowing fluorescent signals to be quantified from whole animals. This device also has a wide-field objective that allows single images of the whole well to be acquired at once (Fig. 1A). To develop drug screening for neuromuscular disease, we used C. eleganstransgenics that co-express GFP and the oculopharyngeal muscular dystrophy (OPMD) protein PABPN1 in body wall muscle cells. Mutant PABPN1 animals show defective motility accompanied by loss of GFP nuclei (20 signals lost in 3-day adults) and muscle cell degeneration (Catoire et al., 2008). These phenotypes are aggravated by sirtuin (sir-2.1/SIRT1) activation and ameliorated by sirtuin inhibition (Catoire et al., 2008; Pasco et al., 2010). Resveratrol, an indirect SIRT1 activator, enhances mutant PABPN1 toxicity whereas EX-527, a selective SIRT1 inhibitor, has the opposite effect (Catoire et al., 2008; Pasco et al., 2010). We used these chemicals to normalize a drug screen assay (Fig. 1B). Synchronised L1 larvae are incubated at 20°C with bacteria and drugs (40 L1 in 50µl/well; 5 wells/point) until they reach adulthood. At 46 hours, FUDR (0.1 mg/ml) is added to prevent egg-laying and hatching. Day-3 adults are immobilized using 2,3-butanedione monoxime (0.1 M, 300 µl/well). Morphometric analysis of images (here 2048×2048) is performed using Image J and a script developed in the laboratory. Average numbers of GFP signals/worm are integrated into a database and subjected to statistical analysis. Drug screening is now ongoing using this assay. Perfect immobilization of animals is required for imaging at 4096×4096 and up. Similar strategies may be used in the development of a variety of suppressor screens.