Chen, Sway P. et al. (2011) International Worm Meeting "How larvae wiggle: functional analysis of the L1 larva locomotory circuit."

Tang, Anji, Chen, Sway P., Wen, Quan, Samuel, Aravinthan D. T.

[International Worm Meeting, 2011]

At all developmental stages, C. elegans navigates its environment by generating and propagating sinusoidal bending waves along its body. The neural circuit controlling the locomotory behavior, however, changes significantly at the late L1 larval stage [1]. Neuroanatomy shows that almost no cholinergic motor neurons innervate the ventral body wall muscles in the L1 larva, raising the question of how the larva is capable of normal locomotion. Here we hypothesize that ventral body wall muscles in the L1 larva contract by default. During the dorsal muscle contracting phase, DB cholinergic motor neurons activate DD GABAergic motor neurons and cause periodic relaxation of the ventral muscles. This hypothesis is supported by two experimental observations. First, we examined the swimming behavior of GABA-deficient mutant unc-25 in the L1 stage and found that the tail consistently curved to one side. Second, we found that wild-type worms expressing GFP in VNC neurons consistently curved to the ventral side after being paralyzed by ivermectin, a drug that silences the motor circuit but has no effect on muscles. Further optogenetic and calcium imaging experiments will be carried out to quantify and relate motor neuron and muscle activity in the free-moving larva. Our approach will build toward a detailed mechanistic model of L1 locomotion. Hopefully, comparison of L1 and adult worm locomotion will shed light on conserved principles of rhythmic motion in general. 1.White, J.G., et al., The structure of the ventral nerve cord of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 1976. 275(938): p. 327-48.

Susoy, Vladislav et al. (2019) International Worm Meeting "Mapping circuit-wide neuronal dynamics to a complex goal-directed behavior in C. elegans."

Venkatachalam, Vivek, Hung, Wesley, Zhen, Mei, Susoy, Vladislav, Samuel, Aravinthan, Whitener, Joshua, Wu, Min

[International Worm Meeting, 2019]

A central question in neuroscience is how brain circuits generate animal behavior. Addressing this question would benefit from knowing the system-wide activity patterns during complex, naturalistic tasks, along with synaptic and cellular features of all relevant neurons. We use the mating behavior of C. elegans males as such a paradigm. The mating behavior is an important self-motivated task, consisting of multiple steps with different temporal dynamics. Using customized microscopes build in our laboratory, we performed simultaneous continuous recordings of the activity of all neurons in the male mating circuit over the entirety of mating, while simultaneously tracking the animal's behavior. We have performed in-depth analyses of seven datasets, lasting 5-10 minutes each, that capture a wide range of mating sub-behaviors. Traces of neuronal activity were extracted for more than 60 identified neurons per dataset. Using these recordings in combination with cell-specific interventions, we were able to assign new specific functions to numerous neurons, as well as follow the progression of neuronal responses from sensory inputs to motor outputs. We have identified and confirmed multiple neurons involved in distinct mating steps including searching, scanning, turning, vulva location, excretory pore detection, insemination, and resting. By integrating the neuronal activity time series across experiments, we inferred an underlying functional network. By comparing the functional network with the known anatomical network of synaptic connectivity, we were able to assess the computational significance of electrical and chemical synapses and the sign and strength of interactions between synaptic partners. Our work creates a framework for a complete predictive model of a complex naturalistic behavior from underlying neuronal and circuit activity and connectivity patterns.

Leifer, Andrew et al. (2011) International Worm Meeting "Shining light on behavior: An optogenetic dissection of neural circuits in freely behaving C. elegans."

Wen, Quan, Samuel, Aravinthan, Clark, Christopher, Leifer, Andrew, Fang-Yen, Christopher, Alkema, Mark

[International Worm Meeting, 2011]

Optogenetics provides a promising new platform for conducting behavioral neuroscience investigations in the nematode Caenorhabditis elegans. The ability to precisely manipulate neural activity in individual neurons in a moving worm allows one to pinpoint the contribution of individual neurons to animal behavior. Previously we developed a high-resolution optogenetic illumination system capable of delivering arbitrary light patterns to targeted cells in a freely moving C. elegans (Leifer and Fang-Yen et al, Nature Methods, 2011). The system, called CoLBeRT, uses a high speed camera and custom computer vision software to monitor the motion of an unrestrained worm. As the worm swims or crawls, the system instructs a digital micromirror device to reflect patterns of blue or green laser light onto specific cellular targets expressing Channelrhdopsin-2 or Halorhodopsin. The system has sufficient accuracy and resolution to stimulate individual mechanosensory neurons while a worm swims. Here we have expanded the CoLBeRT system's capabilities to dissect the motor circuit, mechanosensory circuit and investigate the roles of command interneurons. The system is now able to generate illumination patterns with arbitrary amplitude waveforms and we have adapted the system to work with microfluidic devices. We have used the system to differentiate models of wave propagation in the motor circuit, to study habituation in the mechanosensory circuit and to begin an exploration of the command interneurons. Preliminary evidence suggests that stimulation of the command interneuron AVA modulates the worm's backward velocity.

Fang-Yen, Christopher et al. (2009) International Worm Meeting "The worms crawl in, the worms crawl out: An analysis of C. elegans locomotory gaits."

Wen, Quan, Samuel, Aravinthan, Kawai, Risa, Fang-Yen, Christopher, Wyart, Matthieu, Chen, Sway, Chklovskii, Dmitri

[International Worm Meeting, 2009]

C. elegans exhibits very different locomotory patterns when swimming in fluids and crawling on a solid substrate. Compared with crawling, swimming is characterized by a longer wavelength and higher frequency of undulations. We are interested in understanding how a single circuit composed of command neurons, motor neurons, and muscles is capable of supporting these two locomotory gaits. We estimate that when crawling on moist surfaces, worms are held by capillary forces up to 100,000 times greater than the viscous forces experienced during swimming. To investigate in more detail the effect of this mechanical loading on locomotion, we immerse worms in solutions of varying viscosity and measure their wavelength, frequency, and curvature by machine vision algorithms. We show that worm exhibit a continuous transition from swimming to crawling behavior as viscosity is increased from that of water to a value 5 orders of magnitude larger. Worms in fluids of intermediate viscosity exhibit locomotory patterns intermediate between swimming and crawling. Swimming and crawling can therefore be seen as low-load and high-load limits of a continuum of locomotory patterns. We show that our results can be understood in terms of a biomechanical model of C. elegans locomotion in which the swimming to crawling transition is associated with a transition between elastic-dominated and viscous-dominated dynamics. Next we analyze the behavior of worms undergoing spatial transitions between swimming and crawling behaviors, such that mechanical loading varies over the length of the worm. The analysis of such transitions gives insights into the generation and propagation of the sinusoidal bending wave, and the mechanisms of modulation of locomotory gaits. We show that the worm behavior follows neither strictly local nor strictly global modulation, but rather can be described by a set of rules governing the generation and propagation of undulatory waves.

Calvo, Ana C. et al. (2013) International Worm Meeting "The AIB interneuron is required for thermotaxis."

Hawk, Josh, Samuel, Aravinthan D.T., Venkatachalam, Vivek, Cook, Nathan, Colon-Ramos, Daniel A., Calvo, Ana C.

[International Worm Meeting, 2013]

The nematode Caenorhabditis elegans moves towards the cultivation temperature when placed in a thermal gradient, tracking isotherms once this temperature is reached. AFD is the principal thermosensory neuron and is required to perform all modes of thermotactic behavior. But how one neuron can drive the distinct sensorimotor transformations that underlie movement down or up gradients towards the cultivation temperature vs. isothermal tracking near the cultivation temperature is not understood. To study this question, we explored the downstream synaptic pathways from AFD. AFD has direct synaptic output to only two interneurons, chemical synapses to AIY and electrical synapses to AIB. While AIY role in thermotaxis has been widely studied and its inactivation is known to cause a constitutive cryophilic movement (movement towards colder temperatures), little is know of the AIB role. Using a genetic approach based on reconstituted caspases, we have ablated the AIB interneuron and found that these animals display no preference to move up or down the gradient. Moreover, AIB ablated worms show an increased isothermal tracking behavior: they track isotherms at temperatures where wild type animals show cryophilic or thermophilic behavior. Mutants that lack the gap junction between the AFD and AIB neurons (inx-1) exhibit a similar tracking behavior as the one seen in AIB ablated animals. Taken together, these results suggest that electrical and chemical synaptic pathways from the AFD neuron are differentially used to perform distinct modes of thermotaxis.

Ji, Ni et al. (2015) International Worm Meeting "AIY, a one neuron locus for sensorimotor integration."

Samuel, Aravinthan, Alkema, Mark, Ji, Ni, Venkatachalam, Vivek, Lim, Maria, Rodgers, Hillary, Clark, Christopher, Zhen, Mei, Kawano, Taizo

[International Worm Meeting, 2015]

For an animal to navigate its habitat, the nervous system of the animal must constantly integrate sensory input with feedback from its motor state. Corollary discharge, a phenomenon where signals from the motor circuitry are relayed to upstream circuit components, has been implicated in sensorimotor integration in a variety of animal behaviors. However, the mechanism by which corollary discharge or motor feedback contributes to complex navigational behavior such as thermotaxis is not well understood.To elucidate the loci for sensorimotor integration in C. elegans, we used volumetric imaging to simultaneously track many neurons in the thermosensory and the motor circuitry in animals that freely navigate a spatiotemporal temperature gradient. We observed that the AIY interneurons, which have long been known to play a key role in thermosensory signaling, exhibit activity patterns that correlate with both the thermosensory input as well as the motor behavior of the animal. Through cell ablation and genetic and optogenetic manipulations, we confirmed that the motor-related signal in AIY is corollary discharge in nature. In particular, we identified a series of premotor interneurons that are involved in relaying the motor-related signal to AIY. Through quantitative behavior analysis, we observed that animals with a disrupted corollary discharge pathway exhibit specific deficits in thermotaxis. Finally, we constructed a computational model that explains how the corollary discharge pathway contributes to a biased-random-walk strategy utilized by C. elegans during thermotaxis. Our study reveals a new mechanism by which individual neurons integrate sensory and motor signals to organize navigational behavior. .

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "The core apoptotic executioner proteins CED-3 and CED-4 promote neuronal regeneration in Caenorhabditis elegans."

Slone, Daniel, Hulme, Elizabeth, Shevkoplyas, Sergey, Gabel, Christopher, Samuel, Aravinthan, Driscoll, Monica, Weisberg, Sarah, Pinan-Lucarre, Berangere, Xue, Jian, Whitesides, George

[International Worm Meeting, 2009]

How neurons in their native environments respond to, and recover from, localized physical disruptions such as axon severing is poorly understood. Exciting breakthrough developments in femtosecond laser microsurgery allow precise cutting of individual axons within living Caenorhabditis elegans. Fantastically, some severed neurons have the ability to regenerate, making C. elegans a powerful model for dissecting the genetic requirements of in vivo axonal regeneration. We applied this technology to investigate the role of apoptotic and necrotic genes in the neuronal response to laser severing of ALM touch neurons visualized by pmec-4GFP in the adult C. elegans. Using ced-3 mutants including ced-3(n2433), which encodes a single amino acid substitution that disrupts the caspase active site, we showed that CED-3 caspase, extensively characterized for its role as the essential core executioner protease in apoptosis, promotes efficient regeneration of ALM as well as D-type motor neurons as assessed 24h following axotomy. Time-lapse studies using a microfluidics device further revealed that CED-3 is needed early. We expressed the caspase inhibitor P35 specifically in touch neurons and observed reduced regenerating capacity, indicating that the caspase activity is required inside the severed neuron for its regeneration. In a complementary genetic approach, we found that the regeneration defect caused by ced-3(n2433) was rescued by specific expression of ced-3 in the touch neurons, demonstrating that CED-3 acts cell autonomously for neuronal regeneration. The apoptotic caspase activator CED-4 is required for efficient axonal regeneration, but the upstream apoptotic regulators CED-9 and EGL-1 are dispensable, revealing regulation mechanistically distinct from developmental apoptosis. Regeneration also depends on caspase-related genes csp-1, csp-2 and csp-3 as well as crt-1, a critical necrotic gene encoding the Ca2+-storing ER chaperone calreticulin, which appears to act with CED-3 in the same biological pathway. Our work reveals an unexpected reconstructive role for proteins known to orchestrate cell death. We will present more data about the mechanistic regulation of this regeneration pathway at the meeting. This work was supported in part by the New Jersey Commission on Spinal Cord Research.

Yemini, E.I. et al. (2017) International Worm Meeting "Whole-Brain Imaging with Single-Neuron Identity in C. elegans."

Venkatachalam, V., Yemini, E.I., Hobert, O., Samuel, A.D.

[International Worm Meeting, 2017]

Stereotypy of the C. elegans nervous system affords single-neuron registration across animals, and consequently, robust statistics for neurobehavioral coding and transcriptomics. Fast methods of whole-brain imaging in worm exist, but unfortunately determining the identity of neurons within these volumes remains a bottleneck - requiring a long, difficult, ad hoc process. We have developed a landmarked strain for whole-brain neural identification, NeuroPAL (a Neuronal Polychromatic Atlas of Landmarks). Each neuron is assigned an invariant fluorophore barcode such that color and position specify unambiguous neural identity via the unique 3-tuple (color, position, ganglion). The GFP channel is preserved for reporters of neural activity (GCaMP) and transcriptomics (GFP). Our landmark strain employs 5 fluorophores. To visualize this strain we have used a new microscope, developed by the Samuel lab, with the capability of imaging 9 unique fluorescence channels generated by 4 excitation lines and 4 emission bands. This microscope acquires whole-brain volumes, of 4 fluorophores, simultaneously, at 10Hz. Together, these two innovations permit fast whole-brain imaging, with single-neuron identity and neuronal registration, across an animal populace.

Venkatachalam, Vivek et al. (2015) International Worm Meeting "Pan-neuronal imaging in roaming animals."

Wang, Xian, Tabone, Christopher, Zhen, Mei, Klein, Mason, Chisholm, Andrew, Srinivasan, Jagan, Greenwood, Joel, Clark, Christopher, Samuel, Aravinthan, Alkema, Mark, Mitchell, James, Ji, Ni, Venkatachalam, Vivek

[International Worm Meeting, 2015]

We present an imaging system for panneuronal recording in crawling C. elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ~100 neurons that co-express cytoplasmic calcium indicator GCaMP6 and nuclear localized RFP at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of worms subjected to thermosensory inputs. We uncover a strikingly sparse representation of thermosensory perception involving two neurons (AFDL and AFDR), and a broadly distributed representation of the motor state across the nervous system. Our imaging setup and analysis pipeline, panneuronal imaging in roaming animals, or PANERA, provides a new platform to establish functional maps of sensory to motor transformation in behaving C. elegans and Drosophila larva.

Chen, Sway et al. (2015) International Worm Meeting "Functional and anatomical analysis of the L1 motor circuit."

Schalek, Richard, Laskova, Valeriya, Chen, Sway, Cook, Steven, Suriyalaksh, Manusnan, Lichtman, Jeff, Guan, Asuka, Mitchell, James, Neubauer, Marianna, Witvliet, Daniel, Wen, Quan, Zhen, Mei, Samuel, Aravinthan, Lu, Yangning, Mulcahy, Ben

[International Worm Meeting, 2015]

At all developmental stages, C. elegans navigates its environment by generating and propagating bending waves along its body. The neuronal circuit controlling locomotory behavior, however, changes significantly at the end of the L1 larval stage. During the L1 stage, B- and A-type cholinergic motor neurons innervate dorsal body wall muscles but not ventral muscles. D-type GABAergic motor neurons innervate ventral muscles but not dorsal muscles. Calcium imaging and optogenetic stimulation suggest that D-type neurons are inhibitory at the L1 stage, causing ventral muscles to relax. At the L2 stage and beyond, both ventral and dorsal muscles are symmetrically innervated by cholinergic and GABAergic motor neurons, and phasic excitation coupled to contralateral inhibition is thought to be responsible for the propagation of undulatory waves. So how does the worm produce undulatory behavior at the L1 stage given the profound ventral/dorsal asymmetry in excitatory cholinergic and inhibitory GABAergic inputs? To answer this question, we have reconstructed the L1 motor circuit using serial section electron microscopy. We are now using targeted cellular ablation, calcium imaging, and optogenetics to pinpoint the mechanism for ventral muscle contraction, and understand how alternating waves of excitation and relaxation drive undulation during the L1 stage. Our goal is a detailed mechanistic model of L1 locomotion. Comparing the circuit basis of L1 with adult worm locomotion should shed light on conserved principles of rhythmic locomotion and development of motor circuits.