Gourgou, Eleni et al. (2015) International Worm Meeting "Magnetic field effects on C. elegans locomotive behavior."

Epureanu, Bogdan, Mirzakhalili, Ehsan, Gourgou, Eleni

[International Worm Meeting, 2015]

Despite the extensive work performed on C. elegans sensing a variety of physical stimuli (electrical, thermal, hyper/hypo-osmotic), little is known about their sensing of magnetic field and the effect it has on their physiology or behavior. To elucidate this area, we study worm behavior when exposed to magnetic fields both internal to the organism and external. We used two electromagnets to expose worms to magnetic fields and tracked their locomotion pattern. To track the worms we used two tracking systems, a custom made and a commercially available (WormLab). We tested a number of configurations for the relative position of the magnets and the petri dish, the distance between worms and the magnets as well as on and off time intervals of applied magnetic field. Moreover, in a second set of experiments, worms were fed with superparamagnetic nanoparticles and magnetic microparticles, so as to investigate the effect of secondary magnetic field generated by the internalized particles. Particles of diameters 40nm, 70nm, 100nm and 1um were used. Secondary magnetic field generated by magnetized microparticles was simulated to estimate their magnetic field characteristics. The presence of microparticles in the gut, intestine and fat tissue was verified by fluorescent microscopy, confocal microscopy and SEM. Our results show that 1) iron core magnetic microparticles can be introduced by having the worms feed on a mixture of particles and E. coli OP50. Particles translocate in specific tissues, based on their size and they remain inside the worm's body for a time period reversely analogous to their size, 2) our tracking system offers results comparable with the commercially available system, yet we examined only velocity and distance travelled measurements thus far, 3) the magnetic field used does not seem to have any effect on untreated animals and nematodes fed with 40, 70 and 100nm diameter particles; however, worms containing particles of 1um diameter display changes in their locomotion behavior with their velocity decreased significantly. Therefore we conclude that the magnetic field with the intensity and specification we used does not seem to have an effect, for the time period applied, on the locomotion pattern of wild type animals. Moreover, it takes relatively large particles introduced in worms to observe a significant change to their locomotion parameters. Based on these results, we continue by applying stronger magnetic fields, further analyzing worm behavior to locate other possible changes, monitoring the effects of magnetic fields on specific neuron firing and using different types of particles so as to achieve different localization patterns which may affect differently the nervous system.

Gourgou, Eleni et al. (2021) International Worm Meeting "C. elegans learning strategy in T-mazes and aging-related interventions"

Fretz, Abrielle, Gourgou, Eleni, Goettemoeller, Anne, Hsu, Ao-lin, Chen, Chieh

[International Worm Meeting, 2021]

C. elegans' ability to exhibit associative, non-associative and imprinted memory in the context of chemical stimuli is well studied. We provide evidence for a new type of associative learning, in which nematodes learn to associate food with a combination of proprioceptive cues and information on the structure of their surroundings (maze), perceived through mechanosensation. By using our custom-made Worm-Maze platform, we demonstrate that C. elegans young adults locate food in T-shaped mazes and, following that experience, learn to reach a specific maze arm. The observed learning is a food-triggered multisensory behavior, which requires mechanosensory and proprioceptive input, and utilizes cues about the structural features of nematodes' environment and their body actions. Our findings suggest that C. elegans use a type of response learning strategy to achieve learning in the maze, and that structural features of the environment play a role in the acquired learning. In addition, we show that the observed aging-driven decline C. elegans learning in the maze can still be reversed at mid-age by starvation. Genetically induced dietary restriction (eat-2) and genetic alterations that extend lifespan (eat-2, daf-2) have an impact on nematodes' maze performance. Lastly, we share preliminary results of a computer vision-based, custom- made tracking algorithm, especially developed for use in the challenging, 3-dimensional maze environment. This is the first time that learning in a structured maze environment is methodically portrayed and extensively characterized in C. elegans nematodes.

Gourgou, Eleni et al. (2015) International Worm Meeting "Chemically induced oxidative stress affects ASH neuronal function and C. elegans behavior."

Gourgou, Eleni, Chronis, Nikos

[International Worm Meeting, 2015]

Oxidative stress (OS) is one of the most significant types of stress an organism is exposed to throughout its life. The effect of OS on the nervous system specifically is of great interest, since it has been associated with neurodegenerative diseases. There is also evidence that exposure to herbicides, such as paraquat (PQ), induces OS. However, the effect of OS on the functionality and performance of sensory neurons remains obscure. In vivo about function of OS-exposed animals' sensory neurons. Here, we present our results from experiments in which we exposed wild type C. elegans worms to chemically (PQ) induced OS throughout their life. First, we aim to investigate the effect of OS-exposure on ASH sensory neuron function. To this end, we use a microfluidic device developed in our lab to immobilize worms and deliver the stimulus to their nose, in a highly controlled manner of minimum interference with the nervous system. After delivering the stimulus (glycerol), we record calcium transients from ASH neuron. Our results show that previously OS exposed worms display elevated neuronal excitability compared to non-treated animals, a phenomenon which is abolished when the worms are simultaneously treated with the well-established antioxidant vitamin C. Next, to connect the changes on ASH physiology to a behavior dependent on ASH function, we run the octanol avoidance test, in which worms (untreated, treated, and treated with the antioxidant) are tested regarding their reaction time when exposed to the repellent octanol, a behavior mediated by ASH. Our results show that worms exposed to OS react more slowly than the untreated ones, implying that ASH altered excitability observed with calcium imaging results in a modified behavioral response. In order to further address behavioral changes, we target the locomotion parameters. By using an automated worm tracking system, we analyze locomotion of untreated, treated, and worms to which we had administered the oxidative factor along with the antioxidant. Based on body posture and locomotive parameters measurements, we demonstrate that OS exposed worms, during the first days of their adult life do not display significant differences compared with untreated animals, with the partial exception of day5. The difference observed in the two behavioral expressions, locomotion and octanol avoidance, the first being essentially unaffected by exposure to paraquat whereas the second, directly mediated by ASH, is affected, may reveal a differential impact of oxidative stress on cell specific physiology and subsequent functions of an organism.

Gourgou*, Eleni et al. (2015) International Worm Meeting "Real-time behavioral study of C. elegans by dynamic in situ photopatterning of hydrogel assays."

Bazopoulou, Daphne, Chronis, Nikos, Gourgou*, Eleni, Oliver*, C. Ryan, Hart, A. John

[International Worm Meeting, 2015]

*Equal ContributionsThe use of C. elegans in neuroscience has flourished due to advances in microfluidics. However, traditional microfluidic assays require time-consuming design and fabrication and have limited flexibility during operation. We present a platform for dynamically building microfluidic assays by in situ photopatterning of a bio-compatible hydrogel on NGM plates. This method adds flexibility to the workflow, enabling the researcher to incorporate new features in the assay based on observations as the experiment proceeds. To validate the technique, we first study whether in situ photofabrication of micropillars around swimming C. elegans would influence the worms' velocity and found that going from the open frame, to the micropillar array, to the rippled microchannel, the worm's maximum speed increases 270%. Our method eliminates the need to load worms into a device one by one and sort them into individual chambers, as the chamber can be fabricated in situ around each worm. Next, we fabricate a free-floating lever around a pin anchored to the NGM plate. Worms confined within a frame surrounding this mechanism actively interact with the lever. A variety of mechanisms can be fabricated within the culture environment (one-way gates, floating microparticles, movable isolation chambers, gears) as means to investigate more complicated behaviors. In addition, the researcher can generate freeform input using a tablet, resulting in real-time modification of the assay. Using custom-built LabVIEW, the time between when the pen touches the pad and final PEG-DA photopolymerization is less than 2sec. Last, we demonstrate how worms can be tracked in custom-made mazes to determine their ability to locate food. In the absence of food, the worms choose the left or right ends with equal probability, whereas, when food is placed in one end of the maze, worms choose the leg containing the food almost twice as often. Our photopatterning technique enables rapid and flexible experimentation via micro-scale confinement of model organisms, and in the future could incorporate image analysis and machine learning techniques to acquire large datasets and accelerate breakthroughs in understanding the behavior of model organisms such as C. elegans.

Mirzakhalili, Ehsan et al. (2017) International Worm Meeting "A computational model of the calcium dynamics in Caenorhabditis elegans ASH sensory neuron."

Gourgou, Eleni, Epureanu, Bogdan, Mirzakhalili, Ehsan

[International Worm Meeting, 2017]

C. elegans is widely used as a model system for monitoring stimulus-evoked Ca2+ transients in neurons. The ASH sensory neuron is the subject of several such studies, primarily due to its key importance as a polymodal nociceptor. However, despite the pivotal role of ASH in C. elegans, the overall biology and the characteristics of its Ca2+ transients (e.g., the "off" response), no mathematical model has been developed to describe the full mechanism of ASH Ca2+ dynamics. We propose a phenomenological computational model which captures the Ca2+ transients in the C. elegans ASH neuron upon its activation. The model is built on biophysical cascades that unfold as part of the neuron's Ca2+ signaling events and homeostatic mechanism (e.g., TRPV channels and voltage-gated channels activation, Ca2+ release from intracellular stores, IP3 dynamics, PMCA and SERCA pumps function). The state of the ion channels is described based on Hodgkin-Huxley equations and the remaining molecular states are based on kinetic equations with phenomenological adjustments. We fit the model using experimental data of osmotic stimulus-evoked ASH Ca2+ transients, detected with a FRET sensor (TN-XL), in young and aged worms, both untreated and exposed to oxidative stress. We use a multi-objective genetic algorithm to find the parameters for young untreated worms' data set. Parameters are estimated using a hybrid method that consists of a genetic algorithm and nonlinear least-squares. We use the same approach to fit the model for the other groups of experimental data. We validate the model using data from the literature, from ASH activation by stimuli of several strengths and durations. Finally, we demonstrate how our model can be used to predict the ASH Ca2+ response to stimulation pulses that are challenging to achieve experimentally (stimuli sequences of varying durations/lengths, or ramp stimuli). Our model includes for the first time the changes in ASH cytoplasmic Ca2+ flux observed both upon delivery and withdrawal of the stimulus (i.e., the "on" and "off" responses). This effort is the first to propose a quantitative dynamic model of the Ca2+ transients generating mechanism in a C. elegans neuron, based on essential biochemical pathways of the Ca2+ homeostasis machinery.

Cardoza , Steel et al. (2021) International Worm Meeting "3-dimensional behavioral arenas for C. elegans"

Gourgou, Eleni, Cardoza , Steel, Tse, Lai Yu Leo, Barton, Kira, Branch, Emily

[International Worm Meeting, 2021]

In nature, C. elegans live in a rich 3-dimensional environment. However, their behavior has been assessed almost exclusively on the open, flat surface of NGM (Nematode Growth Medium) plates, the golden standard for C. elegans culture in the lab. We present a method to build 3-dimensional behavioral arenas for C. elegans by directly 3D-printing NGM hydrogel. This is achieved by using a highly customized fused deposition modeling (FDM) 3D-printer, extensively modified to employ NGM hydrogel as ink, i.e., the Parnon Printer. The result is the advancement of 3-dimensional complexity of behavioral assays. To demonstrate the potential of our method, we use the 3D-printed arenas to assess C. elegans physical barrier crossing ability, in the context of aging (young, middle-aged adults), feeding history (fully fed, starved animals) and prior experience (have been or not in the presence of a similar 3D structure before). We also explore the usage of 3D-printed structures to spatially confine C. elegans egg laying behavior. C. elegans behavior in 3-dimensional environments is by definition not possible to explore on standard flat NGM plates. Therefore, the findings reported here would likely not have been brought to light if the Parnon Printer had not been developed. We consider these work a decisive step toward characterizing C. elegans 3-dimensional behavior, an area long overlooked due to technical constrains. We envision our method of 3D-printing NGM arenas as a powerful tool in behavioral neurogenetics and neuroethology.

Gourgou E et al. (2021) STAR Protoc "A maze platform for the assessment of Caenorhabditis elegans behavior and learning."

Hsu AL, Gourgou E

[STAR Protoc, 2021]

Mazes are broadly used to investigate animal decision-making and spatial learning. However, they have been sparsely employed to explore <i>C.elegans</i> behavior and training-improved performance. This protocol describes a highly reproducible, low-cost maze platform, made of the standard, agar-based, nematode culturing material. It can be used to reliably assess <i>C.elegans</i>' maze behavior, and we have recently applied this protocol to establish multisensory learning. Limitations include challenges in locomotion tracking and in distinguishing learning formation versus retrieval. For complete details on the use and execution of this protocol, please refer to Gourgou etal. (2021).

Nektarios Tavernarakis et al. (2006) European Worm Meeting "Bleomycin-Induced Fibrosis in C. elegans?"

Nektarios Tavernarakis, Aggeliki Pasparaki, Eleni Tzortzaki

[European Worm Meeting, 2006]

?. Aggeliki Pasparaki1 Nektarios Tavernarakis1 and Eleni Tzortzaki2. Bleomycin is an antibiotic drug isolated from a strain of Streptomyces verticillus, which has anticancer properties and acts by induction of DNA strand breaks. Due to its cytotoxic properties, the drug has been used to treat many malignant tumors. Bleomycin is also used to induce interstitial pulmonary fibrosis in rodents. Pulmonary fibrosis is a life-threatening condition of unknown molecular etiology in humans. Pulmonary fibrosis affects the alveolar epithelium, the capillary endothelium and the pulmonary basement membrane, where gas exchange is taking place during respiration, and results in abnormal collagen deposition in these tissues. In C. elegans, respiration is achieved by diffusion of oxygen via the intestinal tract, and trough the aqueous film surrounding the body, resembling gas exchange in lung alveoli.. We are investigating the effects of bleomycin on C. elegans survival and physiology. Exposure of worms to bleomycin results in abnormal locomotion, feeding, egg-laying and morphological defects, and lethality. These phenotypes range in severity in a dose dependent manner. The most severe phenotypes are not reversed after removal of the drug. Interestingly, extended exposure to low doses of bleomycin alters the appearance of the cuticle and hypodermis, indicating that similarly to mammals, bleomycin may affect the collagenous exoskeleton of worms by modifying collagen deposition. Thus, genetic analysis in C. elegans may reveal conserved bleomycin targets relevant to the pathogenesis of pulmonary fibrosis in humans.