Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.

Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large-scale assays, as they demand either a time-consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC50), showing a strong linear correlation (R² = 0.91) with the LC50 values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4 > 2,4-dinitrophenol > 3,4-dichloroaniline > SDS > sodium benzoate > EDTA > K2CrO4 ; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale-up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole-organism high-throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds.

Daily variation in melatonin synthesis and arylalkylamine N-acetyltransferase activity in the nematode Caenorhabditis elegans.

Melatonin influences circadian rhythms and seasonal behavioral changes in vertebrates; it is synthesized from serotonin by N-acetylation by arylalkylamine N-acetyltransferase (AA-NAT) and O-methylation by N-acetylserotonin methyltransferase. However, its physiology and function in invertebrate models are less understood. In this work, we studied daily variations in melatonin synthesis and AA-NAT activity in the nematode Caenorhabditis elegans. Under light-dark conditions (LD), a rhythmic pattern of melatonin levels was observed, with higher levels toward the middle of the night, peaking at zeitgeber time (ZT) 18, and with a minimum value around ZT0-6. AA-NAT activity showed a diurnal and circadian fluctuation with higher levels of activity during the early night, both under LD and constant darkness conditions. A peak was found around ZT12 and circadian time (CT) 12. In addition, we investigated whether this nocturnal AA-NAT activity is inhibited by light. Our results show that both white and blue light pulses significantly inhibited AA-NAT activity at ZT18. This work demonstrates the daily fluctuation of melatonin synthesis and AA-NAT activity in the adult nematode C. elegans. In summary, this study takes additional advantage of an extremely useful invertebrate model system, which has only recently been exploited for circadian studies.

The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions.

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.

Circadian rhythms in metabolic variables in Caenorhabditis elegans.

Circadian rhythms govern a wide variety of physiological and metabolic functions in most organisms through neural networks, hormones and gene expression. In this work, we studied the circadian variation in metabolic variables of adult C. elegans such as food consumption, pharyngeal contractions, defecation and oxygen consumption. Feeding behavior was clearly rhythmic under LD conditions, with a non-significant trend under DD conditions. In addition, a daily and circadian variation in muscle contraction of the pharynx was observed. Oxygen consumption also showed a circadian fluctuation with a maximum in the middle of the night (a peak was found around ZT18/CT18). Furthermore, defecation behavior also showed a daily variation in the N2 strain (wild type). This work demonstrates that in the adult nematode C. elegans metabolic variables vary daily. In summary, our results will allow us to take full advantage of this widely used animal model (including research in genetics, ageing and developmental biology) for studies in Chronobiology.

Timing of locomotor activity circadian rhythms in Caenorhabditis elegans.

Circadian rhythms are driven by endogenous biological clocks and are synchronized to environmental cues. The chronobiological study of Caenorhabditis elegans, an extensively used animal model for developmental and genetic research, might provide fundamental information about the basis of circadian rhythmicity in eukaryotes, due to its ease of use and manipulations, as well as availability of genetic data and mutant strains. The aim of this study is to fully characterize the circadian rhythm of locomotor activity in C. elegans, as well as a means for genetic screening in this nematode and the identification of circadian mutants. We have developed an infrared method to measure locomotor activity in C. elegans and found that, under constant conditions, although inter-individual variability is present, circadian periodicity shows a population distribution of periods centered at 23.9+/-0.4 h and is temperature-compensated. Locomotor activity is entrainable by light-dark cycles and by low-amplitude temperature cycles, peaking around the night-day transition and day, respectively. In addition, lin-42(mg152) or lin-42(n1089) mutants (bearing a mutation in the lin-42 gene, homolog to the per gene) exhibit a significantly longer circadian period of 25.2+/-0.4 h or 25.6+/-0.5 h, respectively. Our results represent a complete description of the locomotor activity rhythm in C. elegans, with a methodology that allowed us to uncover three of the key features of circadian systems: entrainment, free-running and temperature compensation. In addition, abnormal circadian periods in clock mutants suggest a common molecular machinery responsible for circadian rhythmicity. Our analysis of circadian rhythmicity in C. elegans opens the possibility for further screening for circadian mutations in this species.

Immunosuppressant calcineurin inhibitors phase shift circadian rhythms and inhibit circadian responses to light.

PP2B is a Ca2+/calmodulin-dependent protein phosphatase that is ubiquitously expressed in mammals. Among other actions, it is an effector mechanism in NMDA-mediated glutamate neurotransmission as well as a regulator of GSK3beta and MAPK signaling cascades. Because all of these mechanisms have demonstrable roles in the control of circadian rhythyms, we hypothesized that PP2B would be a key regulator of rhythm generation and entrainment, and that through inhibition of its phosphatase activity, the circadian system would be affected by immunosuppressant drug therapy. We report here that immunosuppressant drugs (cyclosporin A, FK506) (1) block the circadian responses to light that underlie photic entrainment; (2) produce circadian phase shifts with a characteristic nonphotic profile; and (3) disrupt circadian rhythm expression when applied chronically. These results indicate a role for PP2B in circadian rhythm generation and entrainment. In addition, because rhythm disturbance has been implicated in impairment of both physical and mental health, we suggest that the use of immunosuppressants would be safer and more efficacious if their impacts on circadian rhythmicity were taken into account.

Circadian stress tolerance in adult Caenorhabditis elegans.

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC50 = 340 mM) and oxidative (H2O2 LC50 = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.

An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application.

Automation of simple behavioral patterns, such as locomotor activity, is fundamental for pharmacological and genetic screening studies. Recently, circadian behaviors in locomotor activity and stress responses were reported in the nematode Caenorhabditis elegans, a well-known model in genetics and developmental studies. Here we present a new method for long-term recordings of C. elegans (as well as other similar-sized animals) locomotor activity based on an infrared microbeam scattering. Individual nematodes were cultured in a 96-well microtiter plate; we tested L15, CeMM and E. coli liquid cultures in long-term activity tracking experiments, and found CeMM to be the optimal medium. Treatment with 0.2% azide caused an immediate decrease in locomotor activity as recorded with our system. In addition to the validation of the method (including hardware and software details), we report its application in chronobiological studies. Circadian rhythms in animals entrained to light-dark and constant dark conditions (n=48 and 96 worms, respectively) at 16 degrees C, were analyzed by LS periodograms. We obtained a 24.2+/-0.44 h period (52% of significantly rhythmic animals) in LD, and a 23.1+/-0.40 h period (37.5% of significantly rhythmic animals) under DD. The system is automateable using microcontrollers, of low-cost construction and highly reproducible.