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[
J Neurosci Methods,
2014]
BACKGROUND: While many studies have assayed behavioral responses of animals to chemical, temperature and light gradients, fewer studies have assayed how animals respond to humidity gradients. Our novel humidity chamber has allowed us to study the neuromolecular basis of humidity sensation in the nematode Caenorhabditis elegans (Russell et al., 2014). NEW METHOD: We describe an easy-to-construct, low-cost humidity chamber to assay the behavior of small animals, including soft-bodied invertebrates, in controlled humidity gradients. RESULTS: We show that our humidity-chamber design is amenable to soft-bodied invertebrates and can produce reliable gradients ranging 0.3-8% RH/cm across a 9-cm long x 7.5-cm wide gel-covered arena. COMPARISON WITH EXISTING METHOD(S): Previous humidity chambers relied on circulating dry and moist air to produce a steep humidity gradient in a small arena (e.g. Sayeed and Benzer, 1996). To remove the confound of moving air that may elicit mechanical responses independent of humidity responses, our chamber controlled the humidity gradient using reservoirs of hygroscopic materials. Additionally, to better observe the behavioral mechanisms for humidity responses, our chamber provided a larger arena. Although similar chambers have been described previously, these approaches were not suitable for soft-bodied invertebrates or for easy imaging of behavior because they required that animals move across wire or fabric mesh. CONCLUSION: The general applicability of our humidity chamber overcomes limitations of previous designs and opens the door to observe the behavioral responses of soft-bodied invertebrates, including genetically powerful C. elegans and Drosophila larvae.
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[
Sci Rep,
2020]
In this study, we report a microfluidic device for the whole-life culture of the nematode Caenorhabditis elegans that allows the scoring of animal survival and health measures. This device referred to as the NemaLife chip features: (1) an optimized micropillar arena in which animals can crawl, (2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and (3) ports that allow rapid accessibility for feeding the adult-only population and introducing reagents as needed. The pillar arena geometry was optimized to accommodate the growing body size during culture and emulate the body gait and locomotion of animals reared on agar. Likewise, feeding protocols were optimized to recapitulate longevity outcomes typical of standard plate growth. Key benefits of the NemaLife Chip include eliminating the need to perform repeated manual transfers of adults during survival assays, negating the need for progeny-blocking chemical interventions, and avoiding the swim-induced stress across lifespan in animals reared in liquid. We also show that the culture of animals in pillar-less microfluidic chambers reduces lifespan and introduces physiological stress by increasing the occurrence of age-related vulval integrity disorder. We validated our pillar-based device with longevity analyses of classical aging mutants (
daf-2,
age-1,
eat-2, and
daf-16) and animals subjected to RNAi knockdown of age-related genes (
age-1 and
daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan in the NemaLife chip. Overall, the capacity to generate reliable lifespan and physiological data underscores the potential of the NemaLife chip to accelerate healthspan and lifespan investigations in C. elegans.
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[
MicroPubl Biol,
2020]
Aggregates of the protein tau are the hallmark of several neurodegenerative diseases including Alzheimers disease, frontotemporal lobar degeneration (FTLD-tau), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Picks disease, and chronic traumatic encephalopathy (CTE) (VandeVrede, Boxer et al. 2020). Mutations in the gene coding for tau, MAPT, can cause FTLD-tau, directly linking tau dysfunction with disease (Dickson, Kouri et al. 2011). Another protein, TDP-43, comprises aggregates which are the primary hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), and mutations in the gene coding for TDP-43, TARDBP, can cause disease (Kawakami, Arai et al. 2019). To model tau or TDP-43 proteinopathies, transgenic C. elegans have been generated that express the full-length human protein pan-neuronally. These worms exhibit significant uncoordinated movement on plates and impaired thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). However, tau- and TDP-43- expressing worms are not paralyzed; they still move their heads and have some motility on the plate (coiling, crawling with tail-drag, head swinging) which are not captured in standard crawling or thrashing assays. To assay differences in total activity, we used a WMicroTracker ARENA System (Phylumtech, AR and InVivo Biosystems, USA). The ARENA captures population level activity data by relying on optical interferometry, which uses a large array of infrared LED microbeams to detect both the movement and position of worms on a culture plate. Disruption of an LED microbeam by worm movement is recorded by repeat scans of the 6-well culture plate, and allows for real-time processing. The software identifies changes in the location of disrupted beams between scans and assigns an activity score based on differences identified between each consecutive scan (Simonetta SH). Both tau- and TDP-43- expressing worms had significantly less activity per minute than N2 (Figure 1). Further, we found the ARENA- assessed activity data recapitulated the relative severity of phenotypes among the strains as measured by motility assays. For example, both CK10 (tau V337M) and CK144 (tau WT) have significantly uncoordinated movement when crawling or thrashing in liquid, with CK144 having worse motility than CK10, due to its much higher burden of total tau protein expressed (Kraemer, Zhang et al. 2003). Likewise, CK410 (TDP-43 WT) worms have slightly impaired motility compared with N2 when crawling on a plate, CK423 (TDP-43 M337V) are severely uncoordinated, and CK426 (TDP-43 A315T) have the most severe uncoordinated phenotype. The relative toxicities of these strains stem from the effects of the mutations, as TDP-43 protein expression is relatively even among these transgenic strains (Liachko, Guthrie et al. 2010). Interestingly, the ARENA captures activity of these severely uncoordinated worms that move poorly in motility assays such as crawling on an NGM plate or thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). Therefore, ARENA assessment of aggregate activity may be a more accurate metric for capturing non-locomotor movement of C. elegans that are severely uncoordinated.
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[
Elife,
2023]
Olfactory navigation is observed across species and plays a crucial role in locating resources for survival. In the laboratory, understanding the behavioral strategies and neural circuits underlying odor-taxis requires a detailed understanding of the animal's sensory environment. For small model organisms like C. elegans and larval D. melanogaster, controlling and measuring the odor environment experienced by the animal can be challenging, especially for airborne odors, which are subject to subtle effects from airflow, temperature variation, and from the odor's adhesion, adsorption or reemission. Here we present a method to control and measure airborne odor concentration in an arena compatible with an agar substrate. Our method allows continuous controlling and monitoring of the odor profile while imaging animal behavior. We construct stationary chemical landscapes in an odor flow chamber through spatially patterned odorized air. The odor concentration is measured with a spatially distributed array of digital gas sensors. Careful placement of the sensors allows the odor concentration across the arena to be continuously inferred in space and monitored through time. We use this approach to measure the odor concentration that each animal experiences as it undergoes chemotaxis behavior and report chemotaxis strategies for C. elegans and D. melanogaster larvae populations as they navigate spatial odor landscapes.
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[
PLoS Biol,
2017]
Small, genetically tractable species such as larval zebrafish, Drosophila, or Caenorhabditis elegans have become key model organisms in modern neuroscience. In addition to their low maintenance costs and easy sharing of strains across labs, one key appeal is the possibility to monitor single or groups of animals in a behavioural arena while controlling the activity of select neurons using optogenetic or thermogenetic tools. However, the purchase of a commercial solution for these types of experiments, including an appropriate camera system as well as a controlled behavioural arena, can be costly. Here, we present a low-cost and modular open-source alternative called 'FlyPi'. Our design is based on a 3D-printed mainframe, a Raspberry Pi computer, and high-definition camera system as well as Arduino-based optical and thermal control circuits. Depending on the configuration, FlyPi can be assembled for well under 100 and features optional modules for light-emitting diode (LED)-based fluorescence microscopy and optogenetic stimulation as well as a Peltier-based temperature stimulator for thermogenetics. The complete version with all modules costs approximately 200 or substantially less if the user is prepared to 'shop around'. All functions of FlyPi can be controlled through a custom-written graphical user interface. To demonstrate FlyPi's capabilities, we present its use in a series of state-of-the-art neurogenetics experiments. In addition, we demonstrate FlyPi's utility as a medical diagnostic tool as well as a teaching aid at Neurogenetics courses held at several African universities. Taken together, the low cost and modular nature as well as fully open design of FlyPi make it a highly versatile tool in a range of applications, including the classroom, diagnostic centres, and research labs.
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[
PLoS One,
2019]
An organism's ability to mount a physiological response to external stressors is fundamental to its interaction with the environment. Experimental exploration of these interactions benefits greatly from the ability to maintain tight control of the environment, even under conditions in which it would be normal for the subject to flee the stressor. Here we present a nematode research platform that pairs automated image acquisition and analysis with a custom microfluidic device. This platform enables tight environmental control in low-density, single-worm arenas, which preclude animal escape while still allowing a broad range of behavioral activities. The platform is easily scalable, with two 50 arena arrays per chip and an imaging capacity of 600 animals per scanning device. Validating the device using dietary, osmotic, and oxidative stress indicates that it should be of broad use as a research platform, including eventual adaptation for additional stressors, anthelmintic-drug screening, and toxicology studies.
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[
Nat Methods,
2011]
To quantitatively understand chemosensory behaviors, it is desirable to present many animals with repeatable, well-defined chemical stimuli. To that end, we describe a microfluidic system to analyze Caenorhabditis elegans behavior in defined temporal and spatial stimulus patterns. A 2 cm x 2 cm structured arena allowed C. elegans to perform crawling locomotion in a controlled liquid environment. We characterized behavioral responses to attractive odors with three stimulus patterns: temporal pulses, spatial stripes and a linear concentration gradient, all delivered in the fluid phase to eliminate variability associated with air-fluid transitions. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. We identified both expected and unexpected responses in wild-type worms and sensory mutants by quantifying dozens of behavioral parameters. The devices are inexpensive, easy to fabricate, reusable and suitable for delivering any liquid-borne stimulus.
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[
Nat Chem,
2012]
The helicates--chiral assemblies of two or more metal atoms linked by short or relatively rigid multidentate organic ligands--may be regarded as non-peptide mimetics of -helices because they are of comparable size and have shown some relevant biological activity. Unfortunately, these beautiful helical compounds have remained difficult to use in the medicinal arena because they contain mixtures of isomers, cannot be optimized for specific purposes, are insoluble, or are too difficult to synthesize. Instead, we have now prepared thermodynamically stable single enantiomers of monometallic units connected by organic linkers. Our highly adaptable self-assembly approach enables the rapid preparation of ranges of water-stable, helicate-like compounds with high stereochemical purity. One such iron(II) 'flexicate' system exhibits specific interactions with DNA, promising antimicrobial activity against a Gram-positive bacterium (methicillin-resistant Staphylococcus aureus, MRSA252), but also, unusually, a Gram-negative bacterium (Escherichia coli, MC4100), as well as low toxicity towards a non-mammalian model organism (Caenorhabditis elegans).
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[
PLoS One,
2017]
Pheromone cues are an important component of intersexual communication, particularly in regards to mate choice. Caenorhabditis nematodes predominant rely on pheromone production for mate finding and mate choice. Here we describe a new microfluidic paradigm for studying mate choice in nematodes. Specifically, the Pheromone Arena allows for a constant flow of odorants, including pheromones and other small molecules, to be passed in real time from signaling worms to those making a choice without any physical contact. We validated this microfluidic paradigm by corroborating previous studies in showing that virgin C. remanei and C. elegans males have a strong preference for virgin females over mated ones. Moreover, our results suggest that the strength of attraction is an additive effect of male receptivity and female signal production. We also explicitly examine female choice and find that females are more attracted to virgin males. However, a female's mate choice is strongly dependent on her mating status.
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[
Adv Biol (Weinh),
2021]
AWC olfactory neurons are fundamental for chemotaxis toward volatile attractants in Caenorhabditis elegans. Here, it is shown that AWC<sup>ON</sup> responds not only to chemicals but also to mechanical stimuli caused by fluid flow changes in a microfluidic device. The dynamics of calcium events are correlated with the stimulus amplitude. It is further shown that the mechanosensitivity of AWC<sup>ON</sup> neurons has an intrinsic nature rather than a synaptic origin, and the calcium transient response is mediated by TAX-4 cGMP-gated cation channel, suggesting the involvement of one or more "odorant" receptors in AWC<sup>ON</sup> mechano-transduction. In many cases, the responses show plateau properties resembling bistable calcium dynamics where neurons can switch from one stable state to the other. To investigate the unprecedentedly observed mechanosensitivity of AWC<sup>ON</sup> neurons, a novel microfluidic device is designed to minimize the fluid shear flow in the arena hosting the nematodes. Animals in this device show reduced neuronal activation of AWC<sup>ON</sup> neurons. The results observed indicate that the tangential component of the mechanical stress is the main contributor to the mechanosensitivity of AWC<sup>ON</sup> . Furthermore, the microfluidic platform, integrating shearless perfusion and calcium imaging, provides a novel and more controlled solution for in vivo analysis both in micro-organisms and cultured cells.