Fouad, A. D. et al. (2019) International Worm Meeting "Development of a worm picking robot."

Du, A., Li, Z., Fang-Yen, C., Kassouni, A., Fouad, A. D., Teng, C., Bhirgoo, P. D.

[International Worm Meeting, 2019]

C. elegans' small size and manipulability make it an ideal candidate for automation technologies. Numerous automated methods have been developed for handling, imaging, and sorting worms using microfluidics and flow cells. Common to these methods is a requirement that the worms be handled in liquid, which requires special preparation and limits the phenotypes that can be selected for. To our knowledge, no automated method exists to handle worms on agar plates normally used for C. elegans. Here, we describe a worm picking robot capable of transferring worms on standard worm plates using methods similar to those used for manual worm picking. The robot consists of a motorized 3D gantry positioned above a tray of agar plates. The gantry contains a camera to view the plates and a motorized pick than can be actuated linearly or rotationally. Custom software moves the gantry above a requested plate, removes the lid using a vacuum interface, identifies worms on the plate, and then gently lowers the bacteria-coated pick to the worm by capacitive detection of contact between the pick and the agar surface. To sterilize the pick before and after use, a heating coil is extended to cover the pick tip and activated. We demonstrate that our system can identify and autonomously pick L4 and adult animals and safely transfer them to different plates. We developed a scripting language that allows us to coordinate multistep procedures, such as identifying and transferring worms with a specific behavioral, morphological, or fluorescence phenotypes, and setting up genetic crosses. By automating most worm pushing operations, the system will both increase the productivity of researchers during routine experiments and enable experiments that would be impractical using conventional methods.

Fouad, Anthony et al. (2021) International Worm Meeting "Rethinking the worm pick: Alternative materials and sterilization methods"

Li, Zihao, Fouad, Anthony, Fang-Yen, Christopher

[International Worm Meeting, 2021]

The platinum worm pick, a fixture in C. elegans laboratories for decades, has two drawbacks: (1) the high cost of platinum, a significant problem in many educational settings, and (2) the reliance on an open flame for sterilization, which presents safety hazards. To address the first drawback, we evaluated whether platinum could be replaced with an alternative metal. An ideal worm pick cools quickly after heating, withstands high temperature without degradation, can be flattened and shaped easily, and is inexpensive. With these criteria in mind, we compared 90% platinum, 10% iridium wire (PT9010) with 5 alternatives: stainless steel (SS), Nickel 200, two nickel chromium (Nichrome) alloys, and iron-chromium-aluminum (FeCrAl). To measure cooling rate we built a circuit to resistively heat wires (all 255 microm in diameter) to 800 C and measured the time it took them to cool to 25 C. We found that PT9010 and FeCrAl cooled more rapidly (6-7 s) than the other metals tested (8-9 s). To assay heat resistance we conducted a bending test after 3000 heating cycles of duration 4 s at 800 C. All materials except SS showed good heat resistance, withstanding >50 bends after 3000 heating cycles. SS exhibited poor heat resistance, breaking spontaneously after ~300 cycles. All materials could be easily flattened using standard tools. With regard to cost, all alternative materials were < 0.20 USD/m, as compared to 140 USD/m for PT9010. These results show that all metal alloys tested except for SS represent reasonable, economical alternatives for worm picks. The most promising is FeCrAl which cools as rapidly as platinum, exhibits good heat resistance, and is available at a fraction of the cost. Next, to explore an alternative to flame sterilization, we designed an electric worm pick consisting of a loop of PT9010 or FeCrAl wire attached to a handle containing a rechargeable battery and circuit board. Depressing a button causes current to flow through the loop, heating it to about 800 C within 2 s. A battery charge lasts for ~500 sterilizations. Worm researchers who tested the device reported that the wire loop could be used similar to a worm pick and that electric sterilization promoted faster work since no movements to a flame were necessary. Our device represents a convenient and safer alternative to flame-sterilized worm picks. We are using a similar loop-based worm picking technique in our automated worm picking system (see abstract by Zihao Li et al).

Fouad, A.D. et al. (2017) International Worm Meeting "Distributed rhythm generators underlie C. elegans forward locomotion."

Liu, A., Mark, J.R., Zhen, M., Teng, S., Fouad, A.D., Cornblath, E., Ji, H., Fang-Yen, C.

[International Worm Meeting, 2017]

Coordinated rhythmic movements are ubiquitous in animal locomotory, feeding, and circulatory systems. In many organisms, a chain of neural oscillators underlies the generation of rhythmic waves. In the roundworm C. elegans, proprioceptive feedback plays an important role in wave propagation during forward locomotion. However, it remains unclear whether the locomotor circuit contains more than one rhythm generator. Here, we use targeted optogenetic manipulation and laser ablation experiments to show that multiple sections of forward locomotor circuitry are capable of independently oscillating. When we optogenetically inhibited the muscles, cholinergic neurons, or B-type motor neurons just posterior to the head, we found that the head and tail could simultaneously undulate at different frequencies. We refer to this phenomenon, in which the head frequency decreases while the tail frequency simultaneously increases, as two-frequency undulation (2FU). Worms were capable of 2FU despite ablations of or disruptions to the pre-motor interneurons and most classes of ventral nerve cord (VNC) motor neurons, including subsets of the B-type motor neurons, which are associated with forward locomotion. To confirm that the VNC motor neurons can generate rhythms without synaptic input from head motor circuitry, we developed a method to sever the dorsal and ventral nerve cords in adult C. elegans. Worms in which both cords have been severed in multiple locations were capable of generating robust waves in the mid-body and higher frequency waves in the tail, suggesting that multiple units within the VNC motor circuit are capable of independent oscillation. Finally, we address the decrease in head frequency during 2FU, which is not predicted by models that only consider posteriorward coupling. Using rhythmic optogenetic stimuli, we show that an imposed mid-body pattern is capable of entraining the head to a new frequency. This observation suggests that motor coupling in the VNC motor circuit is bidirectional. Taken together, our results show that like the vertebrate spinal cord, the C. elegans forward motor circuit contains multiple oscillators that normally coordinate their activity to generate behavior. Our work opens the possibility of a genetic and neural dissection of how rhythmic locomotion is generated, propagated, and modulated.

Fouad, A. et al. (2019) International Worm Meeting "Phase response curves for optogenetic manipulation support a threshold switch model for locomotory rhythm generation."

Teng, S., Fang-Yen, C., Li, Z., Liu, A., Alvarez-Illera, P., Yao, B., Ji, H., Fouad, A.

[International Worm Meeting, 2019]

Despite recent advances in understanding which C. elegans neurons generate rhythms during locomotion, the basic mechanisms of rhythm generation remain elusive. To probe the nature of rhythm generation during forward movement, we used optogenetics to briefly perturb muscles and neurons of freely moving animals, and quantified changes in the locomotory phase as a function of the phase of the perturbation within the locomotory cycle. The resulting phase response curves (PRCs) are informative with regard to the mechanisms of wave generation in the motor circuit. We found that NpHR-mediated optogenetic inhibition of anterior body wall muscles causes a PRC with a periodicity of 180 degrees and a sawtooth-like shape, with a gradual linear increase followed by an abrupt drop to a value close to zero. Examination of curvature in a stimulus-triggered average manner showed that the rapid drop of phase shift identified in the PRC corresponded to a change in the tendency for an optogenetically interrupted bending wave to be aborted or continued. We show that the key features of these responses can be explained by a computational model in which the active moment generated by body wall muscles undergoes a switch between fixed values in the dorsal and ventral directions upon reaching thresholds defined by a linear combination of the curvature and time derivative of curvature (see abstract by H. Ji et al).

Fouad, Anthony et al. (2015) International Worm Meeting "Quantitative assessment of C. elegans fat levels using dark field microscopy."

Zhang, Kevin, Fu, Moyu, Fouad, Anthony, Fang-Yen, Christopher, Raizen, David

[International Worm Meeting, 2015]

Measurement of lipids in C. elegans is important for studies of fat metabolism, storage and regulation. Current methods for fat measurement are either limited to fixed worms or require highly complex, specialized equipment. It has long been noted that worms that store higher levels of fats in their intestine have darker intestines when viewed by brightfield microscopy, suggesting that fat is associated with increased optical scattering. Here, we demonstrate a simple method for estimating fat levels in C. elegans using a standard compound microscope. We use dark field images, in which the intestine appears bright against a dark background, to calculate an optical scattering density for each worm. We show that scattering density is strongly correlated with fat levels, as measured by Oil Red O staining, across a wide variety of genetic and nutritional conditions. Finally, we use our method to examine the temporal profile of fat loss during 24 hour periods of starvation in wild-type and mutant adult worms. .

Fouad, A. D. et al. (2019) International Worm Meeting "Automated high-throughput analysis of C. elegans aging using the Worm Collective Activity Monitoring Platform (WormCamp)."

De La Torre, M., Liu, A., De Abreu, C., Xu, J., Hayden, J., Teng, C., Patankar, S., Fang-Yen, C., Churgin, M. A., Fouad, A. D.

[International Worm Meeting, 2019]

C. elegans is a powerful model for investigating the biology of aging. Traditional methods for observing worm aging are slow and dependent on manual observation and manipulation. Our group previously developed an automated multi-well platform (WorMotel) to assay longitudinal C. elegans aging phenotypes of individually isolated worms. Although the WorMotel greatly reduced the amount of manual labor needed to perform aging experiments, scaling of experiments to thousands of conditions for genetic or compound screen is difficult due to the need for manual fabrication of devices and relatively small (240) number of animals per plate. To address these limitations, we developed a complementary method, the Worm Collective Activity Monitoring Platform (WormCamp), which assays aging by monitoring the decline of collective activity of C. elegans populations cultured in standard 24-well plates. To assay the worm populations' aging characteristics, we define a metric based on time required for the cumulative activity distribution for each well to reach a certain fraction of the total cumulative activity. Using validation data derived from WorMotel experiments, we show that this metric provides accurate estimates of lifespan and healthspan. To scale to a large number of conditions, we developed a custom robotic imaging system and software capable of real-time monitoring of lifespan and healthspan in thousands of populations simultaneously. The system consists of a camera and illuminators mounted on a 3D motorized stage positioned above an array of about 100 multi-well plates. The system serially records image sequences from each plate, illuminating it briefly with blue light to stimulate worm activity. We are using the automated system to conduct a whole-genome RNAi screen for genetic interventions that cause changes in lifespan and/or healthspan. The WormCamp method is complementary to the WorMotel method. Since it operates at a population level, the WormCamp provides information on aging with much less detail compared to the WorMotel, but is higher in throughput due to the larger number of animals per plate and the use of standard 24-well plates.

Fouad, A.D. et al. (2017) International Worm Meeting "Efficient and precise ablation of C. elegans cells using pulsed infrared laser cauterization."

Ji, H., Fang-Yen, C., Teng, S., Mark, J.R., Fouad, A.D., Liu, A.

[International Worm Meeting, 2017]

Laser microsurgery has long been a powerful tool for creating specific cellular or circuit lesions in C. elegans. Laser ablations have been typically performed by using a nanosecond or femtosecond pulsed laser to induce local plasma formation in the target cell. In our experience, aberrations in microscope objectives limit our ability to ablate deep structures in animals larger than L2 larvae using traditional laser ablation systems. In addition, the small size of plasma formation makes it difficult to lesion larger structures such as the ventral nerve cord. Here, we demonstrate that C. elegans cells can be efficiently ablated by using a pulsed infrared laser to damage tissues via locally elevated temperature. This method, while less spatially precise than ablation by nanosecond or femtosecond lasers, is highly effective in thick samples like L4 and adult C. elegans. We demonstrate that a single, 0.8 ms pulse from a laser with a wavelength of 1480 nm and peak power of 400 mW, is ideal for killing a targeted neuron but not its immediate neighbors. At this dosage, a neuron 2.5 m away from the target has a ~45% chance of being killed, and a neuron 5 m away has a ~10% chance of being killed. We show that a short train of pulses can reliably sever the ventral nerve cord in adult C. elegans. Hence, our method is a tool that can be used to lesion cells and other structures in C. elegans in cases where traditional microsurgery methods are not practical.

Ji, H. et al. (2019) International Worm Meeting "A novel model of bending wave generation supports a threshold-switch mechanism in the C. elegans motor circuit."

Fouad, A., Ji, H., Fang-Yen, C.

[International Worm Meeting, 2019]

C. elegans forward movement consists of anterior-to-posterior dorsoventral undulations mediated by a neuromuscular circuit. Little is known about how these oscillations are generated or what their intrinsic properties are. To investigate the mechanisms underlying rhythm generation in the forward motor circuit, we applied transient perturbations to undulating worms via inhibitory optogenetic illumination and calculated the induced phase shift as a function of phase at which the perturbations occurred. The worms displayed a sawtooth-shaped phase response curve (PRC) with sharp transitions from phase delay to advance (see abstract by A. Fouad et al). We asked whether the asymmetric shape of the PRC could be explained by a model oscillator using proprioceptive feedback and postural thresholds that switch the active moment of muscles. We found that one such model, in which the switching threshold is a function of the curvature and its time derivative, reproduced the observed PRC shape. To realistically model inhibitory stimuli, we quantified the effects of optogenetic inhibitions on local curvature. We incorporated external loading into the model oscillator and made the following predictions, each of which was experimentally verified: (i) Amplitude of undulation should increase as viscosity increases; (ii) The induced PRC should be shifted to the right as viscosity increases; (iii) Normal undulatory waves display an asymmetry where increases in absolute curvature are slower than decreases. Despite the model's simplicity, it is sufficient to reproduce several key features of locomotion including some that had not been reported previously. Our results are consistent with the idea that C. elegans locomotory oscillators use a threshold-switch mechanism in order to generate and modulate rhythmic motor patterns.

Gao, S. et al. (2017) International Worm Meeting "A compressed C. elegans locomotory circuit utilizes excitatory motor neurons as local central pattern generators for reversal locomotion."

Fouad, A., Huang, Y., Gao, S., Fang-Yen, C., Qi, B., Alcaire, S., Lu, Y, Jin, Y., Kawano, T., Hung, W., Meng, J., Li, Y., Guan, S., Zhen, M., Alkema, M.

[International Worm Meeting, 2017]

Central pattern generators (CPGs), neurons or neural circuits that exhibit and sustain oscillatory activities, drive motor rhythmicity. Through cell ablation, electrophysiology, and calcium imaging, we reveal the CPGs that underlie C. elegans reversal locomotion. We show the following: first, the cholinergic and excitatory A class motor neurons exhibit intrinsic oscillatory activity; second, the intrinsic activity from multiple A class motor neurons suffices for reversal locomotion; third, their oscillatory activity requires intrinsic P/Q/N voltage-gated calcium currents; fourth, attenuation and potentiation of their oscillatory activity by the descending premotor interneuron, via gap junctions and chemical synapses, respectively, determines the initiation and duration of reversal locomotion. Hence, the A class motor neuron themselves serve as local oscillators; regulation of their CPG activities determines the forward versus reversal motor state. These findings exemplify functional compression at the small C. elegans motor circuit: excitatory motor neurons assume the role for rhythm and pattern generation.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.