Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Oral and Poster List

1 Brain-wide calcium imaging reveals widespread and robust attractor-like representation of motor state Harris Kaplan, Tina Schrödel, Saul Kato, Manuel Zimmer 2 4-D imaging of neuronal activities in the whole central nervous system visualizes correlative patterns between multiple neurons Takayuki Teramoto, Yu Toyoshima, Terumasa Tokunaga, Ryo Yoshida, Yuichi Iino, Takeshi Ishihara 3 Dopamine and neuropeptides: parallel pathways to evade and escape aversive stimuli Evan Ardiel, Andrew Giles, Theodore Lindsay, Ithai Rabinowitch, William Schafer, Shawn Lockery, Catharine Rankin 4 Feeding regulation by neuropeptides – a worm opioid system? Mi Cheong Cheong, Young-Jai You, Leon Avery 5 Formation of non-overlapping neuronal territories by mutual repulsion between dendrite arbors in C. elegans Z. Candice Yip, Maxwell G. Heiman 6 An EGF-like transmembrane protein, T24F1.4/c-tomoregulin, mediates sensory neuron dendritic self-avoidance Barbara O’Brien, Timothy O’Brien, Matthew Tyska, David Miller 7 Deconstructing Sensory Neuron Shape Aakanksha Singhvi, Shai Shaham 8 Cilia and extracellular vesicles are signaling organelles Leonard Haas, Juan Wang, Rachel Kaletsky, Maria Gravato-Nobre, April Williams, Jessica Landis, Cory Patrick, Jonathan Hodgkin, Coleen T. Murphy, Maureen M. Barr 9 D2-like signaling attenuates a gap-junction mediated recurrent circuit during male mating Paola Correa, L. Rene Garcia 10 A comprehensive analysis of cell activity in the C. elegans egg-laying behavior circuit Kevin Collins, Michael Koelle 11 New insight into the structure of the C. elegans pharyngeal connectome Steven Cook, David Hall, Scott Emmons 12 Degenerate pathways for excitation of the Caenorhabditis elegans pharynx Nicholas Trojanowski, Olivia Padovan-Merhar, David Raizen, Christopher Fang-Yen 13 Trans-Synaptic Labeling of Specific Neuronal Connections in vivo Muriel Desbois, Steven Cook, Scott Emmons, Hannes Bülow 14 Guanylyl Cyclase Modulation of ASH-mediated Nociceptive Behavioral Sensitivity Michelle Krzyzanowski, Chantal Brueggemann, Mary Bethke, Kimberly Collins, Noelle L’Etoile, Denise Ferkey

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15 SPON-1/F-spondin non-autonomously regulates Q migrations in response to MAB-5/Hox patterning Matthew Josephson, Adam Miltner, Erik Lundquist 16 Axonal fusion in regenerating axons shares molecular components with the apoptotic cell recognition pathway Brent Neumann, Sean Coakley, Rosina Giordano-Santini, Casey Linton, Yi Zhang, Hengwen Yang, Ding Xue, Massimo A Hilliard 17 A Microfluidic Platform for Automated Laser Axotomy​ Sertan Gokce, Sam Guo, Navid Ghorashian, Neil Everett, Travis Jarrell, Aubri Kottek, Alan Bovik, Adela Ben-Yakar 18 The Development and Functional Regulation of the C. elegans Motor Circuit Mei Zhen 19 The Insulin/IGF Signaling Regulators Cytohesin/GRP-1 and PI5K/PPK-1 Modulate Susceptibility to Excitotoxicity in C. elegans Nazila Tehrani, John Del Rosario, Moises Dominguez, Robert Kalb, Itzhak Mano 20 The JBS-Associated E3 Ubiquitin Ligase UBR-1 Regulates Nervous System Development by Maintaining Synaptic Glutamate Homeostasis Jyothsna Chitturi, Maria Lim, Wesley Hung, Abdel Rahman, Jim W Dennis, Mei Zhen 21 Mechanisms underlying inhibition of spontaneous synaptic vesicle fusion by complexin Jeremy Dittman, Rachel Wragg, Daniel Radoff, David Snead, Yongming Dong, Jihong Bai, David Eliezer 22 A gain-of-function UNC-2/CaV2 channel induces behavioral hyperactivity and an imbalance in excitatory-inhibitory signaling Yung-Chi Huang, Jennifer Pirri, Diego Rayes, Shangbang Gao, Yasunori Saheki, Mei Zhen, Cornelia Bargmann, Michael Francis, Mark Alkema 23 Failure of NLP-40 release leads to decreased expulsions in the inx-16 intestinal gap junction mutant Sam McCright, Maureen Peters 24 Genetic analysis of ionotropic acetylcholine receptor function in GABA neurons Alison Philbrook, Michael Francis 25 Cellular and molecular mechanisms of smn-1-mediated neuron-specific degeneration Ivan Gallotta, Alessandra Donato, Nadia Mazzarella, Alessandro Esposito, Justin Chaplin, Ivan Cáceres, Daniel Porto, Paolo Bazzicalupo, Massimo A. Hilliard, Hang Lu, Elia Di Schiavi 26 Dissecting the mechanisms underlying motorneuron disease in C. elegans Maria Dimitriadi, Melissa Walsh, Jill Yersak, Anne Hart 27 Prospects for the neuronal and genetic analysis of economic decisions in the nematode Caenorhabditis elegans Shawn Lockery, Abe Katzen 28 Glia shape URX and BAG sensory dendrites through GRDN-1/Girdin and SAX-7/ L1CAM Ian G. McLachlan, Eizabeth R. Lamkin, Maxwell G. Heiman

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29 CaMKI-dependent regulation of gene expression mediates long-term temperature adaptation in AFD Yanxun Yu, Harold Bell, Dominique Glauser, Miriam Goodman, Stephen Van Hooser, Piali Sengupta 30 Bacterial Secondary Metabolites Alter C. elegans Behavior by Modifying Sites of Neuronal DAF-7 Expression Joshua Meisel, Dennis Kim 31 Sex, age and hunger regulate behavioral prioritization through dynamic modulation ofchemoreceptor expression Renee Miller, Deborah Ryan, KyungHwa Lee, Scott Neal, Piali Sengupta, Doug Portman 32 Multidimensional phenotypic profiling identifies subtle synaptic pattern mutants, and their morphological defects Adriana San-Miguel, Peri Kurshan, Kang Shen, Hang Lu 33 Synaptic position maintenance involved in hypodermal and glial cells Zhiyong Shao, Shigeki Watanabe, Ryan Christensen, Erik Jorgensen, Daniel Colón- Ramos 34 Perlecan antagonizes collagen IV and ADAMTS9/GON-1 in restricting en passant synapse growth Jianzhen Qin, Jingjing Liang, Mei Ding 35 Regulation of microtubule dynamics in synaptogenesis Naina Kurup, Dong Yan, Alexandr Goncharov, Yishi Jin 36 BK Channel Modulation of Withdrawal from Chronic Ethanol Exposure L.L. Scott, S. J. Davis, S. Iyer, R.W. Aldrich, S.J. Mihic, J.T. Pierce-Shimomura 37 Sensory molecules and mechanisms in C. elegans Bill Schafer 38 Sniffing Out Development of Chemotaxis Behavior Laura Hale, Eudoria Lee, Sreekanth Chalasani 39 Neuropeptides and dopamine regulate different behavioral components of non- associative odor learning Akiko Yamazoe, Kosuke Fujita, Yuichi Iino, Yuishi Iwasaki, Kotaro Kimura 40 Single Cell Mass Spectrometry of GABAergic Motor Neurons in the Nematode Ascaris Christopher Konop, Jennifer Knickelbine, India Viola, Colin Wruck, Martha Vestling, Antony Stretton 41 Transcriptional profiling of dissociated adult C. elegans neurons reveals specialized functions and memory components of Insulin/FOXO signaling Rachel Kaletsky, April Williams, Rachel Arey, Vanisha Lakhina, Jessica Landis, Coleen Murphy 42 Developmental history regulates olfactory behavior via RNAi pathways Jennie Sims, Maria Ow, Kyhyung Kim, Piali Sengupta, Sarah Hall 43 Variability in behavioral and neural responses to consistent stimulation Dirk Albrecht

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44 A neural circuit for head-body coordination mediates decision making in C. elegans Ingrid Hums, Harris Kaplan, Fanny Mende, Julia Riedl, Ev Yemini, Michael Sonntag, Richard Latham, Lisa Traunmueller, Saul Kato, Manuel Zimmer 45 Optogenetic cAMP increase enhances transmitter release: depletion of docked and reserve synaptic vesicles, formation of endosomal structures and of putative compound vesicles Wagner Steuer Costa, Szi-chieh Yu, Jana Liewald, Alexander Gottschalk 46 Contrasting responses within a single neuron class enable sex-specific attraction in C. elegans Anusha Narayan, Vivek Venkatachalam, Omer Durak, Neelanjan Bose, Frank Schroeder, Aravinthan Samuel, Jagan Srinivasan, Paul Sternberg 47 Serotonin facilitates efficient foraging in non-uniform environments by mediating an instantaneous slowdown upon re-feeding Shachar Iwanir, Adam Brown, Dana Najjar, Meagan Palmer, Ivy Fitzgerald, David Biron 48 Matching of neuropeptide-receptor couples reveals ancient behavioral modulation by tachykinin signaling Isabel Beets, Lotte Frooninckx, Jan Watteyne, Elien Van Sinay, Olivier Mirabeau, Liliane Schoofs 49 An oscillatory motor circuit optimizes foraging gait in C. elegans Yu Shen, Quan Wen, Connie Zhong, Yuqi Qin, Aravinthan Samuel, Yun Zhang 50 Extracellular matrix components and axon guidance Cassandra Blanchette, Andrea Thackeray, Paola Perrat, Claire Bénard 51 Regulation of neural circuit formation by a Slit-independent Robo pathway Chia-Hui Chen, Dong Yan 52 The Male Anal Depressor Integrates Cell-autonomous Sex Hierarchy Signaling and Sex Specific Exogenous Signals to Achieve Sex differential Morphological and Functional Alterations in C. elegans Xin Chen, Luis Rene Garcia 53 Analysis of the ENU-3 protein family in nervous system development Roxana Florica, Victoria Hipolito, Homai Anvari, Chloe Rapp, Mehran Asgherian, Costin Antonescu, Marie Killeen 54 Amphid cells transiently organize into a supercellular rosette during morphogenesis Ismar Kovacevic, Zhirong Bao 55 Sensory neurons use epithelial mechanisms of morphogenesis to extend their dendrites Isabel I.C. Low, Claire R. Williams, Ian G. McLachlan, Irina Kolotuev, Maxwell G. Heiman 56 An integrated genetic and biochemical analysis of the Heparan sulfate code in Caenorhabditis elegans Kristian Saied-Santiago, Robert Townley, John Attonito, Carlos Díaz-Bálzac, Dayse Cunha, Hannes Buelow

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57 Ryanodine Receptor Channels Mediate Critical Sub-cellular Calcium Signals During Normal and Optogenetically Enhanced Neuronal Regeneration in C. elegans Lin Sun, James Shay, Kevin Roodhouse, Samuel Chung, Melissa McLoed, Christopher Clark, Mark Alkema, Christopher Gabel 58 Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion through UNC-73/Trio, Rac GTPases and UNC-33/CRMP Lakshmi Sundararajan, Adam Norris, Dyan Morgan, Zachary Roberts, Erik Lundquist 59 Dynamics of the developing C. elegans nervous system Amelia White, Anthony Santella, Ismar Kovacevic, Zhirong Bao 60 Neuronal Target Identification Requires AHA-1-Mediated Fine-Tuning of Wnt Signaling in C. elegans Jingyan Zhang, Mei Ding 61 The Visual Detection of odr-1 22G RNAs via a MosSCI Sensor Adriel-John Ablaza, Bi-Tzen Juang, Sanjeev Balakrishnan, Mary Bethke, Chantal Brueggemann, Maria Gallegos, Noelle D. L’Etoile 62 Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in C. elegans Kelli Benedetti, Bi-tzen Juang, Anna Ludwig, Chen Gu, Aarati Asundi, Torsten Wittman, Noelle L’Etoile, Paul Hagerman 63 Serotonergic/Peptidergic Co-transmission in the C. elegans Egg-Laying Circuit Jacob Brewer, Michael Koelle 64 Quantitative analysis of the Caenorhabditis elegans escape from noxious thermal stimuli Jarlath Byrne Rodgers, Byron Wilson, William S. Ryu 65 Opto-genetic and -physiological dissection of the C. elegans escape response reveals new mechanisms in the orchestration of distinct sub-motor programs Christopher Clark, Andrew Leifer, Ni Ji, Jeremy Florman, Kevin Mizes, Aravinthan Samuel, Mark Alkema 66 Pumping off Food (PoffF) reveals a glutamate dependent microcircuit that imposes cue dependent inhibitory tone on the pharynx Nicolas Dallière, Nikhil Bhatla, Robert Walker, Vincent O’Connor, Lindy Holden-Dye 67 Genetic sex of sensory neurons controls attraction to ascaroside pheromones Kelli A. Fagan, Jessica R. Bennett, Frank Schroeder, Douglas S. Portman 68 Magnetosensitive neurons mediate vertical burrowing in C. elegans around the world Andrés G. Vidal-Gadea, Kristi Ward, Celia Beron, Joshua Russell, Jesse Cohn, Nicholas Truong, Adhishri Parikh, Jonathan T. Pierce-Shimomura 69 Neuromodulator network control of a multisensory decision D. Dipon Ghosh, Soonwook Hong, Michael Koelle, Michael Nitabach 70 SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male C. elegans Xiaoyan Guo, Luis Rene Garcia 71 The molecular and circuit basis of thermosensation in Caenorhabditis elegans Vera Hapiak, Harold Bell, Piali Sengupta

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72 Characterizing behavioral responses of adult nematodes to ascarosides Anna Hartmann, Michael O’Donnell, Dongshin Kim, Dirk Albrecht, Piali Sengupta 73 Genetic Analysis of C. elegans Pathogen Avoidance Behavior Alexander Horspool, Howard Chang 74 Regulation of motivational states in C. elegans Changhoon Jee, L. René Garcia 75 Regulation of an Insulin-like Peptide (ILP) network involved in learning Konstantinos Kagias, Diana Andrea Fernandes de Abreu, Antonio Caballero, Joy Alcedo, QueeLim Ch’ng, Yun Zhang 76 Genetic and biochemical studies of the mechanism of neurotransmitter signaling through heterotrimeric G proteins Seongseop Kim, Michael Koelle 77 Downstream regulatory components of the TIR-1/JNK-1 pathway for forgetting in C. elegans Tomohiro Kitazono, Akitoshi Inoue, Takeshi Ishihara 78 Identification of As-nlp-21 and As-nlp-22 peptides in the motor neurons of Ascaris suum Jennifer Knickelbine, Christopher Konop, Colin Wruck, Antony Stretton 79 Identification of new genes involved in Dopaminergic neurons function by cell specific knock-down Ambra Lanzo, Luca Pannone, Marco Tartaglia, Paolo Bazzicalupo, Simone Martinelli, Lucia Carvelli, Elia Di Schiavi 80 Sensory Neurons Enhance Egg-laying Rates Across a Wide Range of Temperatures Samuel Lasse, Miriam B Goodman 81 Dopaminergic neuronal support cells and cholinergic and glutamatergic neurons promote ejaculation and post-ejaculatory behavior in males Brigitte LeBoeuf, L. Rene Garcia 82 Environmentally evoked developmental plasticity of behavior mediated by Insulin- like signaling pathway in C. elegans Harksun Lee, Dae han Lee, Nari Kim, Myungkyu Choi, Junho Lee 83 Two distinct modes of pharyngeal pumping in C. elegans are regulated by food concentration Kyung Suk Lee 84 Transcriptional and developmental regulation of salt associative learning in C. elegans Jana P. Lim, Miriam B. Goodman, Anne Brunet 85 C. elegans core and sex-specific neurons involved in sexual attraction behavior are altered in their differentiation in hlh-3 mutant males Liliana Marquez, Aixa Alfonso 86 Episodic quiescence in fasting C. elegans Richard McCloskey, Christopher Fang-Yen 87 High throughput phenotypic profiling identifies the role of heterotrimeric G-protein signaling pathways in habituation Andrea McEwan, Andrew Giles, Kasper Podgorski, Kurt Haas, Rex Kerr, Catharine Rankin

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88 A male specific neuropeptide, FLP-23, is necessary for male sperm transfer Renee Miller, Inna Hughes, Teigan Ruster, Andy Spitzberg, Steven Husson, Liliana Schoofs, Doug Portman 89 Peptidergic signaling functions in a homeostatic manner to modulate quiescence in C. elegans sleep Stanislav Nagy, Nora Tramm, Jarred Sanders, Shachar Iwanir, Ian Shirley, Erel Levine, David Biron 90 Differing Levels of MAST Kinase Activity Can Code Two Opposing Behavioral Drives During C. elegans Thermotaxis Shunji Nakano, Isabel de Ridder, Takamasa Suzuki, Tetsuya Higashiyama, Ikue Mori 91 Lethargus-quiescence in C. elegans is a systemic brain state under tight control of arousal circuits Annika Nichols, Tomáš Eichler, Saul Kato, Tina Schrödel, Manuel Zimmer 92 A complex neuropeptide signaling cascade inhibits ASH-mediated aversive behavior in C. elegans Mitchell Oakes, Deanna Filppi, Vera Hapiak, Amanda Ortega, Abby Jelinger, Richard Komuniecki 93 The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling Andrew Olson, Michael Koelle 94 DA neurons modulate food related behaviors by signaling through peptidergic AVK and DVA neurons in a distributed neuronal network Alexandra Oranth, Christian Schultheis, Karen Erbguth, Jana Liewald, David Hain, Isabel Beets, Sebastian Wabnig, Wagner Steuer Costa, Alexander Gottschalk 95 Regulation of egg-laying behavior by the conserved EGL-9/HIF-1 hypoxia- response pathway Corinne Pender, H. Robert Horvitz 96 No Abstract Available for this Number 97 Role of FLP-1 neuropeptides on sensory and motor function Daniel Raps, Michelle Sawh, Raubern Totanes, Patrick Loi, Ivor Joseph, Chris Li 98 Branching Out: Determining the function of IL2 neurons in C. elegans dauer and non-dauer animals Alina Rashid, Rebecca Androwski, Nathan Schroeder, Juan Wang, Lenny Haas, Maureen Barr 99 Identification of molecules downstream of the insulin/PI3K pathway involved in the regulation of salt chemotaxis learning Naoko Sakai, Masahiro Tomioka, Takeshi Adachi, Hirofumi Kunitomo, Yuichi Iino 100 Light induces a pharyngeal gag reflex by C. elegans Steven Sando, Nikhil Bhatla, Bob Horvitz 101 Neural basis of plasticity and bidirectionality of klinotaxis Yohsuke Satoh, Hirofumi Sato, Hirofumi Kunitomo, Yuichi Iino 102 Swip-10/Mblac1: Identification of a Novel Regulator of Dopamine Signaling Linked to Glial Control of Extracellular Glutamate Homeostasis Chelsea Snarrenberg, Andrew Hardaway, Sarah Whitaker, Cassandra Retzlaff, Haigang Gu, Zhaoyu Li, Qi Zhang, Shawn Xu, Mausam Ghosh, Michael Robinson, Randy Blakely

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103 Study of transcriptome regulating a dispersal behavior in C. elegans Sangwon Son, Harksun Lee, Junho Lee 104 The AIB interneurons potentially function as a bimodal switch to integrate an array of sensory inputs to modulate nociception in C. elegans Philip Summers, Robert Layne, Bruce Bamber, Amanda Ortega, Richard Komuniecki 105 Novel optogenetic tools for silencing neural activity in C. elegans Megumi Takahashi, Ayako Okazaki, Takashi Tsukamoto, Yuki Sudo, Shin Takagi 106 The AWC and ASI sensory neurons contribute to starvation-dependent plasticity in thermotaxis behavior Asuka Takeishi, Piali Sengupta 107 The roles of biogenic amines on feeding state-dependent thermotactic behavior in C. elegans Satomi Tsukamoto, Shunij Nakano, Ikue Mori 108 An aptf-1 transcription factor is required for RIS neuron to control sleep onset in C. elegans Michał Turek, Ines Lewandrowski, Henrik Bringmann 109 Characterization of a disinhibitory motor circuit in C. elegans Khursheed A. Wani, Beverly J. Piggott, X. Z. Shawn Xu 110 Injection of endogenous As-NLP-22 into intact Ascaris suum causes a decrease in locomotory behavior Colin Wruck, Jennifer Knickelbine, Antony Stretton 111 K+/Cl- Cotransporter KCC-3 regulates thermotaxis behavior in C. elegans Atsushi Yoshida, Shunji Nakano, Takamasa Suzuki, Tetsuya Higashiyama, Kunio Ihara, Ikue Mori 112 Investigating the neural mechanism underlying a hypertonic response in Caenorhabditis elegans Jingyi Yu, Yun Zhang 113 A role for a T-type calcium channel in serotonin-mediated behavior Kara Zang 114 TMC-1 attenuates C. elegans development and sexual behavior in an alien food environment Liusuo Zhang, L Rene Garcia 115 Natural Variation of Pathogen-induced Neuronal Gene Expression in C. elegans Zoë Hilbert, Joshua Meisel, Dennis Kim 116 Mechanisms of Axon Regeneration in the Aging Nervous System Alexandra Byrne, Walradt Trent, Kathryn Gardner, Austin Hubbert, Valerie Reinke, Marc Hammarlund 117 Lesion conditioned axon regeneration in C. elegans Samuel Chung, James Shay, Christopher Gabel 118 Neuronal fusion induced by UNC-70/β-spectrin dependent axonal injury requires the apoptotic recognition pathway Sean Coakley, Brent Neumann, Rosina Giordano-Santini, Casey Linton, Yi Zhang, Hengwen Yang, Ding Xue, Massimo A Hilliard

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119 Evaluation of Cell Death Mechanisms in Nematode Excitotoxicity John Del Rosario, Towfiq Ahmed, JunHyung An, Tauhid Mahmud, Uzair Amjad, Itzhak Mano 120 Role of CREB/crh-1 in Molecular Modulation of Neuroprotection in a C. elegans Model of Excitotoxicity K. Genevieve Feldmann, Itzhak Mano 121 Functional analysis of VPS41-mediated protection from β-Amyloid [GC1] cytotoxicity Edward Griffin, Kim Caldwell, Guy Caldwell 122 Gαq mediates effects of antipsychotic drugs on C. elegans developmental delay/ lethality Limin Hao, Afsaneh Sheikholfslami, Kristin Harrington, Bruce Cohen, Edgar Buttner 123 Impaired mitochondrial morphology in response to an environmental contributor: S. venezuelae metabolite in a C. elegans model of Parkinson’s disease Hanna Kim, Guy Caldwell, Kim Caldwell 124 Age-dependent neuronal changes Anagha Kulkarni-Khandekar, Claire Benard 125 Studying membrane dynamics and EFF-1 fusogen localization during C. elegans axonal regeneration Casey Linton, Rosina Giordano-Santini, Brent Neumann, Sean Coakley, Massimo Hilliard 126 PINK-1 homeostasis and UPS perturbations as a result of a bacterial metabolite link environmental exposure to the genetics of neurodegenerative disease Bryan Martinez, Arpita Ray, Daniel Petersen, Guy Caldwell, Kim Caldwell 127 A Role for the C. elegans Homolog of Retinal Degeneration 3 in a Chemosensory Neuron Luis Martinez-Velazquez 128 A Screen to Identify Regulators of Ciliary Cytoskeleton Stability and Function in Response to Polyglutamylation of Axonemal Microtubules Robert O’Hagan, Winnie Zhang, Maggie Morash, Sebastian Bellotti, Maureen Barr 129 The role of miRNAs at the C. elegans neuromuscular junction: potential SMA modifiers? Patrick O’Hern, Anne Hart 130 C. elegans Models of C9ORF72-linked ALS-FTD Xing Wang, Mochtar Pribadi, Taixiang Saur, Bruce Cohen, Giovanni Coppola, Edgar Buttner 131 One-carbon metabolism genes modulate amyloid-beta toxicity in C. elegans Alzheimer’s disease models Xiaohui Yan, Adam Knight, Kim A. Caldwell, Guy A. Caldwell 132 Neutral cholesterol ester hydrolase I, a downstream modulator of DAF-2 signaling, is protective in C. elegans models of neurodegeneration Siyuan Zhang, Kim Caldwell, Guy Caldwell 133 Identifying cell-autonomous targets of the Hox Transcription factor MAB-5 in directed cell migration Mahekta Gujar, Erik Lundquist

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134 Regulation of neuronal polarity by neuron-glia gap junctions Lingfeng Meng, Dong Yan 135 A novel effector of integrin adhesion complexes is involved in cholinergic synaptogenesis in Caenorhabditis elegans Marie Pierron, Bérangère Pinan-Lucarre, Jean-Louis Bessereau 136 hlh-3 encodes two Achaete/Scute like protein isoforms with different functions Saleel Raut 137 A novel target protects against patterned neurodegeneration in Alzheimer’s disease Sangeetha V. Iyer, Luisa L. Scott, James Sahn, Gabriella Zuniga, Jon Pierce-Shimomura 138 Developmental specification of a polymodal nociceptor in C. elegans Jordan Wood, Denise Ferkey 139 Reconstruction of the L1 nervous system Daniel Berger, Steven Cook, Scott Emmons, David Hall, Douglas Holmyard, David Kersen, Valeriya Laskova, Jeff Lichtman, Ben Mulcahy, Marianna Neubauer, Aravi Samuel, Richard Schalek, Mei Zhen 140 Parallel imaging of C. elegans larval quiescence using the WorMotel Matthew Churgin, Chieh-Chieh Yu, Xiangmei Chen, David Raizen, Christopher Fang- Yen 141 Inducible Protein Degradation Using Trans-Targeting in C. elegans Jesse Cohn, Shameika Wilmington, Andreas Matouschek, Jon Pierce-Shimomura 142 An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells Navid Ghorashian, Sertan Gökçe, Sam Guo, William Everett, Adela Ben-Yakar 143 The use of novel calcium indicators in C. elegans Laura Grundy 144 An Open-Source Analytical Platform for Analysis of C. elegans Swimming Induced Paralysis (Swip) J. Andrew Hardaway, Jing Wang, Paul Fleming, Katherine Fleming, Sarah Whitaker, Alexander Nackenoff, Chelsea Snarrenberg, Shannon Hardie, Bing Zhang, Randy D. Blakely 145 Transgenic C. elegans as a screening platform for anthelmintic development Wenjing Law, Amanda Ortega, Richard Komuniecki 146 Microfluidic devices for rapid quantification of pharyngeal activity by electrophysiological measures Shawn Lockery, Kristin Robinson, William Roberts, Janis Weeks 147 Worm Psychophysics: Targeted Mechanical Stimulation and Automated Behavioral Response Tracking Eileen Mazzochette, Christopher Fang-Yen, Miriam Goodman, Beth Pruitt 148 Automating calcium image analysis Amelia Parmidge, Chantal Brueggemann, Noelle L’Etoile, Jared Young 149 Inducible and titratable silencing of C. elegans neurons in vivo with histamine- gated chloride channels Navin Pokala, Qiang Liu, Andrew Gordus, Cornelia Bargmann

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150 Methods for Studying Nervous System Development in the C. elegans embryo Anupriya Singhal, Peter Insley, Shai Shaham 151 High-throughput reverse genetic screen for synaptic vesicle recycling mutants by optogenetics and Ca++ imaging Sebastian Wabnig, Jana F. Liewald, Alexander Gottschalk 152 Imaging chromatin dynamics at specific loci in the live animal Bo Zhang, Baohui Chen, Jordan D Ward, Bo Huang, Noelle D. L’Etoile 153 Dendritic Arborization in Dauer IL2 Neurons: Role of Surrounding Tissue and Post-Dauer Branch Recovery Rebecca Androwski, Alina Rashid, Nathan Schroeder, Maureen Barr 154 Identifying odor receptors in C. elegans Sherrlyne Apostol, Newman Elizabeth, Sara Nathan, Alesha Cox-Harris, Tan Fanny, Chantal Brueggemann, Noelle L’Etoile, Jared Young 155 Olfactory sensory Neuron Regulation of Physiology in Response to Environment Aarati Asundi 156 Calcium measurements in AWC after adaptation to benzaldehyde Chantal Brueggemann, Noelle L’Etoile 157 A role for muscle-skin interactions in shaping PVD sensory dendrites Kevin Celestrin, Hannes Bülow 158 Noxious stimuli suppress food sensation in ASI Kristen Davis, Young-jai You 159 bHLH factors and insulin signaling are required for feeding-state dependent regulation of chemoreceptor gene expression Matt Gruner, Dominic Valdes, Dru Nelson, Alexander A.M. van der Linden 160 Dissecting the signaling mechanisms underlying the recognition and preference of food odors in C. elegans Gareth Harris, Yu Shen, Heonick Ha, Alessandra Donato, Samuel Wallis, Xiaodong Zhang, Yun Zhang 161 Electrodiffusion model for Ca2+ dynamics of a whole single neuron Yuishi Iwasaki, Sayuri Kuge, Takayuki Teramoto, Takeshi Ishihara 162 Digital transplants: DEG1/MEC4 chimeras reveal functional differences between Degenerin sodium channels Samata Katta, Amy Eastwood, Valeria Vasquez, Miriam Goodman 163 The role of TMC proteins in C. elegans sensory transduction Rhianna Knable, William Schafer 164 Tracking of Ca2+ dynamics in a whole single neuron Sayuri Kuge, Takayuki Teramoto, Takeshi Ishihara 165 Identification of Factors Affecting Cilia Localization of PKD-2 in C. elegans Jamie Lyman Gingerich, Kara Braunreiter, Shelby Hamlin, Casey Gabrhel 166 Defining the cellular circuit of food type-dependent feeding behavior in C. elegans Shashwat Mishra, Roxani Gatsi, Anca Neagu, Joy Alcedo 167 Study of transgenerational inheritance of acquired odor-related traits in Caenorhabditis elegans Fernando Munoz-Lobato, Noelle L´etoile

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168 Comparative genomics reveals novel genes associated with sensory cilia Thomas Sasani, Oliver Newsom, Brendan O’Flaherty, Terese Swords, Johan Henriksson, Elizabeth De Stasio, Peter Swoboda, Brian Piasecki 169 The role of innexins UNC-7 and UNC-9 in mechanosensory neurons Denise Walker, William Schafer 170 The transcription factor pros-1 is expressed in glia and regulates the morphology and function of sensory neurons Sean Wallace, Yun Lu, Shai Shaham 171 Regulation of Synaptic Transmission through Complexin Ishani Basu, Rachel Wragg, Jeremy Dittman 172 Investigating Two Amine Oxidase Domain Containing Genes, amx-1 and amx-2, in Caenorhabditis elegans Reetobrata Basu, Janet Duerr 173 Sink or swim: Discovery of a novel MAP kinase that acts in dopamine neurons to regulate swimming behavior Daniel Bermingham, J. Andrew Hardaway, Sarah Whitaker, Sam Snider, Randy Blakely 174 Unique mechanisms of pH regulation in C. elegans amphid sheath glia Jeff Grant, Laura Bianchi 175 Postsynaptic remodeling of GABAergic motor neurons in C. elegans is transcriptionally regulated by UNC-55 and IRX-1 Siwei He, Alison Philbrook, Michael Francis, David Miller 176 Conserved genes regulate sleep in C. elegans Huiyan Huang, Komudi Singh, Anne Hart 177 Investigating novel targets of the DAF-19 transcription factor in adult-stage C. elegans Alexander Hurlburt, Brian Piasecki, He Zhang, Debora Sugiaman-Trapman, Peter Swoboda, Elizabeth De Stasio 178 A two-tier system of synapse-proximal and synapse-distal neurotransmitter transporters mediates glutamate clearance in C. elegans KyungWha Lee, Jenny Wong, Itzhak Mano 179 The C. elegans RID neuron is a neurosecretory cell that regulates synaptic development and motor behavior Maria Lim, Valeriya Laskova, Jyothsna Chitturi, Douglas Holmyard, Daniel Findeis, Anne Wiekenberg, Jinbo Wang, Ralf Schnabel, Xun Huang, Mei Zhen 180 Investigating the synaptic role of the Gαs pathway Laura Manning, Janet Richmond 181 Regulation of the nicotinic acetylcholine receptor ACR-16 Ashley Martin, Feyza Sancar, Janet Richmond 182 Locating synaptic calcium channels Sean Merrill, Shigeki Watanabe, Jackson Richards, Erik Jorgensen 183 The ASI sensory neurons serve as a peptidergic hub to modulate aversive responses Holly Mills, Vera Hapiak, Rachel Wragg, Amanda Ortega, Abigail Jelinger, Richard Komuniecki xxiv Presenter Underlined Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

184 Serotonin activates a global peptidergic signalling cascade that stimulates ASH- mediated aversive responses Holly Mills, Tobias Clark, Gareth Harris, Amanda Ortega, Richard Komuniecki 185 An unconventional role of a conserved sterol biosynthetic gene, erg-28, in SLO-1 function Kelly Oh, Hongkyun Kim 186 Understanding the role of RIG-3 at the C. elegans neuromuscular junction pratima pandey, nagesh kadam, ashwani bhardwaj, kavita babu 187 A role for neuropeptide signaling in acute nicotine challenge Elizabeth Ronan, Seth Wescott, X. Z. Shawn Xu 188 Examination of the Interplay between Acetylcholine and GABA signaling at the NMJ Jacqueline Rose, Nicole Stankowicz, Amanda Leonti, Parker Stafford, Michael Remington, Katrina Mar, Samuel Moss, Andrew Records-Galbraith 189 Innexins function as plasma membrane channels in native C. elegans touch neurons Rachele Sangaletti, Jeff Grant, Laura Bianchi 190 Autophagy Proteins are Necessary for Synaptic Vesicle Clustering Sarah Hill, Andrea Stavoe, Daniel Colon-Ramos 191 A role for phosphofructokinase-1 (pfk-1) in the maintenance of synaptic vesicle clusters during hypoxia SoRi Jang, Jessica Nelson, Gonzalo Tueros, Katie Underwood, Daniel Colón-Ramos 192 The degenerin family ion channel UNC-8 remodels GABAergic synapses in an activity-dependent pathway Tyne Miller-Fleming, Sarah C. Petersen, Megan Gornet, Ying Wang, Cristina Mattewman, Lu Han, Laura Bianchi, Janet E. Richmond, David M. Miller 193 PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior Han Wang, Derek Sieburth 194 Systematic Phenotypic Characterization of Human 21st Chromosome Gene Equivalents in Caenorhaditis elegans Sarah Nordquist, Allison Griffith, Jesse Cohn, Jonathan Pierce-Shimomura

Presenter Underlined xxv Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

xxvi Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

ABSTRACTS

Abstract # 1 Brain-wide calcium imaging reveals widespread and robust attractor- like representation of motor state Harris Kaplan1, Tina Schrödel1, Saul Kato1, Manuel Zimmer1 1Research Institute of Molecular Pathology (IMP)

Sensory systems have evolved to optimally extract behaviorally relevant information from the environment. However, behavior is usually not a simple function of sensory input; many behaviors occur without obvious sensory stimulation, and even with a repeated stimulus, responses are often variable. These observations have led to the hypothesis that intrinsic neural dynamics are at least as important for behavior as sensory detection of the external world. Such dynamics have previously been recorded either without cellular resolution or as sparse, biased samples. We have recently developed a new platform for whole brain, single-cell resolution calcium imaging in C. elegans. C. elegans exhibits spontaneous, variable motor behaviors; imaging the activity of all of its neurons simultaneously is therefore an ideal approach for measuring intrinsic brain dynamics and their interaction with sensory representations. Whole brain imaging of unstimulated worms revealed that a large proportion of all neurons exhibit spontaneous, synchronous dynamics. Computational methods for multi-dimensionality reduction allowed us to describe these dynamics as oscillations between two attractor-like states. These correspond to two groups of antagonistically active neurons. One group contains the interneurons AVA and AVE, whose activity has previously been associated with reverse locomotion events. By calcium imaging AVA and AVE individually in freely moving worms, we confirmed that they signal only during each spontaneous reversal event. Therefore, AVA/ AVE activities serve as reliable indicators of an otherwise unpredictable motor event. We used this information to map fictive motor output (forward vs. backward locomotion) onto brain-wide activity dynamics of paralyzed worms. This allowed us to predict that many other neurons display increased or decreased activity during reversals. We confirmed this finding for representative neuron classes in freely moving worms. Finally, we tested the effect of sensory stimulation (with oxygen concentration changes) and found that the shapes of the attractor states were only subtly modulated, and their constituent neurons were unchanged. In fact, the major interneuron classes downstream of oxygen sensory neurons already contributed to these states. Instead, the dynamics of the system were altered: the probabilities of switching between forward and backward states were changed in a way that accurately reflects changes in reversal frequency in stimulated, freely moving animals. In conclusion, we found that a large fraction of the worm brain represents motor state, that the involved neurons constitute an intrinsically dynamic attractor-like network, and that sensory stimulation affects behavior by modulating the dynamics of this network. This work shows that intrinsic brain dynamics are fundamentally important for both spontaneous and evoked behaviors.

Session 1 1 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 2 4-D imaging of neuronal activities in the whole central nervous system visualizes correlative patterns between multiple neurons Takayuki Teramoto1, Yu Toyoshima2, Terumasa Tokunaga3, Ryo Yoshida3, Yuichi Iino2, Takeshi Ishihara1 1Kyushu University, Faculty of Sciences, Department of Biology & JST/CREST, 2University of Tokyo, Graduate School of Science, Department of Biophysics and Biochemistry & JST/CREST, 3The Institute of Statistical Mathematics, Research Organization of Information and Systems & JST/CREST

Visualization of information processing by the central nervous system in a live animal has been one of the fundamental challenges of neuroscience. Although many Ca2+ fluorescent biosensors have been developed and utilized to measure the activities of many neuronal cells, performing imaging of the whole-brain in a live animal has been limited due to the complexity of its three-dimensional structure. To achieve Ca2+ imaging of the whole head neurons in C. elegans, we designed a 4-D imaging system based on a piezo positioner and a nipkow-disk type confocal microscope. This imaging system can acquire about 95 frames per a second at three different wavelengths. For image processing of acquired data, we developed a line of programs, which perform positional tracking and segmentation of each neuron. Combining this imaging system and a NLS-tagged ratiometric fluorescent Ca2+ indicator YC2.60 and a NLSx4-tagged mCherry as a position marker, we performed Ca2+ imaging of the whole head neurons in C. elegans, and we succeeded in imaging neural activities of approximately a hundred neurons. Ratiometric snapshots of the neurons showed that each neuron have a variety of ratio value, suggesting that each neuron has a different Ca2+ baseline. By analyzing of temporal changes in ratio of each neuron that are detected and tracked, we found that multiple neurons respond with positively or negatively cross-correlative patterns to odor stimulation. Finally, our 4-D Ca2+ imaging techniques may provide a visualization of whole head neuron activities in C. elegans and will contribute to understanding the neural mechanisms of information processing by the brain.

2 Session 1 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 3 Dopamine and neuropeptides: parallel pathways to evade and escape aversive stimuli Evan Ardiel1, Andrew Giles1, Theodore Lindsay2, Ithai Rabinowitch3, William Schafer3, Shawn Lockery2, Catharine Rankin1 1University of British Columbia, 2University of Oregon, 3MRC Laboratory of Molecular Biology

The literature suggests that ASH mediated responses can habituate. However, failure to avoid some stimuli detected by ASH could be fatal for C. elegans. Why then do the reversal responses habituate? Our data indicate that habituation is part of a strategy to promote dispersal, as constantly switching direction of movement limits displacement. Indeed repeated ASH activation suppresses spontaneous reversals and accelerates forward movement. The avoidance circuit must balance the long-term goal of dispersing, with the short-term goal of evading potentially deadly stimuli. We found that dopamine signaling promotes responding via DOP-4, while a parallel peptidergic pathway promotes dispersal. To investigate ASH habituation we developed three high-throughput learning assays (habituation, dishabituation, and sensitization) using real-time computer vision software for behavioral tracking and optogenetics for ASH stimulation. Two response metrics (latency and reversal duration) displayed a decrement following repeated or persistent ASH activation (habituation). The decrement was readily reversed; tap stimulation facilitated ASH mediated responding from the habituated (dishabituation) or baseline (sensitization) levels via gap junction-dependent (unc-9) output of the body touch receptor neurons. Food and dopamine signaling (bas-1, cat-4, cat-2, trp-4) promoted responding to persistent ASH activation and we identified the D1-like dopamine receptor, DOP-4, as the key mediator. Neuropeptide synthesis mutants (egl-3, egl-21) displayed impaired habituation for a variety of metrics, prompting us to perform an RNAi screen of neuropeptide receptors and precursor genes. Evaluating both spontaneous behavior and ASH-mediated responses and plasticity we identified known and novel loss-of-function phenotypes for posture, locomotion, and learning. Several of the phenotypes have been confirmed with mutants (frpr-3, pdfr-1, pdf-1, nlp-37) and current work is aimed at identifying the sources of the peptides and their sites of action. Furthermore, we have used the multivariate behavioral profiles to predict ligand and receptor partners. We are genetically dissecting behavioral components of habituation and sensitization to understand how a persistent aversive stimulus elicits an optimal escape strategy – minimizing non-essential backward movement and accelerating forward movement.

Session 1 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 4 Feeding regulation by neuropeptides – a worm opioid system? Mi Cheong Cheong1, Young-Jai You2, Leon Avery1 1Department of Physiology and Biophysics, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University

Neuropeptides are essential for the regulation of appetite and body weight. We investigated the peptide signals that control feeding in response to starvation in Caenorhabditis elegans. Here we found that neuropeptides can regulate feeding in eat-2 mutants, which lack MC neurotransmission. eat2 mutants are functionally MC-minus and become starved even in the presence of food. egl3 encodes a proprotein convertase necessary for the maturation of neuropeptides, and egl3 mutants lack almost all neuropeptides. eat-2; egl-3 mutants had a 3-fold decreased pumping rate compared to eat-2 mutants. To identify the specific neuropeptides, we did an RNAi screen of 113 neuropeptide genes, testing whether they affected pumping and growth in an eat-2 mutant background. We found that nlp-24 RNAi decreased eat-2 growth rate and nlp-3 RNAi increased it. nlp-24-encoded peptides have a conserved YGGXX sequence, similar to mammalian opioid neuropeptides. The Komuniecki Lab has shown that nlp-3 signaling is mediated by npr-17, which has sequence similarity to opioid receptors. Morphine and naloxone respectively stimulated and inhibited feeding in starved wild-type worms, but not in worms lacking the G-protein coupled receptor NPR-17. Thus, we suggest that C. elegans has an endogenous opioid system that acts through npr-17, and that opioids regulate feeding behavior. ASI genetically ablated worms did not response to naloxone, and npr-17 is expressed in ASI amphid neurons. This result demonstrates that opioid feeding regulation requires ASI. Together, these results suggest C. elegans may be the first genetically tractable invertebrate opioid model.We are currently testing whether NLP-24 peptide and opioid agonist activation of heterologously expressed NPR-17 and mammalian opioid receptors

4 Session 1 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 5 Formation of non-overlapping neuronal territories by mutual repulsion between dendrite arbors in C. elegans Z. Candice Yip1, Maxwell G. Heiman2 1Program in Neuroscience, Harvard University; Department of Genetics, Harvard Medical School; Boston Children’s Hospital, 2Department of Genetics, Harvard Medical School; Boston Children’s Hospital

The complex mechanisms that control neuronal patterning have classically been defined by two main approaches: mutant analysis to identify molecules, and surgical transplantation to identify cell-cell and cell-tissue interactions. While mutant analysis is widely used in C. elegans, transplant approaches generally have not been available. Here, we use a genetic cell transplantation strategy to define regulative interactions that shape the mechanosensory neuron PVD. PVD normally elaborates a complex branched dendrite arbor that covers the entire body wall. We took advantage of the cell lineage mutant lin-22 to generate ectopic PVD neurons, effectively transplanting four additional PVD neurons at positions evenly spaced along the length of the animal. We used a fluorescence photoconversion approach to visualize the extent of each dendrite arbor. Surprisingly, we found that each lin-22 PVD elaborates a dendrite arbor that is smaller than that of the wild-type PVD, such that it occupies a territory that does not overlap with its neighbors. These non-overlapping arbors are suggestive of dendrite tiling, a patterning mechanism found in more complex nervous systems. We used laser ablation of combinations of PVDs to demonstrate that these non-overlapping territories arise by mutual repulsion between neighbors, consistent with a tiling mechanism. Finally, we showed that this mutual repulsion requires UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5, which have been shown to prevent sister dendrites of wild-type PVDs from crossing, a patterning mechanism called self-avoidance. Importantly, self-avoidance and tiling are molecularly distinct in other systems, and presumably evolved separately. Our results are the first demonstration of mutual repulsion between dendrite arbors in C. elegans, and suggest that the apparently complex phenomenon of dendrite tiling may have evolved by repurposing pre-existing patterning mechanisms for use in a novel context.

Session 1 5 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 6 An EGF-like transmembrane protein, T24F1.4/c-tomoregulin, mediates sensory neuron dendritic self-avoidance Barbara O’Brien1, Timothy O’Brien1, Matthew Tyska1, David Miller1 1Vanderbilt University

The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to noxious or potentially damaging stimuli (e.g., heat, force, cold, toxic chemicals). The receptive field for each sensory neuron is defined by its dendritic arbor in which sister dendrites occupy discrete domains and do not overlap. This phenomenon of dendrite self-avoidance is also widely observed for other neuron types and therefore constitutes a fundamental patterning mechanism during neural development. Two neurons in C. elegans, PVDL and PVDR are model nociceptors for studying the development of dendritic self-avoidance because they exhibit an elaborate but well-characterized dendritic arbor that is readily visible in its location directly beneath the skin. We have previously shown the diffusible cue, UNC-6/Netrin mediates PVD self–avoidance in conjunction with its receptors UNC-40/DCC and UNC-5. An independent genetic screen has now identified a novel component of this pathway that likely functions as a cell surface signal. Mutants of the T24F1.4/c-tomoregulin locus show an elevated fraction of overlapping of PVD dendritic branches (5% in wild type vs. 23% in mutants, p<0.001). c-tomoregulin contains a C-terminal transmembrane domain and a predicted extracellular EGF-like sequence. The mammalian homolog, h-tomoregulin, is highly expressed in the nervous system, but its function is unknown. We hypothesize that c-tomoregulin functions as either a co-receptor or external cue in the UNC-6/Netrin self avoidance mechanism. A T24F1.4::GFP promoter fusion is highly expressed in PVD and other specific neurons in C. elegans. Cell specific rescue experiments are in progress to determine if c-tomoregulin acts in PVD neurons or if it is provided as an external cue from neighboring cells. Future experiments will use pseudo- TIRF microscopy to determine the location of GFP-labeled c-tomoregulin in PVD dendrites as sister dendrite contact evokes self-avoidance. The goal of this work is to elucidate the cell biological pathways that regulate self-avoidance and the role of tomoregulin proteins in this evolutionarily conserved process.

6 Session 1 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 7 Deconstructing Sensory Neuron Shape Aakanksha Singhvi1, Shai Shaham1 1The Rockefeller University

Many sensory neurons sense the environment through microvilli receptive endings. Loss of microvilli leads to impaired sensory perception leading to disorders such as blindness, deafness and dysgeusia, yet we understand very little on how these structures develop. To study microvilli development in sensory systems, we chose the microvilli of the C. elegans AFD neuron as our model. Our molecular genetic analyses uncover novel non-autonomous as well as autonomous modes of regulating microvilli shape. Like receptive endings of sensory neurons across many species, those of the AFD are a composite of cilia and microvilli. We show that these two structures develop independent of each other. Through both genetic and candidate screens, we identified at least eight genes required to make AFD microvilli. To our surprise, at least three of these function in the sheath glia which encases the AFD receptive ending, suggesting that glia plays an instructive role in regulating neuronal shape. Consistent with this, ablations of glia cause a loss of AFD microvilli. We propose the following model which ties four of our mutants in a sequence of events. The sheath glia secretes a cue which regulates the length and number of villi on the AFD. The conserved co–chaperone UNC-23 acts non-autonomously to regulate AFD villi shape, perhaps by regulating folding of this glial cue. Genetic interactions indicate that UNC-23 function is transduced by the receptor guanylyl cyclase, GCY-8 expressed on AFD microvilli. Further, our data suggest that the enzymatic activity of GCY-8 is tightly linked to microvilli shape. Excess cGMP in microvilli – generated either by gain of activity mutations in gcy-8 or loss-of-function mutations in the phosphodiesterases pde-1, 3 and 5, causes loss of AFD microvilli. Finally, genetic interactions suggest that that WSP-1 mediated actin polymerization in microvilli may be regulated by levels of cGMP generated by GCY-8. Taken together, our studies are starting to uncover a novel pathway whereby a glial signal is eventually translated into actin polymerization at microvilli on the neuron. To our knowledge this is the first implication of sensory glia in regulating sensory neuron shape. Our analyses on GCY-8 also provides novel mechanistic insights into the analogous regulation of the shape of outer segments on mammalian rods and cones in the retina by receptor guanylyl cyclase activity.

Session 1 7 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 8 Cilia and extracellular vesicles are signaling organelles Leonard Haas1, Juan Wang1, Rachel Kaletsky2, Maria Gravato-Nobre3, April Williams2, Jessica Landis2, Cory Patrick1, Jonathan Hodgkin3, Coleen T. Murphy2, Maureen M. Barr1 1Department of Genetics and The Human Genetics Institute of New Jersey, Rutgers University, 2Department of Molecular Biology & LSI Genomics, Princeton University, 3Department of Biochemistry, University of Oxford

Cilia and extracellular vesicles (ECV) are two types of signaling organelles. Cilia act as a cellular antennae and function in sensation, with defects resulting in human ciliopathies. ECVs act as intercellular signaling organelles by delivering receptors and functional molecules from donor to recipient cells. Very recently the cilium has been shown to be a source of bioactive ECVs (1, 2). We found that a subset of C. elegans ciliated neurons including six IL2 neurons in both male and hermaphrodite and 21 male-specific ciliated sensory neurons, including the CEM, RnB (n=1~9 but not 6) and HOB neurons, shed and release extracellular vesicles. Using electron tomography, we have shown that ECVs are found in the cephalic lumen surrounding the CEM cilium and ciliary base. There are also ECVs in the extracellular lumen surrounding the IL2 neurons revealed by serial thin section EM by (3).Visualizing ECVs presents a technical challenge due to their small size (100nm). We have developed a system to monitor ECV-release, dynamics, and uptake in living animals by using GFP-tagged ECV cargo such as the TRP polycystin PKD-2. Intraflagellar transport (IFT) machinery and kinesin-3 KLP-6 are required for the transport and release of PKD-2::GFP-containing ECVs to the environment. Wild-type ECVs and their cargo result in a high frequency of tail-chasing behavior, while klp-6-derived ECV spots, whose cargo does not contain PKD-2, are not significantly different from the control spot. These findings indicate that ECVs and their cargo play an important role in animal communication and mating behavior. To study the unique molecular features of the ECV neurons, we performed cell-type specific RNA-seq on the ECV releasing neurons. 295 genes were significantly upregulated compared to whole worm extract. We identified 22 new genes that are specifically expressed in the ECV- releasing ciliated neurons and encode proteins associated with adhesion, signal transduction, and innate immune response. Functional characterization of these genes indicates potential overlap of molecules that mediate mate recognition and immune recognition.

Reference(s) 1. Wood, C.R. et al., The cilium secretes bioactive ectosomes. Curr Biol 23, 906-11 (2013). 2. Wang, J. et al., C. elegans Ciliated Sensory Neurons Release Extracellular Vesicles that Function in Animal Communication. Curr Biol 24, 519-25 (2014). 3. Doroquez D.B. et al., A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans. Elife. 2014 Mar 25;3:e01948.

8 Session 1 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 9 D2-like signaling attenuates a gap-junction mediated recurrent circuit during male mating Paola Correa1, L. Rene Garcia1,2 1Department of Biology, Texas A&M University, 2Howard Hughes Medical Institute

Neuro-modulation of self-amplifying circuits is required to drive the execution of behaviors to their appropriate context. Even though, recurrent neuronal networks are found throughout the C. elegans connectome, the mechanism that fine-tunes reciprocal synapses activity is unknown.Understanding this conundrum is essential, since alterations in circuit integration could lead to extended looped behavioral subroutines, reducing an organism contextual performance and potentially compromising its fitness. Here we use C. elegans male copulation as our model to dissect a complex goal-oriented behavior, which encompasses multiple sub-steps that entail initiation and termination under appropriate circumstances. As the male presses his tail against the hermaphrodite’s vulva, extensive reciprocal innervations of cholinergic sensory-motor neurons (post-cloaca sensila, p.c.s.), hook neurons and their post-synaptic sex muscles execute recurrent and rhythmic copulatory spicule thrusts, ultimately leading to genital penetration. However, distinct signaling mechanisms that restrict repetitive spicule movements to vulva cues are unclear. We found that dopamine (DA) signaling directs rhythmic spicule insertion attempts to the hermaphrodite vulva by dampening electrical coupling of spicule circuit neurons. We identified EMS-induced mutations that block DA regulation, and subsequently found that UNC-7 is an effector of the D2-like signaling pathway. Consistent with this data, UNC-7 is expressed in the male sex-muscles, cholinergic p.c.s. neurons and a hook neuron. Moreover, tissue specific phenocopy of the unc-7(rg396) allele suggests that UNC-7 couples neuronal rather than muscular activity. Via optogenetics, targeted illumination and in copula calcium imaging, we find that p.c.s. neurons excitability is dampened by D2-like signaling, and that in a hypodopaminergic state spurious spicule thrusts last even after artificial p.c.s. stimulation is removed. Our behavioral observations indicate that this neuronal DA/UNC-7 interplay maintains spicule thrusts rythmicity at the vulva, and that such modulation is crucial for optimal progeny siring. Thus, during a looped behavioral routine, such as spicule insertion attempts, DA down- modulates a gap-junction mediated recurrent circuit to decrease any intrinsic residual self- amplifying properties. This dopaminergic feedback ensures reduced inappropriate circuitry stimulation and confines a goal-oriented behavior to a proper context.

Session 2 9 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 10 A comprehensive analysis of cell activity in the C. elegans egg-laying behavior circuit Kevin Collins1, Michael Koelle1 1Yale University

Our goal is to generate, for the first time, a complete understanding of how all the neurotransmitter signaling events within a neural circuit shape the activity of each of its cells and the behavior it directs. The model neural circuit we are analyzing controls C. elegans egg-laying behavior. This circuit has just three neuron types and 16 muscle cells. Past genetic screens have identified signaling proteins that control entry to and exit from ~2 min active phases during which 3-6 eggs are laid, but how these molecules regulate the activity of the cells in the circuit to initiate, sustain, and terminate the behavior has been unclear. We developed tools to record Ca2+ activity in every cell in the egg-laying circuit at high temporal and spatial resolution in freely-behaving animals. The serotonergic HSN motor neurons show periods of rhythmic activity in which Ca2+ transients at the presynaptic termini are phased with body bends. Each egg-laying event follows an HSN Ca2+ transient, and active phases are followed by ~4 min periods of HSN inactivity. Optogenetic activation of HSNs induces activity in the VC neurons and vulval muscles that drives egg laying, similar to that seen in spontaneous active phases. However, HSN activity by itself cannot explain the induction of the active phase since many HSN transients do not lead to egg-laying events. In addition, mutants lacking serotonin or the HSNs still enter active phases, but show uncoordinated muscle contractions. Unlike the HSNs, the cholinergic VC neurons are silent in the inactive phase. During the active phase they show rhythmic Ca2+ transients at their presynaptic termini phased with body bends. We hypothesize that rhythmic HSN activity entrains egg-laying circuit activity to the phasing of body bends. Serotonin released from the HSNs coordinates the muscle response while acetylcholine released from VCs drives contraction and egg laying. We find the uv1 cells are mechanically deformed during egg-laying events, and show a Ca2+ transient following such events. The uv1 cells release tyramine that inhibits egg laying and express a potentially mechanosensitive set of TRPV channels that are required for their function. We hypothesize that both mechanical sensing of unlaid eggs in the uterus, and during egg-laying events, regulate the rhythmic activity we observe in the circuit. Together, we hope to understand how these internal signals modulate neural circuit activity to initiate, sustain, and terminate egg-laying behavior.

10 Session 2 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 11 New insight into the structure of the C. elegans pharyngeal connectome Steven Cook1, David Hall1, Scott Emmons1 1Albert Einstein College of Medicine

The ability of an organism to sense, integrate, and respond to environmental stimuli is dictated by its synaptic connectivity. To comprehend how the behavioral output of an organism emerges from its synaptic connections, full wiring diagrams, or connectomes, must be generated. The pharynx of C. elegans contains a specialized nervous system of 20 neurons plus 9 epithelial, 20 muscle, 9 marginal, and 4 gland cells separated almost entirely from the somatic nervous system. We revisited the ultra-thin TEM image series used to describe the pharynx (Albertson and Thomson, 1976) and performed an independent quantitative reconstruction of all neurons and connected cells. We have generated an adjacency matrix of the pharyngeal connectome that includes anatomical weights for synaptic connections and identification of specific post-synaptic cells. We have scored 761 chemical synapses and 96 gap junctions, which represents approximately a two-fold increase over the existing dataset. These new synapses increase the number of chemical synaptic partners (edges in the network graph) from 53 to 115. The pharyngeal neural network has a shallow processing depth with 16 neurons making neuromuscular junctions, 12 of which contain putative sensory endings. These counts represent respective increases of 4 and 2 cells as reported by Albertson and Thomson. Our new pharyngeal connectome data show small-world network properties, yet suggest differing putative mechanisms of information flow compared to the somatic nervous system of both sexes. The pharyngeal connectome has a lower clustering-coefficient (0.054 vs 0.22) and shorter characteristic path length (1.24 vs 3.48) compared to the complete somatic connectome. Furthermore, our new data suggest that the most shortest-path information, or betweenness centrality, is directed through the I4 interneuron, similar to P. pacificus. By understanding information flow through the C. elegans pharyngeal connectome in greater detail we are capable of generating new testable hypotheses of which neurons are essential for specific pharyngeal behaviors and network function. The pharyngeal connectome, along with the adult male, adult hermaphrodite, and L4 anterior are available on wormwiring.org. Tools for exploring connectivity data include quantitative adjacency matrices, neuron maps, synaptic partner lists, and direct links to electron micrographs.

Session 2 11 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 12 Degenerate pathways for excitation of the Caenorhabditis elegans pharynx Nicholas Trojanowski1, Olivia Padovan-Merhar1, David Raizen1, Christopher Fang-Yen1 1University of Pennsylvania

Degenerate networks, in which structurally distinct elements can perform the same function or yield the same output, are ubiquitous in biology. However, how degenerate neural networks regulate behavior in vivo is poorly understood. A nearly independent network of 20 neurons innervates the pharynx, the rhythmically contracting neuromuscular pump through which C. elegans feeds. Laser ablation studies have provided key insights into the function of this network, but there are multiple neurons for which ablation causes no obvious phenotype, suggesting they may have degenerate functions. To elucidate the functional connectivity of the pharyngeal network, we developed a technique in which individual pharyngeal neurons in immobilized worms are optogenetically manipulated using selective illumination by a laser beam shaped by a digital micromirror device while behavior is quantified by machine vision. Using this method, we found that excitation of the cholinergic MC, M2, and M4 motor neurons causes rapid pumping at rates that mimic behavior on food. Using the synaptic wiring diagram and laser ablation, we have demonstrated that MC, M2, and M4 act directly on pharyngeal muscle to stimulate pumping. Optogenetic inhibition of MC, M2, or M4 decreases pumping rate, confirming a role for these neurons in endogenous pumping rate regulation. Excitation of cholinergic I1 neurons, which connect the somatic and pharyngeal nervous systems, stimulates pumping via MC and M2, and I1 inhibition decreases pumping rate. Previous work indicates that the MC stimulates pumping via nicotinic neurotransmission. Surprisingly, in worms lacking the nicotinic receptor subunit EAT-2, excitation of MC still increases pumping rate, albeit to a reduced level. Excitation of MC also causes pumping in an eat-18 mutant, which lacks pharyngeal nicotinic neurotransmission, but not an unc-17 mutant, which lacks all cholinergic transmission, suggesting that MC can stimulate muscle though a non-nicotinic cholinergic mechanism. MC stimulation does not cause an increase in pumping in eat-18 mutants in the presence of the muscarinic antagonist atropine, nor in double mutants for eat-18 and the gene encoding the GAR-3 muscarinic receptors. Thus, we have identified degeneracy at both the neural and genetic levels in the pharyngeal circuit. We are currently working to understand how the activity of this circuit is altered during sleep and in the presence of neuromodulators.

12 Session 2 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 13 Trans-Synaptic Labeling of Specific Neuronal Connections in vivo Muriel Desbois1, Steven Cook1, Scott Emmons1, Hannes Bülow1 1Albert Einstein College of Medicine

Understanding animal behavior requires the determination and analysis of their neural connectivity. The use of electronmicrographs allows precise determination of all connection between neurons but remains time-consuming and only provides a static image. GRASP (GFP Reconstitution Across Synaptic Partners) and STaR (Synaptic Tagging with Recombination) are in vivo techniques, but the trans-synaptic interaction of GRASP is very strong and STaR relies on colocalization of pre- and postsynaptic labels without a direct trans-synaptic interaction. We thus sought to develop a trans-synaptic labeling method that relies on an enzymatic reaction. We used BLINC (Biotin Labeling of INtercellular Contact), originally developed in cell culture, and adapted it for transgenic use in the nematode C. elegans. BLINC is based on an enzymatic reaction, whereby the E. coli biotin ligase BirA transfers biotin onto a nineteen amino acid acceptor peptide (AP). We fused BirA to NRX-1/neurexin and the AP to NLG-1/ neuroligin, a post-synaptic partner of neurexin. We predicted that, as in cell culture, pre- and postsynaptic expression would result in enzymatic biotinylation of the AP on NLG-1/neuroligin across the synaptic cleft upon coming into proximity with the BirA::NRX-1/neurexin fusion. To detect the biotinylated AP in vivo we transgenically expressed a secreted monovalent streptavidin::tagRFP from coelomocytes, a set of six scavenger cells in C. elegans. My studies show that I am able to label specific synaptic connections between two neurons in vivo. The location and pattern of staining correlate with synapses detected in electron micrographic reconstructions in C. elegans of different ages or genetic backgrounds. Moreover, physical contact between neurons is not sufficient to result in staining. In conclusion, our technique will allow us (1) to study specific neuronal connections in vivo under different experimental conditions and (2) to determine the variability of the neuronal connections across a population of wild-type worms.

Session 2 13 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 14 Guanylyl Cyclase Modulation of ASH-mediated Nociceptive Behavioral Sensitivity Michelle Krzyzanowski1, Chantal Brueggemann2, Mary Bethke2, Kimberly Collins2, Noelle L’Etoile2, Denise Ferkey1 1University at Buffalo, 2University of California San Francisco

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. In the nematode C. elegans, ASH is the primary sensory neuron pair that detects the bitter tastant quinine. We previously found that the cGMP-dependent protein kinase EGL-4 dampens G protein-coupled receptor mediated quinine sensitivity by promoting RGS protein function (Krzyzanowski et al., 2013). egl-4(lof) animals respond better than wild-type animals to 10 mM quinine and avoid dilute levels of quinine (1 mM) that wild-type animals do not respond to. EGL-4 function was necessary and sufficient in ASH to regulate quinine sensitivity, and consistent with the enhanced behavioral output, egl-4(lof) animals exhibited an elevated ASH calcium flux upon exposure to quinine. Although our data suggested cGMP is necessary for EGL-4 function, there are no guanylyl cyclases known to be expressed or function in ASH. We provide evidence here that the guanylyl cyclase ODR-1 acts in a non-cell-autonomous manner to modulate ASH nociceptive sensitivity. Our data suggest that ODR-1 functions in neurons unrelated to quinine detection to provide cGMP for EGL-4 function in ASH. In addition, loss of the gap junction component INX-4 results in behavioral hypersensitivity to dilute quinine. One intriguing possibility is that cGMP could be produced in ancillary sites and delivered to ASH through gap junctions to regulate an animal’s responsiveness to aversive stimuli. If so, these studies could reveal another level of complexity in neural network regulation.

14 Session 2 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 15 SPON-1/F-spondin non-autonomously regulates Q migrations in response to MAB-5/Hox patterning Matthew Josephson1, Adam Miltner1, Erik Lundquist1 1Department of Molecular Biosciences, The University of Kansas

MAB-5/Hox patterns developmental events in the posterior of the animal. For example, it is a determinant for posterior migration of the Q neuroblast descendants. Normally, only QL descendants on the left express mab-5 due to initial posterior migration of QL and canonical EGL-20/Wnt signaling, but expression of MAB-5 in QR descendants on the right, which normally migrate anteriorly, can drive their posterior migration. Thus, MAB-5 is both necessary and sufficient to drive posterior migration of the Q descendants. MAB-5 is expressed in additional cells in the posterior, including seam cells, P cells and body wall muscles, but the effect of this expression on Q descendant migration is poorly understood. To identify targets of MAB-5/Hox in both autonomous and potential non-autonomous roles in Q migrations, we conducted whole-animal RNA-seq of mab-5 loss and gain-of-function mutants (Tamayo et al., 2013). This analysis identified spon-1, which was upregulated in the gain-of-function mab- 5(e1751) mutants. SPON-1 is similar to vertebrate F-spondin, a secreted ECM molecule involved in morphogenesis and cell and axon migrations. spon-1 RNAi suppressed posterior QR migrations in mab-5(e1751)GOF mutants, suggesting that it acts with MAB-5 in posterior migration. We found that the hypomorphic allele spon-1(e2623) caused a lowly penetrant defect in AQR and PQR migration, and suppressed posterior QR descendant migration of mab-5(e1751)GOF. Cell-specific RNAi experiments with spon-1 suggested that it acts non-autonomously in Q migrations, and the spon-1 promoter was not expressed in Q cells. Pspon-1::gfp was expressed specifically in the posterior body wall muscles in early L1 larvae at the time of early Q migrations, and its expression was increased in mab-5(e1751)GOF. However, its expression was not affected by mab-5LOF, consistent with the RNA-seq results, suggesting it might act redundantly with other factors in Pspon-1::gfp expression. In Q cell migration MAB-5 has previously been demonstrated to act cell-autonomously, but our results are consistent with MAB-5 acting non cell-autonomously to pattern the posterior region of the animal to guide Q neuroblast migrations. MAB-5-dependent SPON-1 expression in posterior body wall muscles might inhibit anterior migration or stimulate of posterior migration.

Session 2 15 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 16 Axonal fusion in regenerating axons shares molecular components with the apoptotic cell recognition pathway Brent Neumann1, Sean Coakley1, Rosina Giordano-Santini1, Casey Linton1, Yi Zhang2, Hengwen Yang3, Ding Xue2,3, Massimo A Hilliard1 1The University of Queensland, Queensland Brain Institute, 2Tsinghua University, School of Life Sciences, 3University of Colorado, Department of Molecular, Cellular, and Developmental Biology

Understanding the molecular mechanisms regulating axonal regeneration is essential for the development of effective therapies for nerve injuries. Despite a vast depth of knowledge gained into the regeneration process, we have a very poor understanding of the mechanisms needed to achieve target reconnection. Previously, we and others have demonstrated that target reconnection in C. elegans severed mechanosensory neurons can occur through a process of axonal fusion, with the proximal regrowing fragment recognizing and re-establishing membrane and cytoplasmic continuity with its own separated distal fragment, preventing it from undergoing degeneration (Ghosh-Roy et al. J Neurosci 2010, Neumann et al. Dev Dyn 2011). We have now characterized the process of axonal fusion at the molecular level, uncovering a critical role for molecules previously shown to mediate the recognition of apoptotic cells by neighbouring phagocytes. Using a candidate gene approach, we have discovered that the conserved apoptotic phosphatidylserine receptor, PSR-1, plays an important role in axonal fusion. In animals carrying mutations in the psr-1 gene, the proximal axon regenerates and contacts the distal fragment, but is unable to fuse, as a result of which the distal fragment degenerates. PSR-1 has previously been shown to bind exposed phosphatidylserine (PS) on the surface of apoptotic cells, an “eat-me” signal necessary for recognition and engulfment by phagocytic cells. We find that PS is also exposed on the severed axon where it functions as a “save-me” signal for recognition by the regrowing proximal axon. Interestingly, instead of acting in the CED-2, CED-5, CED-12 phagocytosis pathway, PSR-1 functions in a parallel phagocytic pathway that is mediated by the transthyretin protein TTR-52, and includes NRF-5, CED-7, CED-1, and CED-6. We propose that PSR-1, CED-7, CED-1 and CED-6 all function cell-autonomously, while TTR-52 and NRF-5 are expressed from different tissues, functioning as ligands and bridging molecules to mediate recognition between the regrowing axon and its distal fragment. Thus, our results reveal that recognition between two separated regenerating axonal fragments and recognition between an apoptotic cell and phagocytes share molecular components and mechanisms.

16 Session 2 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 17 A Microfluidic Platform for Automated Laser Axotomy​ Sertan Gokce1, Sam Guo2, Navid Ghorashian3, Neil Everett2, Travis Jarrell2, Aubri Kottek2, Alan Bovik1, Adela Ben-Yakar1,2,3 1University of Texas at Austin, Electrical and Computer Engineering., 2University of Texas at Austin, Mechanical Engineering, 3University of Texas at Austin, Biomedical Engineering

Neurodegenerative diseases and acute trauma to the nervous system are prevalent global health problems. Yet, molecular mechanisms involved in inhibiting or promoting nerve regeneration and degeneration remain to be understood. The nematode C. elegans is an ideal organism to understand these underlying mechanisms and create novel therapies. One successful approach for understanding the underlying mechanisms is to axotomize neurons via precise laser nanosurgery technique to create an in-vivo nerve injuries in living C. elegans. To overcome the challenges of manual worm handling techniques, we have developed a fully automated microfluidic chip platform that can perform laser axotomies on fluorescently labeled neurons in living C. elegans. The microfluidic platform autonomously selects a single animal from a pre-loaded population, immobilizes it in a T-shaped chamber, and then locates and severs the axon of interest in the immobilized animal via using custom developed automation algorithm. The T-shape architecture of the microfluidic immobilization area provides desirable immobilization of the nematodes by guiding them towards a sieve structure and straightening them for the nano-axotomy. Rapid and accurate immobilization of the nematodes was crucial for the full-automation of the axotomy process. The channel shape also allows for decoupling of the staging area from the flushing outlet. We successfully combined efficient and accurate image analysis methodologies with our new microfluidic chip to perform multiple surgeries in a serial manner, with synchronized valve and flow progression facilitating rapid transport and immobilization of individual worms. The automated platform uses image processing algorithms to locate and target axons for ablation at a record rate of less than 17 seconds per worm.

Session 2 17 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 18 The Development and Functional Regulation of the C. elegans Motor Circuit Mei Zhen1 1Lunenfeld-Tanenbaum Research Institute, Toronto, Canada

Fundamental questions remain for the neural circuit basis for motor outputs, one of the most well understood animal behaviors. We study the development and functional maturation of the C. elegans motor circuit. In collaboration with several groups, we are combining molecular, genetic, optogenetic, electrophysiology, and electron microscopy analyses of this small motor circuit, to gain a precise mechanistic understanding on how this defined neural ensemble enables an animal to generate movements.

18 Session 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 19 The Insulin/IGF Signaling Regulators Cytohesin/GRP-1 and PI5K/PPK- 1 Modulate Susceptibility to Excitotoxicity in C. elegans Nazila Tehrani1,2, John Del Rosario1, Moises Dominguez1, Robert Kalb3, Itzhak Mano1,2 1Physiol Pharm & Neurosci, Sophie Davis School of Biomedical Education, City College of New York (CCNY), The City University of New York (CUNY)., 2The Graduate Program in Neurobiology, CUNY, 3Neuroscience Graduate Group, Children’s Hospital of Philadelphia, University of Pennsylvania

Excitotoxicity is a prevalent form of neurodegeneration seen during ischemic stroke, when reduced clearance causes accumulation of Glutamate (Glu) in the synapse and excessive stimulation of postsynaptic neurons. Aging, a critical risk factor for brain damage in stroke, not only increases the chance of having a cerebrovascular event, but also sets the frailty of neurons exposed to excitotoxic insult, affecting their chances to recover or degenerate. The evolutionary conserved Insulin / IGF Signaling (IIS) cascade controls longevity, cellular aging and stress resistance in nematodes and mammals. Blocking the IIS cascade allows the transcription factor FoxO3/DAF-16 to accumulate in the nucleus and activate a transcriptional program that protects cells from a range of insults. We study the effect of IIS cascade on neurodegeneration in a C. elegans model of excitotoxicity, where a mutation in a central Glu transporter (glt-3) in a sensitizing background causes Glu-Receptor –dependent neuronal necrosis. We expand our studies on the role of the IIS cascade in determining susceptibility to excitotoxic necrosis by either blocking IIS at the level of PI3K/AGE-1 or stimulating it by removing the inhibitory effect of ZFP-1 on the transcription of PDK-1. We further show that the components of the Cytohesin/GRP-1, Arf, and PI5K/PPK-1 complex, known to regulate PIP2 production and the IIS cascade, modulate nematode excitotoxicity: mutations that are expected to reduce the complex’s ability to produce PIP2 and inhibit the IIS cascade protect from excitotoxicity, while overstimulation of PIP2 production enhances neurodegeneration. Finally we find that the broad expression of components of this complex extends alsoto postsynaptic neuronal processes that express the key Glu receptor GLR-1. Our observations therefore affirm the importance of the IIS cascade in determining the susceptibility to necrotic neurodegeneration in nematode excitotoxicity, and demonstrate the ability of Cytohesin/GRP- 1, Arf, and PI5K/PPK-1 complex to modulate neuroprotection.

Session 3 19 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 20 The JBS-Associated E3 Ubiquitin Ligase UBR-1 Regulates Nervous System Development by Maintaining Synaptic Glutamate Homeostasis Jyothsna Chitturi1, Maria Lim2, Wesley Hung2, Abdel Rahman2, Jim W Dennis2, Mei Zhen2 1University of Toronto, 2LTRI, Mount Sinai Hospital

Johanson-Blizzard Syndrome (JBS) is an autosomal, recessive congenital disorder characterized by pancreatic exocrine insufficiency and mental retardation. JBS results from mutations in the gene UBR1, an E3 ubiquitin ligase that specifically targets proteins for proteasomal degradation1,2,3. The mechanism by which UBR1 dysfunction leads to JBS is currently unknown. UBR1 substrates that are pathologically relevant to the disease remain to be identified. Here we provide evidence that the C. elegans homolog UBR-1 is present in neurons and regulates synaptic function and motor behaviours. Furthermore, we have identified a glutamate metabolic enzyme as a putative ubr-1 substrate. Using genetics, HPLC and mass spectrometry, we show that reduced UBR-1 activity causes dysfunctional glutamate homeostasis, which leads to defects in synaptic function. Therefore, C. elegans ubr-1 mutants may provide a relevant genetic model to investigate nervous system dysfunction in JBS.

Reference(s) 1. Johanson, A. & Blizzard, R.A. (1971). “Syndrome of congenital aplasia of the alae nasi, deafness,hypothyroidism, dwarfism, absent permanent teeth, and malabsorption”. J. Pediatr. 79, 982–987. 2. Zenker, M. et al (2005). “Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome).” Nature genetics 37(12): 1345-1350. 3. Sukalo, M. et al (2014). “Mutations in the Human UBR1 Gene and the Associated Phenotypic Spectrum.” Human Mutation 35: 1-11.

20 Session 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 21 Mechanisms underlying inhibition of spontaneous synaptic vesicle fusion by complexin Jeremy Dittman1, Rachel Wragg1, Daniel Radoff1, David Snead1, Yongming Dong2, Jihong Bai2, David Eliezer1 1Weill Cornell Medical College, 2Fred Hutchinson Cancer Research Center

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin (CPX) can both promote and inhibit fusion through interactions between its “central helix” and the SNARE complex. Two poorly conserved domains (the accessory helix and the C-terminal domain) on either side of the central helix (CH) are also required for inhibition of spontaneous fusion. We found that the C-terminal domain (CTD) binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. High membrane curvature enhances CPX binding and induces conformational changes in a critical amphipathic region of the CTD. The accessory helix (AH) of CPX contributes to the inhibition of exocytosis but the molecular mechanism for this function remains unknown. Several models have been proposed for the role of AH based on the concept that AH competes with VAMP for a binding site on the SNARE complex. Using a series of AH mutations and chimeras with mouse and fly AH together with NMR and CD spectroscopy, electrophysiology, and behavioral assays, we identified key features of the AH and CH required for inhibition of SV fusion byCPX. We propose that the AH stabilizes the CH through nucleation and propagation of helical secondary structure, thereby facilitating binding of the CH to the SNARE complex.

Session 3 21 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 22 A gain-of-function UNC-2/CaV2 channel induces behavioral hyperactivity and an imbalance in excitatory-inhibitory signaling Yung-Chi Huang1, Jennifer Pirri1, Diego Rayes1, Shangbang Gao2, Yasunori Saheki3, Mei Zhen2, Cornelia Bargmann3, Michael Francis1, Mark Alkema1 1Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA, 2Lunenfeld-Tannenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada, 3Howard Hughes Medical Institute and Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY

Presynaptic voltage-gated calcium channels (CaV2) are crucial regulators of synaptic transmission. C. elegans has a single predicted neuronal CaV2 α1 subunit, encoded by the unc-2 gene. While unc-2 loss-of-function (lf) mutants are sluggish, we identified an unc-2 gain-of-function (gf) mutant, which displays behavioral hyperactivity in locomotion and egg-laying behaviors. Whole cell recordings from HEK cells expressing the CaV2 gain-of- function channel showed activation at a lower membrane potential and a higher current density, indicating an elevated channel activity. unc-2(gf) mutants are hypersensitive to the acetylcholine esterase inhibitor, aldicarb, and show an increased frequency of endogenous cholinergic EPSCs. Surprisingly, unc-2(gf) mutants have a marked reduction of endogenous GABAergic IPSCs resulting in a raised excitation-inhibition ratio. Analysis of synapses showed that unc-2(gf) mutants display enlarged pre-synaptic domains in both cholinergic and GABAergic neurons. However, while we found an increase in the post-synaptic expression of the acetylcholine receptor ACR-16, we found a marked decrease in the expression of the UNC-49 GABA receptor. Imbalanced excitation and inhibition of the nervous system has been associated with several neurologic disorders, such as seizure, epilepsy and migraine. In the human CaV2.1, CACNA1A, gain-of-function mutations result in the Familial Hemiplegic Migraine (FHM). Interestingly, transgenic animals that express unc-2 carrying corresponding FHM mutations recapitulate the behavioral hyperactivity of unc-2(gf) mutants. Therefore, unc-2(gf) mutants may provide insights on circuit malfunction in CaV2 channelopathies that result in excitation- inhibition imbalance.

22 Session 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 23 Failure of NLP-40 release leads to decreased expulsions in the inx-16 intestinal gap junction mutant Sam McCright, Maureen Peters1 1Department of Biology, Oberlin College

The C. elegans defecation motor program is initiated by periodic posterior-to-anterior calcium waves in the intestine. Innexin 16 (inx-16), a pannexin gap junction subunit, is required for functional wave initiation and propagation. The inx-16 mutant’s calcium waves are slow and often initiate in aberrant locations. The motor program contractions are affected differently by inx-16 mutation. The rapidity and orientation of the first motor program contraction, posterior body contraction, is altered in accord with calcium wave dynamics. This contraction is slow and often incorrectly oriented in inx-16 mutants. The last motor program contraction, the enteric muscle contraction, is largely absent. These observations suggest that the mechanisms responsible for releasing the intestinal output signals may vary in their calcium sensitivities. The aim of this project is to investigate the mechanisms underlying the defective enteric muscle contraction in inx-16 mutants. Enteric muscle contraction depends on signaling between the intestine, two GABAergic neurons and enteric muscles. The intestine secretes a neuropeptide-like protein, NLP-40. NLP- 40 responsive neurons instruct the enteric muscles to undergo contraction. NLP-40 release is likely dependent on the calcium wave since the calcium-binding protein synaptotagmin-2 is required for its release. We hypothesized that the abnormal inx-16 calcium waves fail to elicit NLP-40 release. To test this hypothesis we examined NLP-40 release and the functionality of tissues downstream of NLP-40 release, specifically the DVB neuron and the enteric muscles. Optogenetic excitation restored enteric muscle contraction in inx-16 mutants demonstrating the functionality of GABA release and enteric muscle contraction. Calcium imaging suggests that inx-16 mutants largely fail to activate DVB. This failure is likely due to ineffective NLP- 40 release. Levels of secreted NLP-40 are significantly decreased in inx-16 mutants. We conclude that an inx-16 animal’s DVB neuron is able to signal expulsion, but is not activated due to loss of intestinal NLP-40 secretion. These finding suggests that some characteristic of inx-16’s intestinal calcium dynamics cannot stimulate NLP-40 release.

Session 3 23 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 24 Genetic analysis of ionotropic acetylcholine receptor function in GABA neurons Alison Philbrook1, Michael Francis1 1UMASS Medical School

Ionotropic acetylcholine receptors (iAChRs) play essential roles in mediating excitatory signaling in nervous systems ranging from nematodes to humans. Despite the clear importance of iAChRs for normal brain function as well as in various neurological disorders, mechanisms for their biological regulation in the nervous system are poorly defined. We are investigating fundamental iAChR biology using the C. elegans motor circuit as a model

system. Previous work in the Francis lab has identified an iAChR subtype (ACR-12GABA) that is strongly expressed in GABA motor neurons and mediates their activation by presynaptic ACh motor neurons (Petrash et al., 2013). In order to identify mechanisms responsible for the cellular regulation of ACR-12GABA receptors, we sought to establish a system in which we could examine their subcellular localization in individual neurons. We generated a transgenic strain expressing GFP-tagged ACR-12 receptors in the GABA DD motor neurons and focused our efforts on a single neuron, DD1. The DD1 cell body and processes are spatially separated

from the other DD neurons, enabling clear visualization of ACR-12GABA receptor clustering. Interestingly, punctate ACR-12-GFP fluorescence was restricted to a defined spatial domain within ventral DD1 dendrites of adult animals. ACR-12 clusters were concentrated at the tips of spine-like dendritic protrusions in this region and were apposed by presynaptic vesicle markers, consistent with a synaptic localization. In live imaging studies, we found that these postsynaptic clusters were largely immobile, while smaller ACR-12 clusters in asynaptic regions were highly mobile. These results suggest the presence of at least two iAChR populations: those incorporated into mature synapses, and those actively undergoing trafficking to postsynaptic sites. In contrast to adults, ACR-12 clusters were restricted to the dorsal side in L1 animals, consistent with previous studies suggesting that synapses onto the DD neurons undergo remodeling during development. Finally, we found that several additional iAChR subunits are required for normal ACR-12GABA clustering. We hypothesize that these

subunits coassemble with ACR-12 and are essential components of the ACR-12GABA receptor. Thus, our work to date defines fundamental properties of a neuronal cholinergic synapse in vivo. We are now working to investigate molecular requirements for the development and maintenance of these synapses and will present our findings.

24 Session 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 25 Cellular and molecular mechanisms of smn-1-mediated neuron- specific degeneration Ivan Gallotta1, Alessandra Donato2, Nadia Mazzarella1, Alessandro Esposito1, Justin Chaplin2, Ivan Cáceres3, Daniel Porto3, Paolo Bazzicalupo1, Massimo A. Hilliard2, Hang Lu4, Elia Di Schiavi1 1Institute of Biosciences and BioResources, CNR, 2The University of Queensland, QBI, 3BioEngineering Interdisciplinary Graduate Program, Georgia Institute of Technology, 4BioEngineering Interdisciplinary Graduate Program and School of Chemical & Biomolecular Engineering, Georgia Institute of Technology

Smn1 is the gene responsible for Spinal Muscular Atrophy (SMA), a devastating and fatal neurodegenerative disease characterized by progressive degeneration and death of a specific subclass of motor neurons. The lethality associated with loss of function mutations in smn-1, has made the study of its function hard to investigate in any animal model system. In C. elegans two different smn-1 mutants are available, which present some limitation for manipulation and no neurodegeneration (Briese et al., HMG, 2009; Sleigh et al., HMG, 2010). To overcome the limits of classical genetic approaches and investigate the role of smn-1 in the nervous system, we used a neuron-specific RNAi strategy (Esposito et al., Gene, 2007) and silenced smn-1 selectively in the C. elegans GABAergic motor neurons. These animals, viable and fertile, presented an age-dependent degeneration specifically in these neurons, detected as disappearance of presynaptic and cytoplasmic fluorescent markers, and as positive reactivity to genetic and chemical cell-death markers. We demonstrated that genes of the classical apoptosis pathway are involved in the smn-1-mediated GABAergic neuronal death. Remarkably, this phenotype was rescued by the expression of a wild-type copy of the human Smn1, indicating a strong functional conservation between the two genes. To determine the neuron-specific effects of smn-1 depletion, we silenced smn-1 in a different class of C. elegans neurons, the touch receptor neurons. We again observed an age- dependent degeneration, this time preceded by axon retraction with a dying-back phenotype and with a very slow progression toward death. Importantly, we found that, differently from the apoptotic death observed in GABA motor neurons, smn-1-induced degeneration of the touch receptor neurons occurred through a necrotic mechanism, revealing cell-specific molecular machinery activated by lack of smn-1. Using the GABAergic motor neuron degeneration model and a candidate gene approach, we identified three genes that interact with smn-1 to either worsen or rescue the smn-1 induced degeneration depending on whether they carry a loss of function mutation or are overexpressed. Furthermore, we also identified 24 new mutants that suppress the loss of visible GABA motor neurons in smn-1 silenced animals using chemical mutagenesis in a microfluidics based screen. In conclusion, we have developed a new experimental model to investigate SMA in living animals and for the first time in C. elegans we observed neurodegeneration when smn-1 is depleted. We identified the cell death pathways activated by smn-1 silencing and isolated new mutants that suppress the degenerative phenotype in motor neurons. Our results provide a unique new framework to elucidate the molecular mechanisms that underlie SMA.

Session 3 25 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 26 Dissecting the mechanisms underlying motorneuron disease in C. elegans Maria Dimitriadi1, Melissa Walsh2, Jill Yersak1, Anne Hart1 1Dept. of Neuroscience, Brown University, 2Molecular Biology, Cell Biology and Biochemistry Program, Brown University

Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are devastating, progressive neurodegenerative diseases that cause spinal motorneuron dysfunction and death with consequent muscle atrophy. ALS also affects cortical neurons that synapse onto spinal motorneurons and several ALS genes have been implicated in Frontal Temporal Lobe Dementia (FTD) and other neurodegenerative diseases The connections between these seemingly unrelated neurodegenerative diseases suggest that related mechanisms underlie neuronal pathology in these disorders. We are using C. elegans to delineate the proteins and pathways pertinent to motorneuron disease. SMA is usually caused by loss of Survival Motor Neuron (SMN) protein function. Using a previously defined C. elegans model for SMA, we and others have established that loss of the C. elegans SMN ortholog results in progressive neuromuscular defects. Here, using a broad range of approaches, we find that SMN loss in C. elegans causes endocytic defects. To understand these defects and the molecular mechanisms underlying SMA, we have also taken a functional/genetic approach and examined SMA genetic modifiers. Some of these modifier genes were originally defined in human genetic studies; some have been identified and characterized in our work using bioinformatic approaches and functional approaches. Our results suggest that the corresponding proteins may act in an RNP complex with SMN that is critical for motorneuron function and survival. Familial ALS is caused by mutations in numerous genes, including SOD1, TDP-43 and FUS. These proteins are well-conserved across animal species, although there is little clarity as to why they cause late-onset neurological disease. As a complement to the extant ALS overexpression models, we are creating precise models of familial ALS in C. elegans by introducing patient alleles into orthologous C. elegans genes. Using this approach, we aim to define the cellular and genetic mechanisms underlying the earliest perturbations in ALS. We find that patient amino acid changes cause defects in survival and in well-described C. elegans assays of neuromuscular function, albeit less severe defects than those observed in overexpression models. Genetic studies using these precise models should facilitate the identification of the mechanisms underlying the first steps in neuronal dysfunction and death in ALS.

26 Session 3 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 27 Prospects for the neuronal and genetic analysis of economic decisions in the nematode Caenorhabditis elegans Shawn Lockery1, Abe Katzen1 1Institute of Neuroscience, University of Oregon

Economic decisions can be classified as rational or irrational according to the degree to which they maximize the welfare of the decision maker. It is well known that human economic behavior can be rational in some contexts and irrational in others, and that individuals show unique patterns of rational versus irrational decisions. It is perhaps less well known that these patterns exhibit heritability values of up to 40%. This degree of heritability has led to the enthusiastic pursuit of its biological substrate. Unfortunately, however, whole genome association studies chart a vast landscape of genes-of-small-effect. Consequently, there is now considerable pessimism as to the near-term prospects of finding the genes for economic rationality with effects large enough to investigate in practical terms. An alternative approach would be to investigate economic decision making by identifying candidate genes in genetically tractable model organisms, which can accommodate mutations in genes of large effect. This presentation will discuss preliminary experimental results suggesting that it may be possible to investigate the neuronal and genetic basis of economic decisions in C. elegans. Previous work has shown that C. elegans is born without strong food preferences but quickly learns to prefer those foods (species of bacteria) that promote higher rates of growth. Preference is manifested by the amount of time the worm spends foraging in patches of good bacteria (affording high worm growth rate) versus mediocre bacteria (affording moderate growth rate) when equally abundant. My laboratory has recently developed an electro-microfluidic device in which a semi-restrained worm forages between contiguous yet discrete fluid streams containing good and mediocre food. Importantly, we can alter the prices of the two foods by adjusting the concentration of bacteria and we can assess the resulting changes in consumption directly by means of electrodes that monitor muscular impulses associated with swallowing events. Initial results indicate that worms are rational consumers in that they respond as expected to increases and decreases in the relative costs of good and mediocre bacteria. These findings set the stage for analyzing the mechanisms by which value and price are integrated in the nervous system. They also may provide the basis for genetic screens to identify candidate genes required for economic rationality.

Session 3 27 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 28 Glia shape URX and BAG sensory dendrites through GRDN-1/Girdin and SAX-7/L1CAM Ian G. McLachlan1,2, Eizabeth R. Lamkin1,2, Maxwell G. Heiman2 1Program in Neuroscience, Harvard University, 2Department of Genetics, Harvard Medical School; Boston Children’s Hospital

Glial cells closely associate with neurons and guide neuronal migration, morphogenesis, and synapse formation. In C. elegans, all glia ensheathe the dendrite endings of sensory neurons and are required for normal dendrite extension during development as well as normal sensory function in mature animals. We previously identified factors necessary for the extension of glial-ensheathed dendrites. We hypothesized that other, non-ensheathed neurons might also require glia for dendrite extension; we therefore focused on the gas-sensing neurons URX and BAG because they associate closely with glia but are not ensheathed by them. We performed a visual forward genetic screen and isolated ten alleles of two genes that affect URX and BAG dendrite extension: grdn-1, encoding a homolog of Girdin, a cytoskeletal adaptor that localizes to cell-cell contacts in the nervous system; and sax-7, encoding a homolog of the neuron-glia adhesion molecule L1CAM. We defined a 450 bpgrdn-1 promoter that is sufficient for rescue when driving agrdn-1 cDNA, and found that its expression includes the inner labial socket (ILso) glial cells that contact URX and BAG dendrites. Expression of grdn-1 cDNA under the control of other glial, but not neuronal, promoters is sufficient for rescue, indicating it can act solely in glia. By comparison, sax-7 can rescue dendrites when expressed either in glia or in neurons, suggesting a more complex role. Finally, fluorescently tagged GRDN-1 localizes to ILso glial endings, precisely at the point of contact between glia and URX or BAG dendrites. Our working model is that GRDN-1 and SAX-7 are required for formation of specialized neuron-glia contacts between the URX and BAG dendrites and ILso glia, and that the neurons require these glial contacts for dendrite extension.

28 Session 4 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 29 CaMKI-dependent regulation of gene expression mediates long-term temperature adaptation in AFD Yanxun Yu1, Harold Bell1, Dominique Glauser2, Miriam Goodman3, Stephen Van Hooser1, Piali Sengupta1 1Department of Biology and National Center for Behavioral Genomics, Brandeis University, 2Department of Biology, University of Fribourg, 3Department of Molecular and Cellular Physiology, Stanford University

Adaptation in sensory neurons represents a form of experience-dependent plasticity that allows them to retain responsiveness to stimuli over a broad dynamic range. However, whether the response thresholds of sensory neuron are reset on different timescales upon prolonged stimulation, and the underlying mechanisms that mediate this resetting are unclear. Here we investigate the mechanisms by which the AFD thermosensory neurons in C. elegans reset their operating range upon growth of animals at a new temperature (T). The threshold

of temperature-induced neuronal activity in AFD (T*AFD) is set by the cultivation temperature

(Tc). We find that T*AFD is reset on both short and long timescales upon exposure to T>Tc. Prolonged exposure to a new temperature alters expression of AFD-specific guanylyl cyclase genes, which have been shown to regulate T*AFD. We demonstrate that these temperature- dependent gene expression changes require the CMK-1 CaMKI enzyme; CMK-1 translocates to the nucleus upon shift to a higher temperature and phosphorylation by the CKK-1 CaMK kinase. Consequently, cmk-1 mutants exhibit defects in T*AFD adaptation and thermotaxis behaviors. Our results indicate that CaMK1-mediated changes in sensory gene expression contribute to long-term thermal adaptation in AFD. Together, these observations characterize a novel signaling pathway that may operate more broadly to mediate experience-dependent plasticity in the operating ranges of sensory neurons.

Session 4 29 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 30 Bacterial Secondary Metabolites Alter C. elegans Behavior by Modifying Sites of Neuronal DAF-7 Expression Joshua Meisel1, Dennis Kim2 1Biology Department, Massachusetts Institute of Technology, 2Department of Biology, Massachusetts Institute of Technology

Microbes have been increasingly appreciated to exert diverse effects on the physiology and behavior of host organisms, but the molecular mechanisms underlying these interactions remain unclear. Here, we show that Caenorhabditis elegans detects two secondary metabolites produced by Pseudomonas aeruginosa, phenazine-1-carboxamide and the siderophore pyochelin, which alter the neuronal sites of DAF-7/TGF-β expression and modulate host behavior. Upon exposure to P. aeruginosa, a G protein signaling pathway in the ASJ neuron pair activates expression of the neuromodulator DAF-7, which in turn functions through a canonical TGF-β signaling pathway in adjacent interneurons to modulate aerotaxis behavior and promote avoidance of P. aeruginosa. Our data provide a chemical, genetic, and neuronal basis for how the physiology and behavior of a simple animal host can be modified by bacteria, and suggest that secondary metabolites produced by microbes may provide environmental cues that contribute to pathogen recognition and host survival.

30 Session 4 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 31 Sex, age and hunger regulate behavioral prioritization through dynamic modulation ofchemoreceptor expression Renee Miller1, Deborah Ryan2, KyungHwa Lee3, Scott Neal4, Piali Sengupta4, Doug Portman2 1Dept. of Brain and Cognitive Sciences University of Rochester, 2Dept. of Biomedical Genetics Center for Neural Development and Disease University of Rochester, 3Center for Neural Development and Disease University of Rochester, 4Department of Biology National Center for Behavioral Genomics Brandeis University

The adaptive prioritization of behavior requires flexible outputs from fixed neural circuits. In C. elegans, hermaphrodites prioritize feeding, while males will abandon food to search for mates. Consistent with this, males are less attracted than hermaphrodites to the food- related odorant diacetyl. We find that this difference stems from regulation of the diacetyl chemoreceptor odr-10 by the sexual state of a single sensory neuron pair. Moreover, odr-10 is required for efficient food detection, and regulation of this receptor by sex and developmental stage plays a central role in balancing feeding and exploration. Furthermore, starvation transiently reprioritizes feeding over exploration in males by activating odr-10 expression. Well-fed adult males that cannot downregulate odr-10 maladaptively prioritize feeding, impairing their reproductive fitness. Thus, genetic sex, developmental stage and feeding status converge on the expression of a key chemoreceptor to modulate sensory function and dynamically prioritize the feeding and mating drives.

Session 4 31 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 32 Multidimensional phenotypic profiling identifies subtle synaptic pattern mutants, and their morphological defects Adriana San-Miguel1, Peri Kurshan2, Kang Shen2, Hang Lu1 1School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2Howard Hughes Medical Institute, Department of Biology, Stanford University

Genetic screens have largely focused on identifying mutations that cause easily distinguishable, dramatic changes. This has been especially the case when dealing with phenotypes reported by subcellular fluorescent markers, such as synaptic puncta. Multiple genes important for the establishment and maintenance of synaptic sites at stereotypical synaptic domains of axons have been uncovered by isolating mutants with grossly mislocalized synaptic material. Many other genes are believed to be important for efficient neurotransmission, with functions such as regulating the precise number and strength of synaptic connections. Upon perturbation, these are expected to cause subtle rather than severe defects. Subtle changes in micron-sized synaptic puncta are, however, extremely difficult to identify by qualitative inspection. In this work, we perform genetic screens on C. elegans aimed at identifying genes that can cause phenotypes, hidden to the naked eye, in synaptic patterns of the DA9 motor neuron. This neuron, which drives backward locomotion, forms ~ 25 en-passant synapses onto body wall muscles. Such synaptic sites are formed at a precise location along the axon that extends within the dorsal cord. Phenotypic profiling of fluorescently labeled synaptic sites is subject to bias towards morphologies detectable by human observers, such as localization patterns. Without quantitative unbiased analysis, phenotypes undetectable qualitatively are generally overlooked in genetic screens. Phenotypic profiling of synaptic patterns is particularly challenging due to the small size and location of synaptic puncta. Identifying alleles that cause subtle changes in synaptic patterns is thus extremely difficult. Using microfluidics and unsupervised image and data analysis we overcome the main challenges in the search for weak synaptic patterning screens: 1) worm handling and 2) phenotype characterization and classification. We extract phenotypic profiles of fluorescent synaptic patterns, and quantify complex features inaccessible by qualitative observation. We perform fully automated forward genetic screens and isolate mutants with very subtle phenotypes. To account for the heterogeneity present in isogenic populations, statistical analyses based on phenotypic profiling of whole animal populations are performed. Logistic regression models allow populations of true mutants and wild type animals to be discerned, while revealing their most relevant differentiating features. The phenotypes of the mutants found in this work are all extremely difficult to discern by eye, such as smaller or dimmer synaptic puncta. Upon performing a full profile analysis on the mutants identified, weare able to suggest putative altered genetic pathways by analyzing their relationships with a representative collection of mutants of known genetic pathways by hierarchical clustering.

32 Session 4 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 33 Synaptic position maintenance involved in hypodermal and glial cells Zhiyong Shao1, Shigeki Watanabe2, Ryan Christensen1, Erik Jorgensen2, Daniel Colón- Ramos1 1Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine, 2Howard Hughes Medical Institute, Department of Biology, University of Utah

Synaptic contacts are largely established during embryogenesis and are then spatially maintained during growth. To identify molecules involved in synaptic position maintenance, we conducted a forward EMS screen in C. elegans using interneuron AIY as a model. We identified cima-1 (circuit maintenance-1) that is required to maintain synaptic position. In cima-1 mutants, AIY presynaptic contacts are correctly established during embryogenesis, but synaptic position fails to maintain during postdevelopmental growth. cima-1 encodes a solute carrier in the SLC17 family of transporters that includes sialin, a protein that when mutated in humans results in neurological disorders such as salla disease and infant sialic acid storage disease. We found that cima-1 does not function in neurons but rather functions in the nearby hypodermal cells to correctly position glia during postlarval growth. Additionally, our data indicate that CIMA-1 antagonizes the FGF receptor (FGFR), and does so most likely by inhibiting FGFR’s role in epidermal-glia adhesion rather than canonical FGF signaling. Our data suggest that epidermal-glia crosstalk, in this case mediated by a transporter and the FGF receptor, is vital to preserve embryonically derived circuit architecture during postdevelopmental growth.

Session 4 33 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 34 Perlecan antagonizes collagen IV and ADAMTS9/GON-1 in restricting en passant synapse growth Jianzhen Qin1, Jingjing Liang1, Mei Ding1 1Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

In an adult brain, most chemical synapses tend to be retained locally within a rather confined position. Injury, behavioral enrichment, or prolonged sensory stimulation can somehow lift the growth inhibition of adult synaptogenesis. Currently, detailed knowledge of the molecular program that restricts profuse synapse growth remains elusive due to the complexity of the mammalian CNS.

In C. elegans, muscle cells have long neuron-like processes (muscle arms) that run to the nerve bundles in which motor axons reside, and synapses occur en passant between nerve processes and muscle arms. Through genetic screen, we identified that collagens type IV and XVIII and the secreted metalloprotease ADAMTS/GON-1 are critical for growth restriction of en passant synapses in C. elegans. Without these components, ectopic synaptic boutons originated from existing synapses within nerve cord progressively invade into non- synaptic region. We further analyzed the ectopic synapses in more detail by EM. Through reconstruction, we found that the ectopic synapses possess the characteristic features of chemical synapses, including accumulation of synaptic vesicles and darkly-stained active zones, suggesting that the ectopic synapses may contain properly assembled pre-synaptic structures. In addition, the ectopic synapses are surrounded by muscle arms and adjacent to post-synaptic receptor UNC-29. Perlecan/UNC-52 promotes synapse growth and functions antagonistically to collagen type IV and GON-1 but not to collagen XVIII. The growth constraint of synapses correlates with the integrity of the extracellular matrix basal lamina or basement membrane (BM), which surrounds chemical synapses. Fragmented BM appears in the region where ectopic synapses emerge. Further removal of UNC-52 improves the BM integrity and the tight association between BM and synapses. Together, our results unravel the complex role of the BM in restricting en passant synapse growth and reveal the antagonistic function of perlecan on type IV collagen and ADAMTS protein.

34 Session 4 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 35 Regulation of microtubule dynamics in synaptogenesis Naina Kurup1, Dong Yan2, Alexandr Goncharov3, Yishi Jin4 1Division of Biological Sciences, University of California, San Diego, 2Division of Biological Sciences, UCSD; Present address: Duke University School of Medicine, 3Howard Hughes Medical Institute, University of California, San Diego, 4Division of Biological Sciences, Howard Hughes Medical Institute, University of California, San Diego

The presynaptic terminal of a chemical synapse contains a complex network of cytoskeletal elements that are essential for its formation and maintenance. Microtubules (MTs), which are polymers of α- and β-tubulin heterodimers, are an important component of this framework. While the regulation of MT organization and dynamics during axon and dendrite formation has been extensively studied, much less is known about the regulation of MT dynamics at the synapse. Here, we have used the GABAergic D neurons as a model system to understand the role of microtubules in the establishment and maintenance of a pre-synaptic terminal. In particular, this study focuses on the elimination of ventral synapses and subsequent formation of dorsal synapses during the process of DD neuron remodeling. We find that synergistic interactions between a missense mutation in α-tubulin, TBA-1 (tba-1(gf)) (Baran et al., 2010) and loss of the MAPKKK DLK-1 function (dlk-1(lf)) blocks DD remodeling. We show that dlk-1 is specifically required during DD remodeling and the role of the DLK-1 pathway in this process is distinct from its effect on synapse assembly (Nakata et al., 2005, Yan et al., 2009). Using a combination of ultra-structural analysis and live imaging of MT dynamics, we show that an increase in stable MTs is responsible for the defective synaptic remodeling in tba-1(gf) dlk-1(lf). We hypothesize that this increased MT stability affects synaptic vesicle precursor transport. Consistent with this idea, we have identified two novel mutations in the motor protein,unc-116/ kinesin-1, in a genetic suppressor screen of tba-1(gf)dlk-1(lf). We show that these mutations result in a gain of function of the motor, which appear to compensate for the MT defects of tba-1(gf)dlk-1(lf) worms by increasing MT-motor binding affinity. Together, our data uncovers a new role of DLK-1, and provides insights into how the temporal regulation of MT growth underlies DD synapse remodeling.

Session 4 35 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 36 BK Channel Modulation of Withdrawal from Chronic Ethanol Exposure L.L. Scott1, S. J. Davis1, S. Iyer1, R.W. Aldrich1, S.J. Mihic1, J.T. Pierce-Shimomura1 1The University of Texas at Austin, Austin, TX 78712 USA

Neural adaptation to chronic ethanol exposure underlies many of the symptoms of withdrawal from chronic ethanol exposure. The severity of these symptoms in turn is a driving force for relapse. A major goal than is to alter the neural state in withdrawal to more closely match the ethanol naïve state. Both the activity and expression of large-conductance calcium- activated potassium channels change in response to ethanol exposure. Moreover, the activity of these channels influences ethanol behaviors including acute intoxication and tolerance. However, whether the BK channel is a viable target for modulating the extent of withdrawal after chronic ethanol exposure is not yet fully understood. Using the model nematode, Caenorhabitis elegans, we show that BK channel expression is both altered by chronic ethanol and in turn modulates behavioral impairments following withdrawal. Consistent with previous findings in mammalian tissue, we find that in C. elegans neuronal BK channel expression is lower after chronic ethanol treatment. The extent of withdrawal-induced impairments is worse in the absence of functional BK channels, and is rescued by BK channel expression under the endogenous or a pan-neuronal promoter. Conversely, BK channel overexpression improves withdrawal behaviors. These data are consistent with the idea that a reduction in BK channel expression during chronic ethanol exposure leads to an imbalance in the nervous system that contributes to withdrawal symptoms. To probe whether we can improve this imbalance by the application of a BK channel opener during withdrawal, we first found novel peptide BK channel openers. We useda monovalent phagemid display technique combined with functional screening in C. elegans to identify novel BK channel modulators. Further electrophysiological screening verified the ability of several peptides to increase the open probability of mammalian BK channels expressed heterologously. When applied to C. elegans just during withdrawal, these novel BK channel openers improved behavioral deficits in ethanol-withdrawn worms, although the same treatment causes impairments in naïve animals. Together these data indicate that BK channel modulation is part of the neural adaptation to chronic ethanol exposure, and that increasing BK channel activity with genetic or pharmacological manipulations returns neural circuits in ethanol treated animals to a more naïve-like state. (Supported by R01AA020992)

36 Session 4 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 37 Sensory molecules and mechanisms in C. elegans Bill Schafer1 1MRC Laboratory of Molecular Biology, Cambridge, UK

C. elegans has an anatomically simple nervous system, but its sensory modalities parallel those in more complicated animals. Many known sensory transduction molecules are functionally conserved between worms and humans; we have therefore used worm genetics to identify novel receptors and channels involved in senses such as touch and taste and to study their function in vivo. We are also exploring how the neural circuitry integrates information from sensory pathways to control behaviour, with particular interest in the roles of microcircuits involving electrical synapses and extrasynaptic modulation. Recent work has focused on the TMC genes, which encode broadly-conserved multipass integral membrane proteins in animals. The human Tmc1 and Tmc2 are deafness genes required for hair cell mechantransduction; however, channel activity for these proteins has not been demonstrated. C. elegans contains two members of the TMC family. One of these, TMC-1 forms a sodium-sensitive ion channel that functions in polymodal neurons as a sensor for salt chemosensation. The other, TMC-2 functions in mechanosensation. Further characterization of these molecules in vitro and in vivo, as well as analysis of mammalian TMC superfamily members, will be presented.

Session 5 37 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 38 Sniffing Out Development of Chemotaxis Behavior Laura Hale1, Eudoria Lee1, Sreekanth Chalasani1 1Salk Institute for Biological Studies

How does a neural circuit change during development to enable mature behavior? We study the development of chemotaxis behavior to uncover cellular mechanisms of behavioral maturation. We have identified a robust difference in attractive chemotaxis behavior between larval and adult worms. Specifically we observe that animals in larval stages L1, L2 and L3 have reduced attraction to the odors diacetyl and benzaldehyde compared to L4 and adult animals. Interestingly, repulsive chemotaxis responses to nonanone were similar for L3, L4 and adult animals, suggesting circuit-specific mechanisms for maturation. Additional results from analysis of chemotaxis responses of sensory neuron mutants to diacetyl show that L3 and adult worms have different cellular circuits for diacetyl chemotaxis behavior. Collectively these results suggest that changes in the cellular composition of neural circuits between L3 and adult worms may underlie the maturation of chemotaxis behavior. To further characterize differences between L3 and adult cellular circuits we have begun to examine the activity of individual sensory neurons from each circuit. In collaboration with N. Chronis and D. Bazopoulou we developed a novel microfluidic device that traps L3 worms and enables ready imaging of neurons. We used a published microfluidic device to image neurons in adults. Since AWA sensory neurons have been shown to be required for diacetyl chemotaxis behavior in adults we tested whether AWA activity changes in response to presentation of diacetyl. Our initial imaging results show that both L3 and adult AWA cells respond to the addition of diacetyl. Based on our results from sensory neuron mutants we examined the activity of additional neurons in a functional adult circuit. Similar to our behavioral results we found that other sensory neurons responded to diacetyl, suggesting a functional adult circuit that includes multiple sensory neurons. Future imaging work will address whether the functional L3 circuit for diacetyl differs from the functional adult circuit. We will further test the hypothesis that changes in the pattern of activity of neurons within L3 and adult circuits may also underlie behavioral maturation.

38 Session 5 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 39 Neuropeptides and dopamine regulate different behavioral components of non-associative odor learning Akiko Yamazoe1, Kosuke Fujita1, Yuichi Iino2, Yuishi Iwasaki3, Kotaro Kimura1 1Department of Biological Science, Osaka University, 2Department of Biophysics and Biochemistry, University of Tokyo, 3Department of Intelligent System Engineering, Ibaraki University

Multiple neuromodulators are involved in learning. However, the nature of interaction among these neuromodulators—whether they facilitate learning by modulating neural activity independently or cooperatively, for example—is poorly understood. Here, based on quantitative behavioral analysis and genetic studies, we report that non-associative odor learning in worms is regulated by neuropeptide and monoamine signalings, which work cooperatively to affect different behavioral components. We previously reported that avoidance behavior to the repulsive odor 2-nonanone in worms is enhanced after preexposure to the odor as dopamine-dependent non-associative learning: the preexposed worms move away from the odor source for a longer distance than control animals do during the 12-min assay (Kimura et al., J. Neurosci., 2010). From quantitative analysis of 2-nonanone avoidance behavior, we found that, for the enhanced avoidance, the duration of a straight migration (“run”) was increased in the preexposed animals only when they ran away from the odor source in the proper direction (i.e., within ±~40° opposite to the odor source). These direction-specific changes in the run duration might be regulated by changes in worms’ responsiveness to the dC/dt of the odor. Based on genetic analysis, we further found that neuropeptides are required for this preexposure-dependent increases in run duration. Mutations in egl-3 or egl- 21, the genes required for neuropeptide processing, abolished the increases in run duration and in total avoidance distance, suggesting that neuropeptides are required for the formation of preexposure memory in the odor learning. Mutations in cat-2 or dop-3, genes required for dopamine signaling, also abolished the increases in total avoidance distance after preexposure; however, unlike the neuropeptide mutants, the dopamine mutants showed increased run duration as wild-type worms did. Instead, these mutants migrated toward incorrect directions after preexposure, occasionally toward the repulsive odor source, indicating that dopamine signaling is required for regulating the locomotion properly to express the preexposure memory. Thus, neuropeptide-dependent memory formation and dopamine-dependent locomotory regulation are cooperatively required even for the simple non-associative odor learning. Currently, we are trying to identify the sites at which neuropeptide- and/or dopamine- mediated signalings modulate the odor learning.

Session 5 39 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 40 Single Cell Mass Spectrometry of GABAergic Motor Neurons in the Nematode Ascaris Christopher Konop1, Jennifer Knickelbine1, India Viola1, Colin Wruck2, Martha Vestling3, Antony Stretton4 1Department of Zoology, University of Wisconsin-Madison, 2School of Education, University of Wisconsin- Madison, 3Department of Chemistry, University of Wisconsin- Madison, 4Neuroscience Training Program, University of Wisconsin- Madison

The identification of neuronal transmitter phenotypes is an important part of the structural- functional description of the nervous system. Nematodes are well suited for studying transmitter localization since their nervous system only contains about 300 neurons. The morphology of the neurons of A. suum and C. elegans is remarkably similar: neurons in A. suum can be named for their homologs in C. elegans. This similarity extends to the cellular expression pattern of the classical transmitters acetylcholine and GABA. There are 19 ventral cord motor neurons that display GABA-like immunoreactivity in both A. suum and C. elegans as well as 4 ring motor neurons (RMEV,-D, -R, -L). The breakdown in overlapping expression of GABA occurs in the ventral ganglion, where GABA-like immunoreactivity is observed in a bilateral pair of cells in A. suum (AIM or AIY) and RIS and AVL in C. elegans. To date, little is known about the specific cellular expression patterns of neuropeptides in these neurons. Recent improvements in our single-cell dissection protocol along with MALDI-TOF/ TOF mass spectrometry (MS) were used to localize and sequence peptides in the ventral cord inhibitory motor neurons, 4 RME ring neurons, and the AIY/AIM pair. We find three different expression profiles. First, the ventral cord inhibitory MNs and AIY/AIM all express the bioactive peptide As-NLP-22. We have shown that As-NLP-22 has strong and prolonged inhibitory effects on ACh-induced muscle contraction and that it abolishing all spontaneous activity in the muscle strip assay as well as abolishes all locomotory behavior upon injection. Second, the RMEV and RMED express AF7, AF39, AF40 (ortholog of Ce-flp-5) and AF2 (ortholog of Ce-flp-14). Third, RMER and RMEL contain AF5 (Ce-flp-4 ortholog), and peptides from 2 previously unidentifiedAscaris transcripts afp-16 (Ce-flp-26ortholog) and As-nlp-48, a peptide-encoding transcript that had not previously been described in the nematode phylum, yet database searches revealed its existence in EST libraries of several other nematodes. Localization of peptide encoding transcripts was confirmed by in situ hybridization. In contrast to the expression patterns in A. suum, expression of orthologous C. elegans transcripts (determined by GFP reporter constructs; Kim and Lee, 2004; Nathoo et al. 2001) was completely different. Direct tissue analysis of single neurons by MS and MS2 gives specific cellular expression patterns of neuropeptides that are an important in the description of how the nervous system controls behavior.

40 Session 5 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 41 Transcriptional profiling of dissociated adult C. elegans neurons reveals specialized functions and memory components of Insulin/ FOXO signaling Rachel Kaletsky1, April Williams2, Rachel Arey2, Vanisha Lakhina2, Jessica Landis2, Coleen Murphy2 1Princeton University, 2Department of Molecular Biology & LSI Genomics, Princeton University

Characterization of tissue-specific transcriptomes, particularly small tissues that are not well-represented in whole-animal analyses, is critical to understand their functional contributions to organismal phenotypes. While C. elegans is a powerful system to study the coordination of signaling pathways, its outer cuticle prevents the isolation of adult tissues, and therefore its adult tissue-specific transcriptomes. We developed a method to isolate adult C. elegans cells that recapitulates the in vivo state, and have transcriptionally profiled adult neuronal tissue using RNA-seq. Adult neuron gene expression is distinct from embryonic and larval profiles, shifting from developmental processes to adult behavior-related functions, revealing new neuronal genes. Because tissue-specific, Insulin/IGF-1-like signaling (IIS) targets in adults are unknown, we used this method to identify neuronal IIS targets, uncovering several genes required for the extended short-term memory of daf-2 mutants. Adult cell isolation and transcriptional profiling will provide insight into tissue-specific coordination of gene networks in whole animals.

Session 5 41 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 42 Developmental history regulates olfactory behavior via RNAi pathways Jennie Sims1, Maria Ow1, Kyhyung Kim2, Piali Sengupta3, Sarah Hall1 1Department of Biology, Syracuse University, 2Department of Brain Science, DGIST, 3National Center for Behavioral Genomics, Department of Biology, Brandeis University

Exposure to environmental stressors during early development can result in altered adult phenotypes. Larval C. elegans exposed to stressors such as high temperature, limiting food, or high concentrations of dauer pheromone enter the stress-resistant dauer developmental stage; dauer larvae resume development to become reproductive adults (postdauers) when conditions improve. Our previous work showed that postdauer animals exhibit distinct life history traits, global chromatin modifications, small RNA populations, and gene expression profiles when compared to adult animals that bypassed the dauer stage (controls). However, little is known about the epigenetic mechanisms responsible for establishing and maintaining these changes due to developmental history, and how these changes impact adult behavioral phenotypes. We have identified the TRPV channel gene,osm-9 , as a target of environmental programming. OSM-9 is expressed in several sensory neurons and is essential for mechanosensory, osmosensory, and chemosensory behaviors, including the avoidance of high concentrations of ascr#3 mediated by the ADL sensory neurons. We found that in control adults, gfp expression driven under ~350 bp of osm-9 upstream regulatory sequences is observed in the AWA, OLQ, and ADL sensory neurons. However, in postdauer adults, gfp expression is downregulated specifically in the ADL neurons while expression in AWA and OLQ neurons is unaffected. Consistent with this change in expression, we find that the ADL- mediated and osm-9 dependent avoidance of high concentrations of ascr#3 is significantly reduced in wildtype postdauer adults compared to controls, indicating that the differential regulation of osm-9p::gfp is reflective of the expression levels of the endogenous osm-9 gene. Using mutational analyses of promoter sequences, we have identified a ~35 bp cis- acting sequence motif that is required for the differential expression of osm-9p::gfp based on developmental history. To further characterize the underlying mechanisms, we examined the ascr#3 avoidance behavior and osm-9p::gfp expression phenotypes of control and postdauer adults carrying mutations in genes with established functions in dauer formation, RNAi, and chromatin remodeling pathways. Our results suggest that endogenous RNAi pathways regulate osm-9 expression levels via modulation of TGFβ signaling and DAF-3 SMAD, and that the altered expression pattern in turn is maintained through adulthood by alteration of the chromatin state at the osm-9 locus. Our results describe an elegant mechanism by which developmental experience influences adult phenotypes by establishing and maintaining transcriptional changes via RNAi and chromatin remodeling pathways.

42 Session 6 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 43 Variability in behavioral and neural responses to consistent stimulation Dirk Albrecht1 1Worcester Polytechnic Institute

Behavioral responses to a particular stimulus are often highly variable, even upon repeated stimulation of the same animal. To explore the neuronal basis of probabilistic behaviors, we developed a high-throughput microfluidic system for optical recording of neural activity in freely-moving C. elegans as they respond to precise spatiotemporal chemical patterns. The fully-automated widefield imaging system stimulates and records from over 20 animals at once for hours. Experiments rapidly revealed modulation of neuronal calcium dynamics by odor type, concentration, gradient, and duration. We observed consistent neural activity in three different sensory neurons (AWA, AWC, ASH) during probabilistic behavioral responses, suggesting reliable transduction of stimulus information. In contrast, responses in several interneurons (AIY, AVA) correlated with behavior but not with stimulation. A different group of odor stimuli elicited sensory neural activity that varied substantially across isogenic animals but remained consistent across repeated stimulation of an individual, suggesting developmental or epigenetic variations between animals, effects of recent experience or modulatory state, or stochastic effects including gene expression. These observations emphasize the need to examine neural and behavioral responses in many individual animals under repeatable stimulation conditions, and our data suggest optimal experimental conditions to best detect subtle effects of genetic or pharmacologic perturbation of neuronal dynamics. Further studies with this system for high-throughput neuronal imaging in freely-behaving nematodes aim to observe neural signals during dynamic processes such as sensory integration and learning, and to screen for therapeutic compounds that reverse altered neuroexcitability underlying several neurological disorders.

Session 6 43 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 44 A neural circuit for head-body coordination mediates decision making in C. elegans Ingrid Hums1, Harris Kaplan1, Fanny Mende1, Julia Riedl1, Ev Yemini2, Michael Sonntag1, Richard Latham1, Lisa Traunmueller3, Saul Kato1, Manuel Zimmer1 1Research Institute of Molecular Pathology, 2Department of Biochemistry and Molecular Biophysics, HHMI, 3Department of Cell Biology, Biozentrum, University of Basel

Animals navigate through the environment via long-distance travels, which are interspersed with exploratory local search phases. This represents a tradeoff between stereotypy and flexibility: efficient locomotion during travel is ideally achieved through coherent coordination of all body parts, while explorative search relies on deviations in gait and posture. We report on a head-body control system in C. elegans that counterbalances both needs. For traveling distances, worms generate regular body waves, which are coherent undulatory movements across all body parts including the head. However, when we induce local exploratory search phases through stimulation of oxygen sensory circuits, animals down-regulate their regular undulatory crawling in order to slow and to adopt more complex postures. Using eigenworm decomposition, we split worm locomotion into a regular crawling component and a so-called residual motion component. The latter captures all movements deviating from regular undulatory locomotion and can be described as residual head sweeps and body bends added on top of the regular wave. A fraction of head sweeps is coupled to residual body bends, eventually generating head-directed whole-body steering and turning maneuvers. Using genetics and functional imaging of neural activity we characterize a pre-motor circuit including AVK and DVA neurons that utilize FLP-1 and NLP-12 neuropeptides to mediate head-body coordination. This proves to be important for the control of locomotion speed and steering-turning maneuvers. While interfering with flp-1/ AVK results in increased steering-turning due to inappropriately enhanced head-body coupling during fast movement, disturbance of nlp-12 / DVA produces the opposite effects. Neural activity patterns of AVK and DVA in freely moving animals reflect locomotion speed as well as residual head sweeps or body bending events, respectively. Genetic epistasis experiments lead us to a model of mutual interaction of AVK and DVA to fine-tune the degree of head-body coupling. We show that this circuit including AVK and DVA is required for the gradual regulation of head-directed steering and turning upon oxygen concentration changes and therefore is implicated in weathervaning oxygen chemotaxis . This work illustrates how the nervous system combines elementary motion patterns to generate controlled complexity, which enables animals to execute appropriate decisions during navigation.

44 Session 6 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 45 Optogenetic cAMP increase enhances transmitter release: depletion of docked and reserve synaptic vesicles, formation of endosomal structures and of putative compound vesicles Wagner Steuer Costa1, Szi-chieh Yu1, Jana Liewald1, Alexander Gottschalk1 1Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany; Institute of Biochemistry, Goethe University, Frankfurt, Germany

Optogenetic manipulation of 2nd messengers, in contrast to rhodopsin based approaches, does not lead to strong neuronal de- or hyperpolarization, overriding intrinsic activity, but rather exaggerates it by modulating signal transduction. Here, we characterize effects induced by Beggiatoa sp. photoactivated adenylyl cyclase (bPAC) in cholinergic neurons. Behavioral effects of bPAC photoactivation (increased locomotion speed, deeper bending angles) are in agreement with our previous work using Euglena gracilis PAC alpha (EuPACα) [1,2]. Compared to EuPACα, bPAC has high light sensitivity and low dark activity. Moreover, bPAC can be combined with the green-light activated Channelrhodopsin variant C1V1, allowing independent depolarization and enhancement of cholinergic signaling. By electrophysiology, we found an increase not only in miniature postsynaptic currents (mPSCs) frequency (+70%), but remarkably also on mPSC amplitudes (+30%) upon bPAC photostimulation. To understand these phenomena, we analyzed effects of bPAC photo-activation on synapse morphology by high pressure freeze electron microscopy (HPF-EM). As for ChR2 stimulation [3] we observed a bPAC-induced formation of large vesicles, likely endosomal structures, after 30 s and 5 min (+100% and +150% respectively), but not after 5 s of photoactivation. The amount of synaptic vesicles (SV) is reduced after bPAC photoactivation (-40% after 5 min of illumination). Interestingly, this is accompanied by a large reduction of docked SVs of over 50%. These morphological changes together with electrophysiological measurements suggest an enhanced SV vesicle turnover, possibly by speeding up of the docking process through some unknown cAMP/PKA target. The increase in the amount of large vesicles might be coupled to the increased fusion rate postulated above. Intriguingly, we observe the formation of structures that have a morphological appearance of SVs, but are of larger and irregular size. Possibly, these result from intracellular SV fusion, i.e. formation of compound vesicles with higher ACh content, fusion of which may explain the observed increase in mPSC amplitude. Optogenetic manipulation of cAMP coupled with HPF-EM, behavioral and electrophysiological analysis enables analyzing mechanisms of synaptic output modulation necessary for behavior adjustment in complex environments.

Reference(s) 1. Schröder-Lang et al (2007) Nature Methods 4: 39-42 2. Weissenberger et al (2011) Journal of Neurochemistry 116: 616–625 3. Kittelmann et al (2013) PNAS 110, E3007-16

Session 6 45 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 46 Contrasting responses within a single neuron class enable sex- specific attraction in C. elegans Anusha Narayan1, Vivek Venkatachalam2, Omer Durak3, Neelanjan Bose4, Frank Schroeder4, Aravinthan Samuel5, Jagan Srinivasan6, Paul Sternberg7 1Biology Division, Caltech and MIBR/ Dept of Brain and Cognitive Science, MIT, Cambridge, MA, 2Center for Brain Science , Harvard University, 3Neuroscience Graduate Program, MIT, 4Boyce Thompson Institute and Dept of Chemistry and Chemical Biology, 5Center for Brain Science and Department of Physics, Harvard University, 6Dept of Biology and Biotechnology, Worcester Polytechnic Institute, 7Division of Biology, California Institute of Technology

How an animal interprets and navigates its environment is crucial to its survival. In the model organism Caenorhabditis elegans, a class of endogenously produced small molecule signals termed ascarosides mediates a wide variety of social behaviors. C. elegans hermaphrodites secrete small-molecule signals called ascarosides, which attract males. Two of the previously isolated ascarosides ascr#3 and ascr#8, secreted by hermaphrodites are attractive exclusively to C. elegans males in a two-spot behavioral assay. Using the compact C. elegans network and a combination of electrophysiological, calcium imaging, behavioral, genetic and cell ablation techniques, we have analyzed how a population of 4 male–specific sensory neurons (CEMs) necessary for pheromone responses, collectively represent and process sensory information. We examine the sensory representation of pheromones in C. elegans, and how this representation alters the neural circuit thereby affecting the behavior. Male C. elegans exhibit marked concentration preferences for these sex-specific ascarosides. We show that a single cell class, the male-specific CEM neurons, actively maintains these preferences for ascr#8. Ascaroside responses in CEMs can be depolarizing or hyperpolarizing with a defined probability independent of anatomical identity. These opposing responses are tuned to different concentrations with varying kinetics. Worms with one intact CEM show no concentration preference, and reducing synaptic transmission strongly disinhibits all CEM responses. The concentration tuning curves for ascarosides in the CEM pheromonal circuit appear to be not a passive consequence of physical sensory limits but rather actively maintained. Hence, a heterogeneous concentration-dependent sensory representation of ascr#8 appears to allow a single neural class recognizes ranges of sensory cues in C. elegans.

46 Session 6 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 47 Serotonin facilitates efficient foraging in non-uniform environments by mediating an instantaneous slowdown upon re-feeding Shachar Iwanir1, Adam Brown2, Dana Najjar3, Meagan Palmer2, Ivy Fitzgerald2, David Biron4 1The Department of Physics, The Institute for Biophysical Dynamics, The University of Chicago, 2Institute for Biophysical Dynamics, The University of Chicago, 3The University of Chicago, 4The Department of Physics, The James Franck Institute, Institute for Biophysical Dynamics, The University of Chicago

When resources are not uniformly available, fast responses to the changing environment can provide a competitive advantage. The neurotransmitter serotonin (5-HT) has been implicated in mediating C. elegans responses to food. In particular, following starvation 5-HT was shown to mediate the slowdown of locomotion upon encountering food. However, under standard cultivating conditions serotonin deficiency results only in a mild hyperactive phenotype. Here, we show that 5-HT signaling is required for an abrupt onset of the locomotor response to re-feeding and that this instantaneous response enables efficient foraging in a patchy environment. By continuously monitoring locomotion during re-feeding we identified stereotypical wild- type dynamics, the hallmark of which was an abrupt slowdown. In contrast, 5-HT deficient tph-1 mutants and transgenics expressing tetanus toxin in all serotonergic neurons exhibited gradual slowdown dynamics. In all cases, steady state locomotion on food was similar. Thus, 5-HT signaling appears to accelerate the responses to discovering new food rather than mediate an enhanced slowing. The relevance of these rapid dynamics was demonstrated using small patches of food, where the absence of 5-HT signaling visibly reduced the efficiency of foraging. What roles do specific serotonergic neurons play in mediating these responses? We found that both the ADF and NSM neuronal types were required for the wild-type responses, albeit in different ways. Both were activated upon re-feeding but their respective physiological dynamics were distinct. The onset of ADF responses preceded the physical encounter with the food, suggesting a chemosensory mechanism. The responses of NSM were initiated upon encountering the food and lasted for longer periods. Corresponding changes in the dynamics of locomotion were observed when the respective neurons expressed tetanus toxin. In addition, optogenetically activating serotonergic neurons rapidly slowed starved animals, indicating that downstream 5-HT signaling is capable of rapid modulation of locomotion. Taken together, our results suggest a novel role for 5-HT signaling, demonstrate its utility in a non-uniform environment, and show that ADF and NSM act in concert to integrate multiple cues from an external resource.

Session 6 47 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 48 Matching of neuropeptide-receptor couples reveals ancient behavioral modulation by tachykinin signaling Isabel Beets1, Lotte Frooninckx1, Jan Watteyne1, Elien Van Sinay1, Olivier Mirabeau2, Liliane Schoofs1 1Functional Genomics and Proteomics Unit, KU Leuven, Leuven, Belgium, 2Neurobiology and Development Research Unit, Institut Fessard, Gif-sur-Yvette, France

Neuropeptides are key modulators of adaptive behaviors, and represent the largest group of neural messengers with over 250 bioactive peptides predicted in C. elegans. Despite their broad diversity and expression, relatively little is known about how specific neuropeptides function within circuits to modulate behavioral output. Although most are thought to act on G protein-coupled receptors (GPCRs), specific cognate receptors for the majority of neuropeptides remain unknown. We are therefore undertaking a large-scale deorphanization initiative – the Peptide-GPCR project (http://worm.peptide-gpcr.org) – that aims to match all predicted peptide GPCRs of C. elegans to their cognate neuropeptide ligand(s). Using reverse pharmacology, receptor candidates are expressed in a heterologous cellular system with a calcium reporter readout, and challenged with a library of over 260 C. elegans peptides of the established FLP and NLP families. Several novel and evolutionary conserved neuropeptidergic systems were found including a signaling system related to tachykinin signaling in vertebrates. We identified C. elegans homologs of tachykinin neuropeptides through a HMM-based search, and showed that they are able to elicit tachykinin-related receptor activation at nanomolar concentrations. In vivo expression analysis suggests a modulatory role of C. elegans tachykinins in the nociception circuit, and deletion mutants impaired in tachykinin signaling displayed aberrant ASH-mediated aversive responses. Our results suggest an ancient role of this neuropeptide family in pain modulation and behavioral plasticity, which are well-known functions of vertebrate tachykinins. Recent results of our peptide-GPCR deorphanization project and characterization of the C. elegans tachykinin system will be discussed.

48 Session 6 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 49 An oscillatory motor circuit optimizes foraging gait in C. elegans Yu Shen1, Quan Wen2, Connie Zhong3, Yuqi Qin1, Aravinthan Samuel2, Yun Zhang1 1Department of Organismic and Evolutionary Biology, Center for Brain Science, Harvard University, 2Department of Physics, Center for Brain Science, Harvard University, 3Harvard College

The optimal gait for animal locomotion is actively modulated by the nervous system. In C. elegans, the foraging movements are regulated by a network of excitatory and inhibitory motor neurons. Previous studies proposed that the GABAergic RME neurons limit the amplitude of head deflection (McIntire et al. 1993), but the underlying mechanisms remain unclear. Here, we characterize a motor neuron circuit underlying the gait control of head deflection. We show that the calcium activity of RME neurons is correlated with head bending to the same side. This activity, independent of physical displacement of the head, requires acetylcholine release from the head motor neuron SMD. SMD neurons generate cell-autonomous oscillatory activities in correlation with head movement (Hendricks et al. 2012). In contrast to the role of RME neurons, synaptic transmission from SMD neurons promotes head deflection. Furthermore, we find the C. elegans GABA(B) receptor gbb-1/gbb-2 functions in the SMD neurons to restrain head bending amplitude: gbb-1 mutants display exaggerated head deflection as RME-ablated animals, and restoring gbb-1 expression in SMD rescues the behavioral defect. Optogenetic manipulation of RME neuron activity verifies the role of RME in regulating head bending: inhibiting RME increases the bending amplitude, mimicking RME ablation, whereas activating RME decreases the bending amplitude. We propose that interaction between the cholinergic SMD neurons and the GABAergic RME neurons regulates neuromuscular activity. SMD innervates head muscles and signals to RME, which in turn relaxes the contralateral muscles, to facilitate head bending; meanwhile, RME neurons negatively modulate the activity of SMD to limit the bending amplitude through a gbb-1-dependent pathway. Our findings present a parsimonious model in which the interplay of excitatory and inhibitory neurons optimizes the foraging gait in C. elegans.

Reference(s) 1. McIntire SL. et al. The GABAergic nervous system of Caenorhabditis elegans. Nature. 364:337-41. 2. Hendricks M. et al. Compartmentalized calcium dynamics in a C. elegans interneuron encode head movement. Nature. 487:99-103.

Session 6 49

POSTER SESSIONS Memorial Union, Great Hall/Reception Room (4th floor)

Axon Outgrowth and Pathfinding: 50–60 Circuits and Behavior: 61–114, 193 Comparative and Evolutionary Neurobiology: 115 Disease Models and Regeneration: 116–132, 194 Neuronal Cell Fate and Differentiation: 133–138 New Technologies: 139–152 Sensory Signaling: 153–170 Synaptic Function and Modulation: 171–189 Synaptogenesis: 190–192

7:00 PM–10:00 PM Odd Numbered on Tuesday Even Numbered on Wednesday Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 50 Extracellular matrix components and axon guidance Cassandra Blanchette1, Andrea Thackeray1, Paola Perrat1, Claire Bénard1 1University of Massachusetts Medical School

During brain development, migrating neurons utilize guidance cues in the extracellular environment to orient their growth. The spatial and temporal organization of these guidance cues and their receptors is crucial for proper brain assembly. Much is known about how neurons receive guidance cues via specific receptors and transduce them intracellularly to reorient the cytoskeleton and direct growth. However, how the guidance cues themselves are distributed extracellularly for neurons to be guided and how the receptors might be localized within the growth cone is poorly understood. To address these questions with single-cell resolution, we have used the touch receptor neuron AVM as our model. AVM is born post- embryonically and its axon is guided ventrally towards the ventral nerve cord, to then project anteriorly. Seminal work from the Culotti and Bargmann labs has identified the guidance cues and receptors responsible for the ventral guidance of the AVM axon. Since it is well known that the extracellular matrix is important for the diffusion and organization of some morphogens and guidance cues during animal development, we have taken a combined approach of forward genetics and candidate genes to identify extracellular matrix components that might regulate the ventral guidance of the AVM axon. From a screen for mutants that exhibit misguided AVM axons, we have found that the synthesis of glycosaminoglycan chains of the heparan sulfate proteoglycans (HSPGs) is crucial for the ventral axon guidance of AVM. HSPGs play critical roles in development through shaping signaling gradients, facilitating receptor-ligand interactions, and other important processes (Reviewed by Häcker et al., 2005 and Yan & Lin, 2009). Since HSPGs consist of a core protein onto which the heparan sulfate sugar chains are attached, we have set out to determine the role of the main HSPG core proteins in AVM guidance. For this, we characterized AVM ventral axon guidance in a single and multiple mutant analysis of sdn-1, lon-2, gpn-1, agr-1, and unc-52. We found that specific HSPGs are required for the proper ventral axon guidance of AVM. We also found that the heparan sulfate modifying enzymes hst-2, hst-6, and hse-5 play a role in AVM ventral axon guidance, consistent with the findings by the Hobert and Bülow labs that the sulfation and epimerization modifications on the HSPG chains play important roles in axon guidance.

50 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 51 Regulation of neural circuit formation by a Slit-independent Robo pathway Chia-Hui Chen1, Dong Yan2 1Cell and Molecular Biology Ph.D. Program, Duke University, 2Department of Molecular Genetics and Microbiology, Duke University

The formation of proper neural circuits is vital to development and thus normal brain functions. Disruption of neural circuit formation is a major cause for many birth defects such as Down syndrome and Autism spectrum disorders. To gain a better understanding of this process, we use the RME circuit as a model to study the underlying molecular mechanisms. A previous genetic screen for mutants affecting RME circuit formation uncovered a single gene mutation: sax-3. SAX-3 is the only Robo receptor in C. elegans. This evolutionarily conserved protein family has been shown to play important roles in axon guidance, heart development and cancer. Despite the versatile functions of Robo receptors, its interaction with the ligand Slit is critical in most cases. To our surprise, slt-1 mutants do not exhibit the neuronal defects as seen in sax-3 mutants. In addition to slt-1, mutants of eva-1 (a novel Slit receptor) and Netrin-DCC pathway appear normal in RME circuit formation. Further studies showed that the neuronal defects are not cell autonomous, but could be rescued by pan-neuronal expression of sax-3 cDNA, indicating sax-3 may act as its own ligand in this pathway. We have also uncovered a potent suppressor of sax-3 , ju1120, in a separate genetic screen. Together, we show that a novel Robo pathway is essential for neural circuit formation. We will report the results of our analysis for ju1120 at the meeting.

Axon Outgrowth and Pathfinding Poster Session 51 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 52 The Male Anal Depressor Integrates Cell-autonomous Sex Hierarchy Signaling and Sex Specific Exogenous Signals to Achieve Sex differential Morphological and Functional Alterations in C. elegans Xin Chen1, Luis Rene Garcia1,2 1Department of Biology, Texas A&M University, 2Howard Hughes Medical Institute

We studied the C. elegans anal depressor development in males and hermaphrodites to address how a differentiated cell sex-specifically changes its morphology and function prior to adulthood. In both larval male and hermaphrodite, the anal depressor muscle possesses a dorsal-ventrally oriented sarcomere. The sarcomere is dorsally attached to the hypodermis and ventrally attached to the rectum and its contraction facilitates defecation behavior. However, in the adult male, the muscle becomes a copulation muscle, reorganizing the sarcomere and moving its ventral attachment to the spicule protractor muscles. To address when the changes occur in the anal depressor, we used YFP:actin to monitor, and mutant analysis, laser-ablation and transgenic feminization to perturb the cell’s morphological dynamics. In young larva, the muscle of both sexes has similar sarcomere morphology, but the hermaphrodite sex determination system promotes more anterior growth. The male’s anal depressor begins its functional reorganization in L3, by retracting its muscle arm from the neurons of the defecation circuit. Later in L4, the muscle alters its cytoskeleton and sarcomere structure dramatically to become a male mating muscle. The muscle’s ventral region develops a slit that demarcates an anterior and posterior domain. This demarcation is not dependent on the anal depressor’s intrinsic genetic sex, but is influenced by extrinsic interactions with the developing male sex muscles. However, subsequent changes are dependent on the cell’s sex. In L4, the anterior domain first disassembles the dorsal-ventral sarcomere region and develops filopodia that elongates anteriorly towards the spicule muscles. Later, the posterior domain dissembles the remnants of its cytoskeleton, but still retains a vestigial attachment to ventral body wall. Finally, the anterior domain attaches to the sex muscles, and then reassembles an anterior-posteriorly oriented sarcomere. Signaling pathways that regulate cytoskeletal and sarcomere reorganization must work downstream of the sex determination mechanism to regulate those male specific remodeling events. To identify the nature of those signaling pathways, we conducted EMS mutagenesis and RNAi screens, looking for mutants that compromised anal depressor development. We found that mutations in egl-8-encoded PLC beta, contribute to the posterior sarcomere disassembly defects. Additionally, we found that egl-20, which encodes one of the wnt ligands, causes similar defects as egl-8. We are studying the functional relationship between those two genes, to determine if a Wnt-calcium pathway contributes to the male anal depressor reorganization. Taken together, our work identifies key steps in the dimorphic re-sculpting of the anal depressor that are regulated by genetic sex and by cell-cell signaling.

52 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 53 Analysis of the ENU-3 protein family in nervous system development Roxana Florica1, Victoria Hipolito2, Homai Anvari2, Chloe Rapp2, Mehran Asgherian2, Costin Antonescu2, Marie Killeen2 1Molecular Science Graduate program, Ryerson University, 2Dept. of Chemistry and Biology, Ryerson University

The development of the nervous system in C. elegans is directed by guidance cues and their receptors including UNC-6/Netrin and its two receptors UNC-5 and UNC-40/DCC/ Frazzled. Cells and neurons expressing UNC-40 are chemo-attracted towards sources of UNC-6 while those expressing UNC-5 are chemo-repulsed by UNC-6. The DA and DB classes of motor neurons express both UNC-5 and UNC-40 and migrate away from their cell bodies in the ventral nerve cord (VNC) towards the dorsal nerve cord (DNC). In the absence of UNC-5 or UNC-6 the DA and DB motor neuron axons usually exit the VNC but have severe guidance defects. We found that in the absence of both UNC-5 and UNC-40 most axons exit the VNC, suggesting that UNC-40 opposes motor neuron axon outgrowth when UNC-5 is not present. Mutants lacking both UNC-5 and the novel protein ENU-3 often fail to exit the VNC (Yee et al., 2011). Motor neuron axon outgrowth defects are also enhanced by the lack of UNC-5 and any of the five other members of the ENU-3 protein family. Mutants lacking UNC- 6 and ENU-3 also have enhanced DB outgrowth defects suggesting that ENU-3 may work at least partly parallel to the UNC-6 pathway for motor neuron outgrowth. ENU-3 functions in a pathway parallel to UNC-40 and downstream of UNC-6 in guidance of the migrations of the AVM and PVM processes towards the VNC (Yee et al., 2013). Our data indicates that the other members of the ENU-3 family do not enhance the AVM and PVM defects of UNC-40 mutants. The whole UNC-6 pathway co-operates with the SLT-1/Slit pathway in this process. The location of the ENU-3 proteins within cells and the timing of expression of the proteins in C. elegans is currently under investigation.

Reference(s) 1. Yee, C., Florica, R., Fillingham, J., Killeen, M.T. ENU-3 functions in an UNC-6/Netrin dependent pathway parallel to UNC-40/DCC/Frazzled for outgrowth and guidance of the touch receptor neurons in C. elegans. Dev. Dyn. 243: 459-467 2. Yee, C. S., Sybingco, S. S., Serdetchania, V., Kholkina, G., Bueno de Mesquita, M., Naqvi, Z., Park, S.-H., Lam, K., Killeen, M.T. (2011). ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans. Dev. Biol. 352: 243–253.

Axon Outgrowth and Pathfinding Poster Session 53 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 54 Amphid cells transiently organize into a supercellular rosette during morphogenesis Ismar Kovacevic1, Zhirong Bao1 1Sloan Kettering Institute

The bulk of the C. elegans sensory neurons are organized during embryogenesis into 18 sensilla at the nose tip. It has been shown that amphid neurons migrate posteriorly to lengthen the dendrites in a process of retrograde extension (Heiman and Shaham, Cell, 2009). However, the dendrites also must extend anteriorly to the nose tip. As part of our effort toward developing the WormGUIDES (Global Understanding in Embryonic Development) atlas of neuronal morphogenesis, we discovered striking synchronous and rapid anterior extension of dendrites from the sensory neurons, including the amphids. To better understand sensory neuron morphogenesis, we focused on the amphids due to their size and an existing intellectual framework. Our observations revealed that prior to dendrite extension amphid neurons and glia assemble into a layered multicellular rosette with a stereotypical arrangement. The rosette is initially positioned dorsolaterally, anterior to the nascent hypodermis. As the hypodermis migrates anteriorly, the leading edge of Hyp5 appears to engage the rosette vertex, displacing it anteriorly. The leading edge of Hyp5 is locally delayed at the rosette attachment point, presumably due to viscoelastic drag of the amphid bundle. Upon reaching the nose tip, Hyp4 cells insert between the amphid bundle and Hyp5, fusing into a ring. Interestingly, during hypodermal towing of the rosette, the amphid socket cell detaches from the rosette, trails the vertex, and apparently wraps around the processes. Because rosette formation is usually driven by apical constriction of cells, we hypothesize the amphid cells undergo a similar process. We are currently using genetics to dissect the underlying molecular mechanisms for apical constriction and attachment to the hypodermis. We suspect this is a general mechanism for C. elegans head sensilla morphogenesis, and also bears a striking similarity to lateral line development in fish. Our data suggest that rosette formation organizes the neurons and glia into morphogenetic units, which are then positioned at the nose tip by attaching to the advancing hypodermis. This is a robust and scalable method to precisely position large numbers of sensory structures in a confined space.

54 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 55 Sensory neurons use epithelial mechanisms of morphogenesis to extend their dendrites Isabel I.C. Low1, Claire R. Williams1, Ian G. McLachlan1,2, Irina Kolotuev3, Maxwell G. Heiman1 1Department of Genetics, Harvard Medical School; Boston Children’s Hospital, 2Department of Genetics, Harvard Medical School; Boston Children’s Hospital; Program in Neuroscience, Harvard University, 3Universite de Rennes 1

Most neurons and glia are derived developmentally and evolutionarily from epithelia. Here, we show that the major C. elegans sense organ, the amphid, can be viewed as a true epithelium—a continuous sheet of neurons and glia with their apical surfaces exposed to the outside environment—and that its morphogenesis uses mechanisms likely shared by all epithelia. The amphid consists of twelve neurons, each of which extends a single, unbranched dendrite to the nose, and two glial cells, the sheath and the socket, which form epithelial-like junctions with the dendrite endings and with each other. We previously showed that amphid neurons are born at the nose and that the dendrite tips remain anchored there while the cell bodies migrate away, so that the dendrite is stretched between the two. This process is dependent on DYF-7, which is a zona pellucida (ZP) domain protein, a family found at the outward-facing apical surfaces of nearly all epithelia. In dyf-7 mutants, dendrite tips are dragged behind the migrating cell bodies, resulting in severely shortened dendrites. The sheath glial cell travels with the dendrites, while the socket remains at the nose tip. Several lines of evidence have led us to reinterpret these dyf-7 defects as a loss of epithelial integrity. We tagged the secreted ectodomain of DYF-7 and found that it localizes to caps at dendrite tips adjacent to epithelial-like junctions. DYF-7 also localizes to tips of other glial-ensheathed dendrites, all of which are affected in the dyf-7 mutant, but not to non-glial- ensheathed dendrites, which are unaffected in the mutant. When DYF-7 is misexpressed elsewhere in the embryo, it localizes to apical surfaces of other epithelia, notably the gut, suggesting that it binds a ubiquitous component of apical epithelial surfaces. Finally, we directly visualized neuron-glia and glia-glia junctions in the amphid and showed that they contain classic epithelial junction components and, surprisingly, that they remain intact in the dyf-7 mutant although the cells are separated, with only a thin process connecting the sheath and socket. We propose a model whereby DYF-7 forms a matrix at the apical surface and prevents glia from pulling apart under the mechanical stress of cell migration. Our findings indicate that sensory neurons use epithelial mechanisms of morphogenesis and suggest that maintaining epithelial integrity under mechanical stress may be a universal, ancestral role of ZP domain proteins.

Axon Outgrowth and Pathfinding Poster Session 55 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 56 An integrated genetic and biochemical analysis of the Heparan sulfate code in Caenorhabditis elegans Kristian Saied-Santiago1, Robert Townley1, John Attonito1, Carlos Díaz-Bálzac1, Dayse Cunha1, Hannes Buelow1 1Albert Einstein College of Medicine

Cell migration is a hallmark of many biological processes, including development, immunity and malignancies. The extracellular space plays an important role in cell migration, as it enables communication between the migrating cell and the extracellular environment. Heparan sulfate proteoglycans (HSPGs) are diverse extracellular molecules that control cell adhesion, motility and cellular responses by modulating protein signaling. Imaging studies as well as genetic and biochemical data indicate a heparan sulfate code that by way of its molecular complexity regulates cell behavior. However, direct correlation of glycan structure with function remains largely unknown. Using the migration of the motor neuron HSN and the non-neuronal coelomocytes in Caenorhabditis elegans as a paradigm, we report here that the HSPGs syndecan/sdn-1, glypican/lon-2 and perlecan/unc-52 are required independently and non-redundantly for correct migration of the HSN neuron. We further show that some HSPGs are required for promoting whereas others function to inhibit HSN migration. In contrast, syndecan/sdn-1 is the major HSPG required for the stereotypical migration of coelomocytes. Structural and genetic analyses suggest that different HSPGs carry distinct Heparan Sulfate (HS) modification pattern. We propose that the HS code that governs migration of the motor neuron HSN and coelomocytes in Caenorhabditis elegans comprises at least two structurally distinct types of modification patterns that must act in concert for correct cellular migration.

56 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 57 Ryanodine Receptor Channels Mediate Critical Sub-cellular Calcium Signals During Normal and Optogenetically Enhanced Neuronal Regeneration in C. elegans Lin Sun1, James Shay1, Kevin Roodhouse1, Samuel Chung1, Melissa McLoed2, Christopher Clark3, Mark Alkema3, Christopher Gabel1 1Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, 2Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, 3Department of Neurobiology, University of Massachusetts Medical School

Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We have found that localized calcium release from the endoplasmic reticulum (ER) via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Employing laser axotomy of single neurons in C. elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure sub-cellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and is blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to dynamically manipulate cell physiology in the context of neuronal regeneration and links the effect to innate cellular calcium signaling. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth and can be dynamically manipulated for beneficial neurotherapeutic effects.

Axon Outgrowth and Pathfinding Poster Session 57 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 58 Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion through UNC-73/Trio, Rac GTPases and UNC-33/ CRMP Lakshmi Sundararajan1, Adam Norris2, Dyan Morgan1, Zachary Roberts1, Erik Lundquist1 1University of Kansas, 2Harvard University

UNC-6/Netrin directs both attractive and repulsive axon guidance. Much is known about the UNC-6/Netrin in attractive guidance, but less is known about its role in repulsion. UNC- 6/Netrin regulates growth cone protrusion in the repelled VD growth cones, with “attractive” UNC-40/DCC homodimeric receptors stimulating protrusion and “repulsive” UNC-5/UNC-40 receptors inhibiting protrusion (Norris et al., 2011). Both activities of UNC-6/Netrin act in the same VD growth cone (i.e. UNC-6/Netrin stimulates protrusion away from its source and inhibits protrusion near its source). Constitutive activation of UNC-40/UNC-5 repulsive receptors in VD growth cones by expression of a myristoylated version of the UNC-40 cytoplasmic domain (MYR::UNC-40) resulted in small growth cones with severely reduced protrusion. We used a candidate gene approach to identify molecules required for growth cone inhibition mediated by MYR::UNC-40. These studies defined a novel signaling pathway involving the Rac GTP exchange factor UNC-73/Trio, the Rac GTPases CED-10 and MIG-2, and the actin- and microtubule-interacting molecules UNC-33/CRMP and UNC-44/Ankyrin. These molecules were required to inhibit VD growth cone protrusions, and mutations in these genes alone had excessive growth cone protrusion, indicating that they are normally required to inhibit growth cone protrusion. Epistasis studies using activated Rac GTPases MIG-2 and CED-10, which resembled MYR::UNC-40 inhibited growth cones, indicated that UNC-33/CRMP and UNC-44/Ankyrin act downstream of Rac GTPases. UNC-33, UNC-44 and UNC-73 were not required for accumulation of MYR::UNC-40::GFP or full length UNC-5::GFP to growth cones, suggesting that they might directly regulate the cytoskeleton to inhibit protrusion. Preliminary results indicate that this pathway affects microtubule organization in the VD growth cones. In sum, we have defined a new signaling pathway that inhibits growth cone protrusion in response to UNC-6/Netrin receptor activity and that might be involved in repulsive axon guidance. UNC-73/Trio and Rac GTPases have been implicated in attractive axon guidance and growth cone protrusion, but this is the first indication of their role in inhibiting protrusion. CRMP molecules have been broadly implicated in mediating growth cone collapse and cytoskeletal organization in response to Semaphorin, and our results are the first to implicate it in UNC-6/Netrin-mediated growth cone repulsion

58 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 59 Dynamics of the developing C. elegans nervous system Amelia White1, Anthony Santella1, Ismar Kovacevic1, Zhirong Bao1 1Memorial Sloan Kettering Cancer Center

One of the great mysteries of neurobiology is how embryonic neuroblasts differentiate and elaborate processes, which migrate to specific targets where they synaptically interconnect to form functional computational units. The structure and connectivity of the adult C. elegans nervous system has been described at the ultrastuctural level, but the dynamics of the developing connectome are still unknown. The wormGUIDES collaboration aims to produce a systems level description of the developing adult connectome. The wormGUIDES team is using optical sectioning light microscopy techniques with fluorescent protein labels to visualize small sets of neurons in a developing animal, enabling us to track in detail the morphology and movement of the neurons through development. A library of strains, each with a small set of neurons fluorescently labelled is being acquired. We are using computer vision techniques to extract the cell shape of each neuron during cell migration and process outgrowth. To provide a single map of the developing connectome we will integrate segmented neurons from multiple embryos by aligning the location and identification of each cell in the developing embryos over time. We use our automated cell lineaging software, StarryNite to track and lineage all cell nuclei during C. elegans embryogenesis. The reconstructed map of the developing connectome will be available in the WormGUIDES atlas, providing an easily accessible 4D map of the developing embryo to the C. elegans community. An understanding of the dynamics of the development of the C. elegans connectome will provide unique insights into how a nervous system assembles itself by revealing the relative timing of process outgrowth of all classes of neuron together with information on the environment that nerve processes encounter in the course of process out-growth and establishment of synaptic connections. An automated computer vision system for neural development characterization will allow analysis of multiple animals and of mutants with known or suspected neurological defects. This will provide us unique insights as to how nervous systems are formed, the natural variation in this process between animals and how behavioural mutants perturb the structure and connectivity of a nervous system.

Axon Outgrowth and Pathfinding Poster Session 59 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 60 Neuronal Target Identification Requires AHA-1-Mediated Fine-Tuning of Wnt Signaling in C. elegans Jingyan Zhang1, Mei Ding1 1Institute of Genetics and Developmental Biology,Chinese Academy of Sciences

The establishment of functional neuronal circuits requires that different neurons respond selectively to guidance molecules at particular times and in specific locations. In the target region, where cells connect, the same guidance molecules steer the growth of neurites from both the neuron and its target cell. The spatial, temporal, and cell type-specific regulation of neuronal connection needs to be tightly regulated and precisely coordinated within the neuron and its target cell to achieve effective connection. Electrical synaptic transmission through gap junctions is a vital mode of intercellular communication in the nervous system. The mechanism by which reciprocal target cells find each other during the formation of gap junctions, however, is poorly understood. Here we show that gap junctions are formed between BDU interneurons and PLM mechanoreceptors in C. elegans and the connectivity of BDU with PLM is influenced by Wnt signaling. We further identified two PAS-bHLH family transcription factors, AHA-1 and AHR-1, which function cell- autonomously within BDU and PLM to facilitate the target identification process. aha-1 and ahr-1 act genetically upstream of cam-1. CAM-1, a membrane-bound receptor tyrosine kinase, is present on both BDU and PLM cells and likely serves as a Wnt antagonist. By binding to a cis-regulatory element in the cam-1 promoter, AHA-1 enhances cam-1 transcription. CAM-1 is present on BDU and PLM and likely serves as a Wnt antagonist, thus linking transcriptional regulation by AHA-1 to modulation of Wnt signaling. Our study reveals a Wnt-dependent fine- tuning mechanism that is crucial for mutual target cell identification during the formation of gap junction connections.

60 Axon Outgrowth and Pathfinding Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 61 The Visual Detection of odr-1 22G RNAs via a MosSCI Sensor Adriel-John Ablaza1, Bi-Tzen Juang2, Sanjeev Balakrishnan1, Mary Bethke2, Chantal Brueggemann2, Maria Gallegos1, Noelle D. L’Etoile2 1Department of Biological Sciences, California State University East Bay Department of Cell & Tissue Biology, University of California, San Francisco, 2Department of Cell & Tissue Biology, University of California, San Francisco

Caenorhabditis elegans forages for food by distinguishing between various odorants in a dynamic environment. To allow worms to ignore food associated odors that are actually not predictive of food, their olfactory sensory neurons can adapt to persistent innately attractive odors that prove profitless (Colbert and Bargmann, 1995). Adaptation to the odor butanone takes place in the AWC, a paired olfactory sensory neuron (L’Etoile et al., 2002). Odor adaptation is initiated by the translocation of a cGMP-dependent protein kinase, EGL-4, from the cytoplasm into the nucleus of the AWC (L’Etoile et al., 2002; Lee et al., 2010). This translocation depends on decrease in cGMP (O’Halloran et al., 2012). Thus, the activity of ODR-1, a transmembrane guanylyl cyclase, which is required for chemotaxis towards all AWC mediated odorants, must be down regulated for adaptation to occur (L’Etoile and Bargmann, 2000). Nuclear EGL-4 promotes a 22G RNA directed repression of the odr-1 gene thereby initiating long-term odor adaptation (Juang et al., 2013). mut-7 activity, a 3’- 5’ exonuclease, is also implicated in odr-1 22G generation. In addition, a ChIP analysis of HPL-2 was shown to load heterochromatin on the odr-1 locus (Juang et al., 2013). However, there are limitations to qRT-PCR and ChIP analysis. Neither offers a dynamic or cell-specific readout of odr-1 22G RNA function. Here, we show a fluorescent reporter that is capable of visualizing cell specific changes in odr-1 22G RNA. The fluorescence reporter that detects odr-1 22G RNAs are inserted via Mos Single Copy Insertion (MosSCI) (Frøkjaer-Jensen et al., 2008). The sensor has the capability to detect specific odr-1 22G RNA in the AWC olfactory sensory neuron as well as cells throughout the worm such as the germline. The creation of a single copy insertion of an odr-1 small RNA sensor allows us to test our hypothesis that a 22G small RNA-directed pathway is dynamically activated during olfactory adaptation.

Circuits and Behavior Poster Session 61 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 62 Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in C. elegans Kelli Benedetti1, Bi-tzen Juang2, Anna Ludwig3, Chen Gu1, Aarati Asundi1, Torsten Wittman1, Noelle L’Etoile1, Paul Hagerman3 1Department of Cell and Tissue Biology, UCSF, 2Department of Biological Science and Technology, National Chiao Tung University, 3Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X (FMR1) gene; current evidence supports a causal role of the expanded CGG-repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons blocked this plasticity without affecting either the innate odor-seeking response, or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to block olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.

62 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 63 Serotonergic/Peptidergic Co-transmission in the C. elegans Egg- Laying Circuit Jacob Brewer1, Michael Koelle1 1Department of Molecular Biophysics and Biochemistry, Yale University

The C. elegans egg-laying circuit provides a model for studying signaling by the medically important neurotransmitter serotonin. The hermaphrodite-specific neurons (HSNs) have long been thought to release serotonin to excite the active phase of egg laying. However, mutants defective for serotonin biosynthesis are only modestly egg-laying defective. Further, when we use channelrhodopsin to activate the HSN neurons, we see strong stimulation of egg laying even in mutants unable to synthesize serotonin. We hypothesized that the HSNs release a second neurotransmitter in addition to serotonin to help stimulate egg laying. Mutants with defects in neuropeptide signaling have known egg-laying defects, and at least five neuropeptide genes are expressed in the HSNs. We found that the HSNs appear to co-release NLP-3 neuropeptides with serotonin to activate egg laying. We analyzed knockout mutations and transgenic overexpressors for each of the five HSN-expressed neuropeptide genes to see how they affect egg laying. Overexpressing nlp-3 caused a profound hyperactive egg-laying phenotype. nlp-3 null mutants have only a mild egg laying defect. However, double-mutant worms carrying mutations that prevent serotonin and NLP-3 biogenesis show profound, synthetic egg-laying defects, comparable to those of worms lacking HSNs. It therefore appears that the HSNs excite the egg-laying circuit by using serotonin and at least one NLP-3 neuropeptide as co-transmitters. We can now knock out either serotonin or NLP-3 to isolate and study how each single neurotransmitter regulates egg laying behavior. For example, several serotonin receptors are expressed on the egg-laying muscles, but knockouts for these receptors lead to only mild egg-laying defects. However, when we analyze these receptor mutations in combination with an nlp-3 knockout, we observe severe egg-laying defects. We are also working to identify which individual neuropeptides encoded by nlp-3 stimulate egg laying, along with the receptors for these peptides and their sites of action within the egg-laying circuit. The serotonergic/peptidergic co-transmission that we observe in the worm egg-laying circuit may also occur in other circuits. Serotonin appears to be co-released with several neuropeptides in the mammalian brain, and our studies may help to understand the purpose and logic of serotonergic/peptidergic co-transmission in general.

Circuits and Behavior Poster Session 63 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 64 Quantitative analysis of the Caenorhabditis elegans escape from noxious thermal stimuli Jarlath Byrne Rodgers1, Byron Wilson2, William S. Ryu3 1Donnelly Center for Cellular & Biomolecular Research and Dept. of Cell and Systems Biology, University of Toronto, 2Department of Physics, University of Toronto, 3Donnelly Center for Cellular & Biomolecular Research and Dept. of Physics, University of Toronto

When presented with a noxious thermal stimulus, C. elegans exhibit a coordinated sequence of behaviors to escape: the worm executes a reversal, followed by an omega turn, and accelerated forward motion. How these various motor programs are coordinated to optimize the escape, and the sources and levels of stochasticity and control in each component are not well understood. To investigate, we combined a worm-tracking microscope with a galvanometer-steered infrared laser. In real-time, a custom machine vision program processes image data, extracts the worm’s skeleton and identifies the location of the head and tail. This allows the IR laser to be targeted to specific regions of the worm’s body, and the full behavioral response of the worm to be recorded for many minutes at high temporal and spatial resolution. Repeated stimuli can be applied to examine habituation, and stimuli can be applied to the worm’s anterior and posterior simultaneously or in sequence to investigate signal integration. Image- sequences are post-processed and numerous metrics are extracted, including speed, direction, behavioral state and body shape. Using these metrics, we have fully decomposed and quantified the behavioral response of the worm to a noxious thermal stimulus. Several aspects of the escape response scale with stimulus intensity, and by tracking the worm post-stimulus, we show that the behavioral state can remain altered for several minutes, indicating very long-term coordination of motor programs. When confronted with repeated thermal stimuli, C. elegans habituate more quickly to weaker stimuli, consistent with other models of habituation. However, not all components of the behavioral response habituate consistently; notably a short reversal is almost always observed, suggesting multiple control pathways of the thermal noxious escape response. This novel assay provides a strong platform to expand our understanding of how C. elegans encode and respond to their environment. By carefully applying identical and precisely programmed thermal stimuli and leveraging mutant strains and genetic calcium indicators, we are able to unify our characterization of escape and learning across genes, neurons and behavior.

64 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 65 Opto-genetic and -physiological dissection of the C. elegans escape response reveals new mechanisms in the orchestration of distinct sub-motor programs Christopher Clark1, Andrew Leifer2, Ni Ji3, Jeremy Florman1, Kevin Mizes2, Aravinthan Samuel3, Mark Alkema1 1University of Massachusetts Medical School, Department of Neurobiology, 2Princeton University, Lewis Sigler Institute for Integrative Genomics, 3Department of Physics & Center for Brain Science, Harvard University

How does the nervous system orchestrate a compound motor sequence? The C. elegans escape response consists of a defined sequence of behavioral motifs allowing the animal to navigate away from a mechanical stimulus. The escape response consists of four phases: (I) forward locomotion accompanied by lateral head movements; (II) stimulus induced backward locomotion during which head movements are suppressed; (III) a deep ventral bend of the head; (IV) head slide along the ventral side of the body (omega turn); (I) reorientation of forward locomotion in the opposite direction of the stimulus. Our previous work and that of others has provided a framework for the neural circuit that controls this behavior. We used calcium imaging to correlate activity patterns of individual neurons to the temporal sequence of the escape response phases. We used a combination of optogenetics and laser ablations to determine the contribution of individual neurons in the execution of each phase of the escape response. Optical physiology of the AVA, AVD and AVB locomotion command neurons, the RIM and AIB interneurons and the RIV motor neurons reveals unique activity profiles during reversals (II), ventral turns (III) and when the animal reinitiates forward movement (IV). Furthermore, individual activation of these neurons typically only elicits partial responses of the separate phases of escape behavior, while combinatorial activation and inhibition paradigms can attenuate the responses to mimic a touch stimulus. Using our system, we show that suppression of head movements and ventral bending can be uncoupled from the reversal indicating that these behavioral motifs are distinct motor programs. Moreover, we found a role for the RIM and AIB interneurons in the deep ventral bend (III) uncovering a sub-circuit for ventral turning converging onto the SMD and RIV excitatory neck motorneurons. The combination of optogenetics and calcium imaging allows us to dissect a complex behavior into its component motifs and understand how the omega turn is linked to a reversal in the escape response.

Circuits and Behavior Poster Session 65 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 66 Pumping off Food (PoffF) reveals a glutamate dependent microcircuit that imposes cue dependent inhibitory tone on the pharynx Nicolas Dallière1, Nikhil Bhatla2, Robert Walker1, Vincent O’Connor1, Lindy Holden-Dye1 1University of Southampton, 2Massachusetts Institute of Technology

C. elegans feeding behaviour was assayed in intact worms by counting the contraction- relaxation cycles of the pharynx; a single contraction-relaxation cycle is one pharyngeal pump. The behaviour was assayed either on food (Pumping On food; PonF) or following withdrawal from food (Pumping off food; PoffF). PonF occurs at a frequency of 4 to 5 Hz whilst removal from food causes a dramatic reduction in frequency. Investigation of PoffF over a protracted time course during food deprivation revealed this fictive feeding generates at least two distinct pharyngeal states: 1) a steady increase over two hours to a PoffF of 0.5 Hz followed by 2) an erratic fluctuation between low (<0.5 Hz) and high rates (2 Hz). The reduction in PoffF following food removal cannot simply be explained by an absence of an excitatory food-dependent signal as the glutamate deficient mutanteat-4 (ky5) shows a reduced PonF (3-3.5 Hz) but an enhanced PoffF (sustained at 2 Hz) relative to N2 controls. Thus glutamate regulation of the pharynx is context dependent; enhancing PonF and inhibiting PoffF. The latter suggests there is a glutamate dependent decreased pumping that follows food removal. Laser mediated RIP ablation removes the single neural connection between the extrapharyngeal and the pharyngeal nervous system but does not change PoffF suggesting intrinsic regulation of PoffF from within the pharynx. Ablation of the pharyngeal glutamatergic neuron I2 generates an eat-4 like enhanced PoffF suggesting this neuron is a key determinant of the active inhibition that suppresses PoffF. Selective expression of eat-4 (+) in I2 reduces the elevated PoffF of eat-4(ky5) supporting a role for glutamate signalling from this neuron in suppressing pharyngeal activity in the absence of food. This result is reinforced by optogenetic activation of I2. These observations suggest a pharyngeal microcircuit that actively supresses pumping in the absence of food and this is likely mediated by an under-reported sensory function of the I2 neuron. This reinforces an emerging idea that the pharyngeal circuit can perform important sensory and integrating functions that impart profound control on food dependent behaviour executed both within and outside the pharynx (Bargmann et al. 2013). This idea of the pharynx acting as a sensory hub resonates with the autonomous role that the enteric nervous system can play in modulating physiological function in higher organisms.

66 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 67 Genetic sex of sensory neurons controls attraction to ascaroside pheromones Kelli A. Fagan1, Jessica R. Bennett2, Frank Schroeder3, Douglas S. Portman2 1Neuroscience Graduate Program, University of Rochester, 2Department of Biomedical Genetics, University of Rochester, 3Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University

Hard-wired neural circuitry is dynamically modulated to generate variations in behavior. This behavioral flexibility allows animals to respond appropriately to changes in internal states and environmental conditions. The sex chromosomal content, also known as genetic sex, of the nervous system is an internal factor that modulates behavior in many animal species. However, the mechanisms by which genetic sex regulates neural function are largely unknown. In order to better understand these processes, we are investigating the sexually dimorphic attraction behavior displayed by C. elegans males in response to ascaroside pheromones. Through the examination of animals with sexually mosaic neural tissue, we have determined that the neural circuit underlying this sexually dimorphic response is present in both sexes but the circuit remains inactive in hermaphrodites. Subsequent studies have revealed that the genetic sex of a shared sensory neuron may play an important role in the sexually regulation of this circuit. Additionally, we found that neuropeptide signaling is required for male ascaroside attraction. Finally, we have found that a DM domain transcription factor acts in males to promote ascaroside attraction. Together, these data raise the possibility that a DM domain transcription factor may bring about sex-specific differences in neuromodulatory signaling to alter circuit activity and thereby promote male-specific ascaroside attraction.

Circuits and Behavior Poster Session 67 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 68 Magnetosensitive neurons mediate vertical burrowing in C. elegans around the world Andrés G. Vidal-Gadea1, Kristi Ward1, Celia Beron1, Joshua Russell1, Jesse Cohn1, Nicholas Truong1, Adhishri Parikh1, Jonathan T. Pierce-Shimomura1 1Department of Neuroscience; Center for Brain, Behavior & Evolution; Waggoner Center for Alcohol & Addiction Research, Center for Learning & Memory; The University of Texas at Austin, Austin, TX 78712

For millennia humans have used the magnetic field of the Earth to aid in navigation. Only recently have we become aware that other species also use the magnetic field to similar ends. Like us, many animals (e.g. lobsters, turtles, and birds) use the magnetic field to navigate horizontally in their environment. Other organisms, like magnetotactic bacteria, use the magnetic field vector to migrate vertically in their environment. While the list of organisms capable of orienting to magnetic fields (magnetotaxis) has grown steadily for decades, no sensory neuron or transduction pathway has been described for magnetotaxis in any animal. Understanding the neural and genetic bases of magnetic detection remains a longstanding goal of sensory biology. We demonstrate that C. elegans detects and orients to Earth-strength magnetic fields. Magnetotaxis in worms requires the AFDs, a pair of identified ciliated sensory neurons. To our knowledge, these are the first magnetosensitive neurons described for any species. Magnetic transduction appears to involve a cyclic nucleotide-gated ion channel (encoded by the tax-4 and tax-2 genes) and the antenna-shaped villi at the end of the AFD cilia. By studying the magnetotaxis behavior of natural wild isolates from around the world, we find that the innate magnetotaxtic orientation preference tightly relates to optimal burrowing in vertical directions. Up or down preference depends on satiation state and is tuned to the magnetic field properties at specific global origins. We propose a model where AFD neurons integrate environmental information, including the magnetic field of the Earth, to guide C. elegans in vertical migrations in the soil.

68 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 69 Neuromodulator network control of a multisensory decision D. Dipon Ghosh1, Soonwook Hong1, Michael Koelle2, Michael Nitabach3 1Department of Cellular and Molecular Physiology, Yale University, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Cellular and Molecular Physiology, Department of Genetics, and Cellular Neuroscience, Neurodegeneration and Repair, Yale University

To navigate natural environments, animals must process information perceived by multiple senses simultaneously. Precise molecular and cellular mechanisms by which neural circuits integrate multisensory information to influence behavior remain unclear. We designed novel behavioral paradigms to assess principles of decision-making of Caenorhabditis elegans in multisensory environments. We found that neuropeptide and biogenic amine signaling coordinately modulate a neuronal reciprocal feedback pathway to regulate a C. elegans multisensory decision. Our results indicate that in multisensory contexts, neuropeptide Pigment Dispersing Factor-2 (PDF-2) signaling through the PDF Receptor (PDFR) modulates RIM interneuron function; subsequently, RIM secretion of tyramine alters sensory neuron responsiveness through tyramine receptor TYRA-2. Our results suggest that neuromodulators PDF-2 and tyramine regulate multisensory behaviors by tuning interneuron-driven decisions and interneuron-sensory neuron feedback. Importantly, this feedback resembles established concepts of hierarchical multisensory processing in mammals. Thus we demonstrate the utility of the C. elegans model for investigating the molecular and cellular basis of decision- making and conserved features of multisensory processing mechanisms in metazoan nervous systems.

Circuits and Behavior Poster Session 69 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 70 SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male C. elegans Xiaoyan Guo1, Luis Rene Garcia1,2 1Department of Biology, Texas A&M Unisersity, 2Howard Hughes Medical Institute

For a long time, we know that metabolism status contribute to aging. However, the mechanism involved is complicated. C. elegans male mating behavior was used to characterize the behavioral deterioration and physiological change of the neuromuscular circuitry during early aging. Previously, we characterized that the decline of early aging C. elegans male mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation. To further understand the relationship between metabolism alterations and other physiological changes, which ultimately affect the behavioral output, we tested a metabolism regulator’s function in maintaining male mating. Here, for the first time we linked the metabolism status, the ROS production and modification, physiological alteration and behavioral output together. We showed thatthe mating circuits’ functional durability depends on the metabolic regulator SIR-2.1, a NAD+- dependent histone deacetylase. Aging sir-2.1(0) males display accelerated mating behavior decline due to premature hyper-excitability of cholinergic circuits used for intromission and ejaculation. In sir-2.1(0) males, the hyper-contraction of the spicule-associated muscles pinch the vas deferens opening, thus blocking sperm release. The hyper-excitability is aggravated by reactive oxygen species (ROS). Our genetic, pharmacological and behavioral analyses suggest that in sir-2.1(0) and older wild-type males, enhanced catabolic enzymes expression, coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline. However, as a compensatory response to reduce ROS production from enhanced catabolism, anabolic enzymes expression levels are increased as well, resulting in higher lipid synthesis.

70 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 71 The molecular and circuit basis of thermosensation in Caenorhabditis elegans Vera Hapiak1, Harold Bell1, Piali Sengupta1 1Brandeis University

Nervous systems translate complex sensory inputs into defined behavioral outputs via the functions of specific molecules, neurons and circuits; alterations in these pathways lead to behavioral and developmental anomalies. Fundamental processes such as thermosensation require highly sensitive and coordinated mechanisms of detection and integration to maintain organismal homeostasis. In C. elegans, thermosensation is a sophisticated experience- dependent behavior that is mediated by the AFD, AWC and other thermosensory neurons that integrate and translate thermosensory information via a small subset of downstream interneurons to direct appropriate thermotaxis behaviors. The AFD neurons are exquisitely thermosensitive over a wide dynamic range, but their molecular mechanisms of thermotransduction remain poorly characterized. Using reverse genetics and neuron-specific molecular tools, we have begun to identify and characterize novel signaling molecules involved in AFD-mediated signal transduction. Genes identified to date include new components of cGMP second messenger cascades, as well as molecules required for synaptic transmission. Preliminary experiments also suggest that thermal inputs are integrated via a master-slave model whereby AFD coordinates the response ranges of other thermosensory neurons to drive behavior. We are characterizing a peptidergic signaling pathway by which AFD may synchronize the operating ranges of other thermosensory neurons to drive coherent behavioral outputs in defined temperature ranges. These findings will identify and characterize new thermotransduction mechanisms, define novel mechanisms of circuit function, and elucidate how perturbations in such signaling contribute to altered behavior and/or disease states.

Circuits and Behavior Poster Session 71 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 72 Characterizing behavioral responses of adult nematodes to ascarosides Anna Hartmann1, Michael O’Donnell1, Dongshin Kim1, Dirk Albrecht2, Piali Sengupta1 1Brandeis University, 2Worcester Polytechnic Institute

An organism’s ability to detect and respond to environmental stimuli is critical for its survival. C. elegans, like many organisms, utilizes chemosensory information to regulate behavioral, developmental, and reproductive decisions. A complex mixture of small molecules called ascarosides produced by C. elegans plays a vital role in regulating many aspects of the worms’ lifecycle including the dauer-fate decision, reproductive choices, and social behaviors. While several ascarosides have been linked to specific behaviors, and cognate receptors identified for a subset, the functions and molecular mechanisms of ascaroside signaling remain largely unknown. We are using microfluidic devices designed to allow precise spatiotemporal control over stimuli, coupled with worm tracking software, to perform high-throughput, quantitative behavioral analyses of nematode responses to ascarosides. Using these devices, we are able to characterize responses to single ascarosides, as well as examine synergistic effects of multiple chemicals. We are also investigating the extent to which developmental plasticity can modulate adult responses to pheromone components. The high-throughput nature of these assays, combined with detailed analyses of worm behaviors will significantly improve our ability to dissect the neuronal and molecular circuitry involved in pheromone sensation.

72 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 73 Genetic Analysis of C. elegans Pathogen Avoidance Behavior Alexander Horspool1, Howard Chang1 1Department of Biological Sciences, Binghamton University

C. elegans elicits behavior plasticity when encountering microbes. The innate ability to avoid high concentration of pathogenic microbes is critical to confer survival. We have shown previously that E3 ubiquitin ligase HECW-1 and neuropeptide receptor NPR-1 modulate the timing of avoidance when C. elegans is exposed to pathogenic P. aeruginosa. HECW-1 acts in OLL sensory neurons and functions upstream of NPR-1. To identify additional genes involved in C. elegans pathogen avoidance behavior, we performed a screen using EMS mutagenesis. We obtained mutants that elicit accelerated or delayed P. aeruginosa avoidance phenotypes. We are currently mapping these mutations. Based on genetic complementation test, these mutations are not just additional alleles of hecw-1 and npr-1. We are also in the process to determine if these candidates act via hecw-1 and npr-1 to regulate the avoidance behavior. We hope our results will shed new light on the genetic circuitry of stress response in C. elegans.

Circuits and Behavior Poster Session 73 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 74 Regulation of motivational states in C. elegans Changhoon Jee1, L. René Garcia1 1Department of Biology and Howard Hughes Medical Institute, Texas A&M University

Motivation is a goal directed driving force based on physiological needs such as hunger, thirst, and sexual arousal. Pathological changes in the brain’s reward and stress system can direct normal motivated behaviors into compulsive behaviors, such as binge eating, drug seeking, and obsessive sexual behavior. We studied C. elegans copulation behavior to determine how stress can modulate motivational states. C. elegans male mating behavior can be modulated by stress. For example, mating potency declines in aging males and food deprivation stress during early adulthood leads to neuro-adaptive changes, resulting in extending sexual potency in older animals. We developed the mating interference assay (Mi) to quantify the male’s tenacity to copulate, as a proxy read-out for his motivational state. In a dose dependent manner, blue light, a noxious stimulus, can interfere with male copulation. The male’s ability to endure the blue light irritant is correlated with his mating drive. In copula, a mating-deprived male is tolerant to blue light . However, if the male is sexually satiated, he is less tolerant to the blue light irritant during subsequent copulations. To understand how motivational states are modulated, we used the Mi assay to quantify the mating drive of seb-3 mutant male. We found that SEB-3 is a CRF receptor-like GPCR in C. elegans that regulates a conditional response to stress. Relative to wild-type males, a seb-3(gf) allele causes copulating males to endure higher intensities of blue light; however, seb-3(gf) mutants behave like wild-type males under acute stress conditions. In contrast, copulating seb-3 (lf) males display the opposite phenotype, a lower tolerance to the blue light irritant. These observations suggest activated SEB-3 modulates how the copulating male responds to aversive stimulation. Cell ablation and transgenic experiments indicated that one of the SEB-3 sites-of-action is the LUA glutamatergic interneurons, which make recurrent connections with male sensory- motor copulatory neurons. We imaged Ca2+ transients in the LUA neurons in copulating wild type and seb-3 mutant males. In wild-type males, LUA activity increases during vulval location and during spicule insertion attempts. In contrast, seb-3 (gf) males display higher LUA neuronal activity prior to and throughout copulation behavior. Additionally, we found that SEB-3 might promote mating tenacity through NCA family sodium channels. Copulating seb- 3 (gf) males defective for UNC-79, a putative auxiliary subunit of the NCA sodium channel, display reduced endurance to Mi conditions, equivalent to seb-3 (lf) males. Taken together, we suggest that in copping with stress, SEB-3 functions in the LUA neurons to amplify the activity of downstream copulatory neurons in the C. elegans male.

74 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 75 Regulation of an Insulin-like Peptide (ILP) network involved in learning Konstantinos Kagias1, Diana Andrea Fernandes de Abreu2, Antonio Caballero2, Joy Alcedo3, QueeLim Ch’ng2, Yun Zhang4 1Department of Organismic and Evolutionary Biology, Center for Brain Science, Harvard University, 2King’s College London, 3Department of Biological Sciences, Wayne State University, 4Organismic and Evolutionary Biology, Center of Brain Science, Harvard University

In many animals, including humans, insulin and insulin-like peptides (ILPs) act through conserved signaling pathways to regulate neural plasticity. However, the underlying molecular and cellular mechanisms remain largely unknown. Intriguingly, multiple ILPs are encoded in the genomes of many organisms, such as the 40 ILPs in C. elegans, suggesting diversity in the functions of ILPs and potential interaction and combinatorial effects among them, as well as with other factors. Consistent with this idea, we have shown that two C. elegans ILPs, INS- 6 and INS-7, play distinct roles in olfactory learning [1]. Interestingly, the expression of both ins-6 and ins-7 is regulated by environment [2] as well as by other ILP members [3] forming a dynamically interactive and context-dependent network. Here we present our detailed analysis on the regulation of ins-6 and ins-7, and its impact on learning. By using candidate screen approaches we were able to identify factors that can alter the expression of these 2 ILPs in a cell-specific manner and in accordance with the environment. We are now further analyzing the specific role of these factors in different conditions hoping to better understand how the environmental context can impinge upon the ability of organisms to learn.

Reference(s) 1. Chen Z, Hendricks M, Cornils A, Maier W, Alcedo J, et al. (2013) Two insulin-like peptides antagonistically regulate aversive olfactory learning in C. elegans. Neuron 77: 572-585. 2. Cornils A, Gloeck M, Chen Z, Zhang Y, Alcedo J (2011) Specific insulin-like peptides encode sensory information to regulate distinct developmental processes. Development 138: 1183-1193. 3. Fernandes de Abreu DA, Caballero A, Fardel P, Stroustrup N, Chen Z, et al. (2014) An insulin-to-insulin regulatory network orchestrates phenotypic specificity in development and physiology. PLoS Genet 10: e1004225.

Circuits and Behavior Poster Session 75 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 76 Genetic and biochemical studies of the mechanism of neurotransmitter signaling through heterotrimeric G proteins Seongseop Kim1, Michael Koelle1 1Department of Molecular Biophysics and Biochemistry, Yale University

Many neurotransmitters signal by activating heterotrimeric G proteins. Regulators of G protein signaling (RGS proteins) terminate such signaling by activating the intrinsic GTPase activity of Gα subunits. RGS proteins regulate signaling by serotonin and dopamine, neurotransmitters critical to human mental health. However, the purpose of and logic by which RGS proteins regulate neurotransmitter signaling remain poorly understood. Previously, we conducted large-scale genetic screens to isolate mutants that are resistant to paralytic effect of exogenous serotonin. Many of these mutants show a set of defects, including serotonin resistance, hyperactive locomotion, and constitutive egg laying. These

phenotypes are characteristic of a loss of signaling by the inhibitory Gαo G protein GOA-1, or of an increase in signaling by the Gαq G protein EGL-30. We have now used whole-genome sequencing to complete the identification of the causal mutations in all these mutants.

The large majority of the mutations affect the Gαo G protein GOA-1 or the various subunits

(EAT-16, GPB-2, RSBP-1) of the RGS complex that inhibits the opposing Gαq G protein EGL-30. One exception is n3792, which may be a gain-of-function allele of lev-11, encoding tropomyosin. It has been shown that LEV-11 functions in muscles to regulate locomotion and egg laying. Further analysis of n3792 may identify the relationship of tropomyosin function to neural G protein signaling. We are biochemically testing a model that would explain the logic by which RGS proteins

regulate neural G proteins. Gαo and Gαq can act in the same neurons to oppose each other, with Gαo inhibiting and Gαq promoting neurotransmitter release. Our model hypothesizes that each inactive G protein associates with a specific RGS complex and, upon activation, releases this RGS complex to inhibit the opposing G protein. This mechanism would create a binary switch that would allow only Gαo or Gαq to be active at any one time. We have used single copy transgenes to generate functional, epitope-tagged versions of the G proteins and RGS subunits. Currently, we are conducting co-immunoprecipitations to test for the existence of the protein complexes predicted by the model and for their dissociation upon Gα activation.

76 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 77 Downstream regulatory components of the TIR-1/JNK-1 pathway for forgetting in C. elegans Tomohiro Kitazono1, Akitoshi Inoue1, Takeshi Ishihara2 1Graduate School of Systems Life Sciences, Kyushu University, 2Faculty of Science, Department of Biology, Kyushu University

Forgetting is important for animals to avoid memory overflow and interferences between old and new memories, and the molecular mechanisms of the forgetting are still unclear. To elucidate the mechanisms of forgetting, we used one of the behavioral plasticity, the olfactory adaptation in C. elegans as a simple model of memory. We previously showed that the mutants of TIR-1, a p38 MAPK adaptor protein, and its downstream signaling pathway (TIR-1/JNK-1 pathway) exhibit prolonged retention of the olfactory adaptation to diacetyl and isoamylalcohol, which are odorants sensed by AWA and AWC sensory neurons respectively. Our genetic analysis showed that the TIR-1/JNK-1 pathway accelerate forgetting by facilitating the release of the forgetting signals from AWC sensory neurons. In this study, to identify downstream components of the TIR-1/JNK-1 pathway including the forgetting signals, we screened suppressor mutants of a gain of function mutants of tir-1 (ok1052). ok1052 mutants exhibit fast forgetting of the olfactory adaptation probably because the forgetting signals are excessively released in ok1052 mutants. By the suppressor screening of ok1052 mutants, we isolated 71 independent mutants, which showed the prolonged retention of the olfactory adaptation to diacetyl. Based on behavioral analyses, we revealed that these mutants could be classified into two groups; one group showed the prolonged retention of the olfactory adaptation only to diacetyl and another group showed it not only to diacetyl but also to isoamylalcohol. This result suggests that there may be partially different signaling pathway in the forgetting of the olfactory adaptation to these two odorants. Furthermore, some strains also showed the prolonged retention of the salt chemotaxis learning, which is one of the associative learning in C. elegans. To identify the responsible genes of those suppressor mutants, we carried out the whole genome sequencing analysis and single nucleotide polymorphism mapping of mutant strains, and found some novel downstream components of downstream of the TIR-1/JNK-1 pathway. This study enables us to provide new insights on the regulatory mechanisms of forgetting.

Circuits and Behavior Poster Session 77 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 78 Identification of As-nlp-21 and As-nlp-22 peptides in the motor neurons of Ascaris suum Jennifer Knickelbine1, Christopher Konop1, Colin Wruck2, Antony Stretton1 1Department of Zoology, University of Wisconsin-Madison, 2School of Education, University of Wisconsin-Madison

Detailed studies of cellular connectivity and classical transmitter expression have been completed on the motor nervous system of Ascaris suum; however, a complete functional circuit has not yet been described for this relatively “simple” system. One missing piece of information is the neuropeptide content of the motor neurons themselves, which has only recently been examined. Neuropeptides are the largest class of neurotransmitter molecules, playing a variety of roles in the nervous systems of vertebrate and invertebrate animals. The most diverse group is the Neuropeptide-like Proteins (NLPs), whose functions remain largely unexplored. Through the use of single-cell mass spectrometry, our laboratory has identified the major peptide products expressed in the motor neurons of Ascaris. Among these are NLP sequelogs of peptides on the C. elegans nlp-21 and nlp-22 transcripts, which represent the major peptide products of the cholinergic forward-projecting excitatory motor neurons (DE1, DE3, VE1; responsible for anteriorly-propagating waveforms) and the GABAergic inhibitory motor neurons (DI, VI), respectively. Localization of these transcripts was confirmed using in situ hybridization. Preliminary behavioral experiments show a dramatic decrease in acetylcholine (ACh)-induced muscle contraction of Ascaris dorsal muscle in the presence of -8 As-NLP-22 (SLASGRWGLRPamide), with an IC50 of 10 M. Intact worms injected with this peptide exhibited flaccid paralysis in the anterior 1/3 of the worm, where locomotory behavior is most frequent (see accompanying poster by C. Wruck et al.). Muscle exposed to peptides from the As-nlp-21 transcript showed no significant change in ACh-induced contraction compared to controls. Given the dramatic behavioral effects of As-NLP-22, it is crucial that we consider neuropeptides in developing a functional circuit, and also as novel targets for anthelmintic drug development.

78 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 79 Identification of new genes involved in Dopaminergic neurons function by cell specific knock-down Ambra Lanzo1, Luca Pannone2, Marco Tartaglia2, Paolo Bazzicalupo1, Simone Martinelli2, Lucia Carvelli3, Elia Di Schiavi4 1Institute of Bioscience and BioResources, IBBR, CNR, 2Istituto Superiore di Sanità, ISS, 3Department Pharmacology, Physiology & Therapeutics University of North Dakota, 4Institute of Bioscience and BioResources, IBBR

Dopaminergic (DA) neurons are known to be crucial for essential brain functions. Dysfunction of the DA system has been associated with a number of neurological and psychiatric diseases. The DA system also plays a pivotal role in drug addiction, as many drugs of abuse stimulate dopamine release. Apart from Parkinson Disease, most of the disease associated with the DA system results from an imbalance in dopamine neurotransmission with no apparent morphological changes. Deeper insights into DA system is important not only to develop novel therapeutic approaches, but also to understand the different DA neuronal functions. In C. elegans, studies aimed at understanding the molecular mechanisms underlying DAergic function have taken advantage of chemical treatments and of engineered transgenes. This approach is not feasible to study the physiological role played by a gene. On the other hand classical genetic mutations can cause pleiotropic effects or lethal phenotypes in mutant animals. To overcome these limitations, we took advantage of a reverse genetic approach (Esposito et al., Gene, 2007) to obtain an efficient knock-down of control genes (gfp, cat-2, dat-1) specifically in DAergic neurons. Based on literature, we then considered a panel of new genes that may play a key role in DAergic neurons. We started silencing in these neurons the unc-64 gene, which encodes a SNARE protein involved in synaptic vesicle fusion, and whose full depletion causes a lethal phenotype. Animals lacking expression of unc-64 only in DAergic neurons were able to move but presented a pronounced defect in a DA-mediated behavioral assay, known as swimming-induced paralysis (SWIP). Moreover the levels of amphetamine-induced SWIP in these animals were defective and similar to those observed in the dopamine transporter (DAT-1) mutants, suggesting that the silencing of unc-64 specifically in DAergic neurons causes an increase of extracellular dopamine. We took a similar approach with unc-63, an alpha-subunit of levamisole-sensitive nicotinic acetylcholine receptor, expressed in muscles and in unidentified neurons. unc-63 depletion causes a strong uncoordination and a partial resistance to the lethal effects of the nicotinic agonist DMPP (Ruaud and Bessereau, Development, 2006). We silenced unc-63 specifically in DAergic neurons and after exposing these animals to DMPP, we obtained a partial resistance to the lethal effects, which was similar to that observed in unc-63 loss of function mutants. We thus demonstrated that unc-63 unexpectedly mediates nicotine toxic effects in DA neurons. Our data strongly support a new role played by these two genes in the regulation of the DA system. We are now focusing on the characterization of the molecular pathways in which these genes are involved by genetic epistasis and pharmacological approaches.

Circuits and Behavior Poster Session 79 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 80 Sensory Neurons Enhance Egg-laying Rates Across a Wide Range of Temperatures Samuel Lasse1, Miriam B Goodman1 1Department of Molecular & Cellular Physiology, Stanford School of Medicine

For more than 100 years, reports have shown that temperature influences the rate of many animal behaviors. In small ectotherms like C. elegans, temperature could act directly on the motor program (motor neurons and muscle cells), indirectly through specialized thermoreceptor neurons, or both. Nothing is known about the role of thermoreceptor neurons in regulating behavior rates. To investigate this question, we adapted standard C. elegans egg-laying assays and measured steady-state egg-laying rates as a function of temperature, comparing rate-temperature (R-T) curves in wild type (N2) and tax-4 mutants that lack functional thermoreceptor neurons. We found that N2 egg-laying rate is maximal near 25°C and decreases at both cooler and warmer temperatures. This R-T curve has a similar shape to what is reported for other behaviors, such as running speed in lizards. Egg-laying rates in tax-4 mutants are significantly lower and reach their maximum at a cooler temperature (~22.5°C); the shape of the R-T curve is similar to that found in N2 worms. These data indicate that input from the thermoreceptor neurons is required for wild-type performance and that temperature has a direct effect on the egg-laying motor program. Currently, we are investigating how the egg-laying pattern is influenced by temperature. This pattern can be described as a combination of short and long intervals between egg-laying events. We are using a microfluidics based approach to measure how the distribution of intervals changes with temperature (Kopito and Levine 2014). This will provide insight into how each component of the egg-laying circuit is influenced by temperature and the thermoreceptor neurons.

Reference(s) 1. Kopito and Levine (2014). Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues. Lab Chip 2014 Feb 21;14(4):764-70.

80 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 81 Dopaminergic neuronal support cells and cholinergic and glutamatergic neurons promote ejaculation and post-ejaculatory behavior in males Brigitte LeBoeuf1, L. Rene Garcia1 1Howard Hughes Medical Institute, Department of Biology, Texas A&M University

Ejaculation and post-copulatory satiation are poorly understood motivated behaviors. Using optogenetics, calcium imaging, laser ablation and behavioral studies in C. elegans males, we identified that the length of post-copulatory satiation is coupled to successful ejaculation. Additionally, we determined that ejaculation is controlled by cholinergic and glutamatergic neurons that couple ejaculation with the proceeding steps of mating. Sperm movement in males can be broken down into two steps: initiation, when the valve region holding sperm in the seminal vesicle opens, allowing sperm to move to the vas deferens, and release, when sperm transfers out of the cloaca and into the hermaphrodite uterus. We identified that initiation is triggered by the cholinergic SPC sensorimotor neuron. Synapses made with the base of the male’s copulatory spicules allow the SPC to sense when they initially penetrate the hermaphrodite vulva, inducing full spicule insertion via tonic contraction of the spicule- controlling protractor muscles. We determined that initial spicule penetration causes the SPC to not only induce tonic muscle contraction but also trigger sperm movement from the seminal vesicle. Additionally, we showed that the SPV and SPD sensory neurons, whose processes are exposed to the environment at the spicule tip, promote the proper timing of valve opening through the SPC. These neurons control the initiation step of ejaculation, allowing sperm to reach the cloaca opening. The sperm then needs to be released into the hermaphrodite. We identified that this step is regulated by the glutamatergic post cloacal sensilla PCA, through gubernaculum sex muscles that properly adjust the spicules inside the hermaphrodite, allowing sperm transfer to occur. Finally, we showed that the neuronal support socket cells, located around the spicule neurons, use dopamine to promote the sperm release step of ejaculation and regulate the length of post-ejaculatory satiation. Thus, we identified several mechanisms via which behavioral steps are coordinated, culminating in successful sperm transfer and post-ejaculatory satiation.

Circuits and Behavior Poster Session 81 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 82 Environmentally evoked developmental plasticity of behavior mediated by Insulin-like signaling pathway in C. elegans Harksun Lee1, Dae han Lee1, Nari Kim1, Myungkyu Choi1, Junho Lee1 1IMBG, School of Biological Sciences, Seoul National University, Seoul, 151-747, Korea

Behavioral alterations upon environmental changes emerging from neuronal circuits are essential for survival. Many nematode species including the free-living nematode C. elegans exhibit an evolutionarily conserved, developmental stage-specific behavior called nictation: standing and waving in three-dimensional loops on a projection. We demonstrated that nictation is required for transmission of C. elegans to a new niche using flies as carriers, suggesting a role of nictation as a dispersal and survival strategy under harsh conditions. We found that cholinergic transmission in IL2 ciliated head neurons was required for the initiation of nictation. Now we have found that the insulin like-signaling pathway regulates nictation behavior during the pre-dauer period within ASI or ASJ neurons. We also demonstrated that the presence of pathogenic bacteria enhanced the nictation frequency of dauers in the daf-2- dependent manner, suggesting a role of the insulin-like-signaling pathway as an information convergence point for the environmental and developmental plasticity. * Harksun Lee and Daehan Lee contributed equally to this work.

82 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 83 Two distinct modes of pharyngeal pumping in C. elegans are regulated by food concentration Kyung Suk Lee1 1Department of Physics, Harvard University

With growing occurrence of eating disorders, there has been interest in using feeding of C. elegans as a model system to understand molecular mechanism of how food intake is regulated. In this work, using a custom microfluidic device which enables feeding nematodes at a desired concentration of food, we imaged pharyngeal pumping of individual worms at a high time resolution. Automated image analysis of the acquired movies reveals that there are two distinct modes in pharyngeal pumping: regular and irregular. The overall pumping rate is determined by fraction of time spent in each of the two pumping modes. We find that fraction of regular pumping reduces as food concentration is decreased, and that exogenous serotonin restores regular pumping regardless of food concentration. In line with these findings, mutants deficient of serotonin biosynthesis significantly suppress regular pumping even at high food concentration. Thus food abundance is reflected in the maintenance of regular pumping in a serotonin-dependent way.

Circuits and Behavior Poster Session 83 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 84 Transcriptional and developmental regulation of salt associative learning in C. elegans Jana P. Lim1, Miriam B. Goodman1, Anne Brunet1 1Stanford University

Learning and memory depend on the complex interplay of molecular changes that occur on different time scales, including transcriptional and translational control of gene expression. C. elegans are known to exhibit salt associative learning, wherein worms learn to avoid innately attractive sodium chloride. While much of the neuronal circuitry for this behavior has been mapped, and several genetic players have been identified, many important aspects of this behavior remain unknown. Here, we explore the time scales of salt learning acquisition and memory loss in wildtype worms, characterize the necessity of transcription and translation for this behavior, and investigate the learning ability of worms over the course of development. This work aims to uncover novel mechanisms underlying salt associative learning in C. elegans, which should shed light on conserved mechanisms of learning and memory.

84 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 85 C. elegans core and sex-specific neurons involved in sexual attraction behavior are altered in their differentiation in hlh-3 mutant males Liliana Marquez1, Aixa Alfonso1 1University of Illinois at Chicago

HLH-3, a basic helix-loop-helix protein in C. elegans, is a member of the Achaete/ Scute family of transcription factors. Expression of hlh-3 is sexually dimorphic, and both hermaphrodites and males lacking hlh-3 function [hlh-3(lof)] have defects in sex-specific behaviors. To investigate hlh-3’s role in regulating sexual behavior in males, we performed male mating assays for three hlh-3(lof) mutant alleles. hlh-3(lof) males display behavioral defects including abnormal sexual attraction and turning behavior. Sexual attraction is regulated by both core and sex-specific neurons, whereas turning behavior is only regulated by sex-specific neurons. We discovered that neurons in each class are abnormal in hlh-3 mutant males, including the male-specific Ray Type A and CEM neurons, as well as the “core” AWA neurons shared by both males and hermaphrodites. We found that in hlh-3(lof) males, expression of the dopaminergic reporter, cat-2::gfp, was significantly reduced in the Ray type A neurons which are required for the proper execution of turning behavior; suggesting that these neurons are non-functional and consistent with their behavioral defect. Likewise, the “core” AWA and male-specific CEMs, which play a role in detection of hermaphrodite secreted pheromones, were abnormal. In hlh-3(lof) males, characterization of the CEMs using the pkd- 2::gfp reporter revealed that in males less than four differentiated CEMs could be detected. Surprisingly, hlh-3(lof) hermaphrodites retained CEM like cells. Analysis of the “core” AWA neurons was monitored with expression of the odr-10::gfp reporter. We found that the AWA neurons acquired a hermaphrodite-like fate in the hlh-3 mutant males. In summary, hlh-3(lof) males are inefficient in specific mating behaviors because the differentiation and function of cells such as the Ray Type A, CEMs and AWA neurons are altered.

Circuits and Behavior Poster Session 85 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 86 Episodic quiescence in fasting C. elegans Richard McCloskey1, Christopher Fang-Yen1 1University of Pennsylvania Department of Bioengineering

Animals vary their behavioral activity levels in response to various internal and external factors, including circadian, developmental, or nutritional conditions. In C. elegans, quiescence behaviors have been described in association with molting, food satiety, the dauer state, and swimming. Here we report an episodic quiescence behavior in adult worms removed from food (fasting). We used automated imaging and analysis of behavior in a multi-well WorMotel device to quantify movement of worms crawling on NGM agar, swimming in liquid media, or immersed in viscous solutions in which locomotion is intermediate between swimming and crawling. After an initial period of continuous locomotion, worms exhibit bouts of quiescence characterized by cessation of both locomotion and pharyngeal pumping alternating with periods of continuous locomotion and pumping. The average duration of the initial period of continuous locomotion before the first quiescent episode increases as the worm movement becomes more crawling-like: 1.1 ± 0.2 hrs for swimming and 3.1 ± 0.2 hrs for crawling. The dynamics of this episodicity differs depending on the environment: over a 12 h period swimming worms display an average of 3.5 quiescent bouts per hour, each an average of 8.9 minutes in length, while crawling worms display an average of 1.9 bouts/hour, each lasting an average of 22 minutes. In all environments tested the duration of quiescent bouts increases over time. We find that well-fed animals do not exhibit episodic quiescence, suggesting that episodic quiescence is due to fasting. Our results expand on and reinterpret those of Ghosh and Emmons (2008) who observed episodic locomotory quiescence in swimming worms. Our results show that (1) episodic behavior occurs for worms during locomotion in all environments tested, not only during swimming, (2) the quiescence occurs in both locomotory and feeding behavior, and (3) episodic quiescence occurs only in fasting worms. We are testing the hypothesis that episodic quiescence is a response to stressful conditions.

86 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 87 High throughput phenotypic profiling identifies the role of heterotrimeric G-protein signaling pathways in habituation Andrea McEwan1, Andrew Giles2, Kasper Podgorski3, Kurt Haas3, Rex Kerr4, Catharine Rankin3 1Brain Research Centre, 2The Scripps Research Institute, 3University of British Columbia, 4Janelia Farm

Using a novel high-throughput behavioral assay, we collected 13 distinct quantitative measures of movement, sensation and learning (habituation) from 508 known C. elegans mutants. From these data, we identified 6 independent phenotypic components forC. elegans tap habituation that are regulated by non-overlapping subsets of genes. Two approaches were used to identify potential signaling pathways involved in regulating habituation: multivariate analysis to predict potentially undiscovered interactions, and a candidate approach to characterize how known signaling pathways regulate habituation. The multivariate analysis predicted 1075 novel genetic interactions based on similarity in phenotypic profiles. Using the candidate approach, we characterized mutant strains that interacted genetically with one (or both) of the strongest habituation variants, goa-1 and eat- 16. goa-1 and eat-16 are members of the heterotrimeric G-protein signaling pathway and have previously been shown to contribute to the regulation of synaptic release in nervous systems of C. elegans to mammals. Our analysis demonstrates that the Gαi and Gαq signaling pathways share a broad role in regulating habituation whereas the Gαs pathway modulates the rate of habituation.

Circuits and Behavior Poster Session 87 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 88 A male specific neuropeptide, FLP-23, is necessary for male sperm transfer Renee Miller1, Inna Hughes2, Teigan Ruster3, Andy Spitzberg3, Steven Husson4, Liliana Schoofs4, Doug Portman5 1Dept. of Brain and Cognitive Sciences University of Rochester, 2Dept of Biomedical Genetics, 3University of Rochester, 4UK Leuven, 5Dept of Biomedical Genetics Center for Neural Development and Disease

Neuropeptides play important, conserved roles in modulating and optimizing animal behaviors, including reproductive behaviors. C. elegans males execute a complex, stereotyped mating program upon contact with receptive hermaphrodites. Classic neurotransmitters such as acetylcholine and dopamine, as well as neuropeptides, have been shown to mediate various steps of male mating behavior. We identified FLP-23 as a male specific member of the FMRF family of neuropeptides. flp-23 is expressed in two neurons in the male tail tentatively identified as DVE and DVF. flp-23 mutant males, which are morphologically and developmentally normal, are infertile. Specifically, we find that flp-23 males are defective in the sperm transfer step of mating. We are currently taking both candidate and screening approaches to identify a flp-23 receptor. dmsr-1 is one such candidate mutant that we have identified as mating defective. Our work, along with recent evidence from Drosophila, suggests that neuropeptides may be conserved mediators of sperm transfer.

88 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 89 Peptidergic signaling functions in a homeostatic manner to modulate quiescence in C. elegans sleep Stanislav Nagy1, Nora Tramm2, Jarred Sanders3, Shachar Iwanir2, Ian Shirley2, Erel Levine4, David Biron2 1The Institute for Biophysical Dynamics, The University of Chicago, 2Department of Physics and the James Franck Institute, The University of Chicago, 3Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, 4Department of Physics and the Center for Systems Biology, Harvard University

To survive in dynamic environments animals must modulate their behavioral and physiological states. Biological homeostasis invokes an important class of modulatory responses aimed at stabilizing internal conditions. Two signatures of homeostatic compensation were reported in the context of lethargus, the sleep-like state of C. elegans: globally increased quiescence in response to strong mechano-stimulation and a (local) correlation between the durations of consecutive bouts of motion and quiescence in unstimulated animals. Although peptidergic signaling was implicated in regulating quiescence, its role in homeostasis during lethargus remained largely unexplored. In this study, by precisely manipulating the dynamics of quiescence and motion (the two behavioral states within lethargus) we show that motion plays a causal role in prolonging quiescence. Moreover, weak photo- or mechano-stimulation and strong mechanical stimuli reveal two distinct regimes of responses: a micro-homeostatic response and a global response, respectively. In all cases, behavioral responses were similar whether a stimulus interrupted a bout of motion or a bout of quiescence, but distinct between the lethargus and non-lethargus stages. To understand the mechanism underlying micro- homeostasis, we examined the role of neuropeptides in these responses. We found that establishing and consolidating quiescence did not require neuropeptide release, but the loss of a subset of functional neuropeptides strongly suppressed quiescence. Furthermore, the neuropeptide Y receptor homolog, NPR-1, and an inhibitory neuropeptide known to activate it, FLP-18, mediated the modulation of quiescence. Consistent with NPR-1 dependent

activation of Go in transfected mammalian cells, we found that Go signaling was also required for modulating quiescence. Our data suggests that, similar to the homeostatic response to motoneuron imbalance, lethargus quiescence is modulated by excitatory and inhibitory neuropeptides in a combinatorial manner. These findings further strengthen the connections between C. elegans lethargus and mammalian sleep, suggest that bouts of motion and quiescence during lethargus are two distinct sleep-like micro-states, and highlight the importance of neuropeptides in micro-homeostatic responses.

Circuits and Behavior Poster Session 89 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 90 Differing Levels of MAST Kinase Activity Can Code Two Opposing Behavioral Drives During C. elegans Thermotaxis Shunji Nakano1, Isabel de Ridder1, Takamasa Suzuki2, Tetsuya Higashiyama3, Ikue Mori1 1Group of Molecular Neurobiology, Division of Biological Science, Graduate School of Science, Nagoya University, 2JST ERATO, Division of Biological Science, Graduate School of Science, Nagoya University, 3JST ERATO, Division of Biological Science, Graduate School of Science, Institute of Transformative Bio- Molecules, Nagoya University

C. elegans can sense and remember the environmental temperature and navigate themselves toward the cultivation temperature when placed on a thermal gradient. Although studies of the past decades have identified genes and neural circuits involved in thermotaxis, how C. elegans achieves this complex behavior still remains elusive. To further reveal the neural mechanisms that underlie thermotaxis, we undertook genetic screens to look for mutants defective in thermotaxis. Among the isolates we recovered was the mutation nj102, which caused a thermophilic defect. We identified thatnj102 is a reduction-of- function allele of the gene kin-4, which encodes an evolutionarily conserved serine-threonine kinase of the MAST (microtubule-associated serine-threonine) kinase subfamily. To define the null phenotype of kin-4, we utilized CRSIPR-CAS9-mediated genome editing and isolated a putative null allele of kin-4(nj170). The kin-4(nj170) mutation eliminates almost the entire kin-4 coding sequence and, unlike the kin-4(nj102) mutation, caused a cryophilic phenotype. To identify where kin-4 acts, we generated a functional kin-4::gfp translational fusion gene. This kin-4::gfp transgene is expressed broadly in the nervous system, and its expression was observed in neurons previously shown to be important for thermotaxis, including AFD, AWC, AIY and RIA. We performed cell-specific rescue experiments and found that expression of kin-4 in AFD but not in AWC, AIY or RIA rescued the thermotaxis defect of kin-4 mutants, indicating that kin-4 acts in a major thermosensory neuron AFD to regulate thermotaxis. These results suggest that differing reduction levels of kin-4 activity within a single sensory neuron can generate distinct neural codes that endow two opposing behavioral drives. We hope that further analysis will reveal a molecular mechanism by which single sensory neurons generate multiple motor outputs that shape animal behaviors.

90 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 91 Lethargus-quiescence in C. elegans is a systemic brain state under tight control of arousal circuits Annika Nichols1, Tomáš Eichler1, Saul Kato1, Tina Schrödel1, Manuel Zimmer1 1Research Institute of Molecular Pathology

Behaviour can be separated into discreet sustained periods that are a reflection of the underlying brain state. Sleep is one such state and is highly conserved but incompletely understood in terms of regulation, function and mechanism. In mammals we currently do not have the spatial-temporal resolution to measure brain-wide activity at single cell level and therefore cannot determine how individual neurons are contributing to emergent properties of brain states. A newly developed imaging technique allows the combination of brain-wide imaging with the single cell resolution needed for wholly investigating brain states in C. elegans (Schrödel et al. 2013). Recently, the behavioural quiescence seen during the developmental phase of lethargus has been defined as being a sleep-like state inthe worm (Raizen et al. 2008). We have identified an oxygen chemosensory arousal circuit that allows us to rapidly and robustly switch between sleep-like and wake-like states during lethargus: atmospheric oxygen levels induce sustained arousal, while preferred (low) oxygen levels permit sustained quiescence. We have exploited this to image brain-wide activity of neurons during the state transitions. We have discovered that the sleep-like state in C. elegans occurs at a brain wide level and is characterized by sustained quiescence periods occurring concomitantly in almost all neurons. Our unbiased approach identifies quiescence promoting neurons, as well as neurons with characteristic activity patterns between the rapid state transitions. For example, groups of backward premotor interneurons and motor neurons (i.e. RIM, AVA, VA), which during aroused phases show synchronous activity, are uncoupled during brief activity bouts in lethargus. Through behavioural genetics and transgenic cell kills we have confirmed that the oxygen sensory neurons AQR, PQR and URX are required to induce arousal. This, as well as the speed of response indicates that arousal is mediated by a top-down control mechanism. However, falling asleep is characterized by more gradual uncoupling of premotor interneurons suggesting that removal of the arousal signal may allow the brain to relapse back into a (perhaps default) quiescence state. We are currently conducting a candidate behavioural screen for arousal signals. From this we have identified several genes of interest. Using these cutting-edge techniques and the remarkable tractability of the C. elegans system we are dissecting the circuit and endocrine mechanisms of arousal.

Circuits and Behavior Poster Session 91 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 92 A complex neuropeptide signaling cascade inhibits ASH-mediated aversive behavior in C. elegans Mitchell Oakes1, Deanna Filppi1, Vera Hapiak2, Amanda Ortega1, Abby Jelinger1, Richard Komuniecki1 1University of Toledo, 2Brandeis University

Neuropeptides can functionally reconfigure individual microcircuits to differentially modulate a host of complex behaviors. In the present study, we have identified a complex, extrasynaptic, peptidergic signaling cascade that inhibits aversive responses to dilute 1-octanol mediated by the two ASH sensory neurons. The overexpression of the neuropeptide-encoding genes, nlp-44, nlp-15, flp-12 and flp-17in wild type animals, each dramatically inhibit ASH-mediated aversive responses to dilute 1-octanol off food. In contrast, as predicted, nlp-44, nlp-15, flp- 12 or flp-17 null animals respond more rapidly to dilute 1-octanol than wild type animals off food. Extensive genetic analyses and cell-based assays have placed nlp-44 upstream of nlp- 15 and nlp-44/nlp-15 upstream of flp-12/flp-17. NMUR-1 and EGL-6 appear to be receptors for neuropeptides encoded by nlp-15 and flp-17, respectively, based on genetic analysis and direct heterologous characterization (Ringstad). Cell-specific RNAi knockdown and rescue suggest that nlp-15 neuropeptides activate NMUR-1 on the AIB interneurons and the BAG sensory neurons, stimulating the release of flp-12/flp-17 neuropeptides from the BAGs that, in turn, activate EGL-6. As predicted, nmur-1 and egl-6 null animals also respond more rapidly to dilute 1-octanol off food than wild type animals and the overexpression of nmur-1 and egl-6 dramatically inhibits ASH-mediated aversive responses. Together, these data describe a complex, interactive peptidergic signaling cascade that inhibits ASH-mediated aversive responses off food and also modulates a host of additional behaviors. These studies are continuing to 1) identify the receptors for flp-12 and nlp-44 neuropeptides, 2) confirm the ligand specificity of each of the receptors by heterologous expression and 3) characterize the effects of neuropeptide signaling on aversive responses, by cell-based assays and direct electrophysiology. Overall, these studies suggests that at least three different layers of peptidergic signaling act in series to integrate inputs from multiple sensory neurons responding to an array of intrinsic and extrinsic stimuli.

92 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 93 The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling Andrew Olson1, Michael Koelle1 1Yale University, Department of Molecular Biophysics and Biochemistry

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications seem to regulate serotonin signaling. Through large-scale genetic screens for mutants that fail to respond properly to serotonin, we found that C. elegans mutants for either of two subunits of the ELPC ELongator Protein Complex are defective for response to serotonin. Conversely, ELPC overexpressors are hypersensitive to the effects of exogenous serotonin. ELPC is conserved from C. elegans to humans and functions as a cytoplasmic lysine acetylase to reversibly modify other proteins. This is the first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. We used two-dimensional gel electrophoresis to show that in C. elegans lysates,

Gαo, a G protein through which serotonin signals to slow locomotion, exists as a complex series of species of differing charge. Post-translational modification by lysine acetylation alters protein charge and could produce the pattern seen. Our preliminary results suggest that the

series of differentially charged Gαo species seen in wild-type lysates shifts to a less complex pattern of Gαo species in lysates of ELPC mutants. Additionally, we have identified two specific

acetylated lysines in Gαo purified from mouse brain using mass spectrometry. Analysis of Gαo isolated from C. elegans lysates will allow us to determine if worm and mammalian Gαo are

equivalently modified. We hypothesize that ELPC may reversibly acetylate Gαo (and possibly other proteins) to regulate serotonin signaling.

Circuits and Behavior Poster Session 93 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 94 DA neurons modulate food related behaviors by signaling through peptidergic AVK and DVA neurons in a distributed neuronal network Alexandra Oranth1, Christian Schultheis1, Karen Erbguth1, Jana Liewald1, David Hain1, Isabel Beets2, Sebastian Wabnig1, Wagner Steuer Costa1, Alexander Gottschalk1 1Buchmann Institute for Molecular Life Sciences, Institute for Biochemistry, Goethe University Frankfurt, 2Functional Genomics & Proteomics Group, Department of Biology, KU Leuven

Finding a food source and staying in its vicinity is a crucial survival strategy. How neuronal circuits integrate multiple sensory cues from the environment, and from the body, to maintain locomotion towards a food source and to help the animal to remain at it, is a relevant problem, requiring coordination of long-term navigational strategies and “local” fine-control of body movements. We analyzed a network of neurons controlling body posture and locomotion strategy in food related behaviors. We used an unbiased optogenetic approach to characterize the AVK interneurons. We expressed Channelrhodopsin (ChR2) and Halorhodopsin (NpHR) in AVK. Upon photoinhibition in the absence of food, animals showed increased body curvature and highly irregular locomotion, while AVK photostimulation had no effect. OFF food, AVK releases FLP-1 neuropeptides to inhibit subsets of motor neurons and other cells to alter locomotion of the animal. Body curvature is reduced by FLP-1; conversely, when its release is inhibited, curvature increases. We found that AVK is inhibited by the presence of food, and in this situation, i.e. ON food, photoactivation of AVK leads to FLP-1 peptide release and to decreased bending angles. Thus, food modulates the activity state of AVK. Synthetic FLP-1 peptides inhibited cholinergic motor neurons at the NMJ. We identified a FLP-1 receptor required for AVK-FLP-1 effects: NPR-6 is expressed in VC motor neurons, and in further neurons required for chemosensation and feeding, and NPR-6 was activated by FLP-1 peptides in a cell culture assay. The inhibition of AVK by food is in part mediated by dopaminergic (DA) PDE neurons that sense food. Consequently, photostimulation of the DA cells reduced body curvature. PDE neurons innervate and modulate (via the DOP-3 receptor) another neuron affecting body curvature, the proprioceptive DVA cell, which releases excitatory NLP-12 neuropeptides onto motor neurons. Photoexcitation and -inhibition of DVA increased and reduced bending, respectively, thus AVK and DVA work in opposite ways. Both AVK and DVA neurons are coupled to SMB head motor neurons, by chemical synapses (AVK, DVA) and by gap junctions (AVK). Eliminating SMB or gap junctions connecting AVK-SMB, both phenocopied effects of AVK ablation on bending angles. Hence, a network of DA neurons, responding to food, modulates signaling through peptidergic AVK and DVA neurons, that both affect motor neurons in the head and body to alter locomotion behavior.

94 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 95 Regulation of egg-laying behavior by the conserved EGL-9/HIF-1 hypoxia-response pathway Corinne Pender1, H. Robert Horvitz1 1HHMI, Dept. Biology, MIT

Response to changes in levels of oxygen is a fundamental process in human physiology and plays a major role in pathologies as diverse as cardiovascular disease, stroke and cancer.

More generally, the capacity to respond to fluctuations in 2O provides an important adaptation for many organisms, including C. elegans. Diminished access to O2 can elicit metabolic, developmental and behavioral responses. Much remains unknown about the molecular and neural mechanisms underlying behavioral

modifications triggered by chronic exposure to low 2O . We are using the egg-laying behavior

of C. elegans as a model for studying behavioral responses to decreased O2 concentration.

Upon exposure to hypoxic conditions (0.5% O2), worms decrease their egg-laying rate (Miller and Roth, Current Biology 19, 1233, 2009). The conserved prolyl hydroxylase EGL-9 is a key component of the response to hypoxia. egl-9 was originally identified in aC. elegans screen for mutants defective in egg laying. EGL- 9 defines an evolutionarily conserved family of enzymes that hydroxylate the transcription factor hypoxia-inducible factor (HIF-1) using available O2, thus targeting HIF-1 for degradation. Increase in HIF-1 activity as a result of reduced inhibition by EGL-9 under hypoxic conditions is the basis for many C. elegans adaptations to hypoxia, including metabolic and behavioral changes. Additionally, hif-1 loss-of-function (lf) mutations suppress the egg-laying defect of egl-9(lf) mutants. Thus, it is likely that the inhibition of egg laying under hypoxia is controlled by the egl-9/hif-1 pathway. Previous work has demonstrated that the sites of action of EGL-9 for controlling egg laying are in the nervous system and the uv1 cells of the somatic gonad (Chang and Bargmann, PNAS 105, 7321, 2008), but the molecular players controlling egg laying downstream of HIF-1 remain unknown. To find downstream effectors of HIF-1 or parallel pathways that control egg laying in response to hypoxia, we are conducting screens using an egl-9(lf) background to identify second-site mutations that suppress the egg-laying defect of egl-9 mutants. As hif-1(lf) suppresses the egg-laying defect of the egl-9(lf) mutant, we expect mutations in downstream effectors of hif-1 in the control of egg laying to similarly suppress this defect. From a screen of 100,000 haploid genomes, we have identified 17 suppressors of the egl-9 egg-laying defect, at least 13 of which are not alleles of hif-1. These suppressors might represent mutations in new genes required for behavioral adaptation to hypoxia. We are mapping and characterizing these mutants and will continue screening for more suppressors.

Circuits and Behavior Poster Session 95 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 96 No Abstract Available for this Number

96 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 97 Role of FLP-1 neuropeptides on sensory and motor function Daniel Raps1, Michelle Sawh2, Raubern Totanes3, Patrick Loi1, Ivor Joseph1, Chris Li1 1Department of Biology, City College of New York-City University of New York, 2Department of Chemistry, City College of New York-City University of New York, 3Department of Physics, City College of New York-City University of New York

Parasitic nematodes infect over one billion people worldwide. Neuropeptides and their signaling systems are a potential target for anti-parasitic drugs. A large family of FMRFamide- like neuropeptides, also known as FLPs, is present in all nematodes. All FLPs share a common

Arg-Phe-NH2 at their C-terminus. However, little is known about the function of these peptides. We are examining the role of flp-1, which encodes seven distinct FLP peptides and one non- FLP peptide. Previously isolated alleles of flp-1, such as flp-1(yn2), contain deletions that disrupt both flp-1 and a neighboring gene daf-10, which encodes an intraflagellar transport complex A component. Hence, the phenotypes seen in flp-1(yn2) mutants could be due to loss of flp-1 or daf-10. Recently, three new alleles of flp-1 were isolated; one allele is a presumptive null allele, while another allele deletes the coding region corresponding to one to three FLP- 1 peptides and is presumably a hypomorphic allele. The phenotypes of these new mutants showed several differences, presumably because of the varying levels of FLP-1 peptides being expressed. One phenotype, wandering, was found to be a synthetic phenotype due to loss of flp-1 and daf-10. Besides the Caenorhabditis genus, flp-1 sequelogues have been identified in 19 other nematodes, representing 7 clades and including free-living and parasitic nematodes. Hence, understanding the function of flp-1 may provide insights into neuropeptide function in other nematodes, including parasitic nematodes, as well as in mammalian systems

Circuits and Behavior Poster Session 97 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 98 Branching Out: Determining the function of IL2 neurons in C. elegans dauer and non-dauer animals Alina Rashid1, Rebecca Androwski1, Nathan Schroeder2, Juan Wang1, Lenny Haas1, Maureen Barr1 1Rutgers University, 2University of Illinois at Urbana-Champaign

When under stressful conditions C. elegans proceed into dauer, an alternative to the L3 stage. We previously found that morphological changes in the worm are accompanied by neurological changes. In dauer, the four IL2Qs (inner labial 2 quadrant) neurons undergo extensive dauer-specific dendrite arborization along the length of the dendrite. The two IL2L (inner labial 2 lateral) neurons do not undergo arborization along the dendrite, but branch at the nose of the worm forming a crown-like structure [1]. When in dauer, worms perform nictation, a behavior where they stand on their tail and wave around in order to be transported to a more favorable environment. The IL2 neurons have been shown to be important for nictation [2] as well as for the maintenance of the dauer stage. [3] Proprotein convertases (PCs) are responsible for the cleavage of proproteins into their biologically active forms. The human PC family contains seven proteins that cut at dibasic cleavage sites while there are four PCs in C. elegans (KPC-1, BLI-4, AEX-5, and EGL-3). KPC-1 is homologous to furin, a PC essential for mammalian development and associated with numerous pathologies. We previously found that kpc-1 is required for dauer-specific dendrite arborization in the IL2Qs. To determine whether kpc-1 plays a role in dauer recovery, we placed dauers on seeded plates and observed the resumption of pharyngeal pumping four hours after the placement. kpc-1 mutant showed defects in dauer recovery compared to N2. We are currently determining whether genes known to act in the IL2 neurons are important in dauer animals for IL2 branching, recovery, or maintenance. For example, the kinesin-3 klp-6 is exclusively expressed in the IL2 neurons of hermaphrodites, regulates the ciliary kinesin-2 motors [4] and Barr 2011), and is required for nictation behavior [2] klp-6 is not required for branching or dauer recovery, suggesting that these pathways are genetically separable. To further understand IL2 function in dauer and non-dauer animals, we used cell-type specific RNA-seq. We are in the process of characterizing these new candidates.

Reference(s) 1. Schroeder et al (2013). Curr Bio, 23(16), 1527-35. 2. Lee et al (2011). Nat Neurosci, 15(1):107-12 3. Ouellet et al (2008). Dev Bio, 135, 2583-2592. 4. Morsci&Barr(2011). Curr Bio, 21(14):1239-44.

98 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 99 Identification of molecules downstream of the insulin/PI3K pathway involved in the regulation of salt chemotaxis learning Naoko Sakai1, Masahiro Tomioka1, Takeshi Adachi2, Hirofumi Kunitomo1, Yuichi Iino1 1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2Department of Biological science, Faculty of science, Kanagawa University

A major goal in neuroscience is to elucidate the molecular mechanisms for integrating different kinds of information. C. elegans is an excellent model organism for neuroscience research because it exhibits a complex form of behavioral plasticity despite its simple nervous system. Previously, we have found that C. elegans changes responses to external salt concentrations depending on food conditions. When worms are cultivated on a medium that contains sodium chloride (NaCl) and bacterial food, they are attracted to the NaCl concentration at which they are grown. In contrast, after exposure to NaCl under starvation conditions, they learn to avoid the NaCl concentration. This behavioral change is called “salt chemotaxis learning”. We previously reported that the insulin/PI 3-Kinase (PI3K) signaling pathway is involved in salt chemotaxis learning. This pathway is widely conserved among many species including mammals, and regulates a variety of phenomena such as tumor progression and metabolism. Recently, this pathway was reported to be involved also in learning and memory. However, so far, a mechanism by which this pathway regulates neuronal plasticity is poorly understood. In C. elegans, mutants of the insulin/PI3K pathway components show strong defects in salt chemotaxis learning. Although the DAF-16/FOXO-dependent transcription is a major output of the insulin/PI3K pathway in the control of developmental decision and aging, other machinery downstream of the insulin/PI3K pathway has been predicted in salt chemotaxis learning. In this study, we aimed to dissect the molecular mechanisms downstream of the insulin/ PI3K pathway in chemotaxis learning. Through a genetic screen for suppressors of the abnormal behavior in daf-18, which is a negative regulator of the insulin/PI3K pathway, we obtained four suppressors and found that three of them have point mutations in goa-1, which encodes an alpha subunit of Go. Therefore interaction between the Go protein signaling and the PI3K signaling may be important for salt chemotaxis learning. We also investigated the participation in chemotaxis learning of regulator of G protein signaling (RGS) proteins and GPB-2, an ortholog of Gβ, which were previously reported to regulate GOA-1. As a result, RGS mutants and the gpb-2 mutant showed various defects in salt chemotaxis learning, suggesting that they also regulate salt chemotaxis learning along with GOA-1. Furthermore, we performed a candidate screen to identify downstream molecules of AKT- 1 and found that mutants of several members of the TOR pathway show defects in salt chemotaxis learning.

Circuits and Behavior Poster Session 99 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 100 Light induces a pharyngeal gag reflex by C. elegans Steven Sando1, Nikhil Bhatla2, Bob Horvitz1 1HHMI, Department of Biology, MIT, 2HHMI, Department of Brain and Cognitive Science, MIT

Despite lacking the photoreceptor molecules identified in other organisms, C. elegans avoids violet and ultraviolet light (Edwards et al., 2008, PLoS Biol., 6:e198) (Ward et al., 2008, Nature Neurosci. 11:916), suggesting a previously uncharacterized mode of light detection might be present in the worm. This avoidance behavior depends on the gustatory receptor homolog lite-1, but the molecular mechanism of light-detection is unknown. Previous work in our laboratory has shown that the pharynx of C. elegans also responds to light (Bhatla and Horvitz, 19th International C. elegans Meeting, 2013). When exposed to bright light, worms rapidly inhibit pumping via the pharyngeal neurons I1 and I2 and the gustatory receptor homologs lite-1 and gur-3. Bhatla and Horvitz suggested that light acts via generation of an oxidant, perhaps the reactive oxygen species (ROS) hydrogen peroxide. This mechanism of light-detection would be novel. We have observed that during light exposure worms emit bubbles from their mouths, as if reversing pharyngeal flow during a gag reflex. Bubbling occurs several seconds after pumping inhibition, during a transient burst of pumps dependent on the M1 neuron (burst pumping). Using high frame-rate videos, we observed the light-response of worms eating mineral oil. Light reversed the flow of oil in the pharynx, causing spitting. We identified a difference between feeding and burst pumping that might lead to spitting: during feeding, which is comprised of rhythmic contractions of pharyngeal muscle that draw food inwards, the anterior-most region of pharyngeal muscle 3 (pm3) relaxes before the rest of the procorpus, closing the anterior end of the pharynx and trapping food (Fang-Yen et al., 2009, PNAS, 106: 20093); however, during burst pumping the anterior-most region of pm3 remains contracted. This contraction holds the anterior end of the pharynx open, permitting expulsion of material when the corpus relaxes. In some circumstances, light induces vomiting of intestinal contents. We are interested in the cellular mechanisms of the pharyngeal light-response and the molecular mechanism of light-detection. We are pursuing this problem using behavioral and genetic analyses, neuronal ablations, and heterologous expression of lite-1 and gur- 3 in Xenopus oocytes. Studying the pharyngeal light-response might reveal ways the neuromuscular system encodes behavior and also identify molecular pathways for the detection of ROS, physiologically important molecules implicated in many aspects of biology and disease.

100 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 101 Neural basis of plasticity and bidirectionality of klinotaxis Yohsuke Satoh1, Hirofumi Sato1, Hirofumi Kunitomo1, Yuichi Iino1 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo.

Klinotaxis is a behavioral strategy for chemotaxis in C. elegans: The animals detect dorso- ventral (D-V) chemical gradient during forward locomotion and gradually curve towards higher (or lower) concentrations. Tendency of klinotaxis is not fixed but is plastic depending on previous experience. For example, an animal curves to the side with higher NaCl concentrations after cultivation with a high NaCl concentration (positive klinotaxis to NaCl), and curves to the lower side after cultivation with a low concentration (negative klinotaxis to NaCl). Here, we report that C. elegans realizes this plastic bidirectional klinotaxis by differential use of at least two distinct neural circuits. First, we reproduced bidirectional klinotaxis to NaCl by artificial phasic activation of ASER chemosensory neuron, which responds to NaCl concentration decrease, by optogenetics. Activating ASER in synchrony with head swing after cultivation with a high NaCl concentration induced repulsive curving in response to ASER activation, which corresponds to positive klinotaxis to NaCl, and the same phasic activation pattern after cultivation with a low NaCl concentration induced positive curving that corresponds to negative klinotaxis. Next, we examined involvement of the interneurons downstream of ASER by ablating or activating these interneurons. Phasic activation of AIY interneurons induced strong attractive curving in response to AIY activation regardless of previous experience. Ablation of AIY neurons abolished attractive curving induced by ASER activation. However, ablation of AIY failed to affect repulsive curving induced by ASER activation. These results suggest that plastic bidirectional klinotaxis to NaCl is regulated by switching between a microcircuit that contains AIY and those that do not contain AIY. We will further discuss about the involvement of other interneurons that are postsynaptic to ASER.

Circuits and Behavior Poster Session 101 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 102 Swip-10/Mblac1: Identification of a Novel Regulator of Dopamine Signaling Linked to Glial Control of Extracellular Glutamate Homeostasis Chelsea Snarrenberg1, Andrew Hardaway2, Sarah Whitaker3, Cassandra Retzlaff1, Haigang Gu3, Zhaoyu Li4, Qi Zhang3, Shawn Xu4, Mausam Ghosh5, Michael Robinson5, Randy Blakely2 1Neuroscience Graduate Program, Vanderbilt University School of Medicine, Nashville, TN USA, 2Department of Pharmacology, Psychiatry, Vanderbilt University School of Medicine, Nashville, TN USA, 3Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN USA, 4University of Michigan, Ann Arbor, MI, USA, 5Children’s Hospital of Pennsylvania, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Multiple brain disorders are associated with disrupted dopamine (DA) signaling including Parkinson’s disease, schizophrenia and addiction. In C. elegans, an inability to clear synaptic DA results in swimming induced paralysis (Swip). A screen for genes that regulate DA signaling identified two mutants, vt29 and vt33, that exhibit DA-dependent Swip and alter the coding sequence of an unstudied gene, F53B1.6, now named swip-10. Sequence analysis reveals the swip-10 mutations lie within a highly conserved metallo-betalactamase domain. Swip-10 promoter:GFP fusion reporter experiments reveal gene expression in C. elegans glial cells in vivo, consistent with findings that glial-expressedswip-10 genomic DNA and cDNA constructs rescue Swip behavior. Vertebrate glial cells are known to regulate neuronal signaling by control of extracellular glutamate (Glu) homeostasis. Furthermore, after demonstrating that swip-10 animals have elevated DA neuron excitability we hypothesize that swip-10 functions in worm glia to regulate the extracellular Glu that normally acts on worm DA neurons to modulate motor behavior. In support of this hypothesis, eat-4 animals that exhibit defective vesicular Glu release rescue the paralysis of swip-10 animals. Additionally, we found that mutation of multiple Glu transporters generates DA-dependent Swip. Moreover, mutation of the glutamate receptors genes, glr-4, glr-6 and mgl-1, suppresses the swip-10 paralysis phenotype. To explore the connection of these observations to the mammalian central nervous system, we cloned the putative mouse swip-10 ortholog, Mblac1, and overexpressed the protein in 3T3 cells. In this system, we observed a reduction in Na+-independent Glu transport activity - derived from the glutamate/cystine exchanger (XC ). Finally, viral overexpression of Mblac1 in astrocytes alters synaptic Glu signaling when co-cultured with hippocampal neurons. Together, these studies point to a novel mechanism that can influence glial-dependent extracellular Glu homeostasis. Current experiments seek to identify Swip-10/Mblac1 substrates using LC-MS/ MS approaches. Disruptions in glutamate signaling lead to neurological and neuropsychiatric disorders, including diseases that feature disrupted DA signaling. This suggests that further studies of swip-10/Mblac1 may provide insight into the mechanisms disturbed in various brain disorders as well as indicate a novel diagnostic and therapeutic target.

102 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 103 Study of transcriptome regulating a dispersal behavior in C. elegans Sangwon Son1, Harksun Lee1, Junho Lee1 1IMBG, School of Biological Sciences, Seoul National University

Many nematodes show a stage-specific behavior called nictation in which a worm stands on its tail and waves its head in three dimensions. In C. elegans, nictation is a dauer-specific behavior which is an alternative stage for long-term survival in harsh environments like high temperature, starvation, or overpopulation. In the previous study, it was discovered that nictation is initiated by IL2 head neurons (1), and that IL2 neurons show a distinct morphology in dauer stage different from that in nondauer. The different action and morphology of IL2 neurons in dauer stage implies that dauer-specific gene expression is different from that of nondauer. Here I am analyzing IL2 specific transcriptome in dauer through mRNA tagging method (2).

Reference(s) 1. Lee et al., Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons, Nature Neuroscience 15, 107-112 (2012). 2. Takayama et al., Sigle-cell transcriptional analysis of taste sensory neuron pair in Caenorhabditis elegans, Nucleic Acids Research, 1-12 (2009).

Circuits and Behavior Poster Session 103 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 104 The AIB interneurons potentially function as a bimodal switch to integrate an array of sensory inputs to modulate nociception in C. elegans Philip Summers1, Robert Layne1, Bruce Bamber1, Amanda Ortega1, Richard Komuniecki1 1University of Toledo

Sensory inputs are extensively processed and integrated prior to decision-making. In the present study, we have used the C. elegans model to examine how multiple sensory inputs are integrated by the two AIB interneurons prior to the initiation of aversive behavior to dilute 1-octanol mediated by the two ASH sensory neurons. ASH-mediated aversive behavior is complex and can be divided into three phases: the time taken to initiate backward locomotion (BL), the length of the BL and the decision to move forward or reverse after BL is complete. Each phase is modulated independently. For example, on food or 5-HT animals initiate BL rapidly in about 5 s and continue forward after BL is complete, and off food they initiate BL more slowly in about 10 s and reverse and proceed in the opposite direction. Ablation or selective inhibition of the downstream AIBs that receive inputs from the ASHs and an array of additional sensory neurons dramatically decrease the rate of spontaneous reversal, but they increase aversive responses to dilute 1-octanol, i.e., decreases the time taken to initiate BL. In contrast, AIB inactivation abolishes any reversal after BL is complete, suggesting that the modulation of spontaneous reversal is most comparable with the post-initiation responses associated with sensory-evoked behaviors. Importantly, activation/inactivation of the AWC, ASE or ADL sensory neurons also has the capacity to modulate the activity state of the AIBs and hence ASH-mediated reversal. For example, the selective modulation of sensory signaling by 1) the addition of specific ligands, 2) the selective RNAi knockdown or overexpression of eat-4 that encodes a vesicular glutamate transporter essential for glutamatergic signaling and 3) the selective RNAi knockdown or overexpression of individual glutamate receptors in the AIBs, identified by the selective eat-4 overexpression in individual sensory neurons in glutamate receptor null backgrounds and confirmed by selective RNAi knockdown and direct Ca++ imaging and electrophysiology, all have the capacity to modulate AIB signaling and bimodally alter ASH-mediated aversive responses, either by stimulating responses on food from about 10 to 5 s or abolishing food or 5-HT stimulation. These studies are continuing to examine how multiple sensory inputs are integrated by the AIBs in the modulation of both spontaneous and sensory-evoked reversals and may provide useful insights into the altered multi-sensory integration associated with disorders such as autism spectrum disorders, depression and schizophrenia.

104 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 105 Novel optogenetic tools for silencing neural activity in C. elegans Megumi Takahashi1, Ayako Okazaki1, Takashi Tsukamoto1, Yuki Sudo1, Shin Takagi1 1Division of Biological Science, Graduate School of Science, Nagoya University

Archearhodopsin-3 (Arch), a light-driven proton pump activated with green light (~550 nm), can be utilized for neural silencing in animals including C. elegans. To control neural activity under various situations, it is desirable to have optogenetic tools with a variety of altered properties such as action spectrum or ion selectivity. Here we report application of two light-driven proton pumps to C. elegans. One is ArchT, whose light sensitivity is reported to be 3.3 times higher than that of Arch in mammalian neurons. The other is a variant of Arch generated through structure-guided mutagenesis, Mid-Arch, whose absorption maximum shifts to 500 nm. To examine their activity in worms’ cells, each pump was expressed in the body-wall muscles or neurons of the entire body of worms, and the percentage of worms which are paralyzed by light was scored as an index of silencing of the cells. Worms expressing ArchT stopped locomotion when illuminated with a green light, though the silencing activity of ArchT was not significantly different from that of Arch. Worms expressing Mid-Arch also stopped locomotion with light. The 500 nm-light affected worms’ locomotion more efficiently than the 550 nm-light, which is consistent with the results of in vitro studies. These pumps will be helpful to study the function of C. elegans neural networks. In addition to this, we have also confirmed that a novel proton pump derived from a eubacteriumThermus thermophilus, whose activity in eukaryotic cells had not been examined so far, can serve as a light-driven silencer. This is the first report on optogenetic application of “eubacterial” proton pump. Thus, locomotion of C. elegans is a convenient in vivo assay system to assess the utility of candidate molecules as optogenetic tools, which can then be applied to other higher animals.

Circuits and Behavior Poster Session 105 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 106 The AWC and ASI sensory neurons contribute to starvation- dependent plasticity in thermotaxis behavior Asuka Takeishi1, Piali Sengupta1 1Brandeis University

Behavioral responses are regulated by experience-dependent modulation of gene expression and neuronal function. However, the molecular and neuronal mechanisms by which experience modifies behavioral output in response to a given stimulus are not fully understood. Many behaviors in C. elegans are regulated by experience. In particular, the presence of food and feeding status regulates the behavioral strategy of C. elegans on spatial thermal gradients, providing an attractive model system in which to study the mechanisms of experience-dependent behavioral plasticity. While well-fed worms migrate to colder temperatures (negative thermotaxis behavior) when they encounter temperatures higher

than their cultivation temperature Tc, starvation for ≥2hrs suppresses this behavior. We found that the AWC and ASI sensory neurons play a role in mediating this starvation-dependent behavioral plasticity. AWC-ablated animals retain negative thermotaxis behavior even after starvation. Although ablation of ASI neurons alone do not affect this plasticity, ablation of both AWC and ASI neurons abolishes starvation-dependent suppression of negative thermotaxis. Calcium imaging experiments show that the AWC neurons are hyperactivated after starvation, and may regulate thermotaxis behavior via insulin signaling. Current work is aimed at further exploring the mechanisms by which AWC activity is regulated by food deprivation, and how this information is communicated to the circuit to modulate behavior.

106 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 107 The roles of biogenic amines on feeding state-dependent thermotactic behavior in C. elegans Satomi Tsukamoto1, Shunij Nakano1, Ikue Mori1 1Department of Biological Science, Graduate School of Science, Nagoya University, JAPAN

Biogenic amines are important neurotransmitters or neuromodulators by which to regulate behaviors in both vertebrates and invertebrates. We are investigating thermotaxis behavior in C. elegans in order to address as to how biogenic amines regulate neural circuits. After cultivation at a certain temperature with food, the animals migrate to that cultivation temperature, whereas they disperse on the thermal gradient after cultivation without food. A simple neural circuit has been identified for this feeding state-dependent thermotactic behavior (Mori and Ohshima, 1995; Kuhara, Okumura et al., 2008). We previously reported that exogenous serotonin and octopamine can mimic well-fed or food-deprived states, respectively (Mohri et al., 2005), suggesting that endogenous serotonin and octopamine might modulate thermotaxis. In the current study, we found that octopamine-deficient tbh-1 mutants stayed at the cultivation temperature even when they were starved, while serotonin-deficientbas-1 and tph- 1 mutants dispersed from the cultivation temperature in fed condition. Thus, octopamine and serotonin are required for starvation and food signaling, respectively. To identify octopamine receptor required for thermotaxis, we evaluated thermotaxis behavior of mutants for previously discovered octopamine receptor genes (ser-3, ser-6, octr-1, tyra-3) and found that only octr-1 mutants showed a phenotype similar to that of octopamine-deficient tbh-1 mutants. To reveal the molecular mechanism(s) by which monoamines control thermotaxis behavior, we began focusing on PKA (protein kinase A), a kinase known to act downstream of monoamines. We have recently found that two mutants, kin-2(ce179) and gsa-1(ce81), both of which have an elevated activity of PKA, displayed an athermotactic phenotype, suggesting that PKA is involved in thermotaxis. This defect of kin-2(ce179) mutants was rescued by pan-neuronal expression of kin-2 cDNA, indicating that PKA might act in the nervous system to regulate thermotaxis. We are currently attempting to identify the site of action of PKA important for the behavior. Our results so far showed that octopamine and serotonin are physiological modulators of thermotaxis and drive animals to the opposite behavioral states. We believe that further study will elucidate molecular and neural circuit mechanisms of biogenic amine signaling that control behaviors in many species.

Circuits and Behavior Poster Session 107 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 108 An aptf-1 transcription factor is required for RIS neuron to control sleep onset in C. elegans Michał Turek1, Ines Lewandrowski1, Henrik Bringmann1 1Max Planck Institute for Biophysical Chemistry

Sleep or sleep-like behavior is found in all animals that have a nervous system and have been studied carefully. It is characterized by periods of quiescence. Sleep thus appears essential for animals that have a nervous system, and it may be evolutionarily conserved. Quiescence behavior is also found in C. elegans larvae, where it is coupled to development: prior to each of the four molts, larvae go through a phase of behavioral quiescence called lethargus that has long been known but has recently been shown to have properties that define sleep in higher organisms, such as an absence of voluntary movement, reversibility, reduced responsiveness to stimulation, homeostatic regulation, a relaxed body posture, and reduced neuronal activity. Using a genetic screen in C. elegans larvae, we have found that the AP2 transcription factor aptf-1 is required for sleep-like behavioral quiescence in C. elegans. aptf-1 is expressed in only a few interneurons, and expression in GABAergic neuron RIS is required for quiescence. Using calcium imaging, we found that RIS is active specifically at the transition from wake- like to sleep-like behavior. Using optogenetics, we show that aptf-1-expressing neurons can induce sleep-like quiescence. The ability to induce quiescence is conferred by aptf-1. Ablation of RIS caused a dramatic reduction in quiescence and RIS-dependent quiescence requires not GABA but neuropeptide signaling. Thus, sleep-promoting neurons are an important circuit principle that governs sleep-like quiescence of C. elegans. Sleep-like behavior in C. elegans and sleep in higher organisms thus both use common circuit principles to achieve quiescence.

108 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 109 Characterization of a disinhibitory motor circuit in C. elegans Khursheed A. Wani1, Beverly J. Piggott2, X. Z. Shawn Xu1 1Life Sciences Institute, University of Michigan, 2Departments of Physiology, Biochemistry and Biophysics, University of California, San Francisco

Locomotion is a predominant motor program in C. elegans. During locomotion, C. elegans usually moves forward that is periodically interrupted by brief reversals. The neurons that control forward and backward locomotion are well characterized. The command interneuron, AVB, is known to drive forward locomotion, and AVA, AVD, and AVE command interneurons control backward locomotion. We previously showed that in addition to AVA, AVD, and AVE, a disinhibitory circuit comprised of AIB and RIM interneurons also plays a role in the initiation of backward locomotion (Piggott et al., 2011). In this circuit, AIB acts upstream of RIM to relieve the inhibitory effect of RIM on backward locomotion, thereby initiating reversals. We are interested in dissecting the cellular and molecular mechanisms downstream of RIM in the AIB-RIM disinhibitory circuit. To this end, we are systematically identifying the cellular and molecular targets that act downstream of RIM, using genetic, behavioral and optogenetic approaches. Our preliminary observations suggest that both synaptic and extrasynaptic mechanisms take part downstream of RIM, to regulate backward locomotion.

Reference(s) 1. Piggott BJ, Liu J, Feng Z, Wescott SA, Xu XZ (2011). The neural circuits and synaptic mechanisms underlying motor initiation in C. elegans. Cell 147, 922-933

Circuits and Behavior Poster Session 109 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 110 Injection of endogenous As-NLP-22 into intact Ascaris suum causes a decrease in locomotory behavior Colin Wruck1, Jennifer Knickelbine2, Antony Stretton2 1School of Education, University of Wisconsin-Madison, 2Department of Zoology, University of Wisconsin-Madison

The purpose of this study was to examine the effect of the injection of endogenous neuropeptide As-NLP-22 (SLASGRWGLRPamide) on the locomotory behavior of the parasitic nematode Ascaris suum. Our laboratory has previously identified As-NLP-22 in the ventral cord inhibitory motor neurons (DI and VI; see accompanying poster by J. Knickelbine et al. and C. Konop et al.). Whole nematodes were injected with 0.1 mL of either a Ringer control solution (N=9), 10 µM As-NLP-22 (N=7), 1.0 µM As-NLP-22 (N=7), or 0.1 µM As-NLP-22 (N=8). Previous dye injection experiments showed that there is an approximately 10-fold dilution of peptide at the injection site (Reinitz & Stretton, J Comp Physiol A. 1996). After injection, nematodes were transferred to a glass tube with diameter similar to that of the porcine small intestine. They were otherwise unrestricted and allowed to display locomotory behavior. Behavior was observed for 60 minutes post-injection, and photographs were taken every 5 min. Results demonstrate that As-NLP-22 inhibits normal behaviors including maintained body waveforms, propagating waves and associated body propulsion, loops, hairpins, etc., especially within the anterior portion of the nematode where locomotory behavior is most frequent. There is a decrease in inhibitory effects as the As-NLP-22 concentration is reduced, with some nematodes in the 1.0 µM experimental group regaining activity towards the end of 60 minute trials and the majority of nematodes injected with 0.1 µM As-NLP-22 remaining unaffected. After an initial delay, no nematodes injected with 10 µM As-NLP-22 regained anterior function. The doses at which these behavioral responses are observed are comparable to the doses at which As-NLP-22 affects acetylcholine (ACh)-induced muscle contraction (see accompanying poster by J. Knickelbine). Such inhibition of locomotory behavior may make the worm more susceptible to expulsion by intestinal peristalsis, making it a potential target for new anthelmintics.

110 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 111 K+/Cl- Cotransporter KCC-3 regulates thermotaxis behavior in C. elegans Atsushi Yoshida1, Shunji Nakano1, Takamasa Suzuki2, Tetsuya Higashiyama3, Kunio Ihara4, Ikue Mori1 1Division of Biological Science, Nagoya University, 2JST ERATO, Nagoya University; Institute of Transformative Bio-Molecules, Nagoya University, 3Institute of Transformative Bio-Molecules, Nagoya University, 4Center for Gene Research, Nagoya University

C. elegans show thermotaxis, in which animals sense and memorize cultivation temperature and associate it with feeding state. Although previous studies have revealed the neural circuit and molecules important for thermotaxis, how this behavior is achieved at the cellular and molecular level remains to be fully uncovered. To further reveal the mechanism underlying thermotaxis, we have isolated 23 mutants defective in thermotaxis. We began our analysis with nj90, nj94 and nj100 mutants, which exhibit athermotactic phenotypes. Genetic mapping of these mutations revealed that these three mutations mapped to LG II. Complementation tests among these mutants suggest that nj90, nj94 and nj100 are allelic to one another. To identify the gene mutated in these isolates, we performed whole-genome sequencing and looked for mutations present in the genomic region to which nj94 mutation mapped. The result revealed that each of the three mutants possesses a mutation in the single gene, kcc-3, which encodes a cotransporter of potassium and chloride ions. We then performed rescue experiments and found that the introduction of a wild-type kcc-3 genomic DNA into nj94 mutants recovered the thermotaxis defect. In addition, two deletion mutants of kcc-3 displayed abnormal thermotaxis. From these analyses, we concluded that nj90, nj94 and nj100 are alleles of kcc-3 and that KCC-3 plays an important role in thermotaxis. A previous study reported that kcc-3 is expressed in glia-like cells, including sheath and socket cells (Tanis et al., 2009). Our study of kcc-3 may further provide a new finding of glia’s role in the nervous system. Characterization of this new gene likely reveals a novel mechanism by which glial cells regulate the dynamics of the neural circuit, contributing to the generation of thermotaxis behavior.

Circuits and Behavior Poster Session 111 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 112 Investigating the neural mechanism underlying a hypertonic response in Caenorhabditis elegans Jingyi Yu1, Yun Zhang1 1Department of Organismic and Evolutionary Biology, Center for Brain Science, Harvard University

Environmental osmotic changes can interfere with and potentially damage molecular and cellular functions by influencing cytoplasmic osmolarity. Preventing this damage is an important survival skill for animals that live in environments with unstable osmolarity. It is known that in addition to hormonal responses, mammals also use behavioral strategies to maintain a stable cellular osmolarity , such as water-seeking behaviors driven by thirst. However, little is known about the molecular mechanisms or neural circuits underlying these behavioral responses. Previously, it has been shown that Caenorhabditis elegans generates an acute aversive response to high osmotic shock (Bargmann et al. 1990). Here, we show that C. elegans also produces an aversive response when exposed to prolonged hypertonic conditions. Compared with the response to high osmotic shock, this hypertonic response is slower, evoked by lower osmolarity, and involves increased turning rate over time. It does not require the same molecular mechanism as the response to high osmotic shock, since osm-9 mutants show a wild-type response in the hypertonic condition (Liedtke et al. 2003). The behavioral response is specific to the osmotic difference between internal and external environments, and classical mechanosensory or chemosensory mutants do not show any defect. We have tested aquaporin and other channel mutants, and partially mapped the circuit. Currently, we are looking for sensory mechanisms that regulate this behavior.

112 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 113 A role for a T-type calcium channel in serotonin-mediated behavior Kara Zang1 1NYU Medical Center Skirball Institute

Serotonin is a neuromodulator that controls behavioral states, such as appetite and mood. In C. elegans, serotonin released from the hermaphrodite specific neurons (HSNs) drives egg laying. HSNs express the inhibitory G-protein coupled receptor (GPCR) EGL-6, and animals with a gain-of-function (gf) mutation in EGL-6 exhibit excessive inhibition of serotonin release and become bloated with unlaid eggs. To find novel regulators of serotonin signaling, we performed a screen for suppressors of egl-6(gf) and found that a mutation in the T-type calcium channel cca-1 causes strong suppression of the egl-6(gf) phenotype. cca-1(n5209) suppresses the egg-laying defect of egl-6(gf) animals but it causes no overt egg-laying phenotype on its own. We have characterized the effect of the n5209 mutation on channel function and found that n5209 shifts the voltage dependence of activation of CCA-1

currents. Using a Promcca-1::GFP reporter transgene, we found that cca1 is not expressed in the HSNs, but in another neuronal cell-type in the egg-laying system: the ventral cord neurons (VCs). A role for CCA-1 in muscles has been reported (Steger et al. 2005; Shtonda and Avery 2005), but its function in the C. elegans nervous system not known. In other organisms, T-type channels contribute to pacemaker activity and regulate circuit excitability. Therefore, we hypothesize that cca-1 regulates the excitability of the C. elegans egg-laying system and that cca1(n5209) suppresses the egl-6(gf) phenotype by altering the excitability of the circuit in which the HSNs are embedded.

Circuits and Behavior Poster Session 113 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 114 TMC-1 attenuates C. elegans development and sexual behavior in an alien food environment Liusuo Zhang1, L Rene Garcia1 1Howard Hughes Medical Institute, Texas A&M University

Diets affect growth, behavior and lifespan; however, mechanisms involved in coordinating these processes with the potential to adapt in non-optimal food sources are unclear. Here we capitalize on the observation that C. elegans develop and copulate poorly in the chemically- definedCeMM medium, to show that a transmembrane channel-like protein, TMC-1, restrains development and sex in this novel nutritional environment. We found that tmc-1 mutations relax constraints allowing larvae to develop faster and adult males to mate better than wild type. In CeMM, wild type uses tmc-1-expressing pharyngeal MC cholinergic motor neurons and post synaptic pharyngeal EAT-2 acetylcholine receptors to slow development. In addition to the well-described regulation of pharyngeal muscle contraction, we suggest that the MCs might also regulate a stress-induced developmental signaling function facilitated by the pharynx. We found that the pharyngeal expressed microRNA mir-251 is also required to attenuate development, suggesting that the pharynx might use microRNAs to attenuate development in alien nutrient environments. Without tmc-1, the accelerated growth requires insulin-like peptide signaling, and a metabolic response can be measured after 2 hours of CeMM exposure, by increased mRNAs used for catabolism. In addition, we found that TMC- 1 affects post-development behaviors by repressing both appetitive and consumatory male sexual behavior on CeMM. Thus TMC-1 attenuates both growth and sex under nutritional stress, and indicates that single regulators can be genetically modified to promote reproductive fitness to new environments.

114 Circuits and Behavior Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 115 Natural Variation of Pathogen-induced Neuronal Gene Expression in C. elegans Zoë Hilbert1, Joshua Meisel1, Dennis Kim1 1Department of Biology, MIT

Genetic variation is the raw material for natural selection, but little is known about the molecular and neuronal basis of genetic variation in behavior. We have been investigating natural variation in the neuronal expression of a TGF-ß signaling molecule, DAF-7, that plays a pivotal role in the regulation of diverse behaviors of C. elegans such as the dauer developmental decision, reproductive egg laying, and feeding. Normally expressed only in the ASI pair of chemosensory neurons, recent work in our lab (J. Meisel and D. Kim, unpublished data) has demonstrated that daf-7 expression is induced in an additional pair of neurons, the ASJ neurons, upon exposure to the bacterial pathogen, Pseudomonas aeruginosa, and that this change in expression is functionally important in producing a pathogen avoidance behavior. To investigate natural variation in this pathogen-induced response, we have examined a panel of 25 C. elegans wild isolate strains for differences in pathogen-induced daf-7 expression. We observe a wide range in the kinetics of daf-7 expression changes in the response of wild strains to pathogenic bacteria.We have identified distinct genomic loci underlying these phenotypes and are currently working to define the natural polymorphisms that cause genetic variation in the pathogen-induced neuronal expression of daf-7.

Comparative and Evolutionary Neurobiology Poster Session 115 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 116 Mechanisms of Axon Regeneration in the Aging Nervous System Alexandra Byrne1, Walradt Trent1, Kathryn Gardner2, Austin Hubbert1, Valerie Reinke2, Marc Hammarlund1 1Department of Genetics; Program in Cellular Neuroscience, Neurodegeneration and Repair; Yale University School of Medicine, 2Department of Genetics, Yale University School of Medicine

Injured axons lose their ability to regenerate with increased adult age. We found that axon regeneration is inhibited in the adult nervous system by the insulin/IGF1 receptor daf-21. Analysis of mosaic animals revealed that daf-2’s canonical downstream transcription factor, daf-16, regulates regeneration within adult neurons, independently of its role in lifespan determination. Several lines of evidence indicate that daf-16 acts by regulating expression of the essential and conserved regulator of regeneration, dlk-1. First, neuronal DAF-16 binds to the upstream regulatory region of dlk-1. Second, dlk-1 expression is dependent on insulin signaling in aged animals. Finally, regeneration in aged animals is dependent on dlk-1. In addition to identifying dlk-1 as a likely downstream transcriptional target of DAF-16 in neurons, we find that the genome-wide binding pattern of DAF-16 differs in the nervous system from the intestine. This finding suggests that DAF-16 coordinately regulates the expression of its many downstream targets by binding to different regions of the genome with spatial and temporal specificity. Ongoing analysis of these tissue-specific DAF-16 binding profiles may identify neuron specific targets of DAF-16 that mediate its role in neuronal healthspan. Next, we asked whether other components of the insulin pathway regulate axon regeneration. We found that daf-18, which functions within the insulin pathway to regulate lifespan, functions in parallel to the insulin pathway to regulate regeneration. Specifically, although daf-18 acts antagonistically to daf-2 to regulate lifespan, we found that both daf-2 and daf-18 inhibit regeneration. Moreover, daf-18 inhibits regeneration via TOR (Target of Rapamycin) signaling, a function that is independent of daf-16. Together, our findings indicate that axon regeneration in the mature nervous system is regulated by a conserved set of genes and pathways that include dlk-1 and daf-18. Moreover, our data demonstrate that C. elegans is an ideal model to disentangle the precise downstream mechanisms that regulate axon regeneration in the adult nervous system.

Reference(s) 1. Byrne, A.B., et al., Insulin/IGF1 signaling inhibits age-dependent axon regeneration. Neuron, 2014. 81(3): p. 561-73.

116 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 117 Lesion conditioned axon regeneration in C. elegans Samuel Chung1, James Shay1, Christopher Gabel1 1Dept. of Physiology and Biophysics, Photonics Center, Boston University School of Medicine

Employing subcellular-resolution femtosecond laser ablation we demonstrate a strong lesion conditioning effect in C. elegans, where lesion of the sensory dendrite of amphid neurons triggers targeted regeneration of transected axons. We find that this lesion-dependent regeneration readily occurs in Dual Leucine Zipper Kinase/DLK-1 mutant animals that otherwise display no axon regeneration. Similar to mammalian lesion conditioning, pharmacological or genetic disruption of the L-type voltage gated calcium channels stimulates this regeneration in C. elegans. Likewise, fluorescent calcium imaging post surgery demonstrates a reduction in sensory activity following dendrite lesions but no such reduction from an axon lesion alone. We find a link between C. elegans lesion- conditioned regeneration and ectopic axon outgrowth observed in the same sensory neurons. Activity-reducing genetic mutations that cause ectopic outgrowth, including those specifically disrupting cyclic nucleotide mediated sensory transduction, also stimulate lesion-conditioned axon regeneration without a dendrite lesion. Finally, as with ectopic outgrowth, we find that lesion conditioned axon regeneration is mediated by CaMKII/UNC- 43 activity, potentially linking it to pathways modulating developmental neurite outgrowth. Our work demonstrates direct genetic, molecular, and cellular links between three types of axon outgrowth: C. elegans lesion conditioning, C. elegans ectopic outgrowth, and mammalian lesion conditioning. These findings establish C. elegans as a novel model system for the study of lesion conditioned neuronal regeneration and are defining specific molecular pathways that modulate it.

Disease Models and Regeneration Poster Session 117 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 118 Neuronal fusion induced by UNC-70/β-spectrin dependent axonal injury requires the apoptotic recognition pathway Sean Coakley1, Brent Neumann1, Rosina Giordano-Santini1, Casey Linton1, Yi Zhang2, Hengwen Yang3, Ding Xue2,3, Massimo A Hilliard1 1The University of Queensland, Queensland Brain Institute, 2Tsinghua University, School of Life Sciences, 3University of Colorado, Department of Molecular, Cellular, and Developmental Biology

Axonal regeneration is a major component of neuronal repair that follows traumatic injuries of the nervous system. Functional regeneration takes place when the damaged nerve regains contact with its target tissue, however how this occurs is poorly understood. In C. elegans, following transection, a regenerating axon can fuse to its distal, separated axonal fragment to restore the original axonal tract (Ghosh-Roy et al. J Neurosci 2010, Neumann et al. Dev Dyn 2011). This fusion event is highly specific and requires, at least partially, the fusogen EFF- 1. The molecules that regulate the recognition and specificity of this process are unknown. Mutant animals lacking UNC-70/β-spectrin have been shown to present fragile axons that undergo spontaneous breaks and regeneration cycles (Hammarlund et al., JCB 2007). Here we show that loss of UNC-70/β-spectrin induces a novel phenotype of cell-cell fusion between the mechanosensory neuron PLM and PLN, which shares many characteristics with axonal fusion following regeneration induced by laser axotomy. We show that cell-cell fusion, detected as mixing of fluorophores between the two cells, can be modulated by the expression of the fusogen EFF-1 and is regeneration dependent. Using a candidate gene approach, we have discovered that the conserved apoptotic phosphatidylserine (PS) receptor, PSR-1, plays an important role in PLM/PLN fusion. In animals with mutations in psr-1 the rate of PLM/PLN fusion is significantly reduced. PSR-1 has been previously shown to bind exposed PS on the surface of apoptotic cells to mediate recognition and engulfment by phagocytes. Similarly, we observe PS exposure on the axons of PLM in animals lacking UNC-70. We also find similar defects in animals lacking the transthyretin-like protein TTR-52, its receptor CED-1, and the CED-1 downstream effector CED-6. In addition to defects in PLM/PLN fusion in an unc-70 injury model, animals lacking PSR-1, TTR-52, CED-1 or CED-6 also show axonal fusion defects following laser axotomy. Our results reveal a common molecular machinery between axonal fusion and apoptosis where these molecules mediate recognition and reconnection between damaged axonal compartments. We propose that the PLM/PLN fusion phenotype induced by unc-70 dependent injury is a novel paradigm in which to discover and study genes involved in regulating neuronal fusion events following injury.

118 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 119 Evaluation of Cell Death Mechanisms in Nematode Excitotoxicity John Del Rosario1,2, Towfiq Ahmed1, JunHyung An1, Tauhid Mahmud1, Uzair Amjad1, Itzhak Mano1 1Physiol Pharm & Neurosci, City College, The City University of New York., 2MA Program in Biology, City College, The City University of New York.

Excitotoxicity, a necrotic cell death mechanism that is prevalent in brain ischemia, is triggered when Glutamate Transporters (GluTs) are unable to clear Glutamate (Glu) from the synaptic cleft, causing Glu accumulation and overstimulation of the glutamate receptors (GluRs) located on the post-synaptic neuron. The exaggerated stimulation of GluRs causes excessive influx of Ca2+ and Na+ into the post-synaptic cell, leading to necrotic neurodegeneration. The cellular and molecular mechanisms that lead from Ca2+ influx to excitotoxic neurodegeneration remain unclear. Recent reports suggest that conserved pathways such as autophagic cell death contribute to some processes of neurodegeneration. However, the extent of their participation in excitotoxic cell death is still poorly understood. We developed a model of excitotoxicity in the nematode Caenorhabditis elegans by knocking-out the GluT gene glt-3 in a sensitive background. Our recent data shows that blocking autophagy, either through mutation in one of its master-regulators, unc-51, or through pharmacological block using 3-Methyladenine, can slightly reduce excitotoxic neurodegeneration. Monitoring autophagy in vivo by looking at fluorescently labeled LGG-1 expression confirms only a moderate activation of autophagy in nematode excitotoxicity. Blocking apoptosis using a ced-4 mutation has no effect on excitotoxic neurodegeneration. These observations suggest that autophagy has only a minor contribution, while apoptosis has no effect on nematode excitotoxicity. We are currently focusing on other mechanisms that might have a more pronounced role in nematode excitotoxicity. Understanding the molecular mechanisms that regulate excitotoxicity in C. elegans might help us to suggest new strategies to mitigate stroke damage in humans.

Disease Models and Regeneration Poster Session 119 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 120 Role of CREB/crh-1 in Molecular Modulation of Neuroprotection in a C. elegans Model of Excitotoxicity K. Genevieve Feldmann1, Itzhak Mano1 1Physiol Pharm & Neurosci, City College, The City University of New York and The City University of New York Graduate Center

Excitotoxicity is a prevalent type of neurodegeneration, seen most frequently in stroke when an interruption in blood supply to the brain causes the affected brain regions to be deprived of energy. The resulting ischemia causes neurodegeneration by excitotoxicity, a process in which buildup of glutamate (Glu) in the synapses over-excites the post-synaptic neurons. Although excessive Glu signaling triggers excitotoxic neurodegeneration, several lines of evidence suggest that Glu signaling can also activate neuroprotection. In one such scenario, exposing the brain to a mild ischemia induces neuroprotection, so that the effect of subsequent major ischemic events is less devastating, a process called ischemic preconditioning. Ischemic brain preconditioning is mediated by Glu receptors and involves CREB, a transcription factor that mediates many cellular processes and has critical importance in the nervous system. In addition to being important for learning and memory, studies have shown that CREB is a critical mediator of preconditioning, as its activation and stabilization on CRE sequences in the promoter area of different genes activates the neuroprotective program. The pathway for activating CREB under ischemic conditions has been partially elucidated in one mammalian scenario, where the cofactor CRTC seems to give specificity to CREB activation in excitotoxicity. However, the wider applicability of this model in other excitotoxic conditions has not been tested, and many details on this mechanism are missing. Moreover, the subsequent mechanism of CREB-mediated neuroprotection remains completely unknown. We have developed a reliable C. elegans excitotoxicity model, and we now use this system to further understand CREB-mediated neuroprotection. Using mutations in CREB/ crh-1 and CRTC/crtc-1 in our excitotoxicity background I have demonstrated that CREB and CRTC normally suppress excitotoxicity in C. elegans. I am now looking at the effects of CREB’s upstream candidate effectors on excitotoxicity. Furthermore, we are in a position to identify CREB’s downstream targets and the mechanisms underlying neuroprotection, using cell-specific transcriptome profiling in postsynaptic neurons. Understanding mechanisms of neuroprotection in nematode excitotoxicity might help us focus on conserved mechanisms of neuroprotection and allow us to suggest new therapeutic interventions in brain ischemia and stroke.

120 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 121 Functional analysis of VPS41-mediated protection from β-Amyloid [GC1] cytotoxicity Edward Griffin1, Kim Caldwell1, Guy Caldwell1 1University of Alabama, Department of Biological Sciences

According to the Alzheimer’s Association 2012 report, 13% of Americans over 65 years of age, and approximately half of Americans over the age of 85, suffer from Alzheimer’s Disease (AD), resulting in an estimated cost of 200 billion dollars for related health care. Additionally, AD is the most prevalent dementia, and the sixth leading cause of death in the United States. Thus, investigating mechanisms of pathophysiology and identifying potential therapeutic targets for AD is significant. AD is characterized by the formation of plaques, composed primarily of the amyloid- β 1-42 (Aβ) peptide in the brain, resulting in neurodegeneration. Our lab has observed that over- expression of human VPS41 in C. elegans provides neuroprotection from Aβ toxicity, and that depletion of VPS41 in worms expressing Aβ increases the toxicity of Aβ. In yeast, VPS41 has been demonstrated to function in the tethering of vesicles, late endosomes, and AP-3- coated vesicles from the late Golgi, to the lysosome for degradation. Previously, our lab has shown that over-expression of human VPS41 is neuroprotective in a transgenic worm model of Parkinson’s Disease, wherein dopaminergic neurodegeneration is induced by α-synuclein overexpression (Harrington et al., 2012, J. Neurosci.). VPS41-mediated neuroprotection from α-synuclein was ablated by mutation of putative phosphorylation sites in VPS41 and was dependent on its interactions with AP-3δ, RAB7, VPS core proteins, and VPS39. Thus, VPS41 has a role in lysosomal trafficking that impacts neuron survival. The objective of this study is to determine how this specifically relates to cellular processing and ameliorates the impact of neurotoxic proteinaceous products. Here we report the results of a systematic RNAi screen whereby we knocked down the core components involved in lysosomal trafficking and categorized their requirement for Aβ protein toxicity . Our results indicate that endocytic Aβ is trafficked through the Golgi to the lysosome in a VPS41-dependent manner rather than through AP-3-coated vesicles from the late Golgi like α-synuclein. Additionally, preliminary results suggest that the LC3 homologue plays a critical role in Aβ toxicity. In this regard, further analysis of functional effectors of Aβ protein processing via the lysosomal pathway, along with subsequent evaluation in our worm neuronal model of AD (Treusch et al, 2011, Science), will assist in the elucidation of the underlying mechanism involving VPS-41.

Disease Models and Regeneration Poster Session 121 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 122 Gαq mediates effects of antipsychotic drugs on C. elegans developmental delay/lethality Limin Hao1, Afsaneh Sheikholfslami1, Kristin Harrington1, Bruce Cohen1, Edgar Buttner1 1Mailman Research Center, McLean Hospital and Harvard Medical school, Belmont, MA 02478

Our aim is to understand the mechanisms of action of antipsychotic drugs (APDs) using C. elegans as a genetic model. In C. elegans, APD exposure causes developmental delay or lethality, depending on the drug concentration. APDs target neurotransmitter receptors such as dopamine, glutamate, serotonin, and acetylcholine receptors, many of which are associated with Gα proteins. To test whether Gα proteins mediate APD-induced developmental phenotypes in C. elegans, we screened mutants of four types of Gα proteins and found that Gαq (egl-30) reduction-of-function mutants were hypersensitive and that a gain-of-function mutant was slightly resistant to clozapine. To confirm that Gαq activity meditates clozapine’s effects, we analyzed the mutants of Gαq upstream modulators, such as eat-16, ric-8, rsbp-1, rsbp-2, tax-6, cnb-1, egl-10, and unc-43. Our results indicated that increased Gαq activity leads to drug resistance and decreased Gαq activity leads to drug hypersensitivity. We also analyzed downstream components in the egl-30 signaling pathway that regulates acetylcholine release and found that the drug effects are not mediated by these genes. To discover downstream effectors involved in clozapine’s actions, we performed a forward genetic screen for suppressors of egl-30(rf) clozapine hypersensitivity. We obtained 15 suppressors, one of which was more resistant to clozapine than wild type. Eight of the remaining mutants were strong suppressors, and six were weak. All of these strains retained the Egl and Unc phenotypes of egl-30(rf). We have snip-mapped seven suppressors to specific chromosomal regions, and none mapped to regions overlapping known suppressors of clozapine-induced developmental delay. We are performing whole-genome sequencing to identify the mutants.

122 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 123 Impaired mitochondrial morphology in response to an environmental contributor: S. venezuelae metabolite in a C. elegans model of Parkinson’s disease Hanna Kim1, Guy Caldwell1, Kim Caldwell1 1The University of Alabama

Mitochondria dynamically interact via a balance between fission and fusion to maintain organelle morphology. Several hundreds of mitochondria are present in single neuronal projection because of its high-energy demand. As a result, neurons are highly dependent on mitochondria for their function. Defects in mitochondrial complexes can have an affect on mitochondrial morphology, which can result in mitochondrial fragmentation. It has been reported mitochondrial dysfunction is highly associated with neurodegenerative disorders, such as Parkinson’s disease (PD). However, while outstanding progress has been made in discerning genetic associations to PD, the interactions between environmental factors and inherited contributory factors are largely unresolved. We previously identified a species of soil bacteria, Streptomyces venezuelae (S. ven), which produce a secondary metabolite that causes age- and dose- dependent dopamine (DA) neurodegeneration in C. elegans, as well as human DA producing neurons in culture (Caldwell et al., 2009, PLoS One). In this study, we characterize of S. ven metabolite-induced mitochondria dysfunction as it relates to DA neurodegeneration in C. elegans. We found that the S. ven metabolite gradually perturbs mitochondria morphology in body wall muscle cells as worms age and further causes a severe

decline in mitochondria membrane potential (ΔΨm). Previous genetic and pharmacological evidence suggests that the S. ven metabolite influences mitochondrial complex I, but not complex II, in C. elegans (Ray et al., 2014, Cell Death Dis.). We had not, however, examined the impact of the metabolite on complexes III and IV. Yet, a defect in one complex is often associated with altered enzymatic activity of other complexes. For example, complexes III and IV are essential for the stabilization of complex I. Here we report that the S. ven metabolite influences complex IV using genetic approaches. Overall, evidence indicates that this putative environmental contributor to neurodegeneration from a common soil bacterium may cause morphology changes to mitochondria, possibly by causing respiratory chain inhibition and organelle dysfunction.

Disease Models and Regeneration Poster Session 123 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 124 Age-dependent neuronal changes Anagha Kulkarni-Khandekar1, Claire Benard1 1Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA

While the age-dependency of neurodegenerative diseases is well known, the mechanisms by which aging triggers them are largely unknown. We are interested in the molecular mechanisms that help maintain the nervous system throughout life. To understand how the nervous system changes with age and the mechanisms underlying the maintenance of the nervous system during aging, we have carried out a quantitative analysis of morphological changes across several neuronal classes in wild-type worms at different ages. We find that in aged worms ganglia get disorganized, axon bundles become displaced and defasciculated, and novel neurites appear on mechanosensory and dopaminergic neurons. Our findings are largely in agreement with others (1,2,3). Functional decline of motorneurons has been elegantly described as well (4). Our analysis reveals that the kind and timing of age-related changes depend on neuronal type, indicating differential susceptibility of neurons to the stresses of aging. We also have undertaken longitudinal analysis to understand the age- related changes in chemosensory neurons and we find that these neurons displace from the juvenile position in a discontinuous manner. To determine if neuronal maintenance mechanisms impact on the maintenance of the aging nervous system, we have examined the degree of defasciculation, branching and displacement of neurons in sax-7 mutants at different ages. We find that neuronal defects in sax-7 mutants mimic alterations seen in aged wild-type animals, albeit many manifest earlier and can be more severe than in the wild type. To establish how lifespan determination pathways impact on age-related neuronal changes, we have examined the degree of defasciculation, branching and displacement in several neuronal classes in the long-lived mutants clk-1, daf-2 and eat-2, as well as in the short-lived mutant daf-16. Our results indicate that the dynamics of age-related neuronal changes is uncoupled from lifespan determination. Consistent with our results, daf-16 has been found to mediate age-dependent regeneration independently of lifespan (5). Taken together, our results suggest that a combination of intrinsic and extrinsic factors factors, including the class of neurons, their structural context, and lifespan regulators, impact on the state of neurons during aging.

Reference(s) 1. Pan et. al, 2011 2. Tank et. al, 2011 3. Toth et. al, 2012 4. Liu et. al, 2013 5. Byrne et. al, 2014

124 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 125 Studying membrane dynamics and EFF-1 fusogen localization during C. elegans axonal regeneration Casey Linton1, Rosina Giordano-Santini1, Brent Neumann1, Sean Coakley1, Massimo Hilliard1 1The University of Queensland, Queensland Brain Institute

Understanding the molecular mechanisms that mediate axonal regeneration is crucial for developing novel therapies for nerve injury. The C. elegans mechanosensory neurons have been used as a model system to discover some of the molecular and cellular elements that regulate this process. It is now established that regeneration in these neurons can occur through a mechanism of axonal fusion, whereby, following axotomy with a UV laser, the proximal regrowing fragment is able to directly fuse with its severed distal fragment, re- establishing both cytoplasmic and membrane continuity1,2. This pattern of regeneration provides interesting opportunities to study the role of membrane fusion and membrane dynamics in the regeneration process, and potentially harness such mechanisms for therapeutics. Previous studies2, as well as results from our lab, have shown that the C. elegans fusogen EFF-1 is required for axonal fusion during regeneration following axotomy of the PLM mechanosensory neurons. However, little has been characterized about the role of EFF-1 in this process; in particular, it is unclear if this molecule functions cell-autonomously, how it localizes to regulate fusion, and how it relates to overall membrane dynamics during regenerative growth cone formation. Here, by expressing the wild-type eff-1 gene specifically in PLM neurons of eff-1 mutant animals, we show that EFF-1 has a cell-autonomous function in PLM axonal fusion during regeneration. Using structured illumination microscopy (SIM), a super-resolution microscopy technique, we characterise both the membrane dynamics of regrowing axons utilizing a myristoylated version of mCherry, as well as the localization of EFF-1 using a GFP-tagged version of this molecule. These studies were conducted at different timepoints following axotomy to elucidate the role of this fusogen in neurons, and provide a means to understand its spatiotemporal regulation during axonal regeneration. This experimental platform has the potential for genetic manipulations to identify genes regulating localization of EFF-1 and membrane dynamics.

Reference(s) 1. Neumann et al., Dev. Dyn., 2011,2 Ghosh-Roy et al., J. Neurosci., 2010

Disease Models and Regeneration Poster Session 125 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 126 PINK-1 homeostasis and UPS perturbations as a result of a bacterial metabolite link environmental exposure to the genetics of neurodegenerative disease Bryan Martinez1, Arpita Ray1, Daniel Petersen1, Guy Caldwell1, Kim Caldwell1 1The University of Alabama

The causal mechanisms behind neurodegenerative disease are thought to integrate both environmental and genetic susceptibility factors. However, interactions between these contributory influences remain largely unexplored. Here we show a small, environmentally- derived metabolite isolated from a soil bacterium, Streptomyces venezuelae (Caldwell et al., 2009, PloS One), interacts with genetic modifiers of neurodegenerative disease. Firstly, we show that the toxicity of pathogenic proteins expressed in C. elegans neurons (α-synuclein, amyloid-β, and huntingtin) is consistently accelerated in response to metabolite exposure. This response was dependent upon age at treatment and the presence of the pathogenic protein, as young animals (Day 4 post-hatching), control-treated animals, or middle-aged animals (Day 6 through 10 post hatching) without pathogenic protein expression did not exhibit an enhanced neurodegenerative phenotype. To probe the mechanism of this augmented toxicity, we found that exogenous glutathione robustly protects against enhanced α-synuclein stress. Since glutathione is an important endogenous antioxidant in the context of proteostasis, we wished to determine how metabolite toxicity intersects with UPS inhibition. We find that the metabolite acts through a similar pathway, as RNAi or MG132 treatments enhance neurodegeneration such that it becomes intractable to further modulation by the metabolite. As a corollary, this compound also induces aberrant synaptic release dependent on the proteasome target UNC-13, as well as dopamine production (CAT-2), indicating that the UPS may be dysfunctional in metabolite-treated animals. Since cytosolic protein quality control is disturbed, we wished to assess whether mitochondrial protein quality control is also altered. We show that metabolite-treated, as well as UPS semi-depleted animals, are modestly more sensitive to sodium azide than control-treated animals, but are hypersensitive to sodium azide in the absence of a functional mitochondrial unfolded protein response (UPR). To understand these observations in the context of disease, we tested animals bearing mutations in worm homologs of Parkinson’s-linked genes; we find that the metabolite operates in a genetic pathway related to PINK1 (pink-1) dysfunction, but not PARKIN (pdr-1), where it appears to act in parallel. These results advance our understanding of factors linking environmental exposures with genetic susceptibility to disease.

126 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 127 A Role for the C. elegans Homolog of Retinal Degeneration 3 in a Chemosensory Neuron Luis Martinez-Velazquez1 1Skirball Institute, NYU School of Medicine

Retinal Degeneration 3 (RD3) is a small protein necessary for the survival of photoreceptor cells (Friedman, 2006). Mutations that truncate RD3 result in photoreceptor cell degeneration in Leber’s Congenital Amaurosis type 12, a severe form of retinal dystrophy that causes severe loss of vision at an early age. Recent evidence suggests that RD3 is required for proper localization of receptor-type guanylate cyclases to rod and cone outer segments (Azadi, 2010). However, little is known about the function of RD3, where it localizes in vivo (Molday, 2013), and why mutations in RD3 lead to photoreceptor degeneration. We found that the nematode homolog of RD3 is enriched in a subset of ciliated sensory neurons, including

the carbon dioxide (CO2)-sensing BAG neurons. In order to better understand the functions of RD3 in vivo, we used the CRISPR Cas-9 method (Friedland, 2013) to generate loss-of-function alleles. Using these mutant alleles, we have found that the C. elegans homologue of RD3 is required for the proper function of the BAG neurons. BAG neurons share characteristics with retinal photoreceptor neurons, including components of the signaling transduction apparatus, which consists of a receptor type guanylate cyclase, and the cyclic nucleotide gated (CNG) channels TAX-2/TAX-4 (Hallem, 2010). By using live cell and functional imaging techniques, behavioral studies, we will determine the in vivo function of RD3 in a ciliated sensory neuron.

Disease Models and Regeneration Poster Session 127 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 128 A Screen to Identify Regulators of Ciliary Cytoskeleton Stability and Function in Response to Polyglutamylation of Axonemal Microtubules Robert O’Hagan1, Winnie Zhang1, Maggie Morash1, Sebastian Bellotti1, Maureen Barr1 1Rutgers, The State University of New Jersey

When polymerized into microtubules (MTs), tubulins accumulate post-translational modifications (PTMs) such as polyglutamylation. PTMs have been proposed to actas signposts that guide the activities of kinesin and dynein motors, and other proteins that bind MTs. Ciliary MTs are especially prone to post-translational modification, however the roles that MT PTMs play in cilia are largely unknown. The enzymes that regulate post-translational glutamylation of tubulins profoundly affect cilia structure in C. elegans sensory neurons. Loss of CCPP-1, a homolog of murine Ccp1, which deglutamylates MTs, causes the progressive degeneration of amphid and phasmid neuronal cilia. However, ciliary degeneration in ccpp-1 mutants can be suppressed by deletion of ttll-4, ttll-5, or ttll-11, which encode glutamylases that add glutamylation to MTs. These data suggest that excess MT glutamylation destabilizes ciliary MTs, leading to ciliary degeneration. To understand the mechanism by which MT glutamylation regulates ciliary MT stability, we performed a forward genetic screen for suppressors of ccpp-1-induced amphid degeneration. In ccpp-1 mutants, the ciliary degeneration of amphid and phasmid neurons is usually complete by adulthood, such that adults appear completely dye-filling defective. We searched for suppressor mutations by dye-filling the adult F2 progeny of mutagenized ccpp-1 animals and looking for animals with amphids or phasmids that absorbed the dye. We isolated fifteen suppressor mutants, including some that fail to complement ttll-4, ttll- 5, and ttll-11. We hope that our suppressor alleles will illuminate pathways by which post- translational glutamylation regulates MT structure and function.

128 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 129 The role of miRNAs at the C. elegans neuromuscular junction: potential SMA modifiers? Patrick O’Hern1, Anne Hart1 1Brown University

Spinal Muscular Atrophy (SMA) is a neurological disorder characterized by loss of lower motor neurons. This degeneration is almost exclusively caused by mutations in the SMN1 gene that lead to decreased levels of Survival of Motor Neuron (SMN) protein. smn-1 is the C. elegans ortholog of SMN1, disruption of which results in motor defects (Briese et al. 2009). It is unknown how loss of SMN protein perturbs motor neuron function, however evidence suggests that miRNA disruption may play a role (Mourelatos et al. 2003). miRNAs are small non-coding RNAs predicted to regulate protein expression of mRNAs. The RNA helicase Gemin3 is found in numerous complexes with SMN and also pulls down with numerous miRNAs in cultured mouse motor neurons (Dostie et al. 2003). I hypothesize that Gemin3- associated miRNAs are misregulated in smn-1 loss-of-function animals leading to NMJ defects. I have identified C. elegans orthologs of Gemin3-associated miRNAs that regulate neuromuscular junction (NMJ) signaling. Animals lacking SMN or Gemin3 are defective on aldicarb, suggesting NMJ defects. Using tissue-specific rescue analysis and genetic epistasis, I will identify miRNAs that regulate NMJ function in the same pathway as SMN-1. I will also compile a list of potential mRNA targets for candidate miRNAs utilizing online bioinformatics tools, assigning priority to conserved targets with known synaptic function. For each potential mRNA target, I will confirm miRNA regulation and investigate whether expression of these targets is altered in smn-1 animals. These experiments will advance our knowledge of how loss of SMN protein impacts neuronal function and inform our understanding of how miRNA misregulation may contribute to neurodegeneration.

Disease Models and Regeneration Poster Session 129 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 130 C. elegans Models of C9ORF72-linked ALS-FTD Xing Wang1, Mochtar Pribadi2, Taixiang Saur2, Bruce Cohen1, Giovanni Coppola2, Edgar Buttner1 1Mailman Research Center, McLean Hospital & Harvard Medical School, 2David Geffen School of Medicine, University of California Los Angeles

Abnormal expansion of a GGGGCC hexanucleotide (G4C2) repeat in a noncoding region of the gene C9ORF72 is the most common genetic cause of both familial amyotrophic lateral sclerosis (ALS) and familial frontotemporal dementia (FTD). Both gain-of-function and loss- of-function mechanisms have been implicated in the pathogenetics of C9ORF72-linked ALS- FTD. To produce C.e. gain-of-function models, we generated transgenic animals expressing

(G4C2)29::GFP or (G4C2)9::GFP driven by either the hsp-16 promoters for conditional global expression or by the unc-119 promoter for pan-neuronal expression. The extrachromosomal arrays were integrated, and the resulting lines backcrossed. At 20ºC, Phsp-16::(G4C2)29::GFP transgenic animals displayed increased lethality, decreased lifespan, and paralysis. These phenotypes were not observed in wild-type animals and were less severe in animals carrying the Phsp-16::(G4C2)9::GFP transgene. Lethality was alleviated by culturing at 16ºC and was enhanced by various heat-shock protocols. We will conduct a forward genetic screen for suppressors of lethality or paralysis in Phsp-16::(G4C2)29::GFP transgenic animals and, in collaboration with the Coppola laboratory at UCLA, will conduct gene expression profiling experiments on these animals as well. To study loss-of-function mechanisms that may pertain to human ALS-FTD, we backcrossed three mutants alleles of alfa-1, the C.e. homolog of C9ORF72, and performed phenotypic analyses on the resulting strains. We are now conducting gene expression profiling experiments on alfa-1(lf) and N2 animals.

130 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 131 One-carbon metabolism genes modulate amyloid-beta toxicity in C. elegans Alzheimer’s disease models Xiaohui Yan1, Adam Knight1, Kim A. Caldwell1, Guy A. Caldwell1 1Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, 35487

The insulin/IGF-1 signaling (IIS) pathway regulates both aging and proteotoxicity and is well conserved from invertebrates to mammals. Reduction in IIS ameliorates the proteotoxicity associated with amyloid-β (Aβ) aggregation in Alzheimer’s disease (AD), as well as α-synuclein (α-syn) aggregation in Parkinson’s disease C. elegans models. Therefore, certain common metabolic changes mediated by IIS reduction may maintain proteostasis by counteracting α-syn and Aβ misfolding during aging. Previously, we identified that knockdown of gcst-1, encoding glycine cleavage system (GCS) T protein, enhances α-syn misfolding in long-lived daf-2 mutants, suggesting a role for GCS in metabolic change caused by IIS reduction. GCS is a mitochondrial tetrahydrofolate-dependent enzymatic complex involved in glycine catabolism and generation of one-carbon units for downstream metabolism. Notably, alterations in one- carbon metabolism have been associated with age-related AD risk. gcst-1 RNAi exacerbated

C. elegans paralysis caused by temperature-inducible muscle expression of a human Aβ42 transgene. When GCST-1 was overexpressed in a C. elegans neuronal AD model, where human Aβ42 accumulation leads to an age-dependent degeneration of glutamatergic neurons, the neuronal loss was significantly rescued; in parallel, the impaired posterior gentle touch response which is associated with glutamatergic neuronal loss was improved. Another independent but functionally related enzyme, serine hydroxymethyltransferase (SHMT), when knocked down, resulted in similar enhanced paralysis induced by Aβ. The effect of folate on modulating Aβ toxicity was demonstrated by knocking down dhfr-1, which reduces dihydrofolate to tetrahydrofolate, enhanced Aβ-induced paralysis; and trimethoprim, the DHFR- 1 inhibitor, enhanced Aβ glutamatergic neurodegeneration. GCST-1 functions independent of DAF-16; however, gcst-1 RNAi significantly induced ROS production which may lead to the activation of SKN-1. In addition, gcst-1 RNAi was identified to significantly increase α-syn- induced dopamine neuron degeneration in pink-1 mutants, suggesting a role of GCST-1 in mitochondrial stress response, which may also underlie glutamatergic neurodegeneration caused by Aβ. Taken together, our findings on the GCS expand our mechanistic understanding of the well-established, but poorly understood, interaction between folate-dependent one- carbon metabolism and age-associated neurodegenerative diseases.

Disease Models and Regeneration Poster Session 131 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 132 Neutral cholesterol ester hydrolase I, a downstream modulator of DAF-2 signaling, is protective in C. elegans models of neurodegeneration Siyuan Zhang1, Kim Caldwell1, Guy Caldwell1 1The University of Alabama

Aging is the most identified risk factor for Parkinson’s disease (PD), the second most common neurodegenerative disease. We model a cellular aspect of PD by overexpressing human α-synuclein (α-syn)::GFP in the bodywall muscle cells, wherein transgenic animals exhibit α-syn aggregate formation over time. Notably, these α-syn-induced inclusions are greatly reduced by introducing the lifespan-extending daf-2 mutation. To identify components in the daf-2 pathway relevant to α-syn, we perform an RNAi screen for genes that, when knocked down, result in enhanced α-syn misfolding in the daf-2 mutant background. In total, 60 candidates are identified; one of these is Y43F8A.3. Using RT-qPCR, we show that Y43F8A.3 transcription is up-regulated in daf-2 mutant worms and decreases in daf-2, daf-16 double mutant worms. Additionally, selective Y43F8A.3 knockdown in the dopamine (DA) neurons enhance neurodegeneration in an age-dependent manner. With the daf-2 knockdown, DA degeneration is diminished. However, concurrent daf-2 and Y43F8A.3 knockdown abolishes this protection. Further, Y43F8A.3 acts independent of daf-16 as knockdown does not alter DAF-16::GFP localization or exhibit dependence on daf-16 for neurodegeneration. DA overexpression of Y43F8A.3 also attenuates α-syn-induced neurodegeneration. Y43F8A.3 is predicted to encode neutral cholesterol ester hydrolase 1 (NCEH1), which can convert esterified cholesterol to free cholesterol and fatty acids. We hypothesize that neuroprotection by Y43F8A.3 may involve a change of cholesterol levels. To test that, we first introduce a gradient of cholesterol in the medium and find enhanced survival ofwild type (WT) DA neurons with lower cholesterol levels. We then show that the neuroprotection afforded by Y43F8A.3 is dependent on cholesterol. Moreover, when the equilibrium between cholesterol and cholesterol ester is altered by knockdown of the reverse enzyme of cholesterol ester hydrolase, α-syn-induced neurodegeneration is also enhanced. Interestingly, selective knockdown of Y43F8A.3 in the intestine results in DA neuroprotection in a cell non-autonomous manner. Likewise, an enhancement in the unfolded protein response (UPR) is detected when Y43F8A.3 is knocked down. Discerning functional modulators of Y43F8A.3 expression and function will inform our understanding of the interface of metabolism and neurodegeneration as it pertains to PD and other disorders where cholesterol may have a disease-modifying relevance.

132 Disease Models and Regeneration Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 133 Identifying cell-autonomous targets of the Hox Transcription factor MAB-5 in directed cell migration Mahekta Gujar1, Erik Lundquist1 1University of Kansas

The MAB-5/Hox transcription factor is a determinant of posterior migration of the Q neuroblast descendants. To identify transcriptional targets of MAB-5/Hox, we previously used whole organism RNA seq in wild-type, mab-5 loss-of-function, and mab-5 gain-of function mutants (Tamayo et al., 2013). While MAB-5 is expressed in the QL descendants, it is also expressed in other tissues in the posterior of the animal, including P cells, seam cells, and body wall muscles. Our whole-organism RNA seq studies potentially identified MAB-5 targets in all of these tissues, and MAB-5 has non-autonomous roles in Q migrations (see abstract by Josephson et al.). In our current study, we focus on identifying cell-autonomous targets that are regulated by MAB-5 in the Q cells. We generated a mab-5 transgene containing the mab-5 cDNA under the control of the egl-17 promoter, expressed specifically in QL and QR at the time of migration. Pegl-17::mab-5 resulted in posterior QR descendant migrations similar to the mab-5(e1751)GOF allele. We conducted RNA seq on wild-type and Pegl-17::mab-5 animals to identify cell-autonomous targets of MAB-5 in the Q cells. This study identified 76 genes that were up- or down-regulated in Pegl-17::mab-5 compared to wild type. This set was enriched for secreted and transmembrane molecules, as was the set identified in the previous whole organism RNA seq. However, the overlap between sets was small, suggesting that different targets have been identified that might represent cell-autonomous targets that were missed in the previous approach due to mab-5 activity in cells other than the Q cells. Functional validation using RNAi is currently being done to determine which genes suppress mab-5 GOF and which act autonomously in the Q cells.

Neuronal Cell Fate and Differentiation Poster Session 133 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 134 Regulation of neuronal polarity by neuron-glia gap junctions Lingfeng Meng1, Dong Yan1 1Department of Molecular Genetics and Microbiology, Duke University Durham, North Carolina

Electron microscopic studies showed that neuron-glia gap junctions present at different brain regions in mammals1-4.In cultured mammalian embryonic neurons and brain slices, neurons and glial cells are coupled through gap junctions5,6. This coupling declines progressively with neuron maturation and becomes undetectable in postnatal neurons, yet it remains unclear whether these transit gap junctions between neurons and glial cells play any roles in brain development. In C. elegans, RME neurons form gap junctions with six glial cells, GLR cells7. We found that disruption of gap junctions between GLR cells and RME neurons induces mis-location of axonal components to dendrites in RME neurons, and this phenomenon is likely through the change of calcium dynamic in gap junction mutants. Taken together, our studies shed light on the in vivo function of neuron-glia gap junctions, and provide a new mechanism for neuronal polarity.

Reference(s) 1. Brightman MW, Reese TS. Junctions between intimately apposed cell membranes in the vertebrate brain. The Journal of cell biology 1969; 40:648-677. 2. Sloper JJ. Gap junctions between dendrites in the primate neocortex. Brain research 1972; 44:641-646. 3. Berdan RC, Shivers RR, Bulloch AG. Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma. Synapse 1987; 1:304-323. 4. Rash JE, Yasumura T, Dudek FE. Ultrastructure, histological distribution, and freeze- fracture immunocytochemistry of gap junctions in rat brain and spinal cord. Cell biology international 1998; 22:731-749. 5. Alvarez-Maubecin V, Garcia-Hernandez F, Williams JT, Van Bockstaele EJ. Functional coupling between neurons and glia. The Journal of neuroscience : the official journal of the Society for Neuroscience 2000; 20:4091-4098. 6. Froes MM, Correia AH, Garcia-Abreu J, Spray DC, Campos de Carvalho AC, Neto MV. Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures. Proceedings of the National Academy of Sciences of the United States of America 1999; 96:7541-7546. 7. White JG, Southgate E, Thomson JN, Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philosophical transactions of the Royal Society of London Series B, Biological sciences 1986; 314:1-340.

134 Neuronal Cell Fate and Differentiation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 135 A novel effector of integrin adhesion complexes is involved in cholinergic synaptogenesis in Caenorhabditis elegans Marie Pierron1, Bérangère Pinan-Lucarre1, Jean-Louis Bessereau1 1University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534, 69622 Villeurbanne, France, University Pierre and Marie Curie Paris6, 75005 Paris

At the Caenorhabditis elegans neuromuscular junction (NMJ), acetylcholine receptors (AChR) form post-synaptic clusters precisely facing acetylcholine release sites and mediate excitatory transmission. The number and the localization of these receptors are important to regulate the strength of synaptic transmission. To identify novel mechanisms involved in the formation and the maintenance of cholinergic synapses, we performed a genetic screen based on the direct visualization of fluorescently tagged AChR. Mutations causing abnormal AChR localization were identified using rough SNP mapping and whole genome re-sequencing. We identified a novel gene specifically required for the localization of AChR clusters at the NMJs between SAB neurons and head muscle cells. A mutation in this gene leads to the accumulation of multiple AChR clusters at non-synaptic sites of the muscle cell membrane without affecting axonal outgrowth. This gene encodes a protein composed of protein-protein interaction domains and functions cell autonomously. The GFP fusion protein localizes to muscle cell boundaries in regions of contact between adjacent muscle cells, and to dense bodies and M-line in body wall muscle cells in a pattern identical to that observed for PAT- 3/β-integrin. This localization is consistent with its role in other model organism (drosophila, mouse and human culture cells) where it was involved in the stabilization of integrin adhesion complexes. However, immunofluorescence experiment using anti-PAT-3/β-integrin and phalloidin staining in young adults revealed that Pat-3/β-integrin localizes normally to the muscle cell membrane and that the organization of actin filaments is preserved in our mutant. We are currently investigating the role of this protein in AChR localization at SAB synapses. We hypothesize that it exist a link between integrin adhesion complexes and cholinergic synaptogenesis in C. elegans head muscles.

Neuronal Cell Fate and Differentiation Poster Session 135 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 136 hlh-3 encodes two Achaete/Scute like protein isoforms with different functions Saleel Raut1 1University of Illinois at Chicago

We have shown that the gene hlh-3 encodes a basic helix loop helix protein HLH-3, that is required for normal egg-laying behavior. hlh-3 mutants (molecular null alleles - bc248 and tm1688) have an egg-laying defective (Egl) phenotype. We have shown that the mutant phenotype results from abnormal differentiation and axonal pathfinding of the serotonergic hermaphrodite-specific motor neurons (HSNs), which fail to accumulate serotonin and innervate the vulval muscles (Doonan et al., 2008). Our published RT PCR analysis argued that the gene encodes a transcript, herein called hlh-3B, comprised of curated exons 2 and 3. Further analysis of hlh-3(bc277), an allele with a deletion in the region of the 1st putative exon promoted us to reconsider this conclusion. hlh-3(bc277) hermaphrodites show normal egg-laying behavior. Characterization of hlh-3(bc277) hermaphrodites revealed similar HSN axon pathfinding defects as in hlh-3(bc248) and hlh-3(tm1688) animals but no defects in serotonin accumulation. The difference between hermaphrodites harboring hlh-3(bc277) and the other two alleles is that the abnormal HSN axons have branches that appear to synapse on the vulva muscles, suggesting that the hlh-3(bc277) allele might have some residual function. To address the discrepancy between ability to lay eggs and the defects in axon pathfinding, we asked the question whether under normal circumstances the st1 exon, which is missing in hlh-3(bc277) is transcribed and translated. To test this, we fused GFP coding sequences to the hlh-3 promoter region and in frame with the 1st 8 amino acids of the 1st exon to analyze expression in the transgenic animals. We find that this construct is expressed in the embryonic HSNs and not post hatching, which is consistent with the expression pattern seen with the fosmid reporter (HLH-3::YFP). This observation leads us to conclude that in the wild type there is also a longer isoform, which we have named HLH-3A. In the absence of both the isoforms, as in hlh-3(bc248) and hlh-3(tm1688), the HSNs have pathfinding defects and problems with accumulating serotonin. In the absence of HLH-3A only, as in hlh- 3(bc277), the HSNs are pathfinding defective but can accumulate serotonin like WT. Based on the phenotypes, we hypothesize that HLH-3A is important for regulating the expression of genes involved in pathfinding whereas HLH-3B is important for differentiation as measured by accumulation of serotonin.

136 Neuronal Cell Fate and Differentiation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 137 A novel target protects against patterned neurodegeneration in Alzheimer’s disease Sangeetha V. Iyer1, Luisa L. Scott1, James Sahn1, Gabriella Zuniga1, Jon Pierce- Shimomura1 1The University of Texas at Austin, Austin, TX 78712 USA

Alzheimer’s disease (AD) is the sixth leading cause of death in the US, affecting more than 5 million people with sadly no treatment in sight. AD is a neurodegenerative disorder characterized by protein aggregates and selective degeneration of cholinergic neurons. To search for novel targets for treating age-related neurodegenerative diseases like AD, we turned to Sigma receptors. The Sigma-1 receptor was previously found to regulate neurodegeneration and cognitive decline associated with AD. Another receptor in this orphan class, the progesterone receptor membrane component 1 or Sigma-2 receptor (Sig2R) has a well-known role in regulating cancer cell death, but has not been studied as a target for neurodegenerative diseases. To determine whether Sig2R is a useful target for reducing patterned neurodegeneration, we used a C. elegans model of neurodegeneration that expresses a single extra copy of the human Amyloid Precursor Protein (APP) gene. This SC_APP model displays progressive, age-dependent changes in protein regulation in specific cholinergic neurons that subsequently die. In this study we found that a Sig2R null allele (vem-1, vc445 or vc701) significantly reduced the selective degeneration of cholinergic neurons in older adult animals. The presence of either null allele had less effect on baseline neurodegeneration in control animals. We have previously determined that the accumulation of APP protein in our SC_APP model precedes the apoptosis of cholinergic neurons. To ascertain if Sig2R plays a role upstream or downstream of abnormal protein accumulation, we asked if deletion of Sig2R in our SC_APP model altered protein aggregation, specifically the patterned accumulation of APP in the neurons susceptible to degeneration. Results showed that SC_APP animals bearing a deletion of Sig2R showed significantly lower levels of accumulated APP compared to age-matched middle-aged SC_APP adults with a WT copy of Sig2R. Finally, we have started to screen novel molecules that bind Sig2R for the ability to reduce neurodegeneration. We have found two molecules that increase neurodegeneration in both the SC_APP model and control strains. More importantly, we found a molecule that decreases degeneration in the SC_APP model. Moreover, this molecule also reduces APP accumulation in the SC_APP model. Together, our results imply that the deletion of Sig2R restricts neurodegeneration by reducing the inappropriate accumulation of APP that in turn leads to neuron death. Moreover, a drug acting via a Sig2R pathway reduced both protein accumulation and neurodegeneration showing that this highly conserved target may be harnessed for promising therapeutic outcomes in AD and/or other age-related neurodegenerative diseases.

Neuronal Cell Fate and Differentiation Poster Session 137 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 138 Developmental specification of a polymodal nociceptor in C. elegans Jordan Wood1, Denise Ferkey1 1Department of Biological Sciences, State University of New York at Buffalo

All animals rely on their ability to sense and respond to the environment to survive. Nociception (the perception of noxious/painful stimuli) serves an important protective function, eliciting withdrawal and avoidance behaviors in response to potentially damaging stimuli. The primary nociceptors in C. elegans are the two ASH head sensory neurons, which detect multiple forms of aversive stimuli. However, while the sensation of pain is particularly valuable in helping an organism to avoid potentially harmful stimuli, there are still large gaps in our understanding of the mechanisms that govern nociceptor development across species. Based upon previously published experiments from other groups, we hypothesized that the paired-like homeodomain transcription factor UNC-42 functions as a terminal selector of ASH identity. However, our data indicate that although UNC-42 regulates a significant portion of the functionally mature ASH state, it does not function as the sole driver of the terminal ASH identity. In addition to UNC-42, a diverse array of transcription factors are expressed in the ASH neurons, which in combination likely comprise the genetic program underlying ASH specification and thus drive expression of the sensory signaling apparatus that endows the adult ASHs with polymodal nociceptive sensitivity. Using molecular, genetic and behavioral approaches, we have identified and are currently characterizing the function of several transcription factors in ASH specification.

138 Neuronal Cell Fate and Differentiation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 139 Reconstruction of the L1 nervous system Daniel Berger1, Steven Cook2, Scott Emmons2, David Hall2, Douglas Holmyard3, David Kersen1, Valeriya Laskova1, Jeff Lichtman1, Ben Mulcahy3, Marianna Neubauer1, Aravi Samuel1, Richard Schalek1, Mei Zhen3 1Center for Brain Science, Harvard University, 2Albert Einstein College of Medicine, 3University of Toronto and Lunenfeld Institute

The L1 larva has many fewer neurons and neuronal classes than subsequent stages. For example, the motor neurons that innervate adult ventral musculature are absent in L1 animals. Understanding how the L1 stage animals sense and navigate through environments will require mapping its detailed wiring diagram. Here, we report progress in using serial- section electron microscopy of high pressure frozen samples to reconstruct the complete L1 nervous system.

New Technologies Poster Session 139 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 140 Parallel imaging of C. elegans larval quiescence using the WorMotel Matthew Churgin1, Chieh-Chieh Yu1, Xiangmei Chen1, David Raizen2, Christopher Fang- Yen1 1Department of Bioengineering, University of Pennsylvania, 2Department of Neurology, Perelman School of Medicine, University of Pennsylvania

Sleep behavior is universal in the animal kingdom but its regulation is poorly understood. We are using C. elegans lethargus, the period of quiescence accompanying each larval molt, as a model for sleep. Measuring lethargus quiescence requires longitudinal monitoring of individual animals over the course of hours to days with moderately high spatial resolution (~10 microns), making it difficult to quantify this behavior in many animals at a time. To address this limitation, we have developed the WorMotel, a device for longitudinal imaging of up to 48 uniquely identified worms in 3.5 mm diameter wells. The device, made of PDMS (silicone rubber) molded from a 3D-printed master, consists of an array of agar-filled wells optimized for imaging and cultivation of C. elegans. Through continuous imaging at 1 frame per second we capture longitudinal measurements of locomotion for 48 worms from the L3 stage until young adulthood. We validate our method by quantifying quiescence in wild type N2 in addition to mutants with increased (clk-1) and decreased quiescence (kin-2 and egl-4). We screened 16 C. elegans wild isolates for differences in lethargus behavior. We identified strains with increased (JU775) and decreased (MY16) L4 lethargus quiescence compared to N2, and found that these strains had decreased or increased wakeful activity, respectively. These results suggest that these strains have an altered basal level of locomotor activity and thus the measured changes in quiescence may not reflect any true difference in sleep behavior. In contrast, one wild isolate, CX11314, exhibited a decrease in L4 and adult activity alongside a decrease in lethargus quiescence, suggesting a change in sleep. We are currently measuring other sleep phenotypes, such as lethargus sensory response latency. We also report efforts to use the WorMotel to conduct a forward genetic screen for mutations governing lethargus behavior and developmental rate. We expect the WorMotel will be widely applicable to long- term studies of behavior during development and adulthood.

140 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 141 Inducible Protein Degradation Using Trans-Targeting in C. elegans Jesse Cohn1, Shameika Wilmington1, Andreas Matouschek1, Jon Pierce-Shimomura1 1University of Texas at Austin

A system to inducibly degrade a protein of interest would have numerous applications in C. elegans. Most proteins are degraded through the ubiquitin-proteasome system (UPS). This regulated degradation affects numerous cell processes, such as basal protein turnover, signal transduction, and cell cycle control. The degradation signal, or degron, that targets proteins to the proteasome has two distinct components: a proteasome binding tag and an initiation region. The proteasome binding tag is typically a chain of ubiquitin molecules attached to the protein substrate. The proteasome binds the tag and then engages the substrate at the initiation region. Although both components of the degron are usually on the same polypeptide, previous work has shown that they are able to function in trans when on two different subunits of a protein complex. This trans targeting mechanism has so far been successfully adapted to allow for inducibly controlled protein degradation in HEK293 cells. We are currently working to implement this system in C. elegans. The inducible nature of the trans-targeting system along with the ability to express the components of the degron in a combinatorial manner using different promoters, will make this particular technique broadly applicable in C. elegans research.

New Technologies Poster Session 141 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 142 An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells Navid Ghorashian1, Sertan Gökçe1, Sam Guo1, William Everett1, Adela Ben-Yakar1 1The University of Texas at Austin

Automated microfluidic platforms are enabling high-resolution and high-content bioassays on small animal models. An upstream device that automatically delivers different animal populations to these bioassay platforms could facilitate high-throughput drug discovery and genome-wide screens. Current population delivery strategies rely on suction from classic well plates through tubing exposed to air, which causes certain drawbacks: 1) bubble and debris introduction to the sample, which interferes with analysis in downstream systems, 2) experimental throughput reduction due to added cleaning steps, and 3) the need for complex mechanical manipulations of well plate position. To address these concerns, we developed a microfluidic platform that can deliver multiple distinct animal populations from on-chip wells through a multiplexer valve system and used it to deliver C. elegans populatons. This Population Delivery Chip can operate autonomously as part of a relatively simple experimental setup that requires none of the major mechanical moving parts typical of plate-handling systems. The autonomous device setup could serially deliver 16 worm populations from on-chip wells out of a single outlet without introducing debris and bubbles, damaging the animals, or mixing any populations. The device achieved up to 99 % removal of the 100-200 worms preloaded into a given well in under 5 seconds; an order of magnitude faster than current worm delivery methods. This platform could potentially handle similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. This system has led us to develop significantly larger platforms to begin high-throughput screens to discover modulators of neurodegenerative phenomena in C. elegans.

142 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 143 The use of novel calcium indicators in C. elegans Laura Grundy1 1MRC LMB

An important problem in neuroscience is understanding how neural circuits work at the level of molecules and cells. C. elegans provides an opportunity to study these questions in a simple nervous system that is tractable to the use of genetic and molecular approaches. For these reasons it is also a good system to test new genetic tools for studying and manipulating neural activity in vivo. Calcium imaging is a well described technique used for investigating C. elegans neuronal circuitry, for which a variety of calcium indicators have been developed. One such protein is GCaMP, Which, in recent years, has undergone a succession of improvements to enhance its efficiency. We are now at a point where the SNR and photo stability surpass that of FRET- based indicators, such as Cameleon. This larger change in fluorescence intensity allows for lower magnification imaging, as it compensates for the reduction in light entering the microscope under these conditions. Also the response of GCaMP to the influx of calcium is faster than that of FRET- based indicators, further widening the applications of GCaMP. New versions of GCaMP are continually being produced by using a combination of protein structure–guided mutagenesis and semi-rational library screening. The latest in this line of newly produced GCaMPs is GCaMP6 which has been created by Douglas Kim at Janelia Farm. Since optogenetic molecules do not always show the same properties in animals that they do in vitro, it is important to evaluate these molecules efficiently in an in vivo context. We expressed variants of GCaMP6 specifically in the touch neurons ofC. elegans and measured the response to well-defined touch stimuli. As yet the variant GCaMP6f gives the fastest kinetics and surprisingly the biggest fluorescence change. Since 6f has relatively low calcium affinity we wonder if this indicates high baseline calcium in worm neurons, and therefore we are currently testing the effect of mutations that deliberately reduce dissociation constant. The results of this will be presented.

New Technologies Poster Session 143 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 144 An Open-Source Analytical Platform for Analysis of C. elegans Swimming Induced Paralysis (Swip) J. Andrew Hardaway1, Jing Wang2, Paul Fleming3, Katherine Fleming3, Sarah Whitaker1, Alexander Nackenoff1, Chelsea Snarrenberg1, Shannon Hardie1, Bing Zhang2, Randy D. Blakely4 1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN USA, 2Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN USA 37232-8548, 3Electrical Engineering and Computer Science, Vanderbilt University School of Medicine, Nashville, TN USA 37232-8548, 4Departments of Pharmacology, and Psychiatry, Vanderbilt University School of Medicine, Nashville, TN USA 37232-8548

The nematode Caenhorhabditis elegans offers great power for the identification and characterization of genes that regulate behavior. In support of this effort, analytical methods are critical in providing dimensional analyses of subcomponents of behavior. Previously, we demonstrated that loss of the presynaptic dopamine (DA) transporter, dat-1, evokes DA- dependent Swimming Induced Paralysis (Swip), a behavior compatible with forward genetic screens. Standard measurements of C. elegans swimming utilize manual assessments of the number of animals exhibiting swimming versus paralysis. Our approach deconstructs the time course and rates of movement in an automated fashion, offering a significant increase in the information that can be obtained from swimming behavior. Here, we detail the development and implementation of SwimR, a set of tools that provide for an automated, kinetic analysis of C. elegans Swip. SwimR relies on open source programs that can be freely implemented and modified. We show that SwimR can display time-dependent alterations of swimming behavior induced by both genetic manipulations and drug-treatments, illustrating this capacity using various DA signaling mutants and the dat-1 blocker and tricyclic antidepressant imipramine (IMI). We demonstrate the capacity of SwimR to extract multiple kinetic parameters that are impractical to obtain in manual assays. In summary, the SwimR platform is a powerful tool for the deconstruction of worm thrashing behavior in the context of both genetic and pharmacological manipulations that can be used to segregate pathways that underlie nematode swimming mechanics.

144 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 145 Transgenic C. elegans as a screening platform for anthelmintic development Wenjing Law1, Amanda Ortega2, Richard Komuniecki3 1Department of Biological Sciences, The University of Toledo, 2Department of Biology, The University of Toledo, 3University of Toledo

Nematode infections cause significant morbidity and contribute significantly to aloss of Disability Adjusted Life Years. More importantly, in many cases, effective chemotherapy is still not available. Parasitic nematodes also have a devastating economic impact in agricultural settings. Anthelminthic development has been hampered by the lack of cost- effective screening platforms due, in part, to the absence of nematode cell lines, difficulties in maintaining parasitic nematodes in laboratoryand the cost of in vivo screening and pharmcological differences between orthologous receptors from different species. Previously, we and others have demonstrated that exogenous monoamines, such as 5-HT, dopamine and tyramine (TA), each paralyze C. elegans and, where examined, parasitic nematodes. The present study was designed to develop a high-throughput screening platform to identify nematode monoamine receptor agonists in “chimeric” genetically-engineered C. elegans by heterologously expressing 5-HT and TA receptors from the parasites off-target, at sites likely to yield robust phenotypes upon agonist stimulation. Importantly, this approach would maintain the unique pharmacologies of the receptors from individual parasites, but include nematode-specific accessory proteins and a cuticle. Previously, many investigators have rescued a range of behaviors in C. elegans null animals with the expression of proteins from the parasites, validating this approach. We chose to examine pharyngeal pumping and locomotion for off-target expression, as these two behaviors are well characterized, readily assessed by established high-throughput screening assays and have been identified as targets for existing anthelmintics. Specifically, we expressed 1) 5-HT1-like receptors from free-living and parasitic nematodes and their mammalian hosts or 5-HT gated Cl- channels specifically in cholinergic motor neurons of C. elegans quintuple null 5-HT receptor mutants lacking any 5-HT receptors, on the assumption that robust agonist-dependent Gao signaling or hyperpolarization, respectively, would dramatically inhibit ACh release and locomotion and 2) 5-HT or TA gated Cl- channels in pharyngeal or body muscle of C. elegans mutants lacking any 5-HT or TA receptors, respectively, on the assumption that agonist-dependent muscle hyperpolarization would dramatically decrease pharyngeal pumping or locomotion. In all cases, robust selective inhibition was identified, suggesting not only that this approach is useful, but that it may be expanded to other potential anthelmintic targets. This work was supported by a grant fro the Gates Foundation.

New Technologies Poster Session 145 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 146 Microfluidic devices for rapid quantification of pharyngeal activity by electrophysiological measures Shawn Lockery1, Kristin Robinson1, William Roberts1, Janis Weeks2 1NemaMetrix LLC, 2Institute of Neuroscience, University of Oregon

There is growing interest in utilizing alterations in the pumping behavior of the nematode pharynx in screens of the biological activity of a wide variety of compounds. Applications include the search for novel anthelmintics, compounds that prolong health span, and potential drugs to ameliorate the effects of human neuro- and neuromuscular diseases, for which C. elegans is a validated model. In such investigations, pharyngeal pumping is currently measured by slow- motion replay of brief video recordings of feeding worms, during which pumps are counted by a human observer. Because this method is manual, the observation time for individual worms and the number of worms that can be processed per day are severely limited, as is the temporal resolution of pumping behavior. Another limitation is that weak pumps, such as those produced by drugged, aged or diseased worms, can be hard to detect reliably and accurately. Progress in pharynx-based screening would be greatly accelerated by longer observation times and the ability to count pumps automatically at high temporal resolution. To address this issue, we have developed two broad classes of microfluidic devices (elastomeric “chips”) that record electropharyngeograms, the electrical signals emitted by the pharynx, thereby enabling automated quantification of pharyngeal activity with millisecond temporal resolution. In one class of devices, eight or more nematodes can be recorded in parallel. This device can be used after chronic exposure to test compounds, or before, during and after acute exposure to them. In another class of devices, a population of tens to hundreds of worms can be loaded into a reservoir and individual worms can be positioned semi-automatically into an electrical recording module. This type of device is particularly well suited to studies involving chronic compound exposure when large numbers of worms must be processed. In parallel, we are developing an inexpensive, stand-alone system that will allow researchers unfamiliar with electrophysiology to record and analyze pumping behavior automatically in hundreds of worms per day. In addition to accelerating research already in progress, this technology is likely to expand the range and complexity of biological problems that can be addressed by recording pharyngeal activity in a variety of nematode species.

146 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 147 Worm Psychophysics: Targeted Mechanical Stimulation and Automated Behavioral Response Tracking Eileen Mazzochette1, Christopher Fang-Yen2, Miriam Goodman1, Beth Pruitt1 1Stanford University, 2University of Pennsylvannia

The sense of touch is essential to daily life, but is the least understood of all human senses. We use C. elegans to investigate the mechanisms of touch sensation. In classical assays of mechanosensation, an eyebrow hair is used to deliver a so-called gentle touch to freely moving worms. A reversal in the worm’s direction is scored as a positive response. However, these classical assays are non-quantitative and limited in spatial resolution. We have developed a system to enable targeted and calibrated mechanical stimulation by a microelectromechanical cantilever integrated with optical monitoring of behavioral response. Current systems for touch sensation assays are limited to user-controlled targeting of mechanical stimulation to specific positions as well as frame-by-frame user input for analysis (Petzold et al, 2013). Here, we present a technology that will greatly increase experimental throughput by replacing these laborious manual steps with automated machine-vision-based targeting and behavioral analysis. The new tracking system enables us to apply a controlled force (or displacement) to a specific location along the anterior-posterior axis of a freely moving worm and incorporates automated behavioral analysis. To achieve these functions, we designed a novel optical system and software to enable real-time tracking for spatial targeting. The tracking system has two parts: 1) an inverted optical system equipped with oblique lighting that enables us to visualize worms but not the mechanical stimulator; 2) a computer vision algorithm to find the worm and track its skeleton. We plan to apply this system to quantitatively analyze known mechanosensory mutants to understand more about the nature of such phenotypes. The system can also be used to screen for mutations or other manipulations that increase touch sensation.

Reference(s) 1. Bryan C Petzold, Sung-Jin Park, Eileen A Mazzochette, Miriam B Goodman, and Beth L Pruitt, MEMS-based Force-clamp Analysis of the Role of Body Stiffness in C. elegans Touch Sensation, Integrative Biology, 2013 vol. 5(6) pp. 853-864

New Technologies Poster Session 147 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 148 Automating calcium image analysis Amelia Parmidge1, Chantal Brueggemann2, Noelle L’Etoile2, Jared Young1 1Mills College, 2University of California, San Francisco

In recent years, fluorescent imaging has been increasingly used to capture the state of biological systems at different moments in time. For many researchers, analysis of the fluorescent image data has become the limiting factor of this new technique. Wepresent NEPIC, a semi-automated tool for finding and tracking the soma of a single neuron over an entire movie of grayscale calcium image data. When tested on calcium image movies of the AWC neuron in C. elegans, NEPIC correctly identified the neuronal soma in 95.48% of the movie frames, and successfully tracked this soma feature across 98.60% of the frame transitions. Although support for finding and tracking multiple fluorescing Regions of Interest (or fROIs) has yet to be implemented, NEPIC displays promise as a tool for assisting researchers in the bulk analysis of fluorescent imaging data.

148 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 149 Inducible and titratable silencing of C. elegans neurons in vivo with histamine-gated chloride channels Navin Pokala1, Qiang Liu1, Andrew Gordus1, Cornelia Bargmann1 1The Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior and HHMI, The Rockefeller University

We describe a chemical genetic approach for inducibly silencing C. elegans neurons in whole animals, using the histamine-gated chloride channel HisCl1 from Drosophila. C. elegans does not use histamine as an endogenous neurotransmitter. Administration of histamine to freely-moving animals expressing HisCl1 in neurons leads to rapid, potent, and reversible inhibition of neural activity, as assessed by behavior, calcium imaging, and electrophysiology of neurons expressing HisCl1. On its own, histamine has little apparent effect on C. elegans behavior in the absence of HisCl1 transgenes, while HisCl1 transgenes do not cause behavioral defects in the absence of histamine even when expressed pan- neurally from high-copy arrays. Using cell-type specific promoters, we show effects of HisCl1 and histamine on sensory neurons, interneurons, and motor neurons that match their known functions. In addition to outright silencing, the histamine-HisCl1 system can also be used to titrate the level of neural activity, revealing the quantitative relationships between neural activity and behavioral output. The activities of the AVA command interneuron and the DA/VA motorneurons appear to encode reversal length in a dial-like manner. In contrast, the activities of the AIB and AIY interneurons appear to act as switches to trigger and inhibit pirouette events respectively, while having little effect on the events themselves. The histamine-HisCl1 system is effective, robust, and easily applied to individual animals as well as populations. Histamine is inexpensive, and is compatible with nearly any behavioral assay, since it can simply be added to the media. Its ease of combination with light-activated calcium indicators and optogenetic tools should make it a useful addition to C. elegans neurotechnology.

New Technologies Poster Session 149 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 150 Methods for Studying Nervous System Development in the C. elegans embryo Anupriya Singhal1, Peter Insley1, Shai Shaham1 1The Rockefeller University

Glial cells play critical roles in the development of the nervous system in C. elegans (Yoshimura et al, 2008), but neuron-glia interactions during embryogenesis are poorly understood. We propose to study these interactions by large-scale imaging of fluorescent reporters. This study presents three important challenges. First, most cell-specific reporters are not expressed early enough in embryogenesis to visualize the developmental period of process outgrowth. Second, the lack of landmarks and the changing complement of cells makes identification of cells in the C. elegans embryo challenging. Third, development of the nervous system continues after embryos begin to twitch, making it difficult to image cells over time without motion blur. We are developing methods to address these problems. To label cells in the embryo, we are developing a method, based on a previously described setup (Kamei et al, 2009), which uses an infrared laser to target the heat shock response in individual cells and labels them via a phsp-16.2::GFP transgene. We have adapted this method for use in embryonic cells, which are smaller than larval cells and more sensitive to heat toxicity, by using a combination of laser pulsing and specimen cooling to achieve specific induction and cell viability. Using our method, we are able to achieve cell-specific labeling of neuronal and glial precursors at the 200 cell stage, after which the descendants are labeled. To identify cells prior to labeling, we are developing a new computer-automated method for real-time identification which relies on the relative position of cells and not their complete lineal history. Automated cell identification can be applied to cell ablation experiments or embryonic expression profiling of existing reporters. Using our method, we are able to identify cells with ~70% accuracy on average at arbitrary stages prior to the final division, and with higher accuracy at particular developmental time points. Finally, we have built an iSPIM light- sheet microscope (Wu et al, 2011) to allow rapid imaging of embryos with minimal motion blur. We plan to apply these methods to study in quantitative fashion the morphogenesis of neurons and glial cells during the development of the C. elegans nervous system in wild-type animals and mutants.

150 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 151 High-throughput reverse genetic screen for synaptic vesicle recycling mutants by optogenetics and Ca++ imaging Sebastian Wabnig1, Jana F. Liewald1, Alexander Gottschalk1 1Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Germany

Previous screens or analyses of genes involved in synaptic transmission were based on forward or reverse genetic approaches, followed by pharmacological assays with behavioral readouts, or from ChR2 induced neuronal hyperstimulation (Miller, 1996, Sieburth 2005, Kuwahara 2008, Liewald 2008, Vashlishan 2008). Besides being potent tools, these screening approaches were tedious due to low throughput. Attempts to use microfluidic chip systems for automatization (Stirman 2010) were of limited success. Moreover, previous screens could not distinguish between general synaptic transmission defects and defects based on synaptic vesicle recycling. We thus developed a new screening method based on Channelrhodopsin stimulation of cholinergic or GABAergic motor neurons, in combination with simultaneous Ca++ imaging in body wall muscles, using RCaMP as Ca++ sensor (Akerboom 2013). Single animals were analyzed to characterize the approach. To gain high throughput, assays were performed in simple agar-based ‘macro fluidic’ devices allowing bulk measurements of approximately 1700 worms per run. For evaluation, we used different mutants affecting synaptic transmission in cholinergic and GABAergic motor neurons, as well as synaptic vesicle recycling. Since neurons can be stimulated for prolonged periods (> 1 minute) using ChR2, recycling defects become obvious due to an early decay of the Ca++ signal in muscle. To facilitate reverse genetic screening in cholinergic neurons, we generated a new strain for enhanced neuronal RNAi in just these neurons, to avoid lethal phenotypes from the respective protein missing in other tissues, similar to Firnhaber et al., 2013. This was achieved by over expressing SID-1 in neurons of rde-1(ne219) mutants, with a specific RDE- 1 rescue in cholinergic neurons. We performed an RNAi screen of approximately 150 candidate genes previously involved in synaptic transmission, to pinpoint genes likely affecting recycling vs. SV release. Mutants of genes resulting from our RNAi screen showed reproducible phenotypes from imaging up to electrophysiological analysis. Our screening approach allows high throughput rates for characterizing synaptic transmission via RNAi on a conventional epifluorescence microscope equipped with two high power LEDs and a sCMOS camera, along with open source software for acquisition and analysis (µManager, ImageJ). Results on some of the analyzed genes will be presented at the meeting.

New Technologies Poster Session 151 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 152 Imaging chromatin dynamics at specific loci in the live animal Bo Zhang1, Baohui Chen2, Jordan D Ward4, Bo Huang2,3, Noelle D. L’Etoile1 1Departments of Cell & Tissue Biology and Medicine, University of California, San Francisco, 2Department of Pharmaceutical Chemistry, University of California, San Francisco, 3California Institute for Quantitative Biomedical Research (QB3), 4Department of Cellular and Molecular Pharmacology, University of California, San Francisco

Gene expression is regulated dynamically during development. The status of chromatin is an indication and a regulation tool of gene transcription activity in eukaryotes. Studies have shown that some sites of euchromatin can switch to heterochromatin under specific developmental signaling cues, and vice versa (Oberdoerffer & Sinclair, 2007; Trojer & Reinberg, 2007). Previous studies in our lab have demonstrated that in the AWC olfactory sensory neurons of C. elegans, odr-1, which encodes a guanylyl cyclase, is repressed in odor-adapted animals (Juang et al., 2013). In addition, we also observed increased binding of the heterochromatin associated factor HPL-2 at the odr-1 locus in AWC as a result of this adaptation. This suggests that the repression of odr-1 expression might be attributed to chromatin conformation changes. We plan to develop a CRISPR/Cas-based fluorescence imaging technique to facilitate monitoring of the dynamic changes in chromatin structure at specific loci, such as odr-1. By targeting endonuclease-deficient Cas9 tagged with fluorescent proteins to specific sites around a DNA locus, chromatin conformation changes may alter the distance and interaction between these reporters yielding a visible change under the microscope. Our hope is that this tool will allow us to study the chromatin dynamics at specific chromosomal loci in live animals.

152 New Technologies Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 153 Dendritic Arborization in Dauer IL2 Neurons: Role of Surrounding Tissue and Post-Dauer Branch Recovery Rebecca Androwski1, Alina Rashid1, Nathan Schroeder2, Maureen Barr1 1Rutgers, The State University of New Jersey, 2University of Illinois at Urbana- Champaign

Under adverse environmental conditions, C. elegans enters a stress-resistant dauer stage. We discovered that as wild-type animals enter the dauer stage, the six IL2 inner labial sensory neurons undergo a morphological reorganization. This stress-induced plasticity is especially dramatic in the four IL2 quadrant neurons (IL2Qs), which undergo extensive arborization that results in a three-fold increase in total dendritic length. When dauer animals are moved to a favorable environment, they recover from dauer and resume development. During dauer recovery, the bulk of the branches resorb. While IL2 branching is similar to the arborization pattern seen in FLPs and PVDS, the IL2 arborization process is stress-induced, reversible, and occurs within a shorter period of time. We identified several genes necessary for wild-type branching through a genetic screen and a candidate gene approach. kpc-1, a kex2-like proprotein convertase, is required for organized arborization in both dauer IL2 neurons and adult PVD and FLP multidendritic neurons. We show through cell-specific rescue that kpc-1 acts autonomously in the IL2s to modulate branch patterns1. We also found that during dauer, SAX-7, a transmembrane receptor involved in cell adhesion, maintains the integrity of the higher-order branches, but is not necessary for the animals to form branch points along the primary dendrite. Dong et al. 2013 showed that SAX-7 is present in the hypodermis and forms a complex with extracellular proteins DMA-1 and MNR-12, interacting with the PVD neuron to stabilize branch pattern. Therefore, we are interested in whether the SAX-7/DMA-1/MNR-1 complex is important for branching during dauer and how surrounding tissues support these elaborate neurons. Additionally, we have further characterized branch resorption and the remnant branches which persist into adult. Branch resorption appears to be independent from arborization, as kpc-1 and sax-7 animals are able to recover from dauer and resorb their branches, despite their deficiency in forming organized arbors.

Reference(s) 1. Schroeder, NE. et al., Current Biology. (2013), 23(16):1527-35. 2. Dong, X. et al., Cell. (2013), 155(2):296-307.

Sensory Signaling Poster Session 153 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 154 Identifying odor receptors in C. elegans Sherrlyne Apostol1, Newman Elizabeth1, Sara Nathan1, Alesha Cox-Harris1, Tan Fanny1, Chantal Brueggemann2, Noelle L’Etoile2, Jared Young1 1Mills College, 2University of California, San Francisco

Although scientists have been studying olfaction in C. elegans for decades, olfactory receptor proteins remain largely uncharacterized. We are currently using two approaches to address this knowledge gap. A forward genetic screen was carried out to identify genes involved in odor signaling. Mutants were selected if they displayed repeated lack of attraction to benzaldehyde (which is sensed by the olfactory neuron AWC) after exposures to benzaldehyde and E. coli. 27 worm strains were isolated as potentially interesting mutants. We are currently analyzing these mutants for olfactory defects, and have identified two such lines. We are also pursuing localization of candidate proteins. Data generated by Yen-Ping Hsueh in the Sternberg lab identified a set of putative odor receptor genes that are expressed in AWC. We selected eight of these genes for localization analysis and obtained GFP-reporter constructs from the Transgenome Project. We are producing tagged lines and determining which of these proteins localize to the olfactory cilia.

154 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 155 Olfactory sensory Neuron Regulation of Physiology in Response to Environment Aarati Asundi1 1University of California, San Francisco

The balance between proliferation and differentiation of stem cell populations must be responsive to changing molecular and physiological conditions. However, the mechanism behind this plasticity is not well understood. The C. elegans nematode is a tractable organism to study the influence of food sensation on physiology. Recent studies suggest that the number of proliferating germ cells (PGCs), the only stem cell population in the adult C. elegans body, respond to the environment by communication with sensory neurons. Sensory neurons are able to relay information about food abundance to the PGC pool. Specifically, TGF-b signaling from the ASI gustatory neuron promotes a larger PGC pool when food is abundant and a reduced PGC pool when food is scarce [Dalfo et al. 2012]. The olfactory sensory neurons (OSNs), AWA and AWC, are also food-sensing neurons [Chalasani et al. 2007] and thus, may also regulate the PGC pool. Indirect evidence suggests that the OSNs along with ASI reduce lifespan, perhaps via the germline [Alcedo et al. 2004 and Hsin et al. 1999]. We propose that the OSNs may affect the physiology of the C. elegans nematode, as assessed by lifespan and the PGC pool, via secreted molecules. DAPI staining data indicates that functional OSNs are required to maintain proper proliferation of the PGCs. Studies also suggest that the neurons secrete signals to affect behavioral responses to odor. For example, the AWC releases both the neurotransmitter glutamate, which promotes odor chemotaxis, and the neuropeptide NPL-1, which promotes odor adaptation [Chalasani et al. 2010]. Therefore, our hypothesis is that the OSNs are able to regulate the PGC number via secreted molecules.

Sensory Signaling Poster Session 155 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 156 Calcium measurements in AWC after adaptation to benzaldehyde Chantal Brueggemann1, Noelle L’Etoile1 1University of California, San Francisco

Odor sensation and chemotaxis are essential responses that allow C. elegans to locate and move towards food. The paired sensory AWC neurons sense innately attractive odors such as benzaldehyde, butanone or isoamyl alcohol. However, the worm must also be able to ignore profitless odors. Thus, prolonged exposure of the AWC neurons to benzaldehyde in the absence of food leads to adaptation which allows the worm to ignore odors that are not associated with nutrition. We examine the effects of long-term odor exposure on the acute odor response in AWC and downstream interneurons by measuring calcium transients. Our results show that the calcium transients in neither AWC nor AIA neurons do not change after adaptation when compared to naïve worms. We hypothesize that other neurons might be involved in the adaptation process. Therefore we will ablate other neurons using recCaspase. We will screen for neurons involved in adaptation of AWC sensed odors by analyzing their behavior.

156 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 157 A role for muscle-skin interactions in shaping PVD sensory dendrites Kevin Celestrin1, Hannes Bülow1 1Albert Einstein College of Medicine

Complex dendritic arbors facilitate the reception and transduction/integration of a wide variety of environmental stimuli, such as temperature, touch, and pain. Similarly, the complex dendritic arbors of cortical neurons in the central nervous system allow the cortex to function as the center for reasoning, learning and memory. The loss of these complex dendritic arbors represents a hallmark of psychiatric disorders such as schizophrenia and autism spectrum disorders. A wide variety of genes have been implicated in the regulation of dendrite growth including transcription factors, regulators of intracellular transport, and modulators of Golgi- ER and endosomal dynamics. However, the detailed molecular mechanisms and genetic pathways that regulate dendrite branching and dendritic arbor formation remain poorly understood. Specifically, the mechanism of how extracellular factors regulate dendrite development. It has recently been shown that integrins, receptors for ECM proteins, are required for proper dendrite branching in the Drosophila nervous system supporting a significant role for coordinated tissue interaction in dendritic development. InC. elegans it has been shown that neighboring tissue plays a role in dendrite formation via the skin derived cue mnr-1/menorin. In preliminary studies utilizing the highly branched mechanosensory neuron PVD in Caenorhabditis elegans, we have identified a role in PVD development for several genes that are part of a complex that coordinates tissue interactions between the muscle and skin. These findings suggest that these structures may play a role in shaping dendrite arbors. We will report on our progress to elucidate how muscle-skin interactions coordinate PVD dendritic arbor formation.

Sensory Signaling Poster Session 157 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 158 Noxious stimuli suppress food sensation in ASI Kristen Davis1, Young-jai You1 1Department of Biochemistry & Molecular Biology, Virginia Commonwealth University

Satiety quiescence is a behavioral state defined as a lack of movement or feeding following worm satiation. ASI is essential for worms to enter satiety quiescence. Previously we found the presentation of nutrients directly activates ASI. However, the presence of nutrients does not always stimulate feeding behavior. To understand how feeding is controlled by external cues, we treated worms with nutrients mixed with noxious stimuli such as a high concentration of NaCl (4 M - (Chatzigeorgiou, Bang, Hwang, & Schafer, 2013)) or glycerol (1M –(Hilliard, Apicella, Kerr, Suzuki, Bazzicalupo, & Schafer, 2005)). Our preliminary results are that these noxious stimuli override the ASI activation by nutrients, suggesting feeding, a potential result after sensing nutrients via ASI, can be suppressed in the presence of noxious stimuli. Because these noxious stimuli are mostly sensed by ASH neurons, we are currently testing a hypothesis that ASH acts upstream of ASI and suppresses the ASI activation (by nutrients) in the presence of noxious stimuli. If this hypothesis is correct, our next step will be to determine how ASH suppresses ASI; we will determine the neurotransmitter(s) involved and whether ASH directly or indirectly suppresses ASI.

Reference(s) 1. Chatzigeorgiou, M., Bang, S., Hwang, S. W., & Schafer, W. R. (2013). tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature, 494(7435), 95–9. doi:10.1038/nature11845 2. Hilliard, M. a, Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P., & Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. The EMBO Journal, 24(1), 63–72. doi:10.1038/sj.emboj.7600493

158 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 159 bHLH factors and insulin signaling are required for feeding-state dependent regulation of chemoreceptor gene expression Matt Gruner1, Dominic Valdes1, Dru Nelson1, Alexander A.M. van der Linden1 1University of Nevada, Reno

Animals dramatically modify their olfactory behaviors to attractive and noxious odors when starved. This could allow them to alter and optimize their food-search strategies to increase their survival and reproduction. Dynamic changes in the gene expression of chemoreceptors specialized in detecting odors is observed in fish, insects and nematodes, and may be a general mechanism underlying the changes in olfactory behaviors observed in starved animals. How does starvation lead to expression changes of chemoreceptor genes in the chemosensory system? Previously, we found that starvation decreases the expression of a candidate chemoreceptor, srh-234, in the ADL nociceptive neuron. These expression changes in srh-234 are dependent on circuit inputs from the RMG interneuron via Neuropeptide Y receptor, NPR-1, signaling. Recently, we tested whether insulin signaling is also a mediator of the starvation-dependent regulation of srh-234 expression. We found that expression of srh-234 during fed conditions is dependent on DAF-2, which is the sole insulin-like receptor in C. elegans, and its downstream effector, DAF-16. During prolonged starvation the basic helix- loop-helix transcription (bHLH) factors, HLH-30 and MXL-3, modify the transcriptional program of intestinal cells to initiate lipolysis and autophagy. We found that mxl-3(lf) mutants reduce srh-234 expression in fed conditions, whereas DAF-2 is necessary for hlh-30(lf) enhancement of srh-234 expression during starvation. These results may suggest that during starvation transcriptional changes in the intestine are communicated via unknown insulin-like peptide(s) to ADL neurons. To test this hypothesis, we are currently exploring the site of action of the DAF-2/DAF-16 pathway as well as MXL-3/HLH-30 in the starvation-dependent regulation of srh-234. Moreover, sequence analysis of the srh-234 promoter identified putative E-box and MEF2 motifs known to bind bHLH and MEF-2 factors, respectively, and we are determining whether these motifs are required for starvation-dependent regulation of srh-234. Finally, we have identified HLH-2, -3 and -10 as regulators of srh-234 expression. Taken together, our results provide insight into the neural and molecular mechanisms of how expression changes in chemoreceptor genes may contribute to changes in chemosensory behavior as a function of feeding state.

Sensory Signaling Poster Session 159 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 160 Dissecting the signaling mechanisms underlying the recognition and preference of food odors in C. elegans Gareth Harris1, Yu Shen1, Heonick Ha1, Alessandra Donato2, Samuel Wallis1, Xiaodong Zhang1, Yun Zhang1 1Harvard University, 2Queensland Brain Institute, Queensland

Food is critical for survival. Many animals, including the nematode Caenorhabditis elegans, utilize the sensorimotor system to detect and locate preferred food sources. However, the signaling mechanisms underlying food-choice behaviors are poorly understood. Here, we characterize the molecular signaling that regulates recognition and preference between different food odors in C. elegans. We show that the major olfactory sensory neurons, AWB and AWC, play essential roles in this behavior. A canonical Gα protein, together with guanylate cyclases and cGMP-gated channels, are needed for the recognition of food odors. The food- odor evoked signal is transmitted via glutamatergic neurotransmission from AWC and through AMPA and Kainate-like glutamate receptor subunits. In contrast, peptidergic signaling is required to generate preference between different food odors while being dispensable for the recognition of the odors. We show that this regulation is achieved by the neuropeptide NLP-9 produced in AWB, which acts with its putative receptor NPR-18, and by the neuropeptide NLP-1 produced in AWC. In addition, another set of sensory neurons inhibits food odor preference to overall provide a balance between both stimulatory and inhibitory sensory pathways to shape olfactory dependent food preference. These mechanistic logics, together with a previously mapped neural circuit underlying food-odor preference, provide a functional network linking sensory response, transduction and downstream receptors to process complex olfactory information and generate the appropriate behavioral decision essential for survival.

160 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 161 Electrodiffusion model for Ca2+ dynamics of a whole single neuron Yuishi Iwasaki1, Sayuri Kuge2, Takayuki Teramoto2, Takeshi Ishihara2 1Department of Intelligent System Engineering, Ibaraki University, 2Department of Biology, Kyushu University

Recent developments in 4D imaging technique enable us to measure the neural activity in a whole single neuron. In AWCON neuron, for example, Ca2+ response to an olfactory stimulus was slightly different at dendrite, soma and axon (19th IWM, 568C). This result showed that propagation of the neural activity was not passive along the neurite. To understand spatial- temporal Ca2+ dynamics in a whole single neuron, we construct a numerical model of Ca2+ electrodiffusion whose result is able to compare with the Ca2+ imaging data quantitatively. Spatial-temporal variables in our model are the concentrations of Ca2+, Ca2+ buffer molecule, fluorescent protein and +K together with the membrane potential. From theoretical analysis on the following equations, we found that ion as current carrier along the neurite is insufficient when only Ca2+ is considered. Therefore, K+ is introduced as an additional current carrier. Electrodiffusion equations of Ca2+ and K+ are based on the Nernst-Planck equation. That is, temporal changes in the concentrations are driven by diffusions, electrical gradient, chemical reactions with molecules and ionic flux through ion channels. The membrane potential is determined by the law of conservation of charge. Because model parameters of the fluorescent protein are able to determined by affinity and kinetics of Ca2+ indicator such as GCaMP, YC or G-GECO, our model provides “fluorescence intensity” corresponding to the Ca2+ imaging data. From correspondence between our equations and the cable equation under certain assumptions, we estimated electrical conductivity of the neurite. Our electrodiffusion model for Ca2+ dynamics is a theoretical tool to quantitatively investigate the neural activity in a whole single neuron. This research was supported by JST, CREST.

Sensory Signaling Poster Session 161 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 162 Digital transplants: DEG1/MEC4 chimeras reveal functional differences between Degenerin sodium channels Samata Katta1, Amy Eastwood1, Valeria Vasquez1, Miriam Goodman1 1Stanford University

Sensory systems need to be able to detect stimuli over a wide range of strengths. In the case of somatosensation, this is achieved through a set of mechanoreceptor neurons that vary in their operating range. It is not known what determines the sensitivity and operating range of individual mechanoreceptor neurons, however. Here, we focus on the contributions of two closely related sodium channels: MEC4, a poreforming subunit of the mechanotransduction complex in the touch receptor neurons (TRNs), and DEG1, which performs the same function in ASH nociceptors. Mechanoreceptor currents in the TRNs require MEC4 and are activated by forces as low as 50nN. In the ASH neurons, such currents rely on DEG1 and require forces larger than 5μN for activation. To understand how structural domains (identified in 3D crystal structures of a related sodium channel) contribute to such differences, we developed a domainswapping strategy, isolating domains predicted to be important for mechanical sensitivity, and comparing them in a common environment. In particular, we designed chimeric channels consisting of a MEC4 backbone with the extracellular finger and/or thumb domains of DEG1. When expressed in oocytes, chimeras containing the finger transplant are able to form channels, and they display constitutive amiloridesensitive currents that are larger than wildtype, intact MEC4. These changes in the finger domain also affect channel selectivity. In contrast, transplantation of the DEG1 thumb helices into MEC4 produces no detectable current. Preliminary work shows that chimeras containing both finger and thumb display currents that are larger than wild type, but smaller than chimeras with the finger alone. Next steps include asking how such digital transplants affect touch sensation and mechanoreceptor currents by expressing chimeric channels in the TRNS in mec4;mec10 double null mutants. (Reexpressing wildtype MEC4 from a singlecopy MosSci locus restores touch sensation and mechanoreceptor currents to mec4;mec10 mutants.) By improving our understanding of the molecular mechanisms that underlie differences in mechanosensitivity, we will also gain insight into the path mechanical energy follows in the process of mechanotransduction.

162 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 163 The role of TMC proteins in C. elegans sensory transduction Rhianna Knable1, William Schafer1 1MRC Laboratory of Molecular Biology, University of Cambridge

Transmembrane channel-like (TMC) genes encode a family of integral membrane proteins that are broadly conserved between species, with two family members in C. elegans and eight vertebrate members categorized into 3 subfamilies. Members of subfamily A, consisting of TMC1, TMC2 and TMC3, are more restrictively expressed and have been mostly implicated as having roles in sensory transduction. TMC1 and TMC2 are required for hair cell mechanotransduction, with mutations in TMC1 causing hearing loss in humans and mice. The third member of this subfamily, TMC3, has also been suggested as a candidate gene at another deafness locus, but is otherwise largely uninvestigated. In C. elegans, these TMC proteins have been shown to have more diverse functions. While TMC-2 is still implicated in mechanotransduction in some neurons, TMC-1 has instead been shown to play a role in sensing aversive stimuli such as high levels of salt. The relatively high conservation between human, mice and worm TMC proteins, especially in the key TMC domain, hints towards maintenance of some of these functions between species. We have been investigating the function of the TMC proteins in C. elegans, including further characterization of TMC-1 and TMC-2. In addition, we have found that mammalian TMC genes can be functionally expressed in worm neurons such as ASK and IL2, and thus have generated lines that heterologously express murine TMC3 in C. elegans in order to determine its function. We are using calcium imaging of individual neurons and stimulating animals that ectopically express mTMC3 with a range of aversive stimuli in the aim of identifying its agonists and characterizing the function of this largely ignored subfamily member. Results from these experiments will be presented. It is hoped that findings in C. elegans will also direct further studies in mice, and may identify additional functions for TMC proteins in these higher organisms.

Sensory Signaling Poster Session 163 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 164 Tracking of Ca2+ dynamics in a whole single neuron Sayuri Kuge1, Takayuki Teramoto1, Takeshi Ishihara1 1Department of Biology, Faculty of Science, Kyushu University

The nematode C. elegans is an excellent model organism for studying how neuronal circuits activity transform sensory signals. Ca2+ imaging of neurons using Ca2+ sensitive fluorescent proteins, such as GCaMPs and yellow cameleons, has revealed functions of neuronal circuits. To more understand how each neuron transmits sensory signals to next neurons, the characteristic of each neuron should be well understood. However, Ca2+ dynamics in a whole single neuron responding to sensory stimuli are still unclear. We analyzed Ca2+ dynamics in a whole single neuron by a wide-field imaging system, which enabled us to capture series of images of a whole single neuron with more than 54 images per second. For this imaging, we used animals expressing GCaMP6f in sensory neurons by specific promotors in the olfactory chip and observed Ca2+ dynamics depending on stimuli at cilia, a cell body and an axon, simultaneously. We also found that Ca2+ dynamics in a whole neuron were different among neurons. These results suggested that each neuron has unique response to sensory stimuli. We hope that our imaging system makes it possible to analyze precise Ca2+ dynamics of sequential rapid neuronal activation and inactivation in a whole single neuron and that these results are helpful for understanding of the whole neuronal circuit.

164 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 165 Identification of Factors Affecting Cilia Localization of PKD-2 in C. elegans Jamie Lyman Gingerich1, Kara Braunreiter1, Shelby Hamlin1, Casey Gabrhel1 1University of Wisconsin-Eau Claire

Our lab is interested in understanding how primary cilia sense and respond to cues from the cellular environment. To identify genes involved in the ciliary localization of the receptor protein, PKD-2, we are employing both reverse and forward genetic approaches in C. elegans. Using RNAinterference, we have systematically reduced the function of 86.8% of the genes (2126 genes) on chromosome I and have identified approximately 200 genes that affect PKD- 2 localization. Current efforts include further analysis of the cilia phenotypes resulting from reduction of function of these genes and categorization of these genes based on structure, expression pattern, and proposed function. In order to better understand the relationship between receptor localization, cilia structure and cilia function, we have characterized a mutation (my13) that results in not only defective receptor localization, but also defects in cilia-mediated processes. C. elegans homozygous for the my13 mutation exhibit altered sensory behaviors and structural abnormalities of the cilia. We have identified the my13 lesion and found that it is a missense mutation in osm-12, an ortholog of human BBS-7, a gene known to affect human cilia function and to be involved in Bardet-Biedl Syndrome.

Sensory Signaling Poster Session 165 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 166 Defining the cellular circuit of food type-dependent feeding behavior in C. elegans Shashwat Mishra1, Roxani Gatsi2, Anca Neagu2, Joy Alcedo1,2 1Wayne State University, 2Friedrich Miescher Institute for Biomedical Research

Neuropeptides can function either directly or indirectly to modulate synaptic activity. The number of predicted neuropeptides in C. elegans, as well as in humans, is over a hundred, but not much is known about their functions (1). The C. elegans NMUR-1 is a neuropeptide receptor that is a homolog of mammalian neuromedin U receptors (2). It is expressed in the somatic gonad and in sensory neurons, motor neurons and interneurons (2). While nmur-1 has been shown to shorten lifespan only on specific E. coli food sources, nmur-1 can also affect feeding rate in a food type-dependent manner (2). In this study, I will address the cell-specific requirements of nmur-1 in regulating feeding rate in response to different food sources. This approach should delineate a neural circuit that underlies food type-dependent feeding behavior and yield insight into similar mechanisms in mammals where neuromedin U receptors have also been shown to regulate food intake and feeding behavior (3).

Reference(s) 1. Husson et al. (2007) Prog Neurobiol 82: 33–55. 2. Maier et al. (2010) PLoS Biol 8: e1000376. 3. Howard et al. (2000) Nature 406: 70-74.

166 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 167 Study of transgenerational inheritance of acquired odor-related traits in Caenorhabditis elegans Fernando Munoz-Lobato1, Noelle L´etoile1 1UCSF

Animals thrive despite the vagaries of nature by adapting their innate behaviors to suit their circumstances. The ability to inherit the information about a parent’s experiences in a specific environment would represent an obvious advantage for progeny that inhabit the same environment. Though evidence for transgenerational inheritance of epigenetic effects is growing, the molecular mechanisms that would allow a parent’s sensory or behavioral experience to affect subsequent generations are lacking. Our lab has previously found that C. elegans sensory neurons produce small RNAs in response to environmental stimulation. Though these small RNAs arise from the sensory neuron, we find they induce organism- wide increases in small RNA and that this increase is dependent upon the double stranded RNA channel, SID-1. Intriguingly, it is well known that gene-silencing induced by exogenously provided dsRNA occurs not only in the treated animals, but also in the progeny of the treated worms. As a whole, this suggests that extracellular RNAs are responsible for the transfer of information about the environment from sensory neurons to the gonad where epigenetic changes to the germ line would allow transmission to the progeny. These changes would ultimately affect the progeny’s response to its environment. During the development of this project we aim to test these hypotheses.

Sensory Signaling Poster Session 167 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 168 Comparative genomics reveals novel genes associated with sensory cilia Thomas Sasani1, Oliver Newsom1, Brendan O’Flaherty1, Terese Swords1, Johan Henriksson2, Elizabeth De Stasio1, Peter Swoboda2, Brian Piasecki1 1Lawrence University, Department of Biology, 2Karolinska Institute, Department of Biosciences and Nutrition, Center for Biosciences at NOVUM

Cilia/flagella are microtubule-based organelles that facilitate a variety of sensory and motility-specific processes. Because of their widespread phylogenetic distribution and evolutionary conservation in most eukaryotic cells, it is likely that cilia were present in the last eukaryotic common ancestor (LECA). In this study a comparative genomics approach was used to identify ciliary genes that facilitate sensory-specific roles. Using reciprocal basic local alignment search tool (BLAST) analyses, the genomes of organisms that do not make cilia (Arabidopsis thaliana and Saccharomyces cerevisiae) and that retain motile but not sensory cilia (Physcomitrella patens) were subtracted from the genomes of organisms that have retained sensory cilia (Caenorhabditis elegans and Chlamydomonas reinhardtii). These analyses revealed a list of 272 genes that are found exclusively in organisms with sensory cilia but not motile cilia. Importantly, over 9% of the genes on this list have previously been implicated in the sensory cilia-specific roles, thus providing numerous internal positive controls that demonstrate this list is enriched with sensory-specific ciliary genes. A subset of uncharacterized candidate genes from this list are currently being studied in C. elegans, which retains cilia exclusively on a set of neurons termed ciliated sensory neurons (CSNs). Two of these candidate genes, which are found in worms (C. elegans) and algae (C. reinhardtii) but not in moss (P. patens) have been termed wam-1 and wam-2, respectively. We are currently generating a number of promoter- and gene-to-green fluorescent protein (GFP) fusion constructs in order to determine the expression and localization patterns of the proteins encoded by these genes in C. elegans. At this time, expression of wam-1 appears to be localized exclusively in ciliated dopaminergic neurons, while expression of wam-2 has yet to be fully characterized.

168 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 169 The role of innexins UNC-7 and UNC-9 in mechanosensory neurons Denise Walker1, William Schafer1 1MRC Laboratory of Molecular Biology

Gap junctions, composed of connexin (in vertebrates) or innexin (in invertebrates) subunits, allow free (though gated) movement of ions and small signalling molecules between cells, resulting in electrical coupling and the propagation of signals such as calcium waves. They are thus central to the coordinated development and organisation of multicellular organisms. However, the constituent hemichannels can also function independently, as gated channels, connecting the cytoplasm with the extracellular environment, and their opening can be triggered by, for example, changes in extracellular pH, Ca2+ concentration, or mechanical stimulation. An additional subunit family found in vertebrates, the pannexins, which share homology with innexins, are thought to function exclusively as channels1. The touch receptor neurons (TRNs), along with the command interneurons downstream, form part of a complex network of gap junctions and synaptic connections. While ALML and ALMR are not directly connected by gap junctions, they are connected via AVM2. We were therefore interested to investigate the role of gap junction connections in coordination between the anterior TRNs. However, pannexins can function as mechanosensors3,4, while Bouhours et al.5 recently demonstrated that a widely expressed innexin, UNC-7, functions as a hemichannel to regulate neuronal activity. So we were also interested in the possibility that innexin hemichannels could have a more direct role in mechanosensation in the TRNs. We demonstrate that two innexin subunits, UNC-7 and UNC-9 are required for gap junction communication between the anterior TRNs, and that they are required for propagation of the signal from the anterior TRNs that are within mechanoreceptive range, to those that are out of range. We present evidence that, in addition, UNC-7 functions as hemichannels, and that as such it plays an essential role in mechanosensation in both the TRNs and PVD.

Reference(s) 1. Sosinsky et al. (2011) Channels 5:193-7. 2. White et al. (1986) Philos. Trans. R. Soc. Lond. B Biol. Sci. 314:1-340. 3. Richter et al. (2013) FASEB J. 28:45-55. 4. Bao et al. (2004) FEBS Lett. 572:65-8. 5. Bouhours et al. (2011) Mol. Brain 4:16.

Sensory Signaling Poster Session 169 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 170 The transcription factor pros-1 is expressed in glia and regulates the morphology and function of sensory neurons Sean Wallace1, Yun Lu1, Shai Shaham1 1The Rockefeller University

Glia are found tightly associated with neurons throughout animal nervous systems. We are using the amphid sense organ as a model to study how glia regulate the morphology and function of neurons. We previously showed that post-embryonic ablation of the amphid sheath (AMsh) glial cell results in structural defects in the cilia of amphid sensory neurons, and functional defects in chemosensation. We have carried out a post-embryonic RNAi screen to identify glial regulators of neuronal function, through which we identified the Prospero-related transcription factor pros-1. Pros-1 is expressed in AMsh glia, as well as CEPsh glia, and glia of the inner and outer labial sense organs. Pros-1 expression is required in AMsh glia to maintain the integrity of the amphid channel, through which ciliated sensory neurons are exposed to the outside environment, and to maintain the wing-like structure of the glia-enveloped AWC neuron. Pros-1 loss-of-function therefore results in defective chemosensory behaviors. We are currently carrying out additional screens, based on microarray and ChIPseq data, to identify transcriptional targets of pros-1 responsible for these phenotypes, and have identified some candidate target genes. Prospero is a conserved homeodomain transcription factor, with a well-studied role in cell fate determination during embryonic nervous system development. Homologs of Prospero are also expressed post-embryonically in glia in a number of animals, but the function of this gene in differentiated glia has not been addressed. We have found that post-embryonic downregulation of pros-1 expression in glia results in the functional defects described above without affecting glial cell fate, allowing us to investigate novel aspects of the function of this gene in differentiated glia. Our ongoing studies aim to characterize the mechanisms through which pros-1 acts in AMsh glia to regulate amphid sensory neurons. We hope these studies will provide general insights in to how glia regulate the functional properties of the neurons with which they associate.

170 Sensory Signaling Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 171 Regulation of Synaptic Transmission through Complexin Ishani Basu1, Rachel Wragg1, Jeremy Dittman1 1Weill Cornell Medical College

Neurons communicate through specialized structures called synapses. Neurotransmitter filled vesicles fuse with the plasma membrane at the synapse, releasing chemicals that can be detected by the target cell. Modulation of the chemical signaling is essential for behaviors such as learning and memory; however, how transmission is regulated is not well understood. The SNAREs are the minimum proteins essential for vesicle fusion to occur. One of the proteins known to regulate SNARE activity is complexin (cpx-1), a cytosolic protein that has an inhibitory role in spontaneous synaptic fusion across different species. cpx-1 worms are hypersensitive to cholinesterase inhibitor aldicarb. Though complexin acts by binding with the SNARE complex and interacting with the lipid membrane of the vesicle, it is not known how complexin itself is regulated. Heterotrimeric G-proteins are thought to regulate

synaptic transmission. Of these Galphai/o is well known to inhibit synaptic activity. Using a

simple behavioral assay we observed that the Galphao (goa-1) mutant phenocopies cpx-1. This led us to explore the hypothesis that GOA-1 acts through CPX-1 to regulate synaptic activity. Overexpressing goa-1, ectopically expressing a GOA-1 linked GPCR (mAChR 2) in motor neurons and serotonin treatment (which acts through GOA-1 in motor neurons) result in decreased acetylcholine (ACh) secretion. However, in the absence of CPX-1 no such inhibition of secretion is seen. Furthermore, increasing GOA-1 activity has no effect when CPX-1 interactions with either SNARE proteins or synaptic vesicles are disrupted, suggesting GOA-1 mediated regulation of CPX-1 is not dependent on a novel role of complexin. We have identified several residues in the complexin C-terminal domain that are not essential for CPX-1’s inhibitory role but without which GOA-1 is unable reduce ACh release. GOA-1 may be regulating CPX-1 by causing a change in phosphorylation state at these residues. Furthermore, phosphorylation of these residues may regulate complexin’s affinity for highly curved membranes. By using both behavioral and biochemical techniques, we aim to better understand the molecular mechanism of complexin regulation and its effects on synaptic transmission.

Synaptic Function and Modulation Poster Session 171 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 172 Investigating Two Amine Oxidase Domain Containing Genes, amx-1 and amx-2, in Caenorhabditis elegans Reetobrata Basu1, Janet Duerr1 1Ohio University

Monoamines (MAs) affect multiple behaviors in animals. MA homeostasis is critical and is achieved partly by monoamine-oxidase (MAO) enzymes in vertebrates. MAO inhibitors are prescribed for a number of human neurological disorders; unfortunately, they have a large number of adverse side effects. In C. elegans MA trafficking is similar to that in humans, but the degradation of MAs in C. elegans has not been completely characterized. Several MA dependent behaviors are affected by MAOIs in a manner consistent with MAOI inhibition of MA degradation by MAOs. In this project, we have investigated two amine oxidase (AO) domain containing genes, amx-1 and amx-2, by means of heterologous protein expression (in E. coli and P. pastoris). Absorption spectra analysis in presence of the MAOI tranylcypromine indicated that AMX1 and AMX2 bind the redox cofactor flavin adenine dinucleotide (FAD). Biochemical assays were performed with the wild-type (N2) or mutant [amx-1(ok659), amx- 2(ok1235), spr-5(by101) or spr-5(by101); amx-1(ok659)] worm lysates as well as purified AMX1 and AMX2 (two isoforms – AMX2L and AMX2S). Purified AMX1 had very low monoamine oxidase activity in vitro and no significant histone demethylase (HDM) activity. However, AMX1 significantly increased the HDM activity of lysates of amx-1 (ok659), spr-5 (by101) or spr-5(by101); amx-1(ok659). Both AMX2 isoforms had very significant monoamine oxidase activity in vitro, with varied MA and MAOI specificities. This work thus suggests differing roles for these two AO domain containing genes in the worm.

172 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 173 Sink or swim: Discovery of a novel MAP kinase that acts in dopamine neurons to regulate swimming behavior Daniel Bermingham1, J. Andrew Hardaway1, Sarah Whitaker1, Sam Snider1, Randy Blakely1 1Vanderbilt University

The neurotransmitter dopamine (DA) acts across phylogeny to modulate fundamental aspects of physiology and behavior, including movement, appetite, reward and attention. The model system Caenorhabditis elegans is a powerful platform for the discovery and manipulation of genes controlling synaptic function, including genes that control DA production, secretion, inactivation and response. We have performed a forward genetic screen based on a hyperdopaminergic phenotype, “Swimming-induced paralysis” or Swip, displayed by animals with genetic ablation of the dopamine transporter, dat-1. The goal of the screen was to identify mutants that exhibit DA-dependent, dat-1-like Swip with the hope of identifying novel regulators of DA signaling. One such mutant, vt32, was localized by SNP mapping and whole genome sequencing to an uncharacterized gene, here referred to as swip-13. We find that swip-13 mutations result in significantly reduced sensitivity to the neurotoxic dat-1 substrate 6-OHDA, supporting a role for swip-13 in sustaining DAT-1 protein expression, surface trafficking and/or activity. Importantly, DA neuron-specific, transgenic expression of the wild-type swip-13 gene restores normal swimming behavior of swip-13 mutants, establishing expression by DA neurons as the key site of SWIP-13 expression to modulate DA signaling. Fluorescently-tagged, functional swip-13 protein localizes to DA terminals, consistent with a presynaptic role for SWIP-13. Importantly, evidence from a FRAP-based analysis of vesicular fusion in DA neurons indicates normal basal rates of vesicle release in swip-13 mutants, whereas genetic interaction with a dat-1 mutant suggests that swip-13 and dat-1 function in the same pathway. These results further support a role for SWIP-13 in regulating DAT- 1. SWIP-13 protein is highly conserved, likely representing the nematode ortholog of the mammalian atypical MAP kinase ERK7/8. Excitingly, human ERK8 overexpression in human cells increases the uptake capacity of co-expressed DAT. Ongoing efforts seek to uncover the mechanisms by which SWIP-13 and ERK7/8 modulate DA signaling with an eye as to how our findings may provide insights into disorders linked to perturbed DA signaling.

Synaptic Function and Modulation Poster Session 173 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 174 Unique mechanisms of pH regulation in C. elegans amphid sheath glia Jeff Grant1, Laura Bianchi1 1Department of Physiology and Biophysics, Miller School of Medicine, University of Miami

Glia are essential for maintenance of the ionic composition and pH of the synaptic microenvironment. Changes in the intracellular and extracellular pH of both glia and neurons affect the function of several types of ion channels, thus modulating the neuronal excitability. The amphid sheath glia of C. elegans are closely associated with 12 amphid sensory neurons. Our lab has previously shown that the pH sensitive DEG/ENaC channel ACD-1 is expressed in these glia, and that the activity of this channel is involved in modulation of chemosensory behavior in C. elegans. This highlights the likely importance of glial pH regulation in chemosensory signaling. However, to date no studies have been undertaken to determine the mechanisms by which the amphid sheath glia regulate pH. We expressed the GFP-based pH sensor Phlourin under the control of the glial-specific promoter PT02B11.3 to examine the mechanisms of intracellular pH regulation in amphid sheath glia, using in vivo fluorescent pH imaging. Incisions were made in the cuticle of the animals to allow for perfusion of solutions of different compositions over the glia. Analysis of the rate of acid extrusion after an acid-load in these cells revealed that they possess both HCO3--dependent and independent mechanisms of acid removal. Application of the anion exchange blocker DIDS or removal of extracellular Na+ significantly dampened HCO3--dependent acid extrusion in the sheath glia, indicating the likely presence of Na+- coupled HCO3- transporters in these cells. Furthermore, Cl- removal inhibited HCO3-- dependent acid extrusion after an acid load in these cells and also caused a transient alkalization under baseline conditions. These data indicate that Cl-/HCO3- exchange activity is functional in the sheath glia. Interestingly however, HCO3- entry into the glia at baseline pH was not inhibited by removal of Na+ or Cl-, or by several pharmacological inhibitors of known HCO3- entry mechanisms. This suggests that these glia have a unique mechanism of HCO3- flux. We are now performing expression analysis in order to determine the molecular identity of the transporters and/or channels involved in amphid sheath glia HCO3- transport. Once we have identified these proteins, we will determine the role they play in glial pH regulation using RNAi techniques. Furthermore, we will test whether disrupting glial pH regulation by knock- down of these transporters affects sensory perception.

174 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 175 Postsynaptic remodeling of GABAergic motor neurons in C. elegans is transcriptionally regulated by UNC-55 and IRX-1 Siwei He1, Alison Philbrook2, Michael Francis2, David Miller3 1Neuroscience Graduate Program, Vanderbilt University, 2Department of Neurobiology, University of Massachusetts Medical School, 3Neuroscience Graduate program and Department of Cell & Developmental Biology, Vanderbilt University

Neural circuits are actively remodeled during development; however, the mechanisms underlying this process and the timing of rewiring remain largely unknown. The Dorsal D (DD) GABAergic motor neurons of C. elegans undergo extensive remodeling during development. DD synapses initially innervate ventral muscles but are relocated to the dorsal side at the end of the first larval stage (L1). The COUP-TF homolog, UNC-55, functions in Ventral D (VD) motor neurons to block this remodeling program such that VDs maintain output to ventral muscles. Previous studies have focused on the presynaptic components of remodeling DD and VD neurons, whereas the dynamic changes on the postsynaptic side of these remodeling cells have not been investigated. The non-α subunit of the acetylcholine receptor, ACR-12, functions in GABAergic motor neurons to detect input from cholinergic motor neurons in the ventral nerve cord. We used ACR-12::GFP to monitor remodeling in the GABAergic circuit. Initially, ACR-12::GFP is localized to the dorsal processes of DD motor neurons and then relocates to the ventral side at the L1/L2 transition. Similarly, VD motor neurons undergo ectopic remodeling in unc-55 mutants and show strictly ventral ACR-12::GFP puncta in adults. Thus, our findings confirm that presynaptic and postsynaptic markers effectively switch locations in remodeling DD and VD motor neurons and that UNC-55 regulates both of these outcomes. Using cell-specific RNA interference (RNAi), we demonstrated that a downstream target of UNC-55, the Iroquois-like homeodomain protein, IRX-1 is also required for postsynaptic remodeling. We are now actively testing other UNC-55 targets for roles in postsynaptic remodeling. The well-established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems.

Synaptic Function and Modulation Poster Session 175 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 176 Conserved genes regulate sleep in C. elegans Huiyan Huang1, Komudi Singh1, Anne Hart1 1Dept. of Neuroscience, Brown University

Sleep is ubiquitous in animals. Yet, it is unclear how deeply the genes and pathways regulating sleep are conserved across species. Evidence that Notch, EGF and cGMP regulate sleep in multiple species encouraged us to more broadly survey conservation. We shortlisted 19 genes that affect sleep in Drosophila from the literature and tested the impact of their C. elegans orthologs on sleep during the L4-to-adult lethargus. All of the genes tested altered quiescence during the last larval to adult (L4/A) lethargus, and had the expected effect(increased/decreased quiescence) with only two exceptions. This makes it clear that there is deep conservation of molecular mechanisms required for sleep in Drosophila and C. elegans. The striking conservation observed in these two disparate invertebrate animals suggested that conserved genes regulate sleep in all animals. In C. elegans, the level of Notch activity affects both quality and quantity of lethargus quiescence. And, transiently overexpressing the Notch co-ligand, OSM-11, is sufficient to drive inappropriate, anachronistic quiescence in adult animals, which can be suppressed by loss of either Notch receptor or downstream players in Notch signaling. To find pertinent transcriptional targets of Notch signaling and to gain insight into sleep regulation, we undertook a genetic screen to identify suppressors of anachronistic quiescence in adult hsp::osm-11 animals. In this screen, 2122 mutant lines were assessed; 79 independent isolates suppressed the OSM-11-induced anachronistic quiescence. To exclude genes whose loss primarily impacts on locomotion, we next examined endogenous L4/A lethargus quiescence in these 79 strains using 1) an adaption of the Multi-Worm Tracker and 2) the microfluidic chamber-based assay. Twenty-seven strains had defects in endogenous lethargus quiescence. We are working to identify the corresponding genes by whole genome sequencing and will present our results in hand. Given the profound conservation of genes regulating sleep across species, we are confident that genes identified in this C. elegans screen will reveal conserved regulators of sleep across species.

176 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 177 Investigating novel targets of the DAF-19 transcription factor in adult- stage C. elegans Alexander Hurlburt1, Brian Piasecki1, He Zhang1, Debora Sugiaman-Trapman2, Peter Swoboda2, Elizabeth De Stasio1 1Lawrence University, Biology Department, 2Karolinska Institute, Department of Biosciences and Nutrition

Regulatory Factor X (RFX) transcription factors are a conserved family of proteins that regulate gene networks involved in ciliogenesis in both vertebrates and invertebrates. The only RFX transcription factor gene in C. elegans, daf-19, encodes at least four related protein isoforms. Roles for the two smaller isoforms have been identified: DAF-19C is known to regulate genes involved in ciliogenesis of sensory neurons while DAF-19M directs cilia specialization. Quite recently, DAF-19 has been implicated in the regulation of neuronal arborization and in innate immunity. In addition, studies suggest that a larger DAF-19 isoform plays a role in synaptic maintenance. In an effort to better understand the role of DAF-19 in older animals, we undertook a transcriptome comparison of daf-19 mutant and wild-type two- day old adults, in which ciliogenesis and cilia specialization should be completed. Microarray analysis revealed 177 genes to be differentially expressed in daf-19 mutant adults. We have analyzed the expression patterns of a subset of these genes using transcriptional GFP reporters. Expression of several genes in neurons appears to be dependent on daf-19 in adult worms. Interestingly, none of the daf-19 dependent genes identified in this study are known to contain an X-box promoter motif, the DNA sequence targeted by DAF-19C. Our analysis has, therefore, identified novel targets of the DAF-19 RFX transcription factor and suggests that DAF-19 regulates gene expression either together with other DNA binding proteins or at different target DNA sequences.

Synaptic Function and Modulation Poster Session 177 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 178 A two-tier system of synapse-proximal and synapse-distal neurotransmitter transporters mediates glutamate clearance in C. elegans KyungWha Lee1, Jenny Wong1, Itzhak Mano1 1Department of Pharmacology, Physiology, and Neuroscience, Sophie Davis School of Biomedical Education, City College of New York

Tight control of the major excitatory neurotransmitter Glutamate (Glu) is essential for maintenance of precise neural signaling and normal behavior throughout the animal kingdom. The C. elegans nervous system lacks glial barrier that blocks glutamate spillover to adjacent synapses. Intriguingly, we revealed that C. elegans utilizes a two-tier system of Glu clearance, consisted of the role of both synapse-proximal (glt-1 and glt-4 expressed in head muscles and sensory neurons) and synapse-distal (glt-3, glt-6, and glt-7 expressed in the canal cell) glutamate transporters (GluTs). These GluTs seem to have a major role in Glu clearance, as they control Glu-regulated behaviors. We are in the process of looking into physiology of individual GluTs by expressing each one in Xenopus Oocytes and examining its functions. Examining mutant animals, we observe that some behaviors are differentially affected by mutations in proximal vs. distal GluTs. Furthermore, we speculate that Glu increase in some synapses due to glt mutation might result in spillover of Glu between synapses and circuits, and that this spillover may alter behaviors. Indeed, in sodium drop assays, we found that the proximal GluT mutants reveal avoidance to low sodium, in contrast to wildtype’s attraction to it. Low sodium is normally detected by ASE neurons, which transmit the signal to the ASE neurons’ postsynaptic neuron AIB and induce forward mobility. Since in proximal GluT mutants low sodium causes backward mobility, we suspect that the increased level of Glu in these animals’ synapses might have caused ASE-mediated synaptic release of Glu to spilled out of the chemo-attractant circuit and over to neighboring synapses, where Glu excites aversion circuits and generates the unexpected avoidance response. To address if spillover is the mechanism underlying abnormal behaviors, we examine changes in activity of a postsynaptic interneuron utilizing GCaMP imaging (and the Chronis & Bargmann microfluidic chip) upon stimulating a sensory neuron of one circuit and monitoring responses in a neighboring one. Altogether, elucidating mechanisms underlying the maintenance of Glu signaling and synaptic separation in C. elegans will allow us to suggest common strategies for Glu clearance and synaptic separation in other organisms.

178 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 179 The C. elegans RID neuron is a neurosecretory cell that regulates synaptic development and motor behavior Maria Lim1, Valeriya Laskova1, Jyothsna Chitturi1, Douglas Holmyard1, Daniel Findeis2, Anne Wiekenberg2, Jinbo Wang3, Ralf Schnabel2, Xun Huang3, Mei Zhen1 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 2Technische Universität Braunschweig Carolo Wilhelmina, Institut für Genetik, 3Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Complex 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, a neuron with unique morphology along the dorsal nerve cord and that exclusively contains dense core vesicles. We provide several lines of evidence that the RID neuron is a neuroendocrine cell that modulates synaptic development and motor behavior. We further identify unc-39 as a gene that is required for RID development. unc-39 mutants are missing RID and behave similarly to RID-ablated worms. We further describe that unc-39 regulates cell division in the cell lineage that gives rise to the RID neuron. We propose that RID can serve as a genetic model to probe conserved molecular mechanisms underlying neuroendocrine cell development and function.

Synaptic Function and Modulation Poster Session 179 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 180 Investigating the synaptic role of the Gαs pathway Laura Manning1, Janet Richmond1 1UIC Biological Sciences

Previously, studies of C. elegans Gαs pathway mutants demonstrate the importance of this signaling cascade in locomotion (Reynolds et al, 2005). Specifically, the loss-of-function mutants in the Gαs effector adenylate cyclase (acy-1) exhibit profound paralysis, whereas up-regulation of the cAMP-dependent kinase, PKA, via elimination of the regulatory subunit

KIN-2 produces hyperactivity. Despite evidence that the Gαs pathway converges on the core

Gαq pathway that is required for UNC-13-dependent priming of synaptic vesicles, precisely

how the Gαs pathway regulates synaptic function remains to be resolved. To shed light on this issue, we have begun a detailed characterization of synapses in acy- 1(neuron null) and kin-2 loss-of-function mutants. We first examined their sensitivity to the acetylcholine esterase inhibitor, Dylox as an initial readout of synaptic function. Consistent with their opposing locomotory defects we found kin-2 mutants to be Dylox-hypersensitive, whereas acy-1 mutants were Dylox-resistant when compared to wildtype animals. Despite these apparently strong indications of altered acetylcholine release, maximal evoked synaptic response amplitudes, recorded from the neuromuscular junctions (NMJs) of acy-1 and kin-2 mutants were not significantly affected, although a trend toward faster synaptic depression in stimulus trains was observed in acy-1 mutants. Additional experiments under conditions that lower release probability will be conducted to complete this analysis and to examine the potential role of PKA in the regulation of calcium influx, as recently reported at enteric NMJs (Wang and Sieburth, 2013). However, the lack of an obvious synaptic defect is consistent with the preliminary electron microscopy (EM) analysis of synaptic vesicle density and docking at the NMJs of these mutants, which appear close to wildtype. One notable difference that we have observed at the ultrastructural levels is a differential change in the number of dense core vesicles (DCV) at acy-1 and kin-2 synapses, suggesting that the Gαs pathway may preferentially regulate this secretory pathway. This is consistent with previous data, showing that Gαs acts in the same pathway as the DCV priming factor, UNC-31(CAPS) (Charlie et al, 2006). Ongoing experiments will complete this analysis and further probe the potential effects

of the Gαs pathway on peptide release from DCVs.

180 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 181 Regulation of the nicotinic acetylcholine receptor ACR-16 Ashley Martin1, Feyza Sancar1, Janet Richmond1 1University of Illinois at Chicago

At the C. elegans body wall neuromuscular junctions (NMJs) there are two cholinergic ionotropic receptor types, one that is heteromeric and activated by levamisole (LAChR) and one that is homomeric, alpha-7-like, and activated by nicotine (NAChR). LEV-9, LEV- 10, and OIG-4 have been implicated in the clustering of LAChRs, but the expression of the colocalized NAChR appears completely normal when these genes are perturbed. The only receptor subunit known to be required for the C. elegans NAChR is ACR-16, which can form functional homo-pentameric receptors. Previously published data implicates LIN-17, CWN-2, and DSH-1 in an ACR-16 trafficking pathway. However, when we examined ACR-16 localization using an ACR-16::GFP single copy integrant in these mutant backgrounds no change was observed when compared to wild type. Electrically evoked responses at the NMJ were also unchanged from wild type amplitudes. Rapsyn is a possible regulator of the alpha-7 receptor, but we find the evoked responses ofrpy-1 mutants to be the same as wild type. This suggests that other, unidentified proteins play a role in the regulation of the ACR-16 receptor. A forward genetic screen was performed to isolate candidate genes involved in ACR-16 regulation. The screen utilized the single-copy integrant of ACR-16::GFP in an unc-63;acr-16 mutant background to isolate mutants that decrease the level of ACR-16::GFP expression. From this screen, 3 mutants were identified. Electrophysiological recordings demonstrated a reduction in the evoked NMJ responses in these mutants. Further characterization suggested that LAChRs are unaffected as there was no change in response to pressure ejected-levamisole in the mutants and the fluorescence level of RFP-tagged LAChRs was also normal. Behavioral assays performed in an unc-63;acr-16 mutant background revealed a more severe uncoordinated phenotype in all 3 mutant lines when compared to unc-63 alone. This did not appear to be due to a synaptogenesis defect, as immunostaining for the cholinergic vesicular marker, UNC-17 appeared to be wild type. Responses to pressure- ejected nicotine revealed a reduction in amplitude for two of the mutants, while the amplitude remained wild type in the third. This may relate to different roles in the regulation of ACR-16 in these mutants. Whole genome sequencing, using the Variant Densisty Mapping approach, has revealed candidate genes for these mutations, which are currently being characterized.

Synaptic Function and Modulation Poster Session 181 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 182 Locating synaptic calcium channels Sean Merrill1, Shigeki Watanabe1, Jackson Richards1, Erik Jorgensen2 1University of Utah, 2University of Utah, HHMI

Neurotransmission occurs when calcium triggers exocytosis of synaptic vesicles primed at release sites. The amount and duration of free calcium at a release site is determined by the number, position and activity of nearby calcium channels. However, calcium entry through multiple sources within a synapse has been studied only indirectly. Mammals contain at least 10 genes that encode thousands of unique calcium channel isoforms. In C. elegans, unc-68 (RyR), egl-19 (L-type), and unc-2 (N-type) channels are each encoded by a single gene and contribute calcium for synaptic vesicle exocytosis. The precise location of these channels within the ultrastructure of a synapse will lead to a model of their respective functions. We are transgenically attaching to each channel an enzymatic tag that covalently binds organic fluorophores suitable for correlative imaging by super-resolution fluorescence and electron microscopy (nano-fEM). Furthermore, each channel will be measured by biplane 3D super- resolution fluorescence microscopy to determine the colocalization of calcium channels with other synaptic proteins at nanometer resolution. Finally, we expect mutations in vesicle priming proteins unc-13 and unc-10 to affect the location of calcium channels and their adjoining synaptic vesicles.

182 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 183 The ASI sensory neurons serve as a peptidergic hub to modulate aversive responses Holly Mills1, Vera Hapiak2, Rachel Wragg3, Amanda Ortega1, Abigail Jelinger1, Richard Komuniecki1 1The University of Toledo, 2Brandeis University, 3Weill Cornell Medical College

Neuropeptides can function both synaptically and extrasynaptically to reconfigure individual microcircuits and differentially modulate a host of complex behaviors. In the present study, we have identified a role for the two peptidergic ASI sensory neurons in the modulation of aversive responses to dilute octanol mediated by the two ASH sensory neurons. The two ASIs express a wide array of neuropeptide-encoding genes and ASI peptidergic signaling modulates an array of behaviors. For example, the monoamines, tyramine (TA) and octopamine (OA), inhibit various aspects of ASH-mediated aversive responses to octanol that require the expression of ASI neuropeptides encoded by nlp-1/14/18 and nlp- 6/7 /8 respectively (Hapiak et al.,

2013; Mills et al., 2012). The effects of TA and OA are mediated by the Gαq-coupled TA and OA receptors, TYRA-3 and SER-6, respectively. In the present study, we have demonstrated that this ASI-mediated inhibitory peptidergic signaling antagonistically interacts with a second peptidergic cascade that stimulates ASH-mediate aversive responses. This stimulatory cascade is initiated by 5-HT and requires the expression of the neuropeptide receptor, NPR-

17, and the Gαq coupled 5-HT receptor, SER-1, on the ASIs and ASI neuropeptides encoded by nlp-24. For example, aversive responses to dilute octanol are not stimulated by 5-HT in nlp-24 null animals or in animals with either ser-1 or nlp-24 knocked down in the ASIs by RNAi. In contrast, the ASI overexpression of nlp-24 dramatically stimulates aversive responses off food. npr-17::gfp is expressed in a small subset of neurons, including the ASIs, AUAs and PVPs and neuron-selective RNAi knockdown suggests that expression in each neuron pair is required for 5-HT stimulation. Interestingly, ASI NPR-17 overexpression

in wild type animals dramatically decreases TA inhibition, suggesting that the Gαo coupled NPR-17 may be inhibiting the release of the ASI neuropeptides required for TA inhibition. Together, these data suggest that neuropeptide release from the ASIs plays complex role in modulating a host of behaviors and that ASI G-protein signaling may be compartmentalized to selectively modulate the release of individual neuropeptides or groups of neuropeptides. These studies are continuing to confirm these genetic analyses by the subcellular localization of ASI neuropeptides and direct cell-based assays.

Synaptic Function and Modulation Poster Session 183 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 184 Serotonin activates a global peptidergic signalling cascade that stimulates ASH-mediated aversive responses Holly Mills1, Tobias Clark1, Gareth Harris2, Amanda Ortega1, Richard Komuniecki1 1University of Toledo, 2Harvard University

Both 5-HT and neuropeptides stimulate aversive responses mediated by the nociceptive ASH sensory neurons and previous work has demonstrated that three distinct 5-HT receptors, operating at different levels within the ASH-mediated locomotory circuit are essential for 5-HT stimulation. In the present study, we have identified a complex peptidergic signaling cascade involved in the serotonergic stimulation of aversive responses to dilute octanol that requires the neuropeptide-encoding genes, nlp-3 and nlp-24 and predicted neuropeptide receptor, NPR-17. The overexpression of either nlp-24, nlp-3 or npr-17 mimics 5-HT and dramatically decreases the time taken to initiate backward locomotion in response to dilute octanol off food. All three stimulatory phenotypes are absent in an npr-17 null background and extensive genetic analyses places nlp-24 upstream of both nlp-3 and npr-17. Aversive responses in nlp- 3 null animals are not stimulated by 5-HT and the overexpression of nlp-3 in individual pairs of nlp-3 expressing neurons dramatically decreases the time taken to initiate an aversive response off food, suggesting that nlp-3 may be acting humorally/locally and that the absolute levels of humoral nlp-3 dictates the aversive response. Interestingly, only one of the three neuropeptides encoded by nlp-3 (NLP-3C) stimulates aversive responses when injected directly into the pseudocoelomic fluid, not NLP-3A or NLP-3B. In fact, NLP-3A appears to antagonize the action of NLP-3C, i.e., when NLP-3A and 3C are co-injected no stimulation of aversive responses is observed. As predicted nlp-24, nlp-3 and npr-17 null animals all fail to respond to 5-HT in aversive assays. Together, these results suggest that 5-HT stimulates a complex peptidergic signaling cascade to sensitize ASH-mediated aversive responses off food. These studies are continuing to identify and functionally localize the receptor for neuropeptides encoded by nlp-24 encoded neuropeptides.

184 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 185 An unconventional role of a conserved sterol biosynthetic gene, erg- 28, in SLO-1 function Kelly Oh1, Hongkyun Kim1 1Department of Cell Biology & Anatomy, Chicago Medical School, Rosalind Franklin University

The calcium-activated potassium channel, SLO-1, reduces cellular excitability in response to high levels of calcium increases. This physiological property is essential for maintaining calcium homeostasis and proper excitability. To understand how the SLO-1 channel is regulated, we performed a genetic suppressor screen that takes advantage of sluggish, uncoordinated locomotory phenotype of a gain-of-function slo-1(ky399gf) mutant. From this screen, we previously identified the alpha-catulin homologue,ctn-1 , that encodes a cytoskeletal protein involved in localization of the SLO-1 channel at the presynaptic terminals and near dense bodies of muscle. In the same genetic screen, we also identified a cim16 mutation that suppresses the locomotory phenotype of slo-1(gf). However, cim16 mutants do not show the head-bending phenotype, a hallmark phenotype of loss-of-function mutants in genes encoding slo-1 and components of the dystrophin complex. To further understand the regulatory role of cim16 for SLO-1, we cloned cim16 by a combination of genetic mapping and transgenic rescue. cim16 has a mutation in the erg-28 gene, a conserved gene in eukaryotes. ERG-28 is originally identified in yeast as a protein that anchors several ergosterol (sterol found in fungi) biosynthetic enzymes. C. elegans lacks key cholesterol biosynthetic enzymes, and as a result, obtains cholesterol from food source. Although we cannot completely rule out that ERG-28 influences the function of cholesterol-modifying enzymes, our data indicate that ERG-28 is not involved in cholesterol metabolism. First, we found that other mutants defective in genes homologous to cholesterol biosynthetic enzymes cannot suppress the locomotory defects of slo-1(gf). Second, our tissue specific rescue experiments show that neuronal, but not muscle, expression of erg-28 reverts normal locomotion of cim16;slo-1(gf) to the sluggish, uncoordinated locomotory phenotype of slo-1(gf), strongly suggesting that erg-28 has a neuronal tissue specific role, as opposed to a role in the synthesis of diffusible sterol. Consistent with the idea that erg-28 functions in neurons, we found that erg-28 mutant is hypersensitive to aldicarb, an acetylcholinesterase inhibitor, and suppresses aldicarb resistance of slo-1(gf). We found that ERG-28 is specifically localized to the endoplasmic reticulum, suggesting that ERG-28 may be involved in neuronal trafficking of the SLO-1 channels. However, the localization of SLO-1 to presynaptic terminals is not obviously altered by erg-28 mutation. Given that yeast ERG28 organizes several proteins in the ER, we suggest that ERG-28 influences the association of an accessory subunit of SLO-1 or other SLO-1- interacting proteins.

Synaptic Function and Modulation Poster Session 185 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 186 Understanding the role of RIG-3 at the C. elegans neuromuscular junction pratima pandey1, nagesh kadam1, ashwani bhardwaj1, kavita babu1 1IISER

Synaptic plasticity is dependent upon the changes in potentiation that occurs at the synapse. Previous studies have reported that RIG-3, a member of Ig superfamily (IgSF), shows increased paralysis in the presence of the Acetylcholine Esterase inhibitor, aldicarb and functions as an anti-potentiation molecule at the C. elegans Neuromuscular junction (NMJ). It was also previously shown that RIG-3 functions in a CAM-1 (ROR receptor tyrosine kinase) dependent manner and that it controls the localization of ACh (acetylcholine) receptor, ACR- 16 at the NMJ (Babu et al., 2011). Further, CAM-1 also acts as a receptor for Wnt ligands (Green et al., 2008), indicating that the effects of RIG-3 on synaptic function could be a result of changes in Wnt signaling at the NMJ. Wnts are a family of secreted glycoproteins and their secretion depends on the Wntless transmembrane protein, MIG-14 (Myers and Greenwald, 2007; Yang et al., 2008). Wntless/mig-14 mutants were also shown to be resistant to aldicarb- induced paralysis and suppressed the hypersensitivity to aldicarb that was seen in rig-3 mutants (Babu et al., 2011). In our current study, we are in the process of identifying the Wnt through which RIG-3 functions for its role in anti-potentiation at the NMJ. Several prior studies have shown that CAM-1 binds secreted Wnt ligands and functions as a Wnt receptor and that both CAM-1 and Wnts are required for normal synaptic function in C. elegans (Jensen et al., 2012). Genetic and biochemical interaction studies between RIG-3 and CAM-1 are being performed to further characterize the function of RIG-3 at the synapse and to understand the downstream and upstream signals that function through and are required for RIG-3 function at the C. elegans NMJ.

186 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 187 A role for neuropeptide signaling in acute nicotine challenge Elizabeth Ronan1, Seth Wescott2, X. Z. Shawn Xu3 1Life Sciences Institute, University of Michigan, 2Neuroscience Graduate Program, University of Michigan, 3Bernard W Agranoff Collegiate Professor in the Life Sciences, Associate Professor of Molecular and Integrative Physiology, Medical School and Research Associate Professor, Life Sciences Institute

Tobacco use is the leading cause of preventable death in developed countries. Despite this, relapse rates one year following tobacco cessation remains greater than ninety-five percent. While nicotine, the major drug of abuse found in tobacco is a known ligand at nicotinic acetylcholine receptors, mammalian studies continue to suggest an ever-increasing role for neuropeptides in drug addiction. We have previously demonstrated that Bristol N2 worms increase their crawl speed by approximately fifty percent in response to acute nicotine challenge and this nicotine-response behavior is dependent on the nicotinic receptor, ACR-15, as well as the transient receptor potential (TRP) channel homolog, TRP-2. Here, we built upon this previous assay of nicotine-naïve Day 1 worms behaving freely for five minutes on NGM plates in the presence or absence of 100uM nicotine for alterations in crawl speed. Our recent results suggest that a loss of specific neuropeptides blocks locomotor stimulation following nicotine challenge in C. elegans. Moreover, in C. elegans lines lacking properly functioning cognate receptors for these peptides, nicotine has depressant rather than stimulant effects and the depressant quality of this nicotine response persists following impairment of kinase cascades downstream of these neuropeptides. Together, these data suggest that interactions between acetylcholine receptors and the neuropeptide signaling pathways may be effective targets for improving the success rates of tobacco cessation efforts.

Synaptic Function and Modulation Poster Session 187 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 188 Examination of the Interplay between Acetylcholine and GABA signaling at the NMJ Jacqueline Rose1, Nicole Stankowicz1, Amanda Leonti1, Parker Stafford1, Michael Remington1, Katrina Mar1, Samuel Moss1, Andrew Records-Galbraith1 1Western Washington University

Several models have reported modulation of GABA signaling in response to upregulated excitatory receptor activation; however, much of this modulation is indirect via inhibitory interneurons. At the Caenorhabditis elegans neuromuscular junction, GABA and acetylcholine receptors are both found postsynaptically on muscle arms and mobility is mediated by alternating activation of these receptors. Thus the C. elegans NMJ is a site at which direct interaction between excitatory and inhibitory signaling is plausible. Previous studies from this lab have reported an increase in GABA receptor transcripts with RT-PCR, following exposure to the acetylcholine receptor agonist, nicotine, at early stages in development. The current series of studies examine further how GABA and acetylcholine receptor activation may affect both GABA and acetylcholine mediated mobility and receptor expression. Activation of GABA receptors (via application of GABA or from exposure to toluene, a solvent thought to increase GABA signaling; see below), show mobility impairment measured as a decrease in the number of body bends counted over an 80-second period (p<0.05). RT-PCR following acute, high-concentration toluene exposure revealed an increase in unc-47 transcripts (vesicular GABA transporter; p<0.001) and this increase corresponded to elevated levels of synaptobrevin/SNB-1::GFP expression in GABA motor neurons (p<0.05) suggesting that toluene exposure may act by increasing GABA availability. Acute application of 30 μM nicotine alone also produced impaired mobility compared to controls (p<0.05) similar to the toluene- alone condition. Interestingly, when nicotine was applied after toluene exposure, this nullified the toluene-induced mobility impairment (p>0.10 compared to drug-free controls) suggesting mobility may rely on a balance between GABA and cholinergic signaling. Ongoing studies aim to determine if adult and developmental nicotine exposure modifies GABA and acetylcholine receptor expression at both the protein and RNA level. As well, the effects of exposure to levamisole (agonist of the other acetylcholine receptor subtype at the NMJ) are also being examined.

188 Synaptic Function and Modulation Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 189 Innexins function as plasma membrane channels in native C. elegans touch neurons Rachele Sangaletti1, Jeff Grant1, Laura Bianchi1 1Department of Physiology and Biophysics, Miller School of Medicine, University of Miami

Innexins are invertebrate transmembrane proteins that form gap junctions between neighboring cells. In vertebrates, gap junctions are formed by evolutionarily unrelated proteins called connexins. However, innexins share sequence homology with another family of proteins called pannexins. Interestingly, pannexins seem to function exclusively as plasma membrane channels rather than gap junctions. Given the sequence homology between innexins and pannexins, it was postulated that innexins may function both as gap junctions and plasma membrane channels. While some evidence exists that this might be the case, no functional characterization in native cells of innexins as plasma membrane channels has been performed to date. Using a combination of electrophysiological tools and dye uptake assays, we report here that C. elegans touch neurons in situ and in culture express a plasma membrane channel that shares with innexins biophysical and pharmacological features. We show that this channel has a large conductance (1 nS), is non-selective, is activated by mechanical forces, conducts both ions and fluorescent dyes, and is blocked by innexin/pannexin inhibitors. Furthermore, we show that it functions in a voltage-dependent subconductance state that is K+ selective. Our work provides evidence that C. elegans touch neurons express innexins that function as plasma membrane channels rather than gap junctions. We are currently performing single cell RT-PCR experiments to identify the innexin gene that encodes this channel, and pharmacological assays in vivo to establish its function.

Synaptic Function and Modulation Poster Session 189 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 190 Autophagy Proteins are Necessary for Synaptic Vesicle Clustering Sarah Hill1, Andrea Stavoe1, Daniel Colon-Ramos1 1Program in Cellular Neuroscience, Neurodegeneration and Repair and Department of Cell Biology at Yale University

To identify genes required for synaptic vesicle clustering, we conducted a forward genetic screen in C. elegans interneuron AIY. We identified the autophagy gene, atg-9 as required for synaptic vesicle clustering. Autophagy is a cellular degradative process in which a double membrane structure is formed through a stepwise process. There are ~25 known autophagy genes in C. elegans. We examined genetic lesion alleles and determined that 13 autophagy genes are required for synaptic vesicle clustering in the interneuron AIY. We also observe a role for these autophagy proteins in vesicle clustering of other neurons, and have established cell-autonomous rescue for atg-9 in the NSM neuron. To determine how these genes are acting, we examined their localization. ATG-9 is the only multi-pass transmembrane protein implicated in autophagy. In AIY, we observe ATG-9 localization at presynaptic sites and trafficking along the neurite and cell body. The presynaptic localization of ATG-9 is dependent on the synaptic-vesicle-specific kinesin, UNC- 104/KIF1A. In addition, ATG-9 colocalizes with synaptic vesicle markers in mutants affecting synaptic pattering. Together these data suggest that ATG-9 might be present near synaptic vesicles at presynaptic terminals and that autophagy could potentially act in the development or maintenance of synaptic vesicle clusters.

190 Synaptogenesis Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 191 A role for phosphofructokinase-1 (pfk-1) in the maintenance of synaptic vesicle clusters during hypoxia SoRi Jang1, Jessica Nelson1, Gonzalo Tueros1, Katie Underwood1, Daniel Colón- Ramos1 1Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine

Inadequate oxygen supply, or hypoxia, can disrupt and damage neuronal function. We performed a forward genetic screen in C. elegans and identified mutants that exhibit a disruption of synaptic vesicle (SV) clusters in the NSM neuron in response to hypoxia. One of the mutants identified was ola72, an allele of phosphofructokinase-1 (pfk-1). PFK-1 is a rate-limiting enzyme in glycolytic pathway. We demonstrate here that pfk-1 is expressed and required pan-neuronally for the maintenance of vesicle clusters under hypoxic conditions. We also observed that pkf-1 acts cell autonomously in neurons to prevent disruption of synaptic vesicle clusters in response to hypoxia. Based on the pfk-1(ola72) phenotype, we hypothesized that PFK-1 is required for the maintenance of the SV cycle in neurons. To examine this hypothesis, we created pkf-1(ola72);unc-13(e450) double mutants; unc-13 is a presynaptic active zone protein that is essential for exocytosis. We observed that unc- 13(e450) suppresses the pfk-1 mutant phenotype under hypoxic conditions. Together, our data suggest that PFK-1 is required for maintaining the SV cycle during hypoxic conditions.

Synaptogenesis Poster Session 191 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 192 The degenerin family ion channel UNC-8 remodels GABAergic synapses in an activity-dependent pathway Tyne Miller-Fleming1, Sarah C. Petersen2, Megan Gornet2, Ying Wang3, Cristina Mattewman3, Lu Han3, Laura Bianchi3, Janet E. Richmond4, David M. Miller2 1Program in Neuroscience Vanderbilt University Nashville, TN, 2Vanderbilt University, 3University of Miami, 4University of Illinois Chicago

Synaptic networks are extensively remodeled in the developing brain by processes that require neural activity. Developmental changes in neural architecture create a functional nervous system, and defects in synaptic connections are thought to underlie neurological disorders. Members of the DEG/ENaC (Degenerin/Epithelial sodium channel) family are known to modulate plasticity in the mammalian brain, but the molecular events that regulate this effect are poorly defined. To address this question, we have studied a developmentally regulated synaptic remodeling event in C. elegans. During larval development GABAergic Dorsal D (DD) motor neurons reverse polarity by relocating synapses from ventral to dorsal muscles. We have discovered that the DEG/ENaC protein, UNC-8 is necessary for this remodeling process and functions in an activity-dependent pathway. Our results show that UNC-8 is localized ventrally and promotes the removal of ventral DD synapses. The voltage- gated calcium channel subunit, UNC-2, promotes remodeling, indicating synaptic vesicle fusion drives this process. In vitro reconstitution of an UNC-8 channel results in robust cation transport activity that is strongly inhibited by extracellular calcium. Based on these results, we propose a model in which depletion of extracellular calcium by UNC-2 at active GABAergic synapses effectively relieves the calcium block and thereby induces UNC-8 activation. Our results are consistent with a model in which UNC-8 functions as an activity sensor in GABAergic DD motor neurons to trigger deconstruction of ventral synapses in the remodeling process. Future studies will address the cell biological pathways utilized by UNC-8 to effect synapse removal. The results of this study will provide a better understanding of the cellular mechanisms that connect neural activity to synaptic plasticity.

192 Synaptogenesis Poster Session Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 193 PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior Han Wang1, Derek Sieburth1 1Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage- gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

Circuits and Behavior Poster Session 193 Neuronal Development, Synaptic Function & Behavior C. elegans Topic Meeting 2014

Abstract # 194 Systematic Phenotypic Characterization of Human 21st Chromosome Gene Equivalents in Caenorhaditis elegans Sarah Nordquist1, Allison Griffith1, Jesse Cohn1,, Jonathan Pierce-Shimomura1, 1Institute for Neuroscience, University of Texas at Austin

The most common genetic cause of intellectual disability is Down syndrome (DS), currently affecting 1 in 700 live births. Each person with DS presents varying neurological impairments and muscle hypotonia, as well as a host of other medical issues including early- onset Alzheimer’s disease due to the presence of an additional copy of the 21st chromosome. The human 21st chromosome (HSA21) is conservatively estimated to encode 215 protein- coding genes. Only several of these genes have been ascribed to specific DS phenotypes; the remainder, however, are understudied. To gain insight into HSA21 gene functions, we systematically studied loss-of-function phenotypes of HSA21 gene equivalents in the genetic model Caenorhabditis elegans. Excluding 47 genes coding for keratin, approximately 60% of HSA21 genes are represented in C. elegans. Using RNA interference, systematically knocked down each of these genes to characterize pre- and post-embryonic phenotypes. Two-thirds of HSA21 gene equivalents were found to yield phenotypes related to neuronal and/or muscular function or development. To confirm RNAi phenotypes, we further characterized the subset of genes with neuromuscular involvement using loss-of-function mutants. As the first systematic study of HSA21 gene equivalents in any animal, this study may serve as a platform to understand genes that underlie phenotypes in DS.

194 Disease Models and Regeneration Poster Session