Chokshi, Trushal et al. (2009) International Worm Meeting "A Microfluidic Platform for High-throughput Calcium Imaging Assays."

Chronis, Nikos, Chokshi, Trushal

[International Worm Meeting, 2009]

We developed a microfluidic platform for performing high-throughput Ca+2 imaging of chemosensory neurons in C. elegans. The platform automatically loads and unloads single nematodes at the stimulation site and delivers a chemical odor to the nematode''s nose. The platform is interfaced with an image analysis software that assists in collecting high-resolution Ca+2 imaging data without any manual intervention. The automated platform is an essential tool for obtaining repeatable, accurate imaging data from large populations of nematodes and therefore identifying significant statistical trends. We used the microfluidic platform to explore the impact of aging on the chemosensory properties of the ASH neuron. The ASH calcium transients in young nematodes were observed to be statistically different from older nematodes. We envision the use of the proposed microfluidic platform for high-throughput screening of age-resistant mutants or drugs that have anti-aging properties.

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.

Hale, Laura et al. (2015) International Worm Meeting "Developmental changes in composition of chemotaxis circuits may underlie behavioral maturation."

Chalasani, Sreekanth, Chronis, Nikos, Bazopoulou, Daphne, Lee, Eudoria, Hale, Laura

[International Worm Meeting, 2015]

How does the nervous sytem change during development to enable mature behavior? We use the development of chemotaxis behavior as model to study maturation and have shown that L3 larvae respond worse than adults in attractive diacetyl chemotaxis assays. However, L3 larvae are not generally bad at chemotaxis, since they respond similarly to adults in repulsive nonanone chemotaxis assays. To begin to identify neural mechanisms underlying maturation, we mapped both the adult and larval sensory circuits required for attractive diacetyl chemotaxis. Based on behavioral analysis of sensory neuron mutants, we found that the adult sensory circuit contains more neurons than the L3 circuit, suggesting that changing circuit composition may play a crucial role in behavioral maturation. To further characterize differences between the adult and L3 cellular circuits we examined the activity of individual sensory neurons from each circuit. While a microfluidic device for recording neuronal activity from adult worms is published, none had existed for L3 worms. In collaboration with N. Chronis and D. Bazopoulou we developed a novel microfluidic device that traps L3 animals and enables ready imaging of individual neurons. We previously reported that in both adult and L3 worms AWA sensory neurons respond to the addition of diacetyl. Interestingly, our current results show that L3 AWA responses are less reliable than adult responses at a lower diacetyl concentration. We also find, in support of our behavioral results, that different sets of sensory neurons encode diacetyl in adult and L3 animals. Moreover, the functional adult circuit is larger than the L3 one. Collectively, our results show that for adult and L3 worms different cellular circuits are required for attractive chemotaxis behavior, different sensory neurons encode diacetyl, and odors are encoded by cells which are dispensable for behavior. We will present these findings and our results analyzing differences in molecular pathways between L3 and adults.

Chokshi, Trushal V. et al. (2011) International Worm Meeting "Effects of electrical stimulation on the ASH chemosensory neuron in Caenorhabditis elegans."

Chokshi, Trushal V., Chronis, Nikos

[International Worm Meeting, 2011]

Electrical stimulation has been widely used to modulate and study the functionality of the nervous system in-vivo and in-vitro. Here, we characterized the effect of electrical stimulation on ASH neuron in C. elegans and employed it to probe the neuron's age dependent properties. We utilized an automated microfluidic-based platform to acquire calcium imaging data from the ASH neuron in response to an electric current passing through the worm's body. We characterized the ASH neuronal activity in response to electric currents of varying strength and electrical polarity. For positive polarities (the worm's head faces the positive electrode), ASH depolarization was proportional to the magnitude of electric current while for negative polarities (the worm's head faces the negative electrode), it was inversely related. Interestingly, the ASH neuron hyperpolarized for higher negative polarity electric currents. Further, the neuronal activity in response to electrical stimulation showed significant differences across worms of different ages, indicating that the effect of electrical stimulation is age-dependent.

Bazopoulou, Daphne et al. (2011) International Worm Meeting "The impact of longevity controlling genes and stress-inducing environmental factors in the functionality of the ASH chemosensory neuron in aging worms."

Chokshi, Trushal V, Bazopoulou, Daphne, Chronis, Nikos

[International Worm Meeting, 2011]

Understanding the physiology of aged neurons is a particularly challenging task, as aging studies need to be performed in a well-controllable environment and they require a large number of data in order to obtain statistically significantly trends. To address those issues, we have previously demonstrated the use of a microfluidic-based platform for calcium imaging of stimulus-evoked neural responses from single worms in an automated fashion. Extending that work, we performed ASH calcium imaging experiments on long-lived mutants in order to determine the impact of major aging-controlling pathways in the functionality of the nervous system. We found that the daf-2(e1370) mutation, which has already been implicated in lifespan extension and increased stress resistance, has a major effect in the aging of ASH neuron, by preserving the young-like functional characteristics in older ages. In addition, we quantified the effect of stressful conditions, such as heat shock, osmotic stress and hypoxia, in the functionality of the ASH neuron, during aging. Interestingly, a single treatment including a mild heat-stress early in adulthood was able to significantly increase neuronal responses in older animals. We believe that these enhanced neuronal performances are attributed to the hormetic effects of stressors that increase the resistance, improve the survival and cellular function in low doses but have inhibitory or adverse effects at high doses. Finally, we present data from a small scale compound screen that includes FDA approved drugs and dietary supplements with proposed neuroprotective function and compounds that have been reported to have beneficial effects in longevity.

Cregg, James F. et al. (2009) International Worm Meeting "Dissecting the role of serotonin and acetylcholine on egg-laying motor synapses by calcium imaging."

Schafer, William R., Cregg, James F., Chokshi, Trushal V., Chronis, Nikos

[International Worm Meeting, 2009]

Egg-laying is a simple motor program whose regulation integrates responses to diverse sensory cues (food, touch and osmolarity). The principal egg-laying motorneurons (the HSNs) use at least two neurotransmitters, acetylcholine (ACh) and serotonin (5-HT), both of which have been shown to have stimulatory and inhibitory effects on egg-laying. We attempt to tease apart the contributions of these molecules at egg-laying neuromuscular synapses by observing of the effects of exogenous pharmacological agents on both intact and dissected worm preparations with calcium imaging. We have designed a microfluidic chip that allows us to rapidly apply neurotransmitters and other pharmacological agents to intact and dissected worms. Using this approach, we have observed rapid activation of vulval muscle (VM2s) calcium transients by acetylcholine and cholinergic agonists. Indicating that acetylcholine is indeed a potent excitatory neurotransmitter at vulval muscle synapses. We have also observed more sustained activation of muscle activity by serotonin, both in intact and dissected worms. Utilizing the previously characterized serotonin receptor mutants, mod-1, ser-1, ser-4, ser-7, and the newly discovered ser-5 (generously provided by the Komuniecki lab), we have created a collection of strains containing only a single functional receptor subtype, allowing us to assess the contribution of each receptor to vulval muscle modulation by serotonin. Each serotonin receptor confers a unique response in the VM2s to different concentration of 5-HT. One, ser-4, previously characterized as inhibitory, stimulates calcium responses at concentrations of 5-HT three orders of magnitude less that which is commonly used in egg-laying assays. Other receptors display activity at significantly higher concentrations of 5-HT, closer to conditions in previous assays. Loss of any one of the five receptors causes significant loss of sensitivity to 5-HT. Further analysis of receptor phenotypes under varied conditions and in different combinations will be presented.

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.

Bourgeois, Frederic et al. (2009) International Worm Meeting "A microfluidic chip enabling high-throughput in vivo femtosecond laser nanoaxotomy for nerve regeneration studies."

Ghorashian, Navid, Chronis, Nikos, Guo, Samuel, Ben-Yakar, Adela, Bourgeois, Frederic, Hilliard, Massimo, Chokshi, Trushal

[International Worm Meeting, 2009]

The nematode Caenorhabditis elegans is an ideal organism for studying one of the present challenges in neuroscience: nerve regeneration and degeneration. A thorough understanding of these highly dynamic processes requires experimental conditions that minimally affect the animal: no chemicals for immobilization, a reduced number of manipulations, and a reconstituted environment suitable for the animal''s development. We developed a microfluidic device, the ''nanoaxotomy'' chip that fulfills all these criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using this chip, we performed in vivo nanoaxotomy and subsequent time-lapse imaging of regrowing axons in the absence of anesthetics, with the same precision and accuracy as we previously achieved on agar pad with anesthetics. The nanoaxotomy chip is designed in such a way that the worms are serially operated, minimizing the time during which they are immobilized and possibly improving their well-being. Notably, we observed that without anesthetics, axons of both motor and touch neurons can regrow much faster. The severed axons of the touch neurons reconnect to their distal stumps within 1-2 hours whereas those of the motor neurons regrow all the way to their target within 6-8 hours. The advantages of this microfluidic chip over the immobilization techniques previously used in studies of C. elegans, such as anesthesia on agar pads or glue, are: (i) the use of no chemicals other than the liquid growth medium to interfere with the physiological processes of the worms, possibly increasing nerve regeneration success, (ii) the adaptive deflection of the membrane allows the immobilization of the worms from L4 to adult size, (iii) the worms do not need a recovery period after surgery, permitting immediate behavioral study of the post-axotomy functionality, (iv) the sample population is well contained, and experiment conditions are easily reproducible because the trap for surgery and the environment for recovery are on the same chip and finally, (v) the design of the chip is simple enough to be adapted to other organisms or many other kinds of experiments, including ablation, irradiation, stimulation or simply observation, widening the possibilities of high-throughput biological investigations.

Reilly DK et al. (2017) J Vis Exp "Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons."

Lawler DE, Albrecht DR, Srinivasan J, Reilly DK

[J Vis Exp, 2017]

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Chalasani, Sreekanth H. et al. (2009) International Worm Meeting "AWC sensory neurons release two neurotransmitters to regulate exploratory behavior upon removal from food."

Albrecht, Dirk, Bargmann, Cornelia I., Chalasani, Sreekanth H., Kato, Saul

[International Worm Meeting, 2009]

Animals increase their pirouette frequency in response to removal from food stimulus for a period of 15 min. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that AWC sensory neurons become active in response to removal of stimulus, releasing two neurotransmitters (glutamate and a neuropeptide NLP-1). The released glutamate acts to activate AIB and inhibit AIY interneurons, promoting reversals (Chalasani et al 2007). In contrast to glutamate, AWC-released NLP-1 acts on AIA interneurons to suppress reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. AWC calcium responses are modulated in these neurotransmitter mutants, suggesting that feedback pathways affect AWC neuronal activity. References: Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70. Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.