Neuronal Development, Synaptic Function & Behavior C. elegansTopic Meeting 2014 xiii ...

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

Presenter Underlined

xiii

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

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

xiv

Presenter Underlined

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

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 ColónRamos 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 nonassociative 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

Presenter Underlined

xv

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

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

xvi

Presenter Underlined

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

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 Presenter Underlined

xvii

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

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 Insulinlike 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 xviii

Presenter Underlined

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

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 hypoxiaresponse 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 Presenter Underlined

xix

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

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

xx

Presenter Underlined

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

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 Presenter Underlined

xxi

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

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 FangYen 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 histaminegated chloride channels Navin Pokala, Qiang Liu, Andrew Gordus, Cornelia Bargmann xxii

Presenter Underlined

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

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 Presenter Underlined

xxiii

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

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 ASHmediated 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 attractorlike representation of motor state Harris Kaplan1, Tina Schrödel1, Saul Kato1, Manuel Zimmer1 1 Research 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 1 Kyushu University, Faculty of Sciences, Department of Biology & JST/CREST, 2 University 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 1 University 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 1 Department of Physiology and Biophysics, Virginia Commonwealth University, 2 Department 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 1 Program 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 1 Vanderbilt 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, pTc. 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 temperaturedependent 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 1 Biology 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 1 Dept. 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, 4 Department 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 foodrelated 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 1 School 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, we are 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 ColónRamos1 1 Program 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 1 Institute 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 nonsynaptic 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 1 Division of Biological Sciences, University of California, San Diego, 2Division of Biological Sciences, UCSD; Present address: Duke University School of Medicine, 3 Howard 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 1 The 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 calciumactivated 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 used a 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 1 MRC 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 1 Salk 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 1 Department 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 egl21, 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 dopaminemediated 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 1 Department 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 WisconsinMadison The identification of neuronal transmitter phenotypes is an important part of the structuralfunctional 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 unidentified Ascaris transcripts afp-16 (Ce-flp-26 ortholog) 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 1 Princeton 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 1 Department 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 ADLmediated 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 cisacting 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 1 Worcester 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 1 Research 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 1 Buchmann 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 sexspecific attraction in C. elegans

Anusha Narayan1, Vivek Venkatachalam2, Omer Durak3, Neelanjan Bose4, Frank Schroeder4, Aravinthan Samuel5, Jagan Srinivasan6, Paul Sternberg7 1 Biology 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, 7 Division 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 1 The 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 wildtype 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 1 Functional Genomics and Proteomics Unit, KU Leuven, Leuven, Belgium, 2 Neurobiology 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 1 Department 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 1 University 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 postembryonically 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 1 Cell 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 Department of Biology, Texas A&M University, 2Howard Hughes Medical Institute

1

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 1 Molecular 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 UNC6 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 1 Sloan 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 1 Department of Genetics, Harvard Medical School; Boston Children’s Hospital, 2 Department 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-glialensheathed 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 1 Albert 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 1 Department 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 1 University 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. UNC6/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 1 Memorial 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 1 Institute 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 cellautonomously 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 finetuning 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 1 Department of Biological Sciences, California State University East Bay Department of Cell & Tissue Biology, University of California, San Francisco, 2 Department 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 1 Department 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 EggLaying Circuit Jacob Brewer1, Michael Koelle1 1 Department 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 1 Donnelly 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. Imagesequences 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 1 University of Massachusetts Medical School, Department of Neurobiology, 2 Princeton 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 1 University of Southampton, 2Massachusetts Institute of Technology C. elegans feeding behaviour was assayed in intact worms by counting the contractionrelaxation 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 ( 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 RDE1 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 1 Departments 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), 4 Department 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 1 Rutgers, The State University of New Jersey, 2University of Illinois at UrbanaChampaign 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 1 Mills 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 1 University 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 1 University 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 1 Albert 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 GolgiER 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. In C. 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 1 Department 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 1 University 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 helixloop-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 1 Harvard 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 foododor 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 1 Department 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 spatialtemporal 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 1 Stanford 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 1 MRC 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 1 Department 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 1 University 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 PKD2 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 1 Wayne 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 1 UCSF

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 organismwide 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 1 Lawrence 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 1 MRC 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 1 The 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 1 Weill 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 1 Ohio 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), amx2(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 1 Vanderbilt 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 DAT1. 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 1 Department 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 HCO3flux. 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 knockdown 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 1 Neuroscience 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 1 Dept. 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 adultstage C. elegans

Alexander Hurlburt1, Brian Piasecki1, He Zhang1, Debora Sugiaman-Trapman2, Peter Swoboda2, Elizabeth De Stasio1 1 Lawrence 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 twoday 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 1 Department 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 1 Lunenfeld-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 1 UIC 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 acy1(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 1 University 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, LEV10, 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 of rpy-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 pressureejected 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 1 University 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 superresolution 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 1 The 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, NPR17, 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 1 University 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 nlp3 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, erg28, in SLO-1 function Kelly Oh1, Hongkyun Kim1 1 Department 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-1interacting 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 1 IISER

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, ACR16 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 aldicarbinduced 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 1 Life 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 1 Western 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