Laskova, Valeriya et al. (2013) International Worm Meeting "Mapping the entire connectome of C. elegans L1 larvae."

Zhen, Mei, Kasthuri, Bobby, Shalek, Richard, Lichtman, Jeff, Samuel, Aravi, Pfister, Hanspeter, Laskova, Valeriya, Kaynig-Fittkau, Verena, Wen, Quan, Berger, Daniel, Guan, Asuka

[International Worm Meeting, 2013]

To investigate the poorly understood mechanisms of development and function of the nervous system, connections between neurons must be deciphered first. The adult nervous system of C. elegans was mapped to near completion by Dr. John White and his colleagues in 1970s. However, neuronal wiring in C. elegans larvae differs from that of an adult, since it undergoes multiple rounds of neuronal birth, apoptosis and rewiring during development. We utilize an Automatic Tape-Collecting Ultramicrotome (ATUM) to section an entire L1 stage animal into thousands cross-sections, followed by the automated imaging with a scanning electron microscope (SEM) to map an entire neuronal wiring diagram with synaptic resolution. The acquired wiring diagram will guide and be validated by calcium imaging to map the functional connection of the juvenile motor circuit.

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.

Hung, Wesley et al. (2015) International Worm Meeting "The C. elegans RID neuron is a neurosecretory cell that modulates locomotion."

Lu, Yangning, Holmyard, Douglas, McWhirter, Rebecca, Samuel, Aravinthan, Laskova, Valeriya, Zhen, Mei, Ji, Ni, Miller, David M., Chitturi, Jyothsna, Hung, Wesley, Ho, Chi-Yip, Calarco, John, Lim, Maria A

[International Worm Meeting, 2015]

* contributed equallyComplex behaviors are modulated by endocrine neurons, which secrete neuropeptides and hormones. Homeostasis of these neuromodulators is critical, and its disruption has been associated with disease. Despite the ancient and significant role that neuromodulators play in complex behaviors across organisms, a comprehensive understanding of neuroendocrine cell function and their regulation is limited. The C. elegans nervous system provides an excellent genetic platform to investigate neural network functions and behavior. However, despite a fully annotated nervous system, a neuron with dedicated neurosecretory properties has not been identified. Using serial transmission electron microscopy, we identified RID as a potential endocrine cell. RID is the only neuron that extends an axon along the full length of the dorsal nerve cord and almost exclusively contains dense core vesicles, which package and secrete neuromodulators. Using a combination of laser ablation, genetics, and imaging techniques, we provide several lines of evidence that the RID neuron is a neuroendocrine cell that modulates motor behaviors. Using transcriptome profiling of a subpopulation of neurons isolated from both wild-type and unc-39 mutant worms, wherein the RID neuron is absent, we found that the RID neuron is enriched with neuropeptides, including flp-14. We confirm that flp-14 is expressed in the RID neuron and that flp-14 mutants possess locomotor defects similar to RID mutants. Our results demonstrate a neuromodulatory role for the RID neuron in regulating locomotion. We propose that the C. elegans RID neuron may be a useful genetic model to probe for conserved molecular mechanisms underlying neuroendocrine cell development and function. .

Berger, Daniel et al. (2015) International Worm Meeting "The mind of an L1 worm - how do neuronal circuits function and remodel throughout development?."

Samuel, Aravinthan, Emmons, Scott, Neubauer, Marianna, Lichtman, Jeff, Witvliet, Daniel, Chisholm, Andrew, Laskova, Valeriya, Mitchell, James, Mulcahy, Ben, Cook, Steven, Zhen, Mei, Schalek, Richard, Berger, Daniel, Hall, David, Holmyard, Douglas, Kersen, David, Qu, Angie

[International Worm Meeting, 2015]

When humans and worms are born, they have neuronal circuits in place to integrate sensory cues and effect appropriate behavioural or homeostatic responses. During the course of development, a substantial amount of remodelling occurs in the nervous system. New neurons are born and integrated into existing circuits, and existing neurons and circuits are remodelled. The rules of this remodelling are poorly characterised, because in order to investigate neuronal circuit development one needs a connectome-level analysis of the circuit across multiple time points, in multiple animals. This strategy requires serial-section electron microscopy, which has been very low-throughput and labour-intensive.C. elegans represents an ideal organism to perform these studies on, because of their small size and stereotypical development - some of the reasons they were chosen for the original adult serial-section EM reconstruction that culminated in the acquisition of the adult connectome, dubbed 'The mind of a worm'. Since the publication of this landmark paper, new technologies have been developed that improve the fidelity and throughput of the connectome reconstruction, including better fixation methods, increased computational power and more advanced microscopes.We are reconstructing the entire nervous system of multiple L1 worms using serial-section transmission and scanning electron microscopy. The L1 worm is of special interest because of the major remodelling that occurs at the end of L1, including the birth of new major classes of motor neurons. We are finding both conserved and unreported structure and connectivity from the published adult wiring diagram. This dataset will be used as a framework to functionally interrogate the interplay between developing circuits and sensorimotor behaviours, and the mechanisms that allow this dialogue to take place.