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Genetic and molecular analysis of a rhythmic behavior in C. elegans: how neuropeptide signaling conveys temporal information
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Genetic and molecular analysis of a rhythmic behavior in C. elegans: how neuropeptide signaling conveys temporal information
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Genetic and Molecular Analysis of a Rhythmic Behavior in C. elegans: How Neuropeptide Signaling Conveys Temporal Information by Han Wang A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (GENETIC, MOLECULAR AND CELLULAR BIOLOGY) December 2013 Copyright 2013 Han Wang Dedication To my parents and two elder brothers for their full support along the way to my Ph.D. degree ii Ackn owledgements My dissertation work would not be possible without my mentor Dr. Derek Sieburth. I would like to thank him for his guidance, encouragement and support during the past six years. I really appreciate his patience and trust when the projects did not work out during the first two years. He also provides me an excellent lab environment to explore my interests and test my ideas. He is also very helpful to bridge me with collaborators when I need help. I would also like to thank the former and current members from the Sieburth Lab, in particular, Dr. Jason P Chan, Trisha Staab, Kelly Giskis, Bridget LaMonica, and Krishnakali Dasg upta for their help. It is really a good experience for me to work together with them. I am very grateful to my committee, including Dr. Robert Chow, Dr. Huizhong Tao, Dr. Gage Crump and Dr. Sean Curran, for their constructive comments and advice during my annual committee meetings. Also, I would like to thank Dr. Robert Chow for making his lab equipment available for many of my experimen ts. I am also grateful to the following collaborators: James Know les, Liliance Schoofs and Torn Jans sen. I would also like to thank my friends and colleagues at USC for their discussion and technical assistance, including but not limited to: Andrew Weitz, Jung-Hwa Cho, Kassa ndra Kisler Elliott, Litao Tao, Biquan Luo, Zong Wei, Shengzhi Wang, Yuj iao Sun, Feng Wang, Madison Zitting, Reyrnundo Dominguez, Joyce Rohan, Sonia Ming-Yi Lin, Oleg Evgrafov, Andrew Clark, Zack Ram jan, and Ben jamin Berman. Lastly, I would like to thank my greatun cle, greataunt, uncle and aunt for their iii encouragement and advice. I owe my deepest gratitude to my family in China and my girlfriend Minyi Tan for their love and support along the journey. iv Table of Contents Dedication Acknowledgements List of Fignres List of Videos List of Abbr eviations Abstract Chapter 1: Overview and Introduction 1.1 Rhyt hmic Behavior 1.2 Neuropeptide signaling 1.3 C. elegans 1.4 Defecation motor program 1.4. 1 The pacemaker 1.4.2 pBoc 1.4.3 aBoc 11 111 V111 X xu X111 1 1 2 3 6 8 9 11 1.4.4 Exp 12 Chapter 2: Neuropeptide Secreted fr om a Pacemaker Activates Neurons 15 to Control a Rhythmic Behavior 2. 1 Summary 15 2.2 Introduction 16 2.3 Materials and Methods 18 2.4 Results 31 2.4. 1 nl p-40 is required for the Exp step 2.4.2 NLP-40 is secreted from the intestine 31 31 2.4.3 snt-2/ synaptotagrnin fun ctions in the intestine to regulate the 33 Exp step 2.4.4 SNT-2 is the calcium sensor for the release of NLP-40 from 35 the intestine 2.4.5 NLP-40 regnlates rhyth mic calcium influx in the GABAergic 37 v neurons 2.4.6 AEX-2/GPCR is the receptor for NLP-40 39 2.4. 7 NLP-40 is an instructive cue for the depolarization of the 42 GABAergic neurons 2. 5 Discussion 44 2.5.1 How does NLP-40 carry the timing information from the 45 pacemaker? 2.5.2 Calcium-dependent NLP-40 release 46 2.5.3 How does NLP-40 deliver the temporal information to the 46 GABAergic neurons? Chapter 3: PKA Controls Calcium Influx into Motor Neurons during a 54 Rhythmic Behavior 3.1 Summary 54 3.2 Introduction 3.3 Materials and methods 3.4 Results 3.4.1 PKA regulates the Exp step 55 57 64 64 3.4.2 PKA functions in GABAergic neurons to promote the Exp 67 step 3.4.3 PKA acts downstream of Gas in the NLP-40-AEX-2/GPCR 69 peptidergic sigualing pathway 3.4.4 PKA regulates rhythmic calcium influx in the DVB neuron 72 3.4.5 UNC-2/VGCC acts downstream of PKA and mediates 76 calcium influx in the DVB neuron 3.4.6 Other non-voltage-gated calcium channels may also mediate 77 calcium influx in the DVB neuron 3.5 Discussion 81 3.5.1 PKA is essential for presynaptic calcium influx in DVB 81 neurons 3.5.2 UNC-2 and EGL-19 mediate part ofPKA-dependent calcium 82 influx in DVB neurons 3.5.3 The activity of PKA in the GABAergic neurons during the 84 defecation cycle 3.5.4 Using genetically-encoded PKA transgenes to dissect the 86 function of PKA in vivo Chapter4: Conclusions and Perspectives 4.1 Siguificance 4.2 Directly testing rhythmic release ofNLP-40 from the intestine 4.3 Mechanisms of the termination ofNLP-40 sigualing 91 91 92 95 vi 4.4 Directly examining cAMP levels and PKA activity in the DVB neuron 96 4.5 What are other calcium channels that mediate PKA-dependent calcium 97 influx in the DVB neuron? 4.6 What is the permissive signal for the Exp step? 99 4.7 How are the three steps in defecation motor program coordinated? 10 1 Bibliography 10 5 Appendix A1 Publications A2 The legends of the videos 116 116 117 vii List of Figures Figure 1.1: Two sexes of Caenorhabditis elegans 4 Figure 1.2: Defecation motor program in C. elegans 7 Figure 1.3: The siguals controlling the defecation motor program 10 Figure 2.1: nlp-40 mutants lack the Exp step 32 Figure 2.2: NLP-40 undergoes SN T-2 /synaptotagrnin dependent release from the 34 intestine Figure 2.3: snt-2/synaptotagmin functions in the intestine to regulate the Exp step 36 Figure 2.4: nlp-40 is required for rhythmic calcium influx in the GABAergic neurons 38 Figure 2.5: AEX-2 is the receptor for NLP-40 41 Figure 2.6: NLP-40 is instructive for the excitation of the GABAergic neurons 43 Figure S2.1: nlp-40, aex-2 and snt-2 mutants have normal pBoc steps and mild defects 49 in aBoc steps Figure S2.2: Protein aligurnent ofNLP-40 orthologs from different nematode species 50 Figure S2.3: Expression pattern ofSNT-2 and aligurnent of SN T-2 with synaptotagrnin 51 1 homologs from human, mouse and Drosophila Figure S2.4: Some strains used in this study have normal Exp steps 52 Figure S2.5: NLP-40-derived peptides could not activate CHO cells transfected with 53 empty vector Figure 3.1: PKA activity is essential for the Exp step 66 Figure 3.2: PKA functions in GABAergic neurons to regulate the Exp step 68 Figure 3.3: Constitutively active PKA in GABAergic neurons partially bypasses the 70 requirement of AEX-2/GPCR viii Figure 3.4: PKA is necessary for calcium influx in the DVB neuron 73 Figure 3.5: Constitutively active PKA causes ectopic calcium spikes in the DVB 75 neuron and increases calcium spike duration Figure 3.6: unc-2/VGCC functions downstream ofPKA to mediate calcium influx in 78 the GABAergic neurons Figure 3. 7: Other non-voltage-gated calcium channels are required from calcium influx 80 in the GABAergic neurons Figure S3.1: Aligument of C. elegans PKA regulatory subunit (KIN-2a) with mouse 87 PKA regulatory subunit (Ria) Figure S3.2: Aligurnent of C. elegans PKA catalytic subunit (KIN- la) with mouse 88 PKA catalytic subunit (Ca) Figure S3.3: Constitutively active PKA specifically in GABAergic neurons mimics 89 kin-2( /j} mutants Figure S3.4: EGL-19 and UNC-2 do not completely block ectopic calcium spikes in 90 DVB neurons induced by constitutively active PKA ix List of Videos # Video S2.1: Real time calcium imaging in the DVB neuron of wild type 117 Video S2.2: Real time calcium imaging in the DVB neuron of nlp-40(tm4085) mutants 117 Video S2.3: Real time calcium imaging in the DVB neuron of nlp-40(tm4085) mutants 117 with nlp-40 eDNA specifically expressed in the intestine Video S2.4: Real time calcium imaging in the DVB neuron of aex-2(sa3) mutants 117 Video S2. 5: Real time calcium imaging in the DVB neuron of glued nlp-40(tm4085) 117 mutants during P3-1 peptide injection Video S2.6: Real time calcium imaging in the DVB neuron of glued nlp-40(tm4085) 118 mutants during P1 peptide injection Video S2.7: Real time calcium imaging in the DVB neuron of glued nlp-40(tm4085); 118 aex-2(sa3) mutants during P3-1 peptide injection Video S3.1: Real time calcium imaging in the DVB neuron of wild type 118 Video S3.2: Real time calcium imaging in the DVB neuron of aex-2 mutants 118 Video S3.3: Real time calcium imaging in the DVB neuron of unc-25 mutants 118 Video S3.4: Real time calcium imaging in the DVB neuron ofPKA[DN] transgenic 119 animals Video S3.5: Regular calcium spike in the DVB neuron ofPKA[CA] transgenic animals 119 Video S3.6: Ectopic calcium spike in DVB neuron ofPKA[CA] transgenic animals 119 Video S3. 7: Real time calcium imaging in the DVB neuron of unc-2 mutants in normal 119 cycles Video S3.8: Real time calcium imaging in the DVB neuron of unc-2 mutants in 120 incomplete cycles Video S3.9: Real time calcium imaging in the DVB neuron of egl-19; unc-2 mutants in 120 X incomplete cycles Video S3.10: Real time calcium imaging in the DVB neuron of egl-19; unc-2 mutants 120 in incomplete cycles Video S3.11: Regular calcium spike in the DVB neuron of PKA[CA}; egl-19;u nc-2 120 animals Video S3.12: Ectopic calcium spike in the DVB neuron of PKA[CA };egl-19;unc-2 121 animals # These videos are in the separate digital files accompanying this dissertation. They can also be found in the supplemental files in my two papers (publication I and publication 3 in Appendix AI, Page 116). xi aBoc AC AKAP cAMP AKAP CPE Exp GABA GCaMP GPCR pBoc PC PDE PKA PKA[CA] PKA[DN] VGCC List of Abbreviations anterior body wall muscle contraction adenylyl cyclase a-kinase anchoring protein cyclic adenosine monophosphate A-kinase anchoring protein carboxypeptidase E expulsion or enteric muscle contraction garnrna-aminobutyric acid a genetically-encoded calcium indicator G protein-coupled receptor posterior body wall muscle contraction proprotein convertase Phosphodiesterase cAMP-dependent protein kinase or protein kinase A constitutively active PKA dominant negative PKA voltage-gated calcium channel xii Abstract Rhythmic behaviors are those behaviors that occur at regular timing intervals and they are widely observed in animal kingdom. The time intervals are determined by pacemakers and the rhythmic behavioral outputs are generally performed by different tissues, including downstream neurons and muscles. However, how the timing information from the pacemaker is delivered to the downstream effectors remains unclear. To address this question, I study a simple rhythmic behavior in C. elegans: the enteric muscle contraction (Exp) in the defecation motor program. The pacemaker for the defecation motor program is the intestine and the timing is encoded by intestinal calcium oscillations, which drive rhythmical enteric muscle contraction about every 50 seconds. Using an in vivo calcium imaging approach, I fmd that the downstream GABAergic neurons (AVL and DVB) that innervate enteric muscles undergo rhythmic activation that happens immediately before Exp during each defecation cycle, suggesting the timing information from the intestine is transmitted step by step within the circuit controlling enteric muscle contraction. It has been hypothesized that an unknown neuropeptide sigual may be released from the intestine to activate the downstream GABAergic neurons to cause rhythmic enteric muscle contraction. By combing forward genetic screens and whole genome sequencing, I identify a neuropeptide-like J.?.rotein (NLP-40) as the timing sigual from the intestine. First, nlp-40 is essential for enteric muscle contraction. Second, nlp-40 is exclusively expressed in the intestine. Third, NLP-40 is released from the intestine, which is mediated by SNT -2/synaptotagrnin, the xiii putative calcium seusor in the deuse core vesicles (DCV s ), suggesting calcium oscillations in the intestine may trigger rhythmic release ofNLP-40. Fourth, using optogeueti cs and in vivo calcium imaging, I show that NLP-40 does not impact the integrity or development of the circuit that controls euteric muscle contraction. Instead, NLP-40 is both necessary and sufficient for the rhythmic calcium influx in the downstream GABAergic neurons. Fifth, I further demonstrate that NLP-40 triggers calcium influx in the GABAergic neurons by activating its receptor AEX/GPCR. I further delineate the molecular mechanism by which the NLP-40 signaling controls the activation of the GABAergic neurons. Previous studies show that NLP-40 signaling is dependent on cAMP. Here, I develop a genetic technique to either increase or inhibit PKA activity in a tissue specific manner in C. elegans. I demonstrate that PKA is the major target of cAMP in the NLP-40 signaling pathway in the GABAergic neurons to regulate calcium influx. Furthermore, I identify two voltage-gated calcium channels (VGCCs ), UNC-2 and EGL-19 that partially mediate PKA-dependeut calcium influx in the GABAergic neurons. In conclusion, I present evidence to uncover a mechanism by which neuropeptides could function as timing messengers to couple pacemakers to downstream neurons to coordinate the proper execution of rhythmic behaviors. Neuropeptides may eucode timing information via synaptotagrnin-dependent rhythmic release from pacemakers and they activate downstream neurons through binding to their receptors (GPCRs) which leads to PKA signaling cascade to trigger the calcium influx. xiv Chapter 1 Overview and Introduction 1.1 Rhythmic behavior Breathing, walking, swnnmmg and sleep, are examples of rhytlnnic behaviors that underlie many important biological processes. Rhythmic behaviors occur at regular time intervals and the periods can range from seconds to days and even to months. These behaviors are controlled by complex neuronal networks with rhytlnnic activity. There is good evidence suggesting that the time intervals for rhytlnnic behaviors are determined by central pattern generators or pacemakers, which function as biological clocks and control the activation of peripheral effectors to produce rhytlnnic behaviors (Delcomyn, 1980). To understand the principles that control the generation of rhytlnnic behaviors, two basic questions need to be answered: ( 1) How is the timing encoded by the oscillation activity in pacemaker? (2) How is the timing information in the pacemaker delivered to downstream tissues to coordinate the execution of the rhytlnnic behavioral outputs? These two questions are important, because perturbation of either the pacemaker activity or the timing delivery mechanism would disrupt the normal rhythmic behaviors. While the molecular mechanisms underlying the generation of the oscillation activity in the pacemaker have been extensively studied (Marder and Bucher, 2001; Marder and Calabrese, 1996; Selverston, 2010), how the timing information from the pacemakers is reliably transmitted to downstream effectors is less well defined. 1 1.2 N europeptide signaling Neuropeptides consist of a large family of small bioactive peptides that are derived from relatively large precursors. Neuropeptide precursors are first synthesized in endoplasmic reticulum, transported into Golgi apparatus, and then packaged into immature dense core vesicles (DCVs ), which bud from the trans-Golgi network (Kim et a!., 2006; Park et a!., 2009). During the maturation of DCVs, neuropeptide precursors undergo several steps of post-translational modification to generate bioactive peptides: first, they are cleaved by proprotein convertases (PC) at monobasic or dibasic residues to generate small intermediate products; second, the basic amino acids at the C-terminal of these products are removed by carboxypeptidase E (CPE); fmally, those resulting peptides that contain glycine residues at the C-terminal may be further modified through amidation (L i and Kim, 2008). Mature neuropeptides are released from the nervous system or other neuroendocrine cells when DCVs undergo exocytosis. The SNARE complex, which includes synaptobrevin, SNAP25 and syntaxin, is the core machinery that mediates the fusion of DCV s with the cell membrane. Calcium influx is essential for synchronous neuropeptide release and this process is generally mediated by synaptotagrnins, the calcium sensors in DCV s (Voets et a!., 2001 ). After secretion, neuropeptides act as signaling molecules to mediate the communication between different cells or tissues primarily through activating G protein-coupled receptors (GPCRs) on target cells. They generally perform modulatory roles and have relatively long-lasting effects. Neuropeptides usually do not function locally; instead they diffuse across a relatively large distance to regulate the physiology of the target cells that are far apart. 2 Neuropeptide are demonstrated to be involved in diverse biological processes, including rhythmic behaviors. In mammals, the suprachiasmatic nucleus has been established as the central pacemaker for circadian rhythm. Several studies have shown that several neuropeptides, including vasopressin and vasoactive intestinal peptide is critical for the function of suprachiasmatic nucleus (Colwell, 20ll; Freeman and Herzog, 20ll; Kalsbeek et al., 2006; Vosko et al., 2007). Another classic example of the regulation of rhythmic behaviors by neuropeptide signaling is pigment-dispersing factor (PDF), which has been shown to be expressed in the pacemaker and is essential for the rhythmic locomotor activity in Drosophila (Renn et al., 1999). However, because the complex neural circuits underlying these rhythmic behaviors, understanding how neuropeptides from pacemakers control the physiology of the downstream neurons in the output pathway to generate rhythmic behaviors is a daunting task. 1.3 C. elegans Caenorhabditis elegans (C. elega ns) is a small, free-living and non-parasitic soil nematode. It is a roundworm which looks similar to a tiny tube (Figure 1.1). Generally, C. elegans grow as hermaphrodites (XX), which are self-fertilizing and each healthy hermaphrodite adult can produce as many as 300 progenies by itself. The male of C. elegans (XO) allows genetic material to transfer between different strains through genetic crosses with hermaphrodites (Figure 1.1). It is very easy to culture C. elegans on agar plates supplemented with E.coli strain OP50 as food in the laboratory. The life cycle of C. elegans is about 3.5 days at 20°C. The embryo hatches and then undergoes four different larval stages (L 1 to L4) before it reaches 3 Hermaphrod:te Male Figure 1.1 Two sexes of Caenorhalulitis elegmss. Adapted from (Jorgensen and Mango, 2002). Top panel: an adult hermaphrodite, which is about lmm long. The arrowhead indicates the vulva. In the laboratoty, the C. elegans are generally maintained by transferring adult hermaphrodites to new NGM plates with food. Bottom panel: an adult male. The arrow indicates the fan -like structure in the tail, a characteristic of male. 4 adulthood. The adult hermaphrodite is about !tum long and has only 959 somatic cells. The lineage of the somatic cells has been delineated and is found to be invariant between animals (Sulston and Horvitz, 1977; Sulston et a!., 1983). Each of these cells could be directly identified under differential interference contrast (DIC) microscope. Because of its simple anatomy, short life cycle, small size, hermaphroditic lifestyle and genetic amenability, C. elegans was first introduced by Sydney Brenner in the 1960s as a new genetic model organism to study developmental biology and neuroscience. C. elegans is the first multi-cellular organism with its complete genome sequenced in 1998. Despite its small genome (100 million bases, about 1/30 of the human genome), the C. elegans genome has about 20,000 protein-coding genes, similar to the human genome (Hillier et a!., 2005). About 38% of the genes in C. elegans have orthologs in human (Shaye and Greenwald, 2011). The nervous system of the hermaphrodites contains only 302 neurons. The connectome between these 302 neurons has been mapped through reconstruction with electron microscopy imaging serial sections of wild type hermaphrodites: neurons communicate through about 6400 chemical synapses, 900 gap junctions and about 1500 neuromuscular junctions (Altun, 2011). Despite its relatively simple nervous system, C. elegans display complex behaviors, including locomotion, feeding, egg-laying, defecation, chemotaxis, thermotaxis, social behavior, mating, learning and memory (de Bono and Maricq, 2005). Remarkably, C. elega ns shares most, if not all, neurotransmitters with humans. The complete connectivity of the nervous system in C. elegans and the powerful genetic tools available provide researchers a model to understand how the nervous system assembles and functions at the molecular, cellular and circuitry levels, which 5 provides insightful knowledge and helps us to understand how behaviors may be generated in more complex systems. 1.4 Def ecation motor program In the presence of food, C. elegans continuously pumps the food through the pharynx into the intestine lumen and it regularly defecates to expel the digested food. The defecation motor program is a simple rhythmic behavior that occurs about every 50 seconds when worms grow at 20°C and are well fed (Figure 1.2A). It consists of three coordinated muscle contractions: pBoc, aBoc and Exp. Each cycle starts with the contraction of posterior body wall muscles (pBoc ), which pushes the gut contents anteriorly, and then these muscles relax. After 3-4 seconds, anterior body wall muscles contract ( aBoc ), which squeezes the gut contents backward to the pre-anal region. Immediately following aBoc, the enteric muscles (including intestinal muscles, anal depressor muscles and sphincter muscle) contract and open the anus, which leads to the expulsion (Exp) of the gut contents. In wild type animals, all three steps of muscle contraction can be observed under a regular dissecting microscope (Figure 2.1A). Although aBoc and pBoc are not directly associated with the expulsion of the gut contents, they help to maximize the expelled amounts of gut contents. The circuit for the defecation motor program includes the intestine, a pair of GABAergic neurons (AVL and DVB), anterior body wall muscles, posterior body wall muscles and enteric muscles (Branicky and Hekimi, 2006). Because of the easy observation and quantification of the defecation motor program and the simple underlying circuit, it has served as an outstanding model to study many basic biological processes, including generation of calcium 6 A B � pBoc -4- �---- --... Intestine \o Exp Moto r neurons cvcle period I SO sec. Enteric muscles Figure 1.2 The defecation motor program in C. elegans. (A) Diagram of the defecation cycle that contains three stereotyped muscle contractions (modified from (Beg et al., 2008)). Each defecation cycle starts with posterior body wall muscle contraction (pBoc). After about 3 second, anterior body wall muscles contract (aBoc). Immediately after aBoc, enteric muscle contraction occurs, which leads to the expulsion (Exp) of gut contents. These three sets of muscle contractions repeats about every 50 seconds. (B) Diagram showing the circuit that controls Exp. The pacemaker is the intense and it signals to the downstream motor neurons (AVL and DVB). These two neurons innervate enteric muscles and stimulate them to expel the gut contents. 7 oscillations, synaptic transmission in GABAergic neurons, neuropeptide signaling and rhytlunic behaviors since the first systematic genetic analysis by Thomas in 1990 (Beg et a!., 2008; Branicky and Hekimi, 2006; Mahoney et a!., 2008; Thomas, 1990; Wang et a!., 2013). 1.4.1 The pacemaker The pacemaker for the defecation motor program is the intestine itself, which is a tubular structure with a single cell layer consisting of 20 cells that mostly form as bilateral pairs surrounding the intestinal lumen (McGhee, 2007). Genetic evidence suggests that the period of the defecation motor program is encoded by the calcium oscillations in the intestine, which is mediated by ITR-1, the IP3-gated calcium channels in the endoplasmic reticulum. Mutants with the strong loss-of-function allele of itr-1 almost completely lack the defecation motor program, while over-expression of itr-1 leads to a shorter period of the defecation cycle (Dal Santo et a!., 1999) . Recent progress on the development of genetically-encoded calcium indicators allows researchers to visualize this biological clock in vivo. Real time calcium imaging shows that during each defecation cycle, the calcium spike in the intestine first generates in the posterior intestine and it propagates anteriorly within 2-3 seconds (at the speed about 340 Jlllll s) (Teramoto and Iwasaki, 2006). It's clear now that the posterior calcium spike is required for the initiation of the defecation cycle and the posterior-to-anterior calcium wave is also essential for the execution of aBoc and Exp. Disruption of the generation of calcium spikes by injecting an IP3 receptor inhibitor into the posterior intestinal cells mimics the effect of strong loss-of-function allele of itr-1, which completely eliminates the defecation motor program. In contrast, perturbation of the 8 posterior-to-anterior calcium wave by injecting an IP3 receptor inhibitor into the middle part of the intestine only blocks aBoc and Exp (Teramoto and Iwasaki, 2006). Further study reveals that the propagation of calcium spikes across the intestinal cells is likely to be mediated by gap junction (Peters et a!., 2007). Studies aiming to understand how the period of the defecation motor program is controlled have identified several novel modulators of calcium oscillations in the intestine. Now we know that the defecation cycle is not just a passive process that responses to the intake of food. Rather, sensory cues, temperature, aging, ion channels, microRNA and fatty acid composition may work together to integrate enviro rnn ental conditions and the internal physiology of C. elegans and determine the period of the calcium oscillations in the intestine (BoJanowski et a!., 1981; Branicky and Hekimi, 2006; Iwasaki et a!., 1995; Iwasaki and Thomas, 1997; Kemp et a!., 20 12; Liu and Thomas, 1994; Take-Uchi et a!., 1998). The identification of the intestine as the pacemaker for the defecation motor program raises a very interesting question: How does a single pacemaker precisely coordinate the timing of three distinct muscle contractions (pBoc, aBoc and Exp) during each cycle? Previous studies suggest that the intestine may do so by releasing different signal molecules, which acts in different circuits to coordinate these three steps ((Fignre 1.3and (Beg et a!., 2008; Branicky and Hekimi, 2006; Mahoney et a!., 2008; Mcintire et a!., 1993)). 1.4.2 pBoc It is very easy to observe pBoc under the dissecting microscope and thus pBoc has been 9 ? y • ? 1 o o-:·u � � ? GAllA . � ctJ Figure 1.3 The signals controUing the defecation motor program. The diagram is modified from (Zhao and Schafer, 2013).The ITR-1-mediated calcium oscillations in the intestine, which reach the peak about every 50 seconds, encode the timing for the defecation cycle. For pBoc, calcium oscillations in the intestine drive the release of proton through the Na./li exchanger PB0-4; protons then directly activate the posterior body wall muscles by binding to the proton-gated ion channels PB0-5 and PB0-6. For aBoc, the signals from the intestine and from AVL are unknown. For Exp, calcium oscillations in the intestine are proposed to trigger the release of a unknown neuropeptide from dense core vesicles. This unknown peptide is also suggested to bind to AEX-2, a GPCR on the AVL and DVB neurons to cause them to release the neurotransmitter GABA. GABA then activates the ligand-gated cation channel EXP-1 on the enteric muscles to trigger Exp. 10 selected as the marker for the initiation of each defecation cycle. No neurons appear to be required for the pBoc step, suggesting that the intestine may directly signal to the posterior body wall muscles (Beg et a!., 2008). Recent work on pbo mutants, which specifically lack pBoc, shows that protons are the transmitter from the intestine that drive pBoc (Fignre 1.3 and (Beg et a!., 2008)). The calcium oscillations in the intestine may first promote the movement of protons from the intestinal lumen to the cytoplasm of the intestinal cells via NHX-2, a Na + ! H'" exchanger on the apical surface of the intestinal cells. The acidification of intestinal cytoplasm then activates PB0-4, a Na + ; w exchanger on the basolateral surface of the intestinal cells, and leads to large amount of proton eftlux into the posterior pseudocoelom, which is the cavity between the intestine and the posterior body wall muscles (Ffeiffer et a!., 2008). Protons in the pseudocoelom directly cause pBoc by activating the proton-gated ion channel consisting of PB0-5 and PB0-6, both of which are specifically expressed in the posterior body wall muscles (Beg et a!., 2008). These discoveries are very interesting, as they show for the first time that protons could act as a transmitter to cause muscle contraction. 1.4.3 aBoc aBoc, in which the anterior body wall muscles contract and bring the head backward to squeeze the gnt contents to the pre-anal region, occurs in the anterior part of the C. elegans and is not easy to observe together with pBoc and Exp, which both occur in the tail region. In addition, worms regularly move backwards which sometime obscures the observation of aBoc. Thus, the understanding of how aBoc is controlled is lagging, although a few genes, such as unc-16, unc-33, 11 unc-44 and unc-101, that are specifically required for aBoc have been identified from forward genetic screens (Thomas, 1990). Laser ablation experiments establish that AVL, a GABAergic neuron, is the only neuron that is absolutely required for the aBoc, as killing the AVL neuron eliminates aBoc (Mcintire et al., 1993). However, mutants lacking the GABA synthesis enzyme, glutamic acid decarboxylase (UNC-25/GAD), have normal aBoc, suggesting that AVL does not use GABA and it must release a different signal to anterior body wall muscles to trigger aBoc (Mahoney et al., 2008; Mcintire et al., 1993). Furthermore, the signal that mediates the communication between the intestine and the AVL neuron has also not been identified yet (Fignre 1.3). 1.4.4 Exp Exp directly controls the release of gnt contents out of anus. The circuit for Exp includes the intestine, a pair of GABAergic neurons (AVL and DVB), and the enteric muscles (Fignre 1.2B). Defects in Exp lead to the accumulation of the gnt contents in the intestinal lumen, leading to constipation, which manifests in animals with clear posterior halves under the dissecting microscope. Studies on many mutants isolated from forward genetic screens looking for constipated worms have started to reveal the molecular mechanism underlying Exp (Branicky and Hekimi, 2006). Laser ablation of either AVL or DVB only mildly reduces the frequency Exp in the defecation cycle (to 53% and 71%, respectively). In contrast, when both both AVL and DVB are killed, animals become severely constipated and ahnost completely lack Exp, similar to the 12 phenotype of unc-25/GAD mutants (Mcintire et a!., 1993). Both AVL and DVB seem to innervate the enteric muscles, suggesting that AVL and DVB may release the neurotransmitter GABA to activate enteric muscles. This is interesting because in this context, GABA is an excitatory transmitter, which contrasts with its classic inhibitory role. Consistent with this idea, Beg and Jorgensen identified E)IT-las the excitatory receptor on the enteric muscles for GABA (Beg and Jorgensen, 2003). exp-1 encodes a cation-selective ligand-gated ion channel and EXP-1 is enriched in the neuromuscular junction where AVL and DVB innervate enteric muscles. Animals lacking exp-1 have severe defects in Exp, similar to unc-25 mutants. Thus, during the defecation cycle, the GABAergic neurons (AVL and DVB) are likely to release GABA, which binds to EXP-1 to cause Exp (Figure 1.3). A critical question is how the timing information encoded by the calcium oscillations in the intestine is transmitted to the GABAergic neurons (AVL and DVB). Because the processes of AVL and DVB do not directly contact the intestinal cells (White et a!., 1986), it has been proposed that the intestine releases a diffusible sigual to deliver the timing information to these two neurons. Recent studies from other colleagues suggest that this diffusible sigual from the intestine may be a neuropeptide. First, AEX-5/proprotein convertase, which is a processing enzyme for neuropeptide precursors, functions in the intestine to regulate Exp (Mahoney et a!., 2008); second, several factors that are essential for DCV exocytosis (or neuropeptide release), including AEX- l/Munc13-4, AEX-4/SNAP25 and SYN-1/syntaxin, also acts the intestine to control Exp (Doi and Iwasaki, 2002; Mahoney et a!., 2008; Yamashita et a!., 2009); third, AEX-2, a GPCR on the GABAergic neurons, is required for Exp (Mahoney et a!., 2008). AEX-2 may 13 serve as the receptor for the timing signal from the intestine. In addition, the activation of AEX-2 on the GABAergic neurons is demonstrated to act through Gas, adenylyl cyclase and cAMP Like in mammals, the genome of C. elega ns has more than 100 neuropeptide genes. However, the identity of the timing signal from the intestine for Exp is unknown and the downstream signaling pathway of cAMP that leads to the activation of the GABAergic neurons has not yet been defined. In my dissertation, by combing genetic approaches, whole genome sequencing, in vivo calcium imaging technique and behavioral analysis, I identify the neuropeptide (NLP-40) as the timing signaling from the intestine for the Exp and demonstrate that the NLP-40-AEX-2 pathway acts through PKA to activate the GABAergic neurons by controlling calcium influx. My dissertation work, in which I use the rhythmic enteric muscle contraction (Exp) as a model, provides mechanistic insights on how neuropeptides could function as timing messengers to couple pacemaker activity to the activation of target neurons to control rhythmic behaviors. 14 Chapter 2 Neuropeptide Secreted from a Pacemaker Activates Neurons to Control a Rhythmic Behavior 2.1 Summary Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 seconds, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. Here we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We fmd that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagrnin. We identify AEX-2, the G protein-coupled receptor on the GABAergic neurons, as the receptor of NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-4 0-derived peptide depolarizes the GABAergic neurons in vivo. Our results show that 15 NLP-40 carries the timing information from the pacemaker via calcium -dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors. 2.2 Introduction Rhythmic behaviors are widely observed in multicellular organisms. The periods of these rhythms are determined by endogenous biological clocks in pacemakers and range from seconds to even years (Iwasaki and Thomas, 1997; Schibler and Naef, 2005). Genetic, biochemical and electrophysiological studies have shed light on how pacemakers generate biological clocks with different periods (Marder and Calabrese, 1996; Reppert and Weaver, 2002; Stanewsky, 2003). However, how pacemakers impact the physiology of target tissues to generate rhythmic behaviors is largely unknown. Perturbations in the communication between pacemakers and downstream targets can disrupt the orchestrated rhythmic behavioral outputs and can lead to disorders such as insomnia and arrhythmia (Qu, 2010; Sehgal and Mignot, 2011). The C. elegans defecation motor program is a very simple rhythmic behavior with a period of about 50 seconds (Thomas, 1990). It is composed of three stereotypical, sequential muscle contractions: first, the posterior body wall muscles contract (pBoc ); three seconds later, the anterior body wall muscles contract ( aBoc ); next, the enteric muscles contract which leads to the expulsion (Exp) of digested food from the intestine (Fignre 2.1A). Previous studies have shown that the intestine functions as the pacemaker, and the period is set by calcium oscillations 16 in the intestine that peak every 50 seconds (Dal Santo et al., 1999; Nehrke et al., 2008; Peters et al., 2007; Teramoto and Iwasaki, 2006). It has been proposed that the intestine may secrete different signals which act on different circuits to coordinate these three muscle contractions (Branicky and Hekimi, 2006). Among the candidate signals are neuropeptides. Neuropeptides are derived from larger neuropeptide precursors, which are packaged into dense core vesicles (DCVs) where they are cleaved and processed to produce small bioactive peptides (Li and Kim, 2008). Neuropeptides are released when DCVs undergo calcium-dependent exocytosis upon stimulus, which is mediated by the synaptotagmin family of calcium sensors (Pang and Sudhof, 2010). After secretion, neuropeptides activate G protein-coupled receptors (GPCRs) on target cells to regulate diverse biological processes (Salio et al., 2006). While it has been well known that neuropeptides in pacemakers are critical for rhythmic behavioral output (Mertens et al., 2007; Tagh ert, 200 1; Vosko et al., 2007), it is still unclear how neuropeptide signaling establishes rhythmicity in target tissues to generate rhythmic behaviors (Taghert, 2009). The Exp step in the defecation motor program is controlled by a pair of GABAergic neurons, AVL and DVB (Mcintire et al., 1993). These two neurons release the neurotransmitter y-aminobutyric acid (GABA), which activates the excitatory GABA receptor, EXP-1, on enteric muscles to cause muscle contraction (Beg and Jorgensen, 2003). It has been suggested that a secreted signal from the intestine may act through AEX-2, a GPCR, on the GABAergic neurons to control the Exp step (Mahoney et al., 2008). However, the identity of the signal and how it conveys the temporal information from the intestine to the downstream GABAergic neurons are 17 unknown. Here, we report that a conserved neuropeptide-like protein (NLP-40) is required for the Exp step in C. elegans. We show that the calcium oscillations in the intestine drive the release of NLP-40, which is mediated by the calcium sensor, SN T-2/synaptotagrnin. In vivo calcium imaging shows that NLP-40 is the instructive cue for rhythmic calcium influx in the GABAergic neurons by activating its receptor AEX-2/GPCR. We propose a model whereby rhythmic release of neuropeptides encodes temporal information that couples pacemakers to downstream neurons to coordinate rhythmic behaviors. 2.3 Materials and Methods Str ains Strains were maintained at 20°C on NGM plates with E. coli strain OP50 as food. The wild type strain was N2 Bristol. All strains were outcrossed at least four times prior to analysis. Mutant strains were: OJ794 nlp-40(tm40 85) I, 0J l l88 nlp-40(vj3) I, OJ680 unc-13(s69) I, CB1990 dpy-5(e6J)unc-29(e403) I, OJ1441 exp-l(sa6) II, CBlll2, cat-2( elll2) II, GR1321 tph-l(m g280) II, MT13113 tdc-l(n3419) II, OJ1352 snt-2(tml711) III, MT6308, eat-4(ky5) III, OJ1347 glo-1 (zu391) X, OJ1540 aex-2(sa3) X. vj3 was mapped to the left arm of chromosome I (L G I) using standard three-point mapping. Using small nucleotide polymorphism (SNP) mapping, ll out of 82 vj3 non-Dpy recombinants from vj3 dpy-5/CB4856 heterozygotes contained the CB4856 haw2016 polymorphism, placing vj3 at position approximately -17 of LG I. The lesion of vj3 in nlp-40 18 gene was identified by whole genome sequencing with Genome Analyzer II(Illu mina) and analyzed with the MAQgene as previously described (Doitsidou et a!., 2010; Sarin et a!., 2008). The tm4085 allele deletes part of the second exon of nlp-40 (www.wormbase.org). Because the vj3 allele also deletes part of a neighboring gene (data not shown), all further analyses of nlp-40 mutants were done using the tm4085 allele. The tm1711 allele of snt-2 removes the 5 t h exon, and the predicted spliced product introduces a frame shift following the amino acid K210, and creates a stop codon after seven amino acids (www.w orrnbase.org). Str ains with transgenes Transgenic worms were generated by injecting N2, nlp-40(tm4085), snt-2(tm1711) or glo-1 (zu391) with expression plasmids (at 25ng/ J.Ll, unless otherwise stated), together with coinjection markers KP 708( Pttx-3:: RFP at 40ng/fLI) or KP 1106 (Pmyo-2::NL S::GFP at 10 ng/fll) or KP 1368 (Pmyo-2::NLS::m Cherry at 10ng/fLI). The extrachromosomal arrays were integrated into the genome using UV irradiation (with Stratagene UV Stratalinker 2400 at the energy setting at 250f1 JOUL ES x 100). (I) nlp-40 rescue and expression: OJ949 v;Ex330[Pttx-3::RFP, nlp-40 genomic DNA(�8.8kb)]; nlp-40(tm40 85) I, OJ912 v;Ex344[Pttx-3::RFP, Pnlp-40::GF P::NLS, line 1}, OJ913 v;Ex345[Pttx-3::RFP, Pnlp-40::GF P::NLS, line 2}, OJ914 v;Ex346[Pttx-3::RFP, Pnlp-40::GF P::NLS, line 3}, OJ1626 v;Ex368[Pttx-3::RFP, Pges-1::nlp-40cDNA, line 1}; nlp-40(tm40 85) I, OJ1787 v;Ex673[Pttx-3::RFP, Pges-1::nlp-40cDNA, line 2}; nlp-40(tm40 85) I, OJ1470 v;Ex556[Pttx-3::RFP, Pges-1::nlp-40cDNA, line 3}; nlp-40(tm40 85) I, (2) snt-2 rescue and expression: 19 OJ1561 v;Ex595[Pttx-3::RFP, Psnt-2::GFP, line 1}, OJ1821 v;Ex595, · otls348 IV, OH10598 otls348[Punc-47 (300bp)::mC herry, pha-1 (+)}IV, OJ1562 v;Ex596[Pttx-3::RFP, Psnt-2::GFP, line 2}, OJ1700 v;Ex640[Pttx-3::RFP, Psnt-2::GFP, line 3}, OJ1403 v;Ex532[Pmyo-2 ::NLS::GFP, Pges-1::snt-2(wt)cDNA, line 1}; snt-2( ttn1711) III, OJ1404 v;Ex533[Pmyo-2 ::NLS::GFP, Pges-1::snt-2(wt)cDNA, line 2}; snt-2( ttn1711) III, OJ1546 v;Ex585[Pmyo-2 ::NLS::GFP, Pges-1:: snt-2 [D247, 253N}, line 1};snt-2( ttn1711) III, OJ1547 v;Ex586[Pmyo-2 ::NLS::GFP, Pges-1:: snt-2 [D247, 253N}, line 2};snt-2(ttn1711) III, OJ1548 v;Ex587[Pmyo-2 ::NLS::GFP, Pges-1:: snt-2 [D247, 253N}, line 3};snt-2(tt n1711) III, (3) NLP-40 and SNT-2 subcelluar localization and NLP-40 secretion experiments: OJ1660 v;Ex555[Pttx-3::RFP, Pnlp-40::nlp-40 ::YFP at 1ng/ul], OJ1461 v;Ex555; nlp-40(ttn4085) I, OJ1690 v;Ex555; glo-1(zu391) X, OJ1786 v;Ex555; snt-2(ttn1711) III, OJ1701 v;Ex555; v;Ex641; glo-1(zu391)X, OJ1807 v;Ex641[ Pmyo-2 ::NLS::m Cherry, Pnlp-40::snt-2 ::CFP}; glo-1 (zu391) X, (4) The channelrhodopsin-2 experiment: OJ1471 v;Ex541[Pttx-3::RFP, Punc-47(FL)::ChR2(H134R)::GFP at 160ng!Jil}; nlp-40(ttn4085) I, OJ1472 v;Ex541; exp-1(sa6) II, OJ1473 v;Ex541; aex-2(sa3) X, (5) in vivo calcium imaging experiment: OJ1129 v;Ex429[Pmyo-2:: NLS::mCh erry, Punc-47(mini)::GCaMP3.0 at 125ng!Jil} OJ1213 v;Js58[Pmyo-2 ::NLS::m Cherry, Punc-47(mini): :GCa MP 3.0} IV, Note: v;Js58 is an integrant derived from v;Ex429 . OJ1443 unc-13(s69) I; v;Js58 IV, OJ1467 nlp-40(ttn4085)unc-13(s69) I; v;Js58 IV, OJ1468 unc-13(s69) I; v;Js58 IV; aex-2(sa3) X, OJ1607 v;Ex368; nlp-40(ttn4085)unc-13(s69) I; v;Js58 IV, (6) Complementation test experiments: KP3292 nuls152[Pttx-3::RFP, Punc-129::GFP::snb-1] II, For the complementation test between nlp-40(ttn4085) and nlp-40(vj3), the strain used was 20 nlp-40(tm4085)/nlp-40(vj3); nuls1521+. (7) nlp-40 variants rescue experiment: OJ1189 v;Ex455[Pttx-3::RFP; pnlp-40::nlp-40(M1)::YFP, line 1}; nlp-40(tm4085) I, OJ1170 v;Ex450[Pttx-3::RFP; pnlp-40::nlp-40(M1)::YFP, line 2}; nlp-40(tm4085) I, OJ1181 v;Ex452[ Pttx-3::RFP; pnlp-40::nlp- 40(M2)::YFP, line 1}; nlp-40(tm4085) I, OJ1205 v;Ex46 5[Pttx-3::RFP; pnlp-40::nlp- 40(M2)::YFP, line 2}; nlp-40(tm4085) I, OJ1187 v;Ex454[Pttx-3::RFP; pnlp-40::nlp- 40(M3)::YFP, line 1}; nlp-40(tm4085) I, 0J ll85 v;Ex453[Pttx-3::RFP; pnlp-40::nlp- 40(M3)::YFP, line 2}; nlp-40(tm40 85) I, OJ1314 v;Ex502[Pttx-3::RFP; pnlp-40::nlp- 40(M4)::YFP, line 1};;nlp-40(tm4085) I, OJ1315 v;Ex503[Pttx-3::RFP; pnlp-40::nlp- 40(M4)::YFP, line 2};;nlp-40(tm40 85) I, OJ1157 v;EX442[ Pttx-3::RFP, pnlp-40: nlp-21cDNA::YFP, line 1}; nlp-40(tm4085) I, OJ1163 v;EX446[Pttx-3::RFP, pnlp-40: nlp-21cDNA::YFP, line 2}; nlp-40(tm4085) I, OJ1071 v;Ex403[Pttx-3::RFP, pnlp-40::nlp- 40-nlp -21 (C1)::Y FP }; nlp-40(tm40 85) I, OJ1041 v;Ex395[Pttx-3::RFP, pnlp-40::nlp -21-nlp-40(C2)::YFP, line 1}; nlp-40(tm4085) I, OJ1042 v;Ex396[Pttx-3::RFP, pnlp-40::nlp -21-nlp-40(C2)::YFP, line 2}; nlp-40(tm4085) I, OJ1136 v;EX435[Pttx-3::RFP, pnlp-40::nlp21-nlp-40( C3)::YFP, line 1};nlp-40(tm4085) I, OJ1091 v;Ex409[Pttx-3::RFP, pnlp-40::nlp21-nlp-40( C3)::YFP, line 2}; nlp-40(tm40 85) I, OJ1340 v;Ex51 5[Pttx-3::RFP, pnlp-40::nlp21-nlp-40(C 4)::YFP, line 1}; nlp-40(tm40 85) I, OJ1341 v;Ex51 6[Pttx-3::RFP, pnlp-40::nlp21-nlp-40(C 4)::YFP, line 2}; nlp-40(tm40 85) I. (8) Injection ofNLP-40 derived peptides and calcium imaging OJ1569 nlp-40(tm4085) I; v;Js58 IV; OJ1621 nlp-40(tm4085) I; v;Js58 IV; aex-2(sa3) X. P lasmids and Oligos All constructs for transgenes were derivatives of pPD49.26 or pPD96.04 (A. Fire), unless otherwise stated. Plasmid name: promoter: :gene or promoter: :gene::rep orter pHW61: Pges-1: :nlp-40 eDNA pHW64: nlp-40 RNAi clone with nlp-40 eDNA in the RNAi clone vector L4 440 pHW66: Pnlp-40::GFP::NLS pDS292: Pnlp- 40:: nlp-40: : YFP pHW162: Psnt-2::GFP pHW144: Pges-1 ::snt-2 (wt)cDNA pHW157: Pges-1 ::snt-2 [D247,253N] pHW167: Pnlp- 40::snt-2::CFP pHW135: Punc-47(F L)::ChR2::GFP 21 pHW100: Punc-47(mini)::GCaMP3.0 pHW95: Pnlp -40:: nlp-21 cDNA::YFP pDS327: Pnlp-40::nlp-40-nlp-21(C1)::YFP pDS328: Pnlp-40::nlp-21-nlp-40(C2): :YFP pHW91: Pnlp-40::nlp-21-nlp-40(C3): :YFP pHW137: Pnlp-40::nlp-21-nlp-40(C4)::YFP pHW127: Pnlp-40::nlp-40(M1 )::YFP pHW129: Pnlp-40::nlp-40(M2 )::YFP pHW130: Pnlp-40::nlp-40(M3 )::YFP pHW131: Pnlp-40::nlp-40(M 4)::YFP Promoters used in the study nlp-40 promoter (Pnlp-40, 3 511 bp upstream of the ATG of nlp-40 gene, exclusively expressed in the intestine): PCR with 5' primer oHW145: ccccccGCGATCG Cgtgagaagatatcgtacgagg and 3' primer oHW142:ccccccGCGGCCGCgttgattgtgtatgtttggcttactg, digested with AsiSI and Notl. The fragment was cloned to a plasmid derivative of pPD96.04 with AsiSI and Noll inserted in between Sphl and BarnHI. ges-1 promoter (Pges-1 , intestine specific promoter, 1999bp ): PCR with 5' primer oTS84: ccccccGCATGCaactccgaactatgatgacg and 3' primer oTS85 :ccccccGGATCC ctgaattcaaagataagatatgtaatag, digested with Sphl and BarnHI. snt-2 promoter (Psnt-2, 4177bp fragment includes the 791bp upstream of ATG and first exon(93bp), first intron and the first 33bp of the second exon, expressed in intestine and several neurons): PCR with 5' primer oHW242: cccccGCATG Ctggaaacaaaattaacatctcag and 3' primer oHW312: ccccccACCGGTtccaatgtccacgtgttttgg, digested with Sphl and Agel. unc-47 full length promoter (Punc-47(FL), GABAergic neurons specific promoter, 1444bp upstream of ATG): PCR with 5' primer oHW252: ccccccGCATGC atgttgtcatcacttcaaactt and 3' primer oHW189: ccccccGGATCCc tgtaatgaaataaatgtgacgctg, partial digested with Sphl and 22 BamHI. unc-47 mini promote r(Punc-47(mini ), expressed in a small subset of GABAergic neurons, including AVL and DVB. 215bp upstream of ATG): 5' primer oHW202: ccccccGCA TGCCTGCAGcttt cggtttggagagtag and 3' primer oHW189: ccccccGGATC Cctgtaatgaaataaatgtgacgctg, digested with Sphl and BamHI. Genomic DNA and eDNA w;ed in the study nlp-40 genomic DNA for rescue (8867bp, including 3511bp promoter, nlp-40 exons and introns, and the 3'UTR.) : PCR with 5' primer oHW119: gtgagaagatatcgtacgagg and 3' primer oHW120: tactgacgcgtttctcgacg. nlp-40 cDNA (372bp): PCR with 5'primer oHW158: ccccccGCTAGCaaaaAT GAAACTCGT AA TTCTGCTATC and 3' primer oHW161: ccccccGGTACCttattggaattgattacgagcacg, digested with Nhel+ Kpnl; PCR with 5 primer oHW158 and 3' primer oHW160: ccccccACCGGTttggaattgattacgagcacg for NLP-40: :YFP fusion protein, digested with Nhel+ Agel. The nlp-40 eDNA used in the study contained a silence mutation (A to G at position 183 from ATG). snt-2 (wt)cD NA(lll Obp): PCR with 5'primer oHW273: ccccccGCTAGCaaaaATGTGGGCGACCGGAG CAATC and 3' primer oHW274: ccccccCCA TGGtta gtcatcatccttctttttctcc, digested with Nhel+Nco; PCR with 5' primer oHW273 and 3' primer oHW307: ccccccCCCGGGgtcatcatccttctttttctcc for SNT-2:: CFP fusion protein, digested with Nhel+ Xrnal. 23 snt-2[D247,253N]: created with overlap PCR using wild type snt-2 cDNA as template. First round: oHW273 ( 5')+oHW304(3 ') gatgAatgttggtgg ttcttcaAatccatacg cgtatggatTtgaagaac caccaacatTcatc and oHW303(5')+oHW274(3') ; second round: 5' primer oHW273 and and 3' primer oHW274, digested with Nhel+ Nco I. YFP and CFP (867bp): They shared the same primers sequences, PCR with 5' primer oDS283: ggggggaccggtAGT AAAGGAGAAGAACTTT TCACTGG and 3' primer oDS289: ggggggggtaccttaTTTGTATAGTTCATCCA TGCC, digested with Agel and Kpnl. GCaMP3.0: PCR with 5' primer oHW214: ccccccGCGATCGCAAAAatgggttctcatcatcatcatc and 3' primer oHW215: ccccccGCGGCCGCtt acttcgctgtcatcatttg. Digested with As iS I and Notl, was cloned into a derivative of pPD49.26 with AsiSI and Notl sites were inserted between Nhei and Kpnl. nlp-21 cDNA(492bp): PCR with 5' primer oDS620: ccccccGCTAGCccaccATGCGT AA TTCACTTTTCAC and 3 'primer oDS621: ccccccACCGGTgtcgtctagtcttcctggcttg for NLP-21:: YFP fusion protein, digested with Nhel+ Agel. nlp-40 Ml [ (24' GLE) to AAA, (70' ggtctcgag) to (gctgccgcg)], created by QuickChange site-directed mutagenesis (Stratagene) , using 5' primer oHW223: catcggctccggcagctgccgcggagaagctgcgtgc and 3' primer oHW224: gcacgcagcttctccgcggcagctgccggagccgatg, digested with Nhei + Agel. nlp-40 M2 [(62' FVP) to AAA, (184' tttgttcca) to (gctgctgca)], created by QuickChange site-directed mutagenesis (Stratagene) , using 5' primer oHW227: 24 agccgacacattccttggggctgctgcacagaagagaatggtcgc and 3' primer oHW228: gcgaccattctcttctgtgcagcagccccaaggaatgtgtcggct, digested with Nhel+ Agel. nlp-40 M3[(71' WQP) to AAA, (211' tggcagccg) to(gcggcggcg)], created by QuickChange site-directed mutagenesis (Stratagene) , using 5' primer oHW229: cagaagagaatggtcgcggcggcggcgatgaagcggtcgatga and 3' primer oHW230: tcatcgaccgcttcatcgccgccgccgcgaccattctcttctg, digested with Nhei + Agel. nlp-40 M4[( 109'PEE) to AAA, (325'ccggaggaa) to (gcggcggca)],created by QuickChange site-directed mutagenesis (Stratagene) , using 5' primer oHW233: gcctcggagtcaacgcggcggcagttttggcggatc and 3' primer oHW234: gatccgccaaaactgccgccgcgttgactccgaggc, digested with Nhei + Agel. nlp-40-nlp-21 (C1), created with overlap PCR using wild type nlp-40 and nlp-21 eDNA as templates. First round: oHW273(5')+oHW304(3') gatgAatgttggtggttcttcaAatccatacg cgtatggatTtgaagaac caccaacatTcatc and oHW303(5')+oHW274(3') ; second round: 5' primer oHW273 and 3' primer oHW274, digested with Nhel+ Agel. nlp-21-nlp-40(C2), created with overlap PCR using wild type nlp-40 and nlp-21 eDNA as templates. First round: oHW273(5')+oHW304(3') gatgAatgttggtggttcttcaAatccatacg cgtatggatTtgaagaac caccaacatTcatc and oHW303(5')+oHW274(3') ; second round: 5' primer oHW273 and 3' primer oHW274, digested with Nhel+ Agel. nlp-21-nlp-40(C3), created with overlap PCR using wild type nlp-40 and nlp-21 eDNA as templates. First round: oDS620(5') +oHW208(3') cgaccattctcttctgtggaacaaaAC GGGCTCCGCCGCGCTTC from nlp-21 eDNA and oHW207(5') 25 GAAGCGCGGCGGAGCCCG Ttttgttccacagaagagaatggtcg +oHW160(3') from nlp-40 eDNA; second round: 5' primer oDS620 and 3' primer oHW160, digested with Nhei+Agei. nlp-21-nlp-40(C4), created with overlap PCR using wild type nlp-40 and nlp-21 eDNA as template. First round: oHW273(5')+oHW304(3') gatgAatgttggtggttcttcaAatccatacg cgtatggatTtgaagaac caccaacatTcatc and oHW303(5')+oHW274(3') ; second round: 5' primer oHW273 and 3' primer oHW274, digested with Nhel+ Agel. Plasmids fOr the CHO cellular assay fOr NLP-40 and AEX- 2 binding exper iment The backbone of the following two plasmids is piRES2-DsRed2 (Clontech). pHW148: pCMV -IRES2-GCa MP3.0, which was used as negative control in Figure S2.5. This plasmid was created by replacing IRES2-DsRed2 (Xrnai+Noti) of the plasmid piRES2-DsRed2 with IRES2-GCaMP3.0 overlap per product (first round: oHW184 (5'): caaaatcaacgggactttcc +oHW277(3 '): gatgatgatgatgagaacccatggttgtggccatattatc from plasmid piRES2-DsRed-2 and oHW276 ( 5 '): gataatatggccacaaccatgggttctcatcatcatcatc +oHW215(3 '): ccccccGCGGCCGCttacttcgctgtcatcatttg from pHWlOO, then overlap per with 5' primer oHW184+ 3' primer oHW215, digested with Xrnal +Not!. pHW149: pCMV-sigual sequence -aex-2 cDNA-IRES2-GCaMP3.0. The sigual sequence was desigued to improve the targeting of AEX-2 into the CHO cell plasma membrane. aex-2 eDNA with 30 bp sigual sequence (upcase sequence in oHW183) from secreted alkaline phosphate was per with 5' primer oHW183: ccccccgctagcccaccA TGCTGCTGCTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCT GGGCatgaactcaacggacattattg and 3' primer oDS723: cccccGGTACCctacatatcacaccgtaaggg, 26 digested with Nhei+Kpni, and then cloned it into piRES2-DsRed2 vector. The IRES2-DsRed2 in the resulting plasmid was replaced with IRES2-GCaMP3.0 overlap PCR product (see pHW148). Behavioral assays The defecation motor program was scored as previously described (Liu and Thomas, 1994; Thomas, 1990). Individual adult worms (about 24 hours after L4 ) were transferred to fresh NGM plates seeded with OP50 bacterial lawn and were allowed to calm down. Each worm was examined for 10 consecutive defecation cycles, and the pBoc, aBoc and Exp steps were recorded using the Etho program (James Thomas Lab website: http://de pts.washington.edu/jtlab/software/otherSoftware.html) (L iu and Thomas, 1994; Mahoney et a!., 2008). The parameter (Exp per cycle) for each individual worm was calculated as the ratio ofExp over pBoc. For each genotype, 8 to 10 individual worms were assayed. Unpaired two-tail Student's t test with unequal variance was used to determine statistical significance between two samples. Channelrho dopsin exp eriments The assay was performed as previously described (Mahoney et a!., 2008). The extrachromosomal array v;Ex54l[Pttx-3::RFP, Punc-47(F L)::ChR2(H l3 4R)::GFP} was used to activate the GABAergic neurons with blue light. Briefly, L4 transgenic worms were transferred into NGM plates spotted with OP50 bacteria containing 500 f1M all-trans retinal. After about 24hours in dark, the defecation cycle was observed under a Leica MS5 fluorescent stereomicroscope equipped with a standard GFP filter set and an x-Cite series 120 excitation light source (EXFO). Only young adults with visible green fluorescence of ChR2: :GFP in the DVB 27 neuron were scored. Each worm was first scored for 10 consecutive defecation cycles with 1-5 seconds of blue light pulse just after the pBoc step (when the posterior body wall muscles start to relax). Then the same worm was allowed to recover in the dark condition for 1 to 2 hours and scored for another 10 consecutive defecation cycles without any blue light pulses. For each genotype, 9 to 10 worms were scored. Paired two-tail Student's t test was used to determine statistical significance between two conditions of the same genotype. Fluorescence Imaging and analy sis Fluorescence imaging was done using a Nikon eclipse 90i microscope equipped with a Nikon Plan Apo 100x oil objective (N.A. � 1.40) and a Nikon Plan Apo 40x oil objective (N.A.� l.O) and a Photometries Coolsnap ES 2 camera. Worms were first immobilized with 30mg/ml 2, 3-Butanedione monoxime (BDM, Sigma) in M9 buffer, and then mounted on 2% agarose imaging pad. Image stacks were captured and maximum intensity projections were obtained using Metamorph 7.0 software (Universal Imaging). Fluorescence imaging in the intestine was performed in glo-1 (zu391) mutant background, which has normal Exp steps (Fignre S2.4) to reduce intestinal auto-fluorescence (Hermann et al., 2005). The coelomocyte assay was performed as previously described (Sieburth et al., 2007). Unpaired two-tail Student's t test with unequal variance was used to determine statistical significance between two samples. In vivo calcium imaging We created a stable transgenic line (v;Js58 [pmyo-2:: NLS::m Cherry, Punc-47mini::GCaMP 3.0}) with GCaMP3.0 specifically expressed in AVL and DVB neurons using the unc-47 mini promoter (215bp upstream of ATG) (Eastman et al., 1999). v;Js58 has 28 normal Exp steps (Figure S2.4) The strains used in Figure 2.4B to 2.4F, contained the unc-13(s69) mutation, because unc-13(s69) mutants are almost completely paralyzed (Richmond et al., 1999), but still had normal Exp steps (Figure S2.4). Live imaging of young adults was performed on NGM-agarose plates with food topped with coverslip, using a Plan Apo 40x oil objective (N.A.= l.O) and a standard GFP filter set. In this configuration, the worms continuously consumed the food and had normal defecation cycles. Only worms that had normal pharyngeal pumping and also oriented laterally (with the left side toward the objective) were imaged. For each worm, fluorescence in the DVB neuron was recorded for 250 seconds at 4 frames per second (typically 30-50ms exposure time with 3x3 binning). We quantified the GCaMP3.0 fluorescence in the synaptic region of the DVB neuron, where the axon enters the ventral cord (White et al., 1986), in each individual defecation cycle. The average fluorescence (F) of GCaMP3.0 was calculated as the average fluorescence of a region of interest encompassing the synaptic region of the DVB neuron minus the background fluorescence of a similar area. The average GCaMP3.0 fluorescence of the 10 frames before the initiation of pBoc in each cycle was defmed as F0, the fluorescence change (�) of the frames within the same cycle was calculated as �=F -F0. In cycles without the Exp step, no visible calcium spikes were observed three seconds after pBoc. nlp-40 and aex-2 mutants occasionally displayed passive Exps, due to the accumulating pressure in the intestinal lumen, which occur at random time. In these cases, calcium influx preceding these passive Exp steps may also be observed (nlp-40: 2 out of 2 passive Exp, n=52 cycles; aex-2: 2 out of 4 passive Exp, n=51 cycles). 29 In vitro cellular assay fOr AEX- 2 receptor activation byNLP-40-derived peptides Chinese hamster ovary cells (CHO-K l), stably expressing the mitochondrially targeted apo-aequorin (mtAEQ) and the human Ga16 subunit were used for the Ca 2+ measurements and cultured in Ham's Fl2 medium (Sigma) containing 10% fetal bovine serum (FBS), 100 UI/ml of penicillin/streptomycin, 250 flg/ml Zeocin and 2.5 flg/ml fungizone (Arnphoterin B). Cell lines were split every 3 days (1:15) and grown at 37°C in a humidified atmosphere of 5% C02 in air. CHO/mtAEQ/Gru6 cells were transiently transfected with the aex-2 eDNA construct (pHW149) or the empty vector (pHW148) using the FuGENE 6 transfection reagent (Promega), according to the manufacturer's instructions. Intracellular calcium was monitored as previously described (Mertens et al., 2005). Briefly, cells expressing the receptor were collected 2 days post-transfection in BSA medium (DMEM/HAM's Fl2 with 15 rnM HEP ES, without phenol red, supplemented with 0.1% BSA) and loaded with 5 f!M coelenterazine h (Invitrogen) for 4 h to reconstitute the holo-enzyme aequorin. After a tenfold dilution, cells (25,000/well) were exposed to synthetic NLP-40-derived peptides (Genscript, NLP-40 Pl: APSAPAGLEEKL; NLP-40 P3-l: MVAWQPM; NLP-40 P3-2: VAWQ PM. All three peptides are >95% purity) reconstituted in BSA medium. The calcium response was recorded for 30s on a Mithras LB 940 luminometer (Berthold Technologies) in quadruplicate. After 30s, Triton X-100 (0.1 %) was added to the same well as a positive control and as a measure of the total cell Ca 2+ response. BSA medium was used as a negative control and 1 f1M ATP was used to check the functional response. Cells transfected with the empty vector (pHW148) were used as a negative control. EC50 values were calculated from dose-response 30 curves, constructed usmg a computerized nonlinear regresswn analysis, with a sigmoidal dose-response equation (Sigmaplot 9.0). 2.4 Results 2.4.1 nl p-40 is required for the Exp step The gene nlp-40 (neuropeptide-like protein 40) was identified in a forward genetic screen for genes that regulate synaptic transmission (see Materials and Methods). Two independently isolated nlp-40 mutants, vj3 and tm4085, both of which delete significant portions of the nlp-40 coding region (Figure 2.1D and Materials and Methods), displayed distended intestinal lumens, nearly complete elimination of Exp and reductions in aBoc (Figure 2.1B, 2.1C and S2.1B). However, both pBoc frequency and calcium oscillations in the intestine were normal (Figure S2.1A and data not shown). The constipated phenotype and Exp defects of nlp-40 mutants could be fully rescued by a transgene containing nlp-40 genomic DNA (Figure 2.1B and 2.1C). In addition, knockdown of nlp-40 expression by RNA interference (RNAi) also produced similar Exp defects (Figure 2.1C). Thus, nlp-40 is necessary for the execution of the Exp step. nlp-40 is predicted to encode a 123-amino-acid neuropeptide precursor protein, which is highly conserved in nematodes. The NLP-40 precursor contains a predicted sigual sequence and three dibasic consensus cleavage sites predicted to serve as processing sites to generate four small peptides (P1 to P4) (Figure 2.1E and S2.2). 2.4.2 NLP-40 is secreted fr om the intestine Examination of the expression pattern of nlp-40 using a functional, endogenous nlp-40 31 A C LO 8 wl.'t:J' J•,:pe ·� -=· · � � ... .,.. .. , m u • .. M ••• 0 ,A nlp-40 ---- _,: - ;...;.. _ .;;.;; ..,;... - E •lm4085 -·-------- vj3 NlP.tlO pncursoo- • • • ;: Pl r F'l P:} P4 Ub liD ID lil.U- �e 2.1 n�-IOII'Wimtf lacJctht E'P ri.tp. (A) Dilt�Wl oftht ddt cation cycle it C. ekp», which nputs tvti)" 5 0 se conds (adapud from(Mthomy tt t1.,2008 )). Each cyclt is intiutdwih postt rior body wan musclt contta ctim �Boc),U\d 3 stcords ltur, U'lltrior bodyvnll musdts c ordrut (tBoc) ,whkh is immtdilttly followtd by tr�ttru musclt cortraction � t� (Elop) stt p). Tht two GABAtrtJ,c nturons,AVL md DVB, which cordroltht � rup ,trt indiuted bymowhuds. (B) RtFts t:l'lUlM: DIC itmgts of 1ht posterior ircttstints of young adult WOimS wilh indictttd gmotypts. 'lht tnlugtd space wilhin 1ht inttstitW lnntn (black lints) indicnts thu. tht b.Jtmn is dimndtd h nlp-40 mubl'lts. (C) Quud' i cWonc4tht � frtqueruyinyout'lgadult \11Clm6 oftht hdkaud gmotypts. �40 gDNArtscut dtnous t$-40�4085) lWWd. u'lintl> t�ssing gmomic �40 tnnsguus. ·� ptr cycle"is ddined u 1ft rWo of � owr pBoc. (D) Uu gtnt m..: turt of nlp-40 md W positior6 of b vi3 U\d �4085 ddttiolls trt indi:ttt d. 'lht ngion labtltd nlp-40rtprtsents h gt:nOl::llt DNA frtpmm (9Jpp ltmmral Won:nWon) ustd for rtscut . (E) Schtmuk oftht NLP-40 prott in,which is a ntUropt ptii t prtcursorwilh 123 tmino adds. Tht s�lstqutnet is shown and mowhuds hdicatt 1ht thrtt cons tnSUS davtgt stu by pro-protein convtrusts. NLP-40 is Jndicted to yitlifour mnllptptidts (Pl to P4). Tht mtm U\d rundc'd tiiOrs m shown. Asurisks (-.., indicw s�icU'lt difitrtnct : p < 0 .0005 in 9J.ldtnt's t·te rt. 31 promoter to drive the expression of GFP with a nuclear localization sequence (NLS) revealed that nlp-40 was exclusively expressed in intestinal cells (Figure 2.2A). Expression of nlp-40 eDNA under the control of a heterologous intestine-specific promoter completely rescued the Exp defects of nlp-40 mutants (Figure 2.2B). To determine whether NLP-40 is secreted from the intestine, we expressed a functional NLP-40:: YFP fusion protein in the intestine. This fusion protein adopted a highly punctate pattern of fluorescence on the basolateral surface of the intestinal cells (Figure 2.2C and 2.2D). YFP fluorescence was also detected in six coelomocytes (Figure 2.2C and 2.2E). They are scavenger cells in C. elegans whose function is to take up proteins, which are secreted from other tissues, by bulk endocytosis (Fares and Grant, 2002). Thus, our results are consistent with the notion that NLP-40 is packaged in dense core vesicles (DCV s) and is secreted from the intestine. 2.4.3 snt-2/synaptotagmin fu nctions in the intestine to regulate the Exp step If calcium oscillations in the intestine instruct the execution of rhythmic Exp by triggering NLP-40 secretion, we reasoned that NLP-40 release would be calcium dependent. To test this idea, we determined whether synaptotagrnin, which is the principal calcium sensor for synchronous exocytosis of synaptic vesicles and DCVs (Pang and Sudhof, 2010), mediates the Exp step and NLP-40 release from intestine. In C. elegans, there are seven genes encoding synaptotagrnin family members, snt-1 to snt-7 (www.wormbase.org). Only RNAi-mediated knockdown of snt-2 produced detectable Exp defects, reducing the Exp frequency to approximately 40% (Figure 2.3A and data not shown). snt-2 (tm l711) mutants, which contain a 33 A DIC hP.ad rn t P. sti n e .. . n l p -4 0 p r on 'IO t er .. GF P .. Nl5 c • • • • A A A .a. A • .1. • so IJffi • F 8 1.0 .. � 0.8 8. 0.6 k 0.4 G 0.2 0.0 1.2 g 1.0 � �- 0 ]: 0.8 :1 .� ¢:iij c. E 0.6 � � :: z 0-0 4 -; . � z 0.2 0.0 �e 2.2 NLP·.W um.. £otf SNT-2/�tCI'tap\in dt��tn&:m nltasefrom. the mtstint. (A) Tht t�tssionpatl2m of nlp-40 . RtFtstntatM DIC U\dfluMtsc tntimags:s c4 m L3 stagt \I10im t>p'tssing a tnr6cq,timal nporur of nlp-40, h whi:h GFP::NLS i> driwn by 1ht nlp-40 tndogmous JC'om:4tr fr� (35Ubp q�strum of ATG). \lAW mowluds indicttt tht rrudti of ircttstitW celk. (B) Qu.ardjfkuion of 6\t � frtqumcy in )'0\D'Ig adults wih 1ht indi:wd gtnol;)'pts. 1ft Wstinalnscut dmous tnnsgmk r�P·40(1:m4085) \I10ilr6 t�ssing nlp-40 eDNA unda h MstiM·sptcit'ic gts·l promoter. (C) D�ml showing ptptidt t>prtsstd 1ht Wstint em bt statttd md Ul<tn up by cotlolmcyus (grtm w.ls) through bul< tndocytos:is. (D) �strctWvt Dragt of NLP-40::YFP localizWm on tht buolmnl surface of tht flriWm msmt (son» punua ut irdictttd by mows ). (E) md (F) �strctWvt imlgt ofh porurior coe bmocyus (outlintd by a dashed lint) h wili l;)'Pt (E) U\d mL·2 ll1lWdS (F). Flmrtscent patchts vrilhin tht cell nfhcts secnt2d NLP-40::YFP Ul<tn up by 1ft cotlolmcyte. (G) Quardjt'i cWon of t\t t'Wrtgt NLP.40::YFP fb.wrtscmct in cotlomocyus of L4 WOimS c4 wild l)'pt (n=25), md s:rd.·2(1ml111) lllWr4S 1)\=24). 'nit mun U\d sta1\lkd mor ut shown. J.swi;ks (. -.. ) irdi:ue s�i: trct dfftrmc t ctp < 0 .0005 in StW.trd.•s t·ttrt. 34 862bp deletion that is predicted to introduce an early stop codon in snt-2 before the C2B domain (Materials and Methods), displayed similar Exp defects (Figure 2.3B). A transcriptional reporter of snt-2, in which GFP was driven by the endogenous snt-2 promoter, was expressed throughout the intestine, as well as in several neurons in the head and tail (Figure 2.3C). However, GFP fluorescence was not detected in the two GABAergic neurons, AVL and DVB, which control the Exp step (Figure S2.3A and S2.3B). Expression of snt-2 eDNA specifically in the intestine rescued the Exp defects of snt-2 mutants (Figure 2.3B). Thus, we conclude that snt-2/synaptotagrnin functions in the intestine to regulate the Exp step. 2.4.4 SNT-2 is the calcium sensor for the release of NLP-40 fr om the intestine Functional SNT-2: :CFP fusion proteins adopted a punctate pattern of fluorescence on the basolateral surfaces of intestinal cells that co-localized with NLP-40: :YFP puncta ( 90.4o/ o± 1.9% co-localization, n�l3 worms) (Figure 2.3D). To directly test whether snt-2 regulates the secretion ofNLP-40 from the intestine, we examined the accumulation ofNLP-40::Y FP in coelomocytes. The intensity of YFP-tagged neuropeptide fluorescence in coelomocytes is a measure of the efficacy of neuropeptide secretion in C. elegans (Sieburth et a!., 2007; Speese et a!., 2007). NLP-40:: YFP fluorescence in coelomocytes was reduced by approximately 50% in snt-2 mutants, compared to wild type controls (Figure 2.2E, 2.2F and 2.2G). Thus, SNT-2 is associated with the DCV s containing NLP-40 and snt-2 is required for normal secretion of NLP-40 from the intestine. SNT-2 contains two calcium binding domains, C2A and C2B. The C2B domain contains 35 A 1.0 n {! 0 . 3 iS " � n . r. " o!'i fiA "· ' (I.U � �;;, ,.i# ·· "" ... . ... · f. · c B n.� , . ���� " ' il'' 1'> " � \l (, � a c• 0.:0 "" "' "' ' .'? .�':'.- :t : : � ..,-r*' '· D , � , t: • .. �� .... � · - .i' 'Y- � :¢ ' ,y · :v ·II' ,.,:: if . .. :l � ; · ., �e 2.3 snt-2-tyrupl� fundi(IJW in 'Bl.e intesti\t to n£tl.Ue ih.t E,;p mp. (A) tnd (B) Quud' k Won of tht E� frtqutruy c4 RNAi·trtate d )'0\D'Ig adults (A) md in )'0\D'Ig a dulls with indicwd gmol;ypts (B). (C) Tht t�ss:ionp tllt mof $'t2·2. Rtprtstrdati\� fb.lmsctntimtgt of maduh.t'Prtssing a .m�-2 tnnsapww nporur, in whi:h GFP � drinn by tht .n-�-2 JC'om:lttr frtptnL (4111 bp. from 192bp upstrum of ATGto 33 bp oftht second txon of $nl·2 gmt). Alrowhtads ivlicttt fluortscenu in htad tlnd Wl (D) Co-bctli:Jation of NLP-40::YFP (ftlst colortd ingrttl\1 md SNT-2::CFP !Jalst colored innd) h 1ht inttstint �ingh na)>-4'0promoter). 'lht mow Micttts 1ht cotlom:lcyu, whi:h onlyttkts up NLP-40::YFP. RigN. ptntls: 5x tmga'lit'katim c4 1ft indkwd ngims (Whtd rtcunglts). SC!lm co-loctli:P.dpunctun indicated bymowhuds. &ttl' Os indi:ttt s�kUd. difftrt l\(ts: "'P <ODS. '""'"' p < 0 .0005 in S1udtr4.\ t·ttst. 36 all five conserved aspartic acid residues (Figure S2.3C), which are the key residues that coordinate calcium (Bai and Chapman, 2004). To determine whether calcium binding is critical for SN T-2 function, we mutated two aspartic acid residues in the C2B domain of SNT-2 corresponding to the residues m Drosophila synaptotagrnin I that are required for calcium-dependent synchronous transmitter release (Mackler et a!., 2002). Expression of this calcium-binding defective SNT-2 (referred to as snt-2 [D247, 253N], Figure S2.3C) in the intestine failed to rescue the Exp defects of snt-2 mutants in three independent transgenic lines (Figure 2.3B). Thus, SN T-2 is likely to be an important calcium sensor for NLP-40 release from the intestine. The residual NLP-40 secretion observed in snt-2 mutants may be mediated by other calcium sensors or by calcium-independent NLP-40 release. 2.4.5 NLP-40 regulates rhythmic calcium influx in the GABAergic neurons Once released from the intestine, NLP-40 could control Exp by either activating the GABAergic neurons or by directly activating the enteric muscles. To distinguish between these possibilities, we tested whether optogenetic activation of the GABAergic neurons could bypass the requirement of nlp-40. It was previously shown that activation of channelrhodopsin-2 (ChR-2) in the GABAergic neurons with blue light in the presence of all trans-retinal but not in its absence could restore Exp to mutants lacking aex-2, the GPCR on GABAergic neurons (Mahoney et a!., 2008). We found that activation of ChR-2 in the GABAergic neurons rescued the Exp defects of nlp-40 mutants to a similar extent as aex-2 mutants, but failed to rescue the Exp defects of mutants lacking exp-1, the excitatory GABAreceptor on enteric muscles (Figure 2.4A). 37 A B Ul "" nvnMt!r¢n Synaptic �.on II..' ( " " {; O.!i - •• u ll: '·' n . • n . > 0.0 h !l :. b gi'lt c w i f tl r yp( • 0 tai p 4al .l 1 . 5 / XP • t . � ;:. • ;:. pllO< .. •"'" < ' l � � \ " · ' ... �� ' 'TI !Yit (si l O " .0 . 5 5 w " T i me: (sl E Jli?..Jil ; i lft�.) (i r>. 1f / Otve" F aex-1 ,z 1.5 rc • IS - . ..,, ,.. � "' , ... .(1 (1 . � \ <Q u . ... ... . • • 0 -· � - . - (1 . � .:) < Ti�TC b ) 10 " .OS '> Dll e(s l 111 " �e 2A n/p-40 is n(flirtd W rhytbtd( <alciJ.ft in1lux in. the CAB A4qi< n.eurms. (A) Quud' icWon oftht � frt qutn(y h �\l'lg adulrs wih indicau d gtnOI;)'p ts . In tht prtsenct of tll·h'an.s ntiu.l, A 1-5 second blut lighl.pulst Uta pBoc wu used to activatt b G.ABAtrgic M.Jror�S t'9;1ftssing ChR:2::GFP �ing t\t .�mc-4'7 full lenglh prom:�ttr ), as prMously rtportt d (l.4WA\ty tt al, 2008) . (B) �ssim oftht ctlcilmindicuor, GCaMP3.0 intht DVB M.Jron ('rils 58). 'lbp: schtm.Wc mwhg*'t cell body, tmn md syruptic ngim (boxtd) of tht DVB fturon in b tail l4ddlt U\d Bottom: nprtst� snapshots from a rul·timt vidto showing kruse h GC&MP3.0 fluortsctnet in tht S)'lUptic ngimrigld.btfort h Elop rup. Woims ut or� r��td mwiorto>mrdtht ltftud dorstlsidt on h top oftht itmgt. (C to F) I«Ftsnu.titt trues showing clungts of GCaMP3.0 fluortsctnct h DVB syntpst s duringtht dtftcuion proctss in worms witl hdic tttd gtnol;)p ts. JW.ctlcilm imtging in (B) to (F) wu pa:f on:nt d in tht wc- 13�69) lmlW'I1 background (Set Mwritls U\d Mtfrods). lnttstintlrtscut dmous tnnsgmic tip-40�4085) WOimS t�tssing nlp-40 eDNA unda h ircttstht·sptcific ge$·1 promoter. 1ft mm md rundard trrorS trt shown. Asttr isks (--, hdicttt si9Ui:mt d iff ert:nus: '""''"' P4l.0005 inpairtd tYIO·swplt tu st. 38 Therefore, NLP-40 does not function to activate the muscles directly, but instead is likely to participate in the activation of the GABAergic neurons during the Exp step. To determine whether nlp-40 activates the GABAergic neurons, we performed calcium imaging of GABAergic neurons during the defecation cycle using GCaMP3.0, a genetically-encoded calcium indicator (Tian et a!., 2009). Expression of GCaMP3.0 in the DVB neuron allows the visualization of the DVB cell body and the synaptic region where the DVB axon enters the ventral cord and innervates the enteric muscles (Figure 2.4B) (White et a!., 1986). In wild type worms, we observed a single robust calcium spike in the synaptic region of the DVB neuron (and also often in the cell body) that began approximately three seconds following each pBoc step, peaked immediately before each Exp step, and returned to baseline about two to three seconds following Exp. Calcium spikes were not observed at any other time in the cycle (51 out of 51 cycles in 14 worms, Figure 2.4B, 2.4C, Video S2.1 and data not shown). These results show that DVB neurons undergo rhythmic activation that correlates with the Exp step. nlp-40 mutants displayed no calcium spikes in DVB neurons (0 out 46 cycles in 14 worms, Figure 2.4D and Video S2.2), except for two calcium spikes that were associated with two passive Exp steps (see M). Expression of nlp-40 eDNA in the intestine completely restored rhythmic calcium transients to nlp-40 mutants (35 out of 35 cycles in 12 worms, Figure 2.4E and Video S2.3). Thus, the GABAergic neurons are rhythmically activated and this rhythmic activation is dependent on NLP-40 from the intestine. 2.4.6AEX-2/GPCR is the receptor fo r NLP-40 39 We hypothesized that AEX-2/GPCR might be the receptor on the GABAergic neurons for NLP-40, since aex-2 mutants phenocopied nlp-40 mutants for defects in the Exp and aBoc steps (Figure 2.4A, S2.1 and (Mahoney et al., 2008)) and calcium influx in DVB neurons (0 out of 47 cycles in 12 worms, Figure 2.4F and Video S2.4). To directly test this hypothesis, we first determined which of the four predicted mature peptides derived from NLP-40 (P l to P4, Figure 2.1E) mediates the Exp step. To do this, we generated four NLP-40 variants (Ml to M4, Figure 2.5A), each of which contains missense mutations in one of the four predicted mature pep tides. Only the NLP-40 variant carrying mutations in the P3 peptide (termed M3), was not able to rescue the Exp defects of nlp-40 mutants (Figure 2.5A). Next, we generated chimeras between NLP-40 and NLP-21 (C l to C4, Figure 2.5A). nlp-21 encodes a neuropeptide precursor that is expressed in the intestine (Nathoo et al., 2001) but does not regulate the Exp step (data not shown). Only chimeras (C2 and C3) that included the P3 peptide region could rescue nlp-40 mutants (Figure 2.5A), whereas chimeras that did not contain P3 were not functional (C l and C4). Thus, the region including the P3 peptide is both necessary and sufficient for NLP-40 function. P3 encodes a seven-amino-acid peptide, MVAWQPM (referred to as P3-l). This peptide and a possible partial degradation product of it, VAWQPM (referred to as P3-2), have previously been biochemically identified by mass spectrometry of C. elegans peptide enriched extracts (Husson et al., 2007). Finally, we tested whether the P3 peptides could activate AEX-2/G PCR in a heterologous CHO cellular assay system. In this system, activation of the GPCR is coupled to release of intracellular calcium, which leads to an increase of aequorin luminescence (Mertens et al., 2005). 40 A NLP•40 Ml .• , Ml M4 NIP-21 Pl·L: MVA\VQPM Pl-2:: VA.WQPM ., . / .. .; Cl ! i i � � � � 55 i C2 C3 (4 E><p rescue i<lildl:l • .. .. B " ' " .g " 1 .. '" « " " ' . .. LQ�(N I.P40) , h1 Pi.etre 2.5 AEX·2 U Bit r«fll't<>r f«< NLP-40. (A) SU'wfure.fundion arWyril d NLP·.W vuhnk Pl-P4 dtnotts 1ht four pndicttdpeptidts dtrivtd from NLP.40,bastd on tht cons tnSUS dlbasi: cltaon;t silts (bruk mowhtads). Ht11:htdrtg:ions nprtstnt �1 stqutnets. P3 ma.y givt rise to two ptptidts as drunni\td by mus sptctr omttty , P3·1 md P3·2. Ml-M4 nprtstr4S vuW4s wilh thrtt constcutivt nsiiuu rtplutd wilh almint � tsttr W<). Cl-C4 rt�ftsents cl'litmn.s wilh NLP-21 tt t\t indica�d posilions . lncomplru rtscut r4 somt vuW4s maybe dut to indfi: itntproctssing ofmu�ar�tprouins. (B) Dost·rtsponst curvts of CHOhd.AEQIG.,t. cells tuns:ftcttd wilh AEX-2/GPCR md truttd wilh h indktttd syrdhtti( NLP-40pt ptii ts: NLP-40 Pl: PJISPJIAGLEEKL; NLP-40 P3·1: MVAWQPM; NLP-40 P3·2 : VAWQ PM. Ptrctrd. activWon is bued on c alcium dtptndtrd. uqucti\ lumintscenct. U\t mm tnd rundardtirors m shown. 41 We found that both P3-1 and P3-2 peptides were able to activate AEX-2/GPCR. The NLP-40 P3-1 peptide (EC50=3.39 nM ± 1.17 nl\.1) was a more potent ligand for AEX-2/GPCR, compared to the NLP-40 P3-2 peptide (EC50=26. 73 nM ± 1.18 nl\.1) (Figure 2.5B). In contrast, NLP-40 P1 (AP SA PAGLEEKL), which was not required for nlp-40 function (Figure 2.5A), failed to activate AEX-2/GPCR at all concentrations tested (Figure 2.5B). Also, CHO cells transfected with the empty vector that did not include aex-2 eDNA did not generate responses to any of the three peptides tested (Figure S2.5). Taken together, we conclude that AEX-2/G PCR is the receptor for the NLP-40 P3 peptides. 2.4.7 NLP-40 is an instruc tive cue fo r the depolariza tion of the GABAergic neurons If NLP-40 is the timing signal that delivers the temporal information encoded by the calcium oscillations in the intestine to the downstream GABAergic neurons, we predict that NLP-40 would be an instructive cue for the excitation of GABAergic neurons. To test this prediction, we examined whether acute delivery of the NLP-40 P3-1 peptide to the GABAergic neurons in vivo would be sufficient to activate them. To mimic the endogenous release ofNL P-40 into the pseudocoelom, we injected the synthetic NLP-40 P3-1 peptide into the pseudocoelom of immobilized nlp-40 mutants, and recorded calcium response in GCaMP3.0-expressing DVB neurons. We found that the NLP-40 P3-1 peptide consistently induced a single calcium spike in DVB neurons 3.5±0.60 (mean± s.d.) seconds following injection (10 out of 10 injections, n= lO worms) (Figure 2.6A, 2.6B and Video S2.5). The onset delay and duration of the NLP-40 P3-1 induced calcium spikes in DVB were similar to those induced by endogenously released NLP-40 42 A B c 0 � �i 1 -" �'· � · · - � .. 3 - · 1 --� r... ...:: ::::. .,_ · 0 . 2 ; ' 1 0 lS 0.6 � �0 . .1 <I 0.2 , t p - 40 Pl - 0 t- ---: :<=�= � ·0.1 5 1. 0 1 5 ... .2 "'� --. ; .. - � � 0.2 fllir 4 0 ; aex - 2 P3-1 - " +-----�---------- ' , . ;:-nrne (s)...., • lr1testln�: GABAergic neurons: Enteric muscle$: �e 2.6 NLP·.ul is inriructive f(IJ'tht e� oftht GABAtqic J'ltW'(oJ'lf. (A) Left: brigtll fidd imlgt mvmg h injtctimnttdlt instrt2d into 1ft pstudocotlom of WOimS thuhtvt bttn itmm bili:Jtd by glling. Middlt tnd Rig'd: nprtstnativt smpshots from a rtal·tint vidto showing in:rust in GCtMP3.0 flumsctnct in h ceD body U\d axon � mows ) of tht DVB nt uron wilhin 4 secords titer injection of NLP-40-dtrivtd bioactivt ptptidt P3·1 in �40; vJls5B uUntls (GCaMP3.0 in DVB ntUr ons). 'MUtt tst.trW<s (.., nprutnt wto-flumscew:t from tht inttrtint. (B) to (D) R!prtstrdatilt tracts showing dwigts of GCtMP3.0 fhortsctl"l(t h tht cell body of tht DVB u uron in wm:ns wilh iwfrtttd gtnot)pts tfttr injection wilh t\t hdktttd NLP-40-dtriltd ptptidts. (E) M:ldd of tht circuit cordrollirlg h � st2p. Each dtftcatt.n cydt is iniliwd wth a calcium spi<t i\ 1ht inttstint. SNT-2/�on DCVs stnsts tht in:nut inctl d.nn mdpromotts h rtltast of NLP-40 dtrivtd ptptidts from. tht Wstint. Once statttd, NLP-40 activus ts receptor AEX-2/GPCR., which is coupltd tc the !lCii•:r.ti<�n ai th.:- l1 $ a d � : l ) l:t:c :ycla;�:.: (ACY 1 tCI �nldr . .:c .:A v:r. Th.:- :a.:1ivmia:1 �i Af.X-2/GPCR triggtrs ctkilm influx in AVL tnd DVB n.tUrOnS. 'lhtse tY10 neurons become ex dled tndrtltase GABA, which h tim\ bhds to tht exchtory GABA receptor, EXP · l . uui hls leads to 1ht enteric lWSd cordractions (�). 'lht downstnan effectors of cAMP m unlcnown. 43 (Figure 2.4C), suggesting the injected NLP-40 P3-l recapitulates endogenous NLP-40 function. In contrast, injecting the NLP-40 P3-l peptide into nlp-40; aex-2 double mutants failed to elicit any calcium responses (0 out of 8 injections, n�8 worms) (Figure 2.6D and Video S2. 7). In addition, injection of the NLP-40 Pl peptide, which did not activate AEX-2 in vitro, also failed to elicit any calcium responses in DVB neurons (0 out of 7 injections, n�7 worms) (Figure 2.6C and Video S2.6). Thus, we conclude that NLP-40 is instructive for the excitation of the GABAergic neurons. 2.5 Discussion In this study, we demonstrate that NLP-40 is the timing sigual that couples the calcium oscillations in the intestine to the rhythmic activation of the downstream GABAergic neurons during the defecation motor program in C. elega ns. Based on our work and previous studies (Branicky and Hekirni, 2006; Mahoney et a!., 2008; Nehrke et a!., 2008; Peters et a!., 2007; Teramoto and Iwasaki, 2006), we propose a model for the control the Exp step (Figure 2.6E). During the defecation motor program, calcium oscillations in the intestine drive calcium-dependent rhythmic secretion of NLP-40, which 1s mediated by the calcium sensor SNT-2/ synaptotagrnin. Once released, NLP-40 binds to its receptor, AEX-2/G PCR, which activates Gas and adenylate cyclase to produce cAMP and thus triggers calcium influx to depolarize the downstream GABAergic neurons. These neurons then release GABA, which activates its receptor EXP-1 on the enteric muscles to drive the Exp step. Therefore, rhythmic release of neuropeptides may be a mechanism by which temporal information is encoded to 44 mediate the communication between pacemakers and the downstream neurons to control rhythmic behaviors. 2.5.1 How does NLP-40 carry the timing information fr om the pacemaker? Our results support the model that NLP-40 carries timing information from the intestine via calcium-dependent rhythmic release (Figure 2.6E). First, we fmd that the calcium sensor SNT-2/s ynaptotagrnin resides on NLP-40-containing DCVs and mediates the release of NLP-40 from the intestine, and that calcium binding of SNT-2/s ynaptotagrnin is required for the Exp step (Figure 2.2G, 2.3B and 2.3D). Second, DVB neurons undergo NLP-40 dependent rhythmic activation and NLP-40 can instruct the activation of DVB neurons (Figure 2.4C, 2.4D, 2.6B and Video S2.1, S2. 5). The short period (about 50 seconds) of the defecation cycle makes regulation of NLP-40 release by transcriptional or translational control unlikely. Therefore, we speculate that during the defecation cycle, calcium oscillations in the intestine may drive rhythmic release of NLP-40 through SNT-2/s ynaptotagrnin. Interestingly, capacitance measurements in rat gonadotropes have shown that calcium oscillations can induce rhythmic exocytosis (Tse et a!., 1993). In addition, immunohistological evidence suggests that cyclic release of the neuropeptide pigment-dispersing factor (PDF) from the pacemaker may be important for circadian locomotor activity in Drosophila (Park et a!., 2000). Thus, rhythmic release of neuropeptides may represent a general mechanism by which timing information is transmitted from pacemakers. 45 2.5.2 Calcium-dependent NLP-40 release SNT-2 is most similar to the mammalian synaptotagmin 1, 2, and 9 family members, which mediate fast, synchronous secretory vesicle secretion in the brain (Gustavsson and Han, 2009). It is interesting to note that the relatively mild Exp defects observed in snt-2 mutants differ from the phenotype of nlp-40 mutants. In the absence of snt-2, NLP-40 release from the intestine may be coupled less effi ciently or be uncoupled from the calcium oscillations. Nonetheless, the residual NLP-40 released in snt-2 mutants may drive the Exp step if it reached a certain threshold to activate AEX-2/GPCR in some cycles. In principle, SN T-2 could function redundantly with one or more of the six other C. elegans synaptotagmin family members. Alternatively, other types of calcium binding proteins may mediate the residual NLP-40 release in snt-2 mutants. One interesting candidate is AEX-1, a Munc 13-4 like protein, which is a C2 domain containing protein that functions in the intestine to regulate the Exp step (Doi and Iwasaki, 2002). However, it has not been tested whether calcium binding of the C2 domain in AEX -1 is required for its function. 2.5.3 How does NLP-40 deliver the temporal information to the GABAergic neurons? Many studies have established a classic neuromodulatory role of neuropeptides in which neuropeptides fine-tune the excitability of neurons and circuits (Bargmann, 2012; Chalasani et al., 2010; Hu et al., 2011; Root et al., 2011). However, our results suggest that NLP-40 can act more like a classic neurotransmitter by depolarizing the GABAergic neurons through its receptor 46 AEX-2/GPCR. First, the rhythmic calcium spikes in DVB neurons are abolished in nlp-40 and aex-2 mutants (Figure 2.4C, 2.4D and 2.4F). Second, injection of the NLP- 40-derived P3-l peptide into the pseudocoelom is sufficient to reliably elicit a calcium transient in the DVB neuron within a few seconds, and this response is dependent on AEX-2/G PCR (Figure 2.6B and 2.6D). Third, it is unlikely that classic neurotransmitters are involved in activating DVB neurons during the defecation cycle, since mutants defective in the biosynthesis, transport or release of classical neurotransmitters (such as unc-13/lv1u ncl3, eat-4 for glutamate, cat-2 for dopamine, tph-1 for serotonin, tdc-1 for tyramine and octopamine, unc-17 for acetylcholine) have grossly normal Exp frequency (Figure S2.4 and (Miller et al., 1996)). Therefore, NLP-40 delivers timing information by serving as an instructive cue for the excitation of the GABAergic neurons via its receptor AEX-2/GPCR. NLP-40 P3-l injection caused consistent calcium responses in DVB but failed to induce robust enteric muscle contractions (Exp) (data not shown). This is likely due to the additional unknown permissive signal proposed by Mahoney et al., which restricts the Exp step from occurring outside of a small window within a few seconds of the pBoc step (Mahoney et al., 2008). Our results show that this permissive signal most likely controls the refractory property of enteric muscles, since peptide injection (presumably at any time in the cycle) always elicited a calcium response in DVB. Thus, the refractory period may be a mechanism by which the robust rhythmicity of Exp, instructed by NLP-4 0/AEX-2 signaling in the GABAergic neurons, 1s ensured. That neuropeptides are involved in controlling the Exp step has been implied by the 47 identification of a well-conserved set of proteins involved in the processing and secretion of neuropeptides, including AEX-5/pro-protein convertase, AEX-4/SNAP25, AEX- l/Munc13-like protein, and SYN-1/syntaxin, which are necessary for the Exp step (Doi and Iwasaki, 2002; Mahoney et a!., 2008; Yamashita et a!., 2009). Our results extend these studies by identifying the peptide and providing mechanisms by which the peptide is released from the pacemaker and activates its downstream targets. The identification of additional downstream components in this GPCR signaling pathway will promote further understanding of how neuropeptides can instruct rhythmic behaviors. 48 A B ••• 1.0 - 1.0 " 0.8 ' � v 0.8 .!!! v 1:; - 0.6 ., • � g 0.6 .l! <> 0 . 4 • u 0 m fl:l 0.4 0.2 0.2 0.0 o.o Jlitwt S21 nlp-40, c.o.-2 end.� II'Whfttt hlvtt nr.nnal pBo< fitp m4m.l.4 ddtdtin aB� riep . (A) and (B) QmnriYt.li:Jn d. ll'lqwney � p&c Ulld dro< i\ )'(Q\C •$ll wums will i'ldicc.td � s. 1ht m c tM stlndard con: ae shcMb. Asurisks indk tilt cl&d'icuc. ttjfermc ts: • P < 0 .05, -P < 0.0005 ir.\ 9:udaC. �, l t1 .u . C l<Ofl"OIIU•l r; l>"�"'n"'"1 r; l; ioJ •• I' ..... 1.: ("1-;q;tn'\" (; · � J;: c.;ni: a c 1> " "'" ' " � '" 1 l)t C b ! i(J Q � H b ) C d •1 � n l . 'l< 1) ;. �.: :.:a � .: n:- ::.1 s� , , , ,, �e S22 PNtEin tligunad of NI.P · .W orihP!oJf frt111-.. H-m mm.dod.e �petief. Pink color indbtu idtntity U\d grttnrtJC'tS trllS similuity. Black mowhttds indicu.t cons tnSUS cltawgt silts by proprw in c onwrtast ,md Pl to P4 indicat2 stht prt di:ttd cltawgt produ:ts of NLP-40 . so A 11�3d OIC s . 1r;: : r :m� ·.:,1 r; ·� • (ir.s:.,.�r�:: <- )• JV) tf r 1CI-' r, "'ll • · � · � ' f. " c l;."now, ;�yl.. :t,1; ""'"'" ... \lyl� 2'14 l•rv:.;q>t.�la . .. . ,.u �::1 t.:: C.O:(I>l:ll'l' � 1\t • ..! :tt9; I u -... ,, tyl. J.;J t.'•"!l$� 1<0/t' • • ) :! r·� " "' i' - 1" $1;.�·•·1 .H11 ( ,.. · ,.._ 1."' ;nt-? ;P1 l t o u m B \ o1 f · -' - r, ' r� V, f h•f' ........ J/ t r _; lrt o· r ;.-. ,:.,. _ 11: ( . • t 1!1 J ,. , rn ( h L t 1 I 'd i ' .. .. .. $Jt; �':! !1':2 :l • l �· Sl3 J!xin uion pdl<ln <.<! SNT-2 and alipw4"" <.<! SNT-2 W1h 'Yft'Pic� 1 h.,....,f' fr(IJI\hurMn, 11\owe md Drosupi'D lfl. (A) U\d (B) hmgts of tht hudngion (A) U\d tail ngim (B) of mimW CO·t�ssing GFP drivtn by $nl·2 promoter md mc:htii)' in G.ABAtrgk lWJrorcs (otlJ3tiB). (C) SNT-2 is COr6trvtd. 'lbp: schtmWc of tht C. elegm» SNT-2 J®ttin wih m N·UtmirW tr� domain(TM) md tY10 c tlciumbinding domtins, ClAU\d C2B . Bottom: aJi3'cmtmoftht ClB doma.inof C. elegam SN'f.2 wih symptota9Wn 1 hol:mlogs from lumu\, mous t U\d Dn»ophila, showing constrvuion ofta fivt cakium-binding .Asp nsihlts (mowhtads). Uu tY10 opm trrOWht ads indicate t\t .Asp nsiduts tlW.wtn tr&UWtd to &:nin SNT-2 [D241 , 253N) . Sl 1.0 0.8 .!! " >· 0 0 . 6 � 3 OA 0.0 Hpre $24 SW!le rtrdu we4tn'IW ftullyhn-enormal.E,;p f'tttN, Qwrd'ktt.ionr». Elilfnqutncy in young ldult wmns with hi hticc.e4 pnol)pts. gk> Jwu used to ndu:t p 'bu. �golmd fluonsctn: t . tn·l3 wu �td to it:t:mobilia worn¥ for in n · l'O uldum � YjlsSI k 11'C.'lsgtnic W«mS with Get.WP3D sptefi: illy tlqftSs.td i\ IIJI.L ..S.I>VBntwons.Noht Utb ttda\W .-.: sipWc-udydilf�ec. .fi'<ewildl;vpt i\ SU!tta \ HW L. 1ht 1:DUn fi SlaJ'ldardtli'CI'S Ut shc:Mn. 100 NlP..O P1 • • NlP- 40 P3·1 ., , NLP-40PH c 0 .. ; > "' (.> .. <( ... "' 0 - 10 •• ·' .. LOG(NLP-40], M �e S25 NLP·40·daivedp �!��ti.de1 (CI\M not actin:tt CHO ulls tnnsf'« ttd '..at\ fi:J'l'lPI:y Vtd(ol'. Dost·nsponst curvt s of CHOhri.AEQJU.,. cells tnru:ftcted wilh � vtcw tnd truttd Y1i&\ i\t indktttd syrdhtti( NLP-40pt ptii ts: NLP-40 Pl: PJISPJIAGLEEKL; NLP-40 P3·1: MVAWQPM; NLP-40 P3·2 : VAWQ PM. Ptrctrd. activWon is bued on c alcium dtptndtrd. uqucti\ lumintscenct. U\t mm tnd rundardtirors m shown. 53 Chapter 3 PKA Controls Calcium Influ x into Motor Neurons during a Rhythmic Behavior 3.1 Summary Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-I, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA trans genes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and 54 EGL-19, but not CCA-1 function downstream ofPKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels. 3.2 Introduction Cyclic adenosine monophosphate ( cAJ\1P) is a potent second messenger that plays an important role in cellular responses to extracellular signals to regulate a wide array of biological processes. In the nervous system, cAJ\1P has been implicated in controlling axon guidance, axonal regeneration, sensory function, learning and memory (Ghosh-Roy et al., 2010; Pifferi et al., 2006; Silva et al., 1998; Song et al., 1997). cAJ\1P signaling is also critical for the execution of rhythmic physiological processes such as heart beating and circadian rhythm in a variety of organisms (Hell, 2010; Levine et al., 1994; Mahoney et al., 2008; Shafer et al., 2008). However, the mechanism by which cAJ\1P controls rhythmic outputs remains unclear. cAJ\1P is synthesized by adenylyl cyclases (ACs ), which are activated by G protein-coupled receptors (GPCRs) that are coupled to the heterotrimeric G protein a subunit, Gas (Sassone-Corsi, 20 12). Work in a variety of cell types has shown that cAJ\1P has three major molecular targets: cyclic nucleotide-gated (CNG) channels, exchange proteins directly activated by cAJ\1P (Epac) and cAJ\1P- dependent protein kinase (PKA) (Fignre 3.1A and (Sassone-Corsi, 2012)). CNG channels are non-selective cation channels that are critical for the excitability of certain sensory neurons (Kaupp and Seifert, 2002). Epac proteins are guanine exchange factors 55 for the small G protein Rap, and have been shown to regulate cardiac function and insulin secretion (Gloerich and Bos, 2010). PKA is a conserved serine/threonine kinase that has been implicated in a wide array of biological processes, including cell growth, neural function, cell differentiation and metabolism (Skalhegg and Tasken, 2000). In neurons and neurosecretory cells, PKA regulates the release of neurotransmitter and neuropeptides (Seino and Shibasaki, 2005). PKA activity has also been implicated in the execution of rhythmic behaviors, such as sleep and circadian locomotor activity in the fly (Joiner et a!., 2006; Majercak et a!., 1997). PKA phosphorylates many substrates in excitable cells. For example, in cardiac muscles, PKA phosphorylates the ryanodine receptor and the L-type calcium channel to regulate heart beating (Hell, 2010; Reiken et a!., 2003). In neurons, several synaptic proteins, such as RIM- la, synapsin and tomosyn are reported as PKA substrates that regulate neurotransmitter release (Baba et a!., 2005; Seino and Shibasaki, 2005). In addition, it has been shown that PKA can phosphorylate calcium channels in hippocampal neurons, which may account for PKA-dependent modulation of neurotransmitter release and gene expression (Hell et a!., 1995). However, it is unclear how PKA impacts the physiology of neurons to regulate rhythmic behavioral outputs. The C. elegans defecation motor program is a simple rhythmic behavior that occurs about every 50 seconds (Thomas, 1990). Each cycle contains three sequential muscle contractions: the posterior body wall muscle contraction (pBoc ), anterior body wall muscle contraction ( aBoc ), and enteric muscle contraction (Exp, which leads to the expulsion of the gut contents). The period of the defecation cycle is controlled by a pacemaker in the intestine, and the Exp step is initiated by the release of a neuropeptide-like protein NLP-40 from the intestine. NLP-40 instructs the 56 excitation of a pair of GABAergic neurons (AVL and DVB), which in tum release the neurotransmitter GABA to trigger the Exp step (Beg and Jorgensen, 2003; Branicky and Hekirni, 2006; Dal Santo et a!., 1999; Wang et a!., 2013). NLP-40 activates the GPCR AEX-2 on the GABAergic neurons, which is coupled to the heterotrirneric G protein a subunit GSA-1/Gas, leading to the activation of adenylyl cyclase and the production of cAMP (Mahoney et a!., 2008; Wang et a!., 2013). However, the molecular targets of cAMP in the GABAergic neurons are not known and how cAMP signaling impacts G ABA release to mediate the Exp step is unclear. In this study, we find that KIN -1, the C. elega ns homolog of the PKA catalytic subunit, is essential for the rhythmic contraction of enteric muscles. By genetically manipulating the activity of PKA specifically in the GABAergic neurons, we establish that PKA is the downstream target of cAMP in the peptidergic signaling pathway that controls enteric muscle contraction. Furthermore, using in vivo calcium imaging, we find that PKA activates the DVB neuron by promoting calcium influx at presynaptic terminals, and that the voltage-gated calcium channels, UNC-2 and EGL-19, partially mediate PKA-dependent calcium influx. Thus, our results suggest that PKA signaling can control rhythmic behaviors by regulating presynaptic calcium entry. 3.3 Materials and Methods Str ains Strains were maintained at 20°C on NGM plates with E. coli strain OP50 as food. The Bristol strain N2 was used as reference strain. The strains used in this study: OJ1672 cng-2(tm4267) IV, FX05036 cng-4/che-6 (tm 5036) IV , OJ1748 tax-2(p671) I, KJ5562 tax-4(p678) 57 111; cng-3(jh l13)1V ; cng-1 (jhlll) V, RB830 epac-l(ok655) Ill, DG3393 tnExl09; kin-l(ok338) I, OJ1540 aex-2(sa3) X, OJ1603 v;Js76 [Pttx-3::RFP, 40ngl pl; Punc-47(FL):: kin-2a(G 310D), 50 ngl pl] V, OJ1601 v;Js77 [Pttx-3::RFP, 40ng lpl; Punc-47(FL):: kin-2a(G 31 OD), 50 ngl pl] IV, KG421 gsa-] (ce81) I, OJ1908 gsa-] (ce81) I; v;ls77 1V, OJ1854 v;lsl 02 [Pmyo-2::NLS::GFP, JO ngl pl; Punc-47(FL)::kin-J a(H9 6R, W205Q), 50ngl pl] V, OJ1858 v;Jsl03 [Pmyo-2:: NLS::GFP, JO ngl pl; Punc-47(FL)::kin-l a(H9 6R, W205Q, 50ngl pl)] I, OJ1 896 v;Jsl 031; v;Js77 IV, OJ1909 v;lsl 02 V; aex-2(sa3) X, OJ1910 v;lsl 031; aex-2(sa3) X, OJ680 unc-13(s69) I, OJ1213 v;ls58 [Pmyo-2 ::NLS::m Cherry, 10 ngl pl; Punc-47(mini)::G CaM P3, 125ngl pl] IV, OJ1443 unc-13(s69) I; v;JS58 IV , OJ1468 unc-13( s69) 1; v;JS58 IV ; aex-2(sa3) X, OJ1759 unc-13(s 69) 1; v;Js58 IV ; v;ls76 V, OJ1859 unc-13(s69) I; unc-25( el56) Ill; v;ls58 JV, OJ1917 unc-13(s69) I; v;ls581V ; v;Jsl 02 V, OJ1526 unc-2(ljl) X; OJ1899 v;Jsl 031; unc-2(ljl) X; OJ1919 unc-13(s69) I; v;Js58 IV ; unc-2( /jl)X , JD21 cca-l(adl 650) X , OJ1911 egl-19( n582) 1V , OJ1925 egl-19( n582) 1V ; unc-2(/ jl) X, OJ1924 v;lsl031; egl-19(n5 82) 1V ; unc-2( /jl)X , OJ1 351 v;ls64 [Pmyo-2::N LS::m Cherry, 10 ngl pl; Punc-47(mini): :GCaMP 3, 125ng lpl] 11, OJ1905 unc-13(s69) I; v;Js64 11, OJ1918 v;Js64 11; egl-19(n5 82) IV ; unc-2( /jl) X, OJ1923 unc-13(s69) I; v;ls64 11; v;lsl 02 V, OJ1949 v;ls6411; egl-19(n5 82) IV ; v;ls 102 V; unc-2( /jl) X, OJ794 nlp-40(tm4085 ) I, OJ1524 kin-2 (cel79) X, OJ1525 nlp-40(tm4085) I; kin-2 (cel79) X, OJ185 5 nlp-40(tm40 85) I; v;Is 102 V, OJ1856 nlp-40(tm4085 ) v;lsl031. cng-2(tm4267) mutants contain a 330 bp deletion and a single nucleotide (g) insertion (www.wormbase.org), which is predicted to generate a fr ame shift that truncates CNG-2 at the C-terminu s. cng-2(tm4267) was originally reported to be sterile and lethal (Nlitani Lab). However, 58 we found that after outcross cng-2(tm4267) mutants were viable. Behavioral assay (or the defec ation motor pr ogram The defecation motor program was analyzed as previously described (Liu and Thomas, 1994; Thomas, 1990). Briefly, L4 stage hermaphrodites were transferred to a new plate. After about 20-24 hours, each worm was transferred to a fresh NMG plate and let to settle down for at least five minute s. Ten consec utive defecation cycles were scored for each worm using the Etho program software (http :/ /depts .washingt on.edu/ jtlab/software/ other Software.htrnl) (Liu and Thomas, 1994). Only the pBoc and the Exp steps were scored, omitting the aBoc step. 8-10 worms were assayed for each genotype. "Exp per cycle" for each worm was calculated as the ratio of Exp over pBoc. The results were present as mean ± sem for each genotyp e. Unp aired two-tail Student's t-test with unequal variance was used to examine the significant difference between two different genotyp es. Molecular biology The backbone of the plasmids constructed below was pP D49.26 (A. Fire). Punc-47(F L), the 1444 bp full length unc-47 promoter, which was exp ressed in all GABAergic neurons in C. elegans (Mahoney et a!., 2008), was amplified from N2 genomic DNA using 5' primer: cccccc GCA TGCatgttgt catcacttcaaactt and 3' primer ccccccGGAT CCctgtaatgaaataaatgt gacgctg. The PCR product was partially digested with Sphl and BamHI, and cloned into the MCSI of a derivative plasmid of pP D49.26. Punc-47(mini), the 215 bp unc-47 mini promoter, which was only exp ressed in a subset of GABAergic neurons ( 4 RME, RIS, AVL and DVB neurons) (Eastman et a!., 1999), was 59 amplified from N2 genomic DNA with 5' primer cccccc GCATGCCTGCA Gctttcggtttggagagtag and 3' primer ccccc cGGA TCCctgtaatgaaataaatgtgacgctg. The PCR product was digested with Sphl and BamHI, and cloned into the MCSI of a derivative plasmid of pPD49.26 (with AsiSI and Notl inserted between the Nhel and Kpnl in MCS II). GCaMP3 (1353bp), the genet ically-encoded calcium indicator (Tian et al., 2009), was cloned fr om a plasmid with GCaMP3 sequence (a gift from Robert Chow, USC) by PCR with 5' primer cccccc GCGATCGCAAAA atgggttctcatcatcatcatc and 3' primer ccccccGCGGCC GCttacttc gctgtcatcatttg. The PCR product was digested with AsiSI and N otl, and was cloned into of a derivative of pP D49.26 that had unc-47 mini promoter in MCS I and had AsiSI and Notl sites inse rted between Nhel and Kpnl in MSCII to generate the plasmid pHWlOO: Punc-47(mini) : :GCaMP3. The wild type cDNA ofkin-2a (1101 bp) was cloned from an N2 cDNA library (synt hesized using NEB Protoscript RT -PCR kit) with 5' primer ccccccGCTA GCAAAAatgtcgggt ggaaacgaagag and 3' primer cccccc GGTA CCttaggtcatcagtttgacgta tgag. The Gly310 residue in KIN -2a was corresponding to the Gly324 residue in the site B of the mouse PKAre gulatory subunit that will generate dominate negative PKAwhen it is mutated to Asp (Correll et al., 1989). The kin-2a (G310D) variant was generated by two-step overlapping PCR using the following primers: pair 1 ( 5' primer ccccccGCTA GCAAAAatgtcgggt ggaaacgaagag and 3' primer gaagaagag cgatttcGTcgaaatag tccgacattccaag and pair 2 (5' primer cttggaatgtcggactatttc gACgaaatcgc tcttcttc and 3' primer 60 cccccc GGTAC Cttaggtcatcagtt tgacgta tgag). The final overlapping PCR product was digested with Nhei and Kpnl , and then cloned into the MCS II in a derivative plasmid from pP D49.26 that contained the unc-47 full length promoter in MCS I to generate plasmid the pHW154: Punc-4 7(FL) : :kin-2a(G3 !0D). The wild type cDNA of kin- la (10 80bp) was cloned from N2 cDNA library with 5' primer cccccc GCTA GCAAAAatgctcaagtttc tgaaacc and 3' primer cccccc GGTA CCttaaaactcggcaaactctttg. The His96 and Trp205 residues in KIN- la are corresponding to the His87 and Trp l96 in the mouse PKA catalytic subunits which would generate consti tutively active PKA when they are mutated to Gin and Arg, respectively (Orellana and McKnight, 1992). The KIN- la (H96R, W205Q) variant was created by two-step overl apping PCR using the following primers: pair I( 5' primer ccccccgctagcAAAAatgctcaagtttct gaaacc and 3' primer gaatgcgcttttcgttcaacg tTtgctccacttgcttgagttttac ), pair 2 ( 5' primer gtaaaactcaagcaagtggagcaAacgtt gaacgaaaagcgcattc and 3' primer tctggtg tgccgcaca atgtccTcgttc gtcctttgacacgtttc) and pair 3 ( 5' primer gaaacg tgtcaaaggacgaac gAggacattgt gcggcacaccaga and 3' primer cccccc GGTA CCttaaaactcggcaaactcttt g).The final overl apping PCR product was digested with Nhei and Kpnl, was cloned into the MCS II in a derivative plasmid from pP D49.26 that contained the unc-47 full length promoter in MCS I to generate the plasmid pHW173: Punc- 47(FL): :ki n-l a(H96R, W205Q). Sequencing was performed to confirm the mutations kin-2a(G3 !0D) and kin-] a(H96R, W205Q) in the pHW154 and pHW173, resp ectively. 61 Generation o(transg enic animals Microin jection of exp ression plasmids into the gonad of C. elegans was performed to generate tran sgenic animals with extr aclno mosomal arrays, according to the standard procedure (Mello et a!., 1991). Generally, total DNA concentration of the in jection solution was lOOng/fLI (using the plasmid pBluescript to fill up if needed). The extraclnomosomal arrays were integrated into the genome to generate stable transgenic worms using UV irradiation. The integrated transgenic lines were outcro ssed at least 6 times. The plasmid pHW154 (Punc-4 7(FL) ::k in-2a(G3 lOD)) was in jected at 50ng/fLI, together with the co-in jection marker plasmid KP708 (Pttx-3: :RFP) at 40ng/fll to generate the array v;Ex582 [Pttx-3::RFP, 40ng/f1l; Punc-4 7(FL) :: kin-2a(G3 !0D), 50 ng/fll]. This array was integrated into genome to generate the PKA[DN] transgenic strains (v ;Js 76 and v;Js77) with dominant negative PKA specifically expressed in GABAergic neuron s. The plasmid pHW173 (Pun c-47(FL): :ki n-l a(H96R, W205Q)) was in jected at 50/f!l, together with the co-injection maker KP1106 (Pmyo-2::NLS::GFP) at !Ong/fLI to generate the array v; Ex709 [Pmyo-2: :NLS :: mCherry, !Ong/f!l; Punc-47( FL) :: kin-l a(H96R, W205Q), 50 ng/fll]. This extrachomosomal array was integrated into genome to generate the PKA[CA] transgenic strains (v ;Js l0 2 and v;Js l0 3) with constitutively active PKAspecifically exp ressed in GABAergic neu rons. The plasmid pHWIOO (Punc-47 (mini) ::G CaMP3) was in jected at 125ng/fLI, together with co-injection marker plasmid KP1368 (Pmyo-2::NLS: :mCherry) at !Ong/fll to generate the array v;Ex429 [Pmyo-2::NLS: :mCherry, !Ong/f1l; Punc-4 7(mini )::GCaMP3 ,l25ng!J1l]. This array was 62 integrated into genome to generate transgenic strains v;Js58 and v;Js64, which expressed GCaMP3 in AVL and DVB neu rons. In vivo calcium imaging The calcium imaging experiment was performed as previously described (Wang et aL, 20 13). We used two independent transgenic strains ( v;Js58 and v;Js64) with AVL and DVB neurons expres sing GCaMP3 to perform in vivo calcium imaging on DVB neu rons. Both strains had normal Exp steps (data not shown) . Although unc-13(s69) mutants had a longer cycle length (78.5±11.4s, mean ±SD, n�S), they were also most completely paralyzed and still had normal Exp steps (Wang et aL, 2013). Thus, unc-13(s69) mutation was included in all strains to immobilize animals for calcium imaging, except for those strains with elg-19(n5 82);unc-2(/ jl) double mutations, because the elg-19( n582); unc-2(/jl) double mutants were almost completely paralyzed. Young adult worms were transferred to NGM-a garose plate seeded with the food OP50. These agarose plates were topped with cover slides and imaged under a Nikon eclipse 90i microscope equipped with a Nikon Plan Apo 40x oil obj ective (N.A .�J.O), a standard GFP filter and a Photometries Coolsnap ES 2 camera. Only those worms kept continuous pumping and posi tioned laterally with the left side pointing to the objective were selected for imaging. Time lapse imaging was obtained using Metamorph 7.0 software (Universal Imaging) . Each worm was recorded for 250s at 4 frames per second (3x3 binning, exposure time ranging fr om 5 ms to 80 ms dependent on the baseline GCaMP3 fluorescence in DVB neuron in each individual worm). Unlike DVB, AVL neuron is located in the head, so we could not routinely perform the calcium imaging on AVL and observe the Exp step in the same field. However, during some experiments, we did 63 observe that AVL fired at the same time as DVB in coiled worms in which we could see AVL and Exp in the same field. The quantification of the GCaMP3 fluorescence intensity in the synaptic region of DVB neurons using Metamorph 7.0 software (Universal Imaging) was performed as previously described (Wang et a!., 2013). The synaptic region of DVB neurons was manually selected as region of interest (ROI) and the average of the GCaMP3 fluorescence intensity for each frame was recorded. Meanwhile, a similar area near the tail region was used as background fluorescence for each frame. The GCaMP3 fluorescence (F) was defined as the (ROI - background). The average of the GCaMP3 fluorescence in the first 10 frames was used as baseline fluorescence F0. For each fr ame, the change of the GCaMP3 was presented as M/ F0� (F-F0)/F0 . The duration of each calcium spike was defined as the time differ ences between the first frame in which the GCaMP3 fluorescence increa sed and the frame where the GCaMP3.0 fluorescence returned the baseline. Ectopic calcium spikes describe those calcium spikes that did not happen within 10 seconds after pBoc. 3.4 Results 3.4.1 PKA regulates the Exp step To identify cAMP eff ectors that mediate enteric muscle contraction, we first examined C. elegans mutant worms of putative cAMP targets for defects in the Exp step. The C. elegans genome encodes six CNG chann els: cng-1, cng-2, cng-3, cng-4/che-6, tax-2 and tax-4 (Bargmann, 64 1998), some of which have well characterized roles in sensory transduction (Kaupp and Seifert, 2002), whereas the functions of the other channels are largely unknown. Putative null or loss- of -function mutations in any of these CNGs caused no obvious defects in the defecation motor program period or in the execution of pBoc or Exp (Figure 3.1B and data not shown) . The C. elegans genome encodes a single Epac homolog, epac-1. Putative null epac-1 mutants had no defects in the defecation motor program, including in the Exp step (Figure 3.1B and data not shown). Thus, CNG channels and Epac are unlikely to be the cAMP targets that control the execution of the Exp step. The PKA catalytic subunit is encoded by a single gene, kin-1, in C. elegans. kin-l(ok338) loss- of -function mutants, which contain a 763 bp deletion in kin-] that removes part of the catalytic domain, die during embryogenesis. The lethality of kin-] (ok338) animals can be partially rescued by mosaic exp ression of wild type kin-] trans genes (Kim et a!., 2012). We found that a fraction of mosaic kin-] ( ok338) animals that survive to adulthood had distended intestinal lumens indicative of constipation. These animals lacked the Exp step in nearly all defecation cycles but both execution ofpBoc and cycle length were normal (Figure 3.1B and 3.1C and data not shown) . The Exp defects seen in constipated kin-] mosaic worms were almost identical to those of animals lacking nlp-40 or aex-2 /GPCR, which are components of the peptidergic sigualing pathway that activates the GABAergic neurons (Figure 3.1B and 3.1C and (Wang et a!., 20 13)). Taken together, we conclude that PKA activity is absol utely required for the generation of rhythmic enteric muscle contractions and that PKA is likely to be a ma jor, if not the only, downstream target of cAMP during the Exp step of the defecation motor program. 65 A B • . : NLP- 40 1.0 • AEX-2/G CR � 0.8 CNG cha� nnels G !s ;/ Ep ac l ? AC . l cAMP > (.) � 0.6 Q. X w 0. 4 c R c wild type 1 ? PKA 10 5 20 5 30 5 40 5 p.x . ... ... ' .......... ' .......... � ......... : .. . p .. x ....................................... . ... p .. x .................................... . ....... p .. x ................................ . ....... p ... x ............................... . .......... p .. x ............................. . ....... p.x ................................. . ........... p .. x ............................ . .............. p.x .......................... . ................. p .. x ...................... . ................. p .. x ........... . 0.2 kin-1/PKA mosaic 10 5 20 5 30 5 40 5 p . .... . .. . � .. 00. 0 ••• � .. .. .. 0 . . � ......... : . .. .. p ....................................... . . ... p ...................................... . . ....... p .................................. . . ........ p ................................. .. ........... p ............................... . .............. p ........................... . . . ............... p ......................... .. ................. p ........................ .. . .................. p ...................... .. . .................. p ...................... .. aex-2/GPCR 10 5 20 5 30 5 40 5 p . ..... ... � ..... ... . � ... ... ... � ......... : .. . ..p .................. ..................... . . . ......... p ............. ................... . . .......... p .............................. . . . ............ p ............................ .. . ............... p ......................... .. . ............. p ............................ . . .............. p ........................... . . .............. p .......................... .. . .............. p ........................... . . .............. p ............... .. *** Figure 3.1 PKA activity is essential for the Exp step. (A) Diagram of the NLP-40-AEX-2/GPCR peptidergic signaling pathway acting in the GABAergic neurons that controls rhyth mic release of GABA during the enteric muscle contraction (Exp) step of the defecation motor progr am. The potential cAMP targets in this peptidergic pathway are indicated. The PKA holoenzyme is a tetram er with two regulatory subunits (R) and two catalytic subunits (C). (B) Quantif ication of the Exp step in young adult worms with the indicated genoty pes. "Exp per cycle" is defined as the average of the ratio of Exp to pBoc. (C) Representative ethograms of ten consecutive defecation cycles of young adult worm s with the indicated genoty pes. The kin-1 mosaic represents constipated worms segregated from the strain DG 3393: kin-l (o k338 ) I; tnEx l09. Each dot represents ls . "p" stands for the pBoc step and "x" indicates the Exp step. aBoc is omitted. Means and standard errors are shown. Asterisks (***) indicate significant difference from wild type: p < 0.005 in Student's t-test. 66 3.4.2 PKA fu nctions in GABAergic neurons to promote the Exp step To determ ine whether PKA functions in GABAergic neurons to control the Exp step, we generated worms exp ressing dominant negative PKA transgenes specifically in these neu rons. The PKA holoe nzyme is a tetramer composed of two regulatory (R) subunits and two catalytic (C) subunits (Figure 3.1A). When cAMP levels are low, R subunits bind to and inhibit the activity of C subunits ; when cAMP levels increase, two cAMP molecules bind to each of the R subunits at two distinct sites (site A and site B), leading to the dissoci ation of the PKA holoenzyme and activation of the C subunits (Skalhegg and Tasken, 2000). It has been shown that a single amino acid substitution (G324D) in site B of the mouse regulatory subunit (Ria) abolishes cAMP binding and prevents the dissociation of the PKA holoenzyme in vitro (Correll et a!., 1989). Ex pression of this mutant R subunit in mice generates a dominant negative effect on PKA activity in vivo (Willis et a!., 2011). kin-2 encodes the sole R subunit in C. elegans, and KIN-2 shares 74% overall sequence similarity with mouse Ria, and 97% similarity in the cAMP binding sites (Figure S3. 1). We mutated the corresponding Gly residue to Asp (G310D) in the B site of the KIN-2a isoform (referred to as PKA[DN], Figure 3.2A and S3.1) and exp ressed this construct specifically in GABAergic motor neurons (using the full length unc-47 promoter) (Mahoney et a!., 2008; Wang et a!., 2013). Two independently generated PKA[DN] transgenic lines (v;Js76 and v;J s7 7) displayed distended intestinal lumens, and dramatically reduced cycles in which Exp occurred (Figure 3.2 B and 3.2D and data not shown). This phenotype was similar to, but not as severe as that of the mosaic kin-] mutants , likely due to variable exp ression of the unc-47 67 A Dominant negative PKA PKA[DN]: KIN-2a (G31 0D) R c 0 inactive vjls77 (PKA[DN]) 10 s 20 s 30 s 40 s I I I I p ......................................... . ... p ...................................... . ........ p ................................. . ...... p ................................... . ..... p .... x ............................... . ......... p ................................ . ......... p ................................ . ............. p ............................ . ......... p ................................ . ............. p ............................ . .......... p ................ . B 1.0 Q) 0.8 (} >. (.) Cii 0.6 c.. c.. Lrl 0 . 4 *** 0.2 gsa-1(gf)/Gas 10 s 20 s 30 s 40 s I I I I p ..... x ........ .......................... . .... p ...... x ............................. . .......... p .. ..... x ...................... . . ................. p ...... x ............... . .................. p . . ....... x ............ . . ................ p ..... x ................ .. ................. p ...... x ............... .. . .............. p .... x ................... .. ............. p ..... x .................... .. . .......... p ..... x ...................... .. ........ p .... .. x ...... .. c 1.0 � 0.8 >. (.) Cii 0.6 c.. c.. Lrl 0. 4 0.2 0.0 gsa-1(gf)/Gas; PKA[DN] 10 s 20 s 30 s I I I p ........................... . .. ........ p ................. . .. ............ p ............ .. .. ............. p ............ . .. ........... p .............. . .. ......... p ................ . .. .... p ..................... . .. ... p ...................... . . p ........................ p .. .. .. X ....................... . .. ..... p ................... .. Figure 3.2 PKA fu nctions in GABAergic neurons to regulate the Exp step. (A) Diagram showing the construction of dominant negative PKA (PKA[DN] ). "R" and "C" indicate the PKA regulatory and catalytic subunit, respectively. "x" represents the substitution (G 310D) in the site B of the regulatory subunit KIN-2a, which presumably blocks cAlvlP binding and prevents its dissociation with PKA catalytic subunit. (B) and (C) Quantification of the Exp step of young adults with the indicated genoty pes. PKA[DN] denotes PKA dominant negative transgenic wonns (v jls76 and vj ls 77) in which the mutated regulatory subunit kin-2a( G310D) was exp ressed specifically in GABAergic neurons using unc-47 full length promoter. gsa-1 (gf) is a gain-of-function allele (ce81} of gsa-1/ Gas. (D) Representative ethogr ams of ten consecutive defecation cycles of young adult worms with the indicated genoty pes. vj ls 77 is used for PKA[DN] in (D). Each dot represents ls . "p" stands for the pBoc step and "x" indicates the Exp step. aBoc is omitte d. Means and standard errors are shown. Asterisks (***) indicate significant difference from wild type in (B) and gas-1 (gf)/Gas in (C): p < 0.005 in Student's t-test. "n.s." indicates no significant difference between indicated groups. 68 promoter in DVB neurons (data not shown). Thus, PKA activity is required in GABAergic neurons to promote the Exp step . 3.4.3 PKA acts downstream of Gus in the NLP-40-AEX-2/GPCR peptidergic signaling pathway To determine whether PKA functions in the peptidergic signaling pathway activated in the GABAergic neurons that control the Exp step, we examined whether PKA[DN] transgenes could block Exp in animals in which this pathway is constitutively active. gsa-1/Gas gain-of -function (gs a-] (gf)) mutations restore Exp to aex-2/GPCR mutants , consist ent with a role for gsa-1/Gas downstream of aex-2/GPCR (Mahoney et a!., 2008). However, gsa-] (gf) mutations failed to restore Exp to animals expressing PKA[DN] (Figure 3.2C and 3.2D). These data show that the effects of the AEX-2/GPCR peptidergic sigualing pathway on Exp are dependent on PKA in the GABAergic neur ons. For reasons that are unclear, the gsa-] (ce81); PKA[DN} double mutants had a slig htly shorter cycle length compared with either single mutant (Figure 3.2 D and data not shown) . To independently confirm that PKAfu nctions in the AEX-2/GPCR peptidergic sigualing pathway to regulate the Exp step, we generated a constitutively active kin-1 /PKA variant (referred to as PKA[CA]) and examined whether PKA[CA] transgenes could bypass the requirement of nlp-40 or aex-2/GPCR. The His87 and Trpl96 residues in the mouse PKA catalytic subunit lie at the interf ace between the R and C subunits and are necessary for the interaction between them (Kim et a!., 2005). H87Q, Wl96R substitutions disrupt this interaction resulting in a 69 A B 1.0 Constitutively active PKA PKA[CA]: KIN-1 a(H96Q, W205R) a> � 0.8 (.) active c PKA[CA} 10 s 20 s 30 s 40 s 50 s p ... x . .. .' ......... � .......... ' . .. ..... . .' . .... .. ... ' .. . . .... p ... x ............. � ............................. . ....... p ...... . . x ................................... . .. p ................................................. . .. p .. . x ............................................. . p .. x ................................................ . .. p .. x ..... . ..... .. � ................................ . ... p . ... . x .......................................... . ....... p .. x ......................................... . ............ p ... x ................................... . ............ p ... x ........................... .. � 0.6 a. Jj 0.4 PKA[CA}; PKA[DN] 10 s 20 s 30 s 40 s 50 s I I I I I p . ... x ........................................... . ... p .. . x ......................................... . ......... p ... x ................................... . . .......... p . .. x ................................. . ........ p .. . . x ................................... . ........ p.x ...................................... . ......... p ... x ................................... . ................. p .. . x ........................... . ............ p . .. x ................................ . ....... p . .. x ..................................... . ........ p .... x .............. . *** ** ** PKA[CA}; a ex- 2/GPCR 10 s 20 s 30 s 40 s p .... .. . ' .......... ' ........... ! .......... ' .... . .. .... p ......... ............................ . .. .......... p .............................. .. .. ............ p ............................. . .................. p . .. x ..................... . .. ................. p . .. x .................... . .. ................... p ...................... . .. ................... p ...................... . .. ................... p ...................... . .. ...... � .... p .............................. . .. .... p ....... .......... . Figure 3.3 Constitutively active PKA in GABAergic neurons partially bypasses the requirement of AEX-2/GPCR. (A) Diagram showing the construction of the constitutively acti ve PKA (PKA[CA]). "R" and "C" indicate the PKA regulatory and catalytic subunit, respectively. The two asterisks (*) represent the two mutations (H96Q, W205R) in the PKA catalytic subunit KIN-la, which presumably disrupt its association with the regulatory subunit but do not affect its enzymatic activity. (B) Quantification of the Exp step of young adults with the indicated genoty pes. PKA[CA] denotes PKA constitutively active transgenic worms (vjlsl 02 and vjlsl 03) expressing the mutated catalytic subunit kin-1 a(H96Q, W205R) in the GABAergic neurons using the unc-47 full length promoter. vjlsl 03 and vjls 77 were used for the PKA[CA];PKA[DN] strain. (C) Representative ethograms of ten consecutive defecation cycles of young adult worms with the indicated genoty pes. Each dot represents ls . "p" stands for the pBoc step and "x" indicates the Exp step. Ectopic Exp steps are indicated by ";:(. Means and standard errors are shown. Asterisks indicate significant differences between indicated grou ps: ** P < 0.01, *** P < 0.005 in Student 's t-test. 70 consti tutively active catalytic subunit that increases PKA activity in vivo (Li et a!., 1995; Orellana and McKnight, 1992). The C. elegans KIN- la isoforrn shares 91% amino acid identity with the mouse PKA catalytic subunit (Fig ure S3.2). We made the corresponding substitutions in the KIN- la isoforrn (H96Q, W205R) (Figure 3.3A and S3.2 ), and generated two independent transgenic lines (v ;Js l0 2 and v;J sl0 3) expr essing KlN- la(H96Q, W205R) specifically in GABAergic neurons (using the full length unc-47 promoter). Several pieces of evidence support the notion that PKA[CA] transgenes confer cons titutive PKA activity. First, animals expressing PKA[CA] tran sgenes had gro ssly normal defecation motor programs (Figure 3.3B and 3.3C) but occasionally displayed "ectopic" Exp following normal Exp steps ( 4 out of 100 cycles in v;J sl 02 and 10 out of 100 cycles v;Js l0 3. p�O.ll and 0.03, respe ctively, n�10, one tail t-test, compared to wild type) (Figure 3.3C). Ectopic Exp steps were also observed in kin-2(cel 79) mutants (5 out of 100 cycles, p�0.09, n�10), which carry a mutation in the PKA regulatory subunit that is predicted to increase PKA catalytic activity (Schade et a!., 2005). Second, ectopic Exp steps are also observed in gsa-1/ Ga.s gain-of-function mutan ts, which mimic constitutive peptidergic activation of the pathway in GABAergic neurons (Mahoney et a!., 2008). Finall y, PKA[CA] transgenes completely suppre ssed the Exp defects caused by PKA[DN] expers sion (Figure 3.3B and 3.3C), demonstrating that PKA[CA] is not inhibited by the PKA regulatory subunit. We found that PKA[CA] transgenes partially restored Exp to ae.x-2/GPCR and nl p-40 mutants (Figure 3.3B, 3.3C, and S3.3). Similarly, kin-2 loss-o f-function mutations restored Exp to nl p-40 mutants to a similar extent as PKA[CA] transgenes (Figure S3.3). Constitutively active 71 acy-J/a denylyl cyclase tran sgenes in GABAergic neurons have also been reported to partially rescue the Exp defects of aex-2/GPCR mutants (Mahoney et a!., 2008). Taken together, these results are consistent with the idea that cAMP generated by the NLP-40-AEX-2/GPCR peptidergic signaling pathway in the GABAergic neurons activates PKA, which drives Exp by promoting GABArelease. 3.4.4 PKA regulates rhythmic calcium influx in the DVB neuron We previously found that during the defecation motor program, the axons of DVB neurons display robust, rapid calcium transients that peak just before each Exp step, suggesting that rhyth mic presynaptic calcium influx in DVB drives rhythmic GABA release fr om DVB neu rons. Using a genetically- encoded calcium indicator, GCaMP3 (Tian et a!., 2009), we found that fluorescent spikes in DVB axons began about 3 seconds following the pBoc step, reached maximal intensity about I second later (immediately before each Exp step), and returned to baseline within 2 seconds (Fignre 3.4A, 3.4 B and 3.4C and Video S3. 1 and (Wang et a!., 2013)). In most cases, calcium transients appeared to initiate in the synaptic region of DVB neurons and often would spread along the axon to the cell body. In wild type anima ls, the "normal" pattern of pBoc-calcium transient-Exp was highly reproducible, occurring 100% of the time (23 cycles, 11 animals, Fignre 3.4C and 3.4D). In mutants lacking aex-2/GPCR, only about 9% of cycles adopted a normal pattern ( 4 out of 44 cycles, 11 ani mals), and the remaining 91% of cycles lacked both the calcium transients and Exp ( 40 out of 44 cycles, 11 animals, Fignre 3.4C and 3.4 D and Video S3.2). In contrast, in unc-25 mutants , which lack the GABA biosynthetic enzyme 72 A B before pBoc (t = 0 sec) r � before Exp (t = �4 sec) ·� 3. 0 wild type o 2.0 .... Exp u.. u: <l 1.0 � Boc c � 0.0 ,. '- -1.0 20 40 3 lwild type If 2 p s e c u: 1 "" <l 0 .. -· -1 5 u.. 2 pBoc U:1 � <Jo , 10 o 3 1 ae x -2 -1 5 10 Time (s) 60 80 15 15 D 1.0 rJ) 0.8 Q) l5 >. () - 0.6 0 c Q) 0.4 () Q; a... 0.2 .... Exp Exp · p � · � D pBoc __._ , __ _j !J. F/F 0 3s .... Exp pBoc � \ pBoc � 100 120 140 160 180 Time(s) l1: 1 pBo � o i 1 un c-2 5 <l 0 I ee� -1 5 u.. 2 pBoc u: 1 � <l o = 10 o 3 1 PK A[D N] -1 5 10 Time (s) 200 15 ......, 15 Figure 3.4 PKA is necessary for calcium influx in DVB neurons. (A) Expre ssion of the genetically-encoded calcium indicator, GCa:MP3 in DVB neurons (vjls 58). Top: diagram of the DVB neuron in the tail region. Synapse means the neuromuscular junction where the DVB neuron innervates enteric muscles. .Middle and Bottom: two snapshots from a real-time imaging video of wild type animals showing an increase in flu orescence in synaptic region of DVB neurons, as indicated by the white arrow, right before the Exp step. (B) A representative trace of the GCa:MP3 flu orescence in the synaptic region of DVB neurons in wild type animals showing DVB neurons are rhyt hmically activated during three consecutive defecation cycles. Note that the cycle length is longer than 50 seconds, likely due to unc-13( s69) mutation, which was used to immobilize the worms for calcium imaging (See Materials and Methods for details). (C) Representative trac es of the GCa:MP3 flu orescence in the synaptic region of DVB neurons in worms with the indicated genoty pes. The observed pBoc step and Exp step are indicated by arrows and arrowheads, respectively. (D) Classification of the different patterns of pBoc, fluorescent spikes in DVB and Exp in worm s with the indicated genoty pes. vjl s76 was used for the PKA[DN] strain. In (D), wild type: 23 cycles in 11 animals; unc-25: 44 cycles in 11 animals; aex-2: 44 cycles in 11 animals; PKA[DN] : 30 cycles in 12 animals. 73 glutamic acid decarboxylase (GAD) (Jin et a!., 1999), most of the cycles without Exp steps still produced a calcium spike (3 1 out of 33 cycles without Exp, 11 animals, Figure 3.4C and 3.4 D and Video S3.3). These results show that the calcium transients is dependent on the peptidergic signaling pathway but occurs independently of GABA release from DVB neuron s, and are consist ent with the idea that the calcium spikes in the synaptic region of DVB neurons drive the Exp step by triggering GABArelease. Our behavioral results show that PKA activity may be necessary and sufficient for GABA release fr om the GABAergic neuron s. In principal, PKA could control GABA release directly by acting on the synaptic vesicle machinery at presynaptic terminals or indirectly by regulating the excitability of the GABAergic neuron s. To distinguish between these two possi bilities, we examined whether PKA[DN] or PKA[CA] trans genes impacted GCaMP3 fluorescence spikes in DVB neurons during the defecation motor program. PKA[DN] transgenes produced a calcium pattern that was similar to that observed in aex-2/ GPCR mutan ts: 80% of cycles lacked both calcium transients and the Exp steps (3 1 cycles, 12 anima ls, Figure 3.4C and 3.4 D and Video S3.4). On the other hand, PKA[CA] transgenes caused "ectopic" fluorescent transients in DVB neurons, which occurred once or twice in between cycles on average (22 regular calcium spikes and 26 ectopic calcium spikes observed during 24 cycles, 11 animals, Figure 3.5A, 3.5B and 3.5C and Video S3.6). These "ectopic" fluorescent transients were occasionally associa ted with an ectopic Exp (6 out of the 26 ectopic calcium spikes, 11 animals, see Discussion). The duration of both the "normal" and "ectopic" fluorescent transients in PKA[CA] tran sgenic animals was 74 A 3.0 o2.0 L.l.. ........ L.l.. <l l.O 0.0 ·1.0 B 3 0 L.l.. 2 ........ 1 L.l.. <l 0 ·1 3 0 2 L.l.. ;:;:. 1 <l 0 ·1 PKA[CA] pBo \ 20 40 wild type pBoc \ 5 10 c 3.5 § � 3 "(3 (f) ro g 2.5 UN ectopic calcium spike � Q; 2 o o.. .2 � 1.5 0.. ..>< .9' 5. !Il (f) 1 60 80 100 160 0.5 Time (s) D 15 20 25 wild type PKA[CA] n.s. PKA[CA] regular calcium spike pBoc � 5 10 15 20 25 - · 25 wild type PKA[CA] PKA[CA] regular ectopic calcium calcium E spike spike 0 lL u: <l 3 n.s. 2 1 0 wild type PKA[CA] PKA[CA] regular ectopic calcium calcium spike spike Figure 3.5 Constitutively active PKA causes ectopic calcium spikes in DVB neurons and increases calcium spike duration. (A) A representative trace of the GCaMP3 fluo rescence in the synaptic region of DVB neurons in transg enic worms expressing PKA[CA] (v jlsl 02) during two consecutive defecation cycles. An ectopic calcium spike in DVB neurons was observed, as indicated by the bracket. Note that the cycle length is longer than 50 seconds, likely due to unc-13( s69 ) mutation, which was used to immobilize the worms for calcium imaging (See :Materials and Methods for details). (B) Representative trac es of the GCaMP3 fluo rescence in the synaptic region of DVB neurons in worm s with the indicated genoty pes. The observed pBoc step and Exp step are indicated by arrows and arrowhe ads, respectively. (C) Av erage frequency of ectopic calcium spikes in DVB neurons during a 250-second imaging period in wild type animals (0.0±0.00 events per 250 seconds, n=lO animals) and vj ls l02 (2.4±0.69 events per 250 seconds, n=ll anim als). (D) and (E) the duration and amplitude of regular DVB calcium spikes and ectopic calcium spikes in wild type and PKA[ CA] ( vj ls 1 02) anim als. Means and standard errors are shown. Asterisks indicate significant diff erences from wild type: ** P < 0.01, *** P < 0.005 in Student's t-test. "n.s." indicates no significant difference between indicated grou ps. 75 sig nificantly longer compared to wild type controls (Fignre 3.5D and Video S3.5 and S3.6). However, the amplitude of calcium transients was not significantly different (Fignre 3.5E, wild type: 6F/F0�1.8 3±0.17, n�30; PKA[CA] : normal calcium transients 6F/F0�2.37±0.22, n�2J, p�0.06; ectopic calcium tran sients, 6F/F0�2.08±0.22, n�26, p�0.39, two tail t-test, compared to wild type). Taken together, we conclude that PKA is esse ntial for the generation of calcium transients in DVB neurons during the defecation cycle. In addition, PKA activity is sufficient to generate calcium transients and may pos itively regnlate the duration but not the amplitude of calcium trans ien ts. 3.4.5 UNC-2NGCC acts downstream of PKA and mediates calcium influx in the DVB neuron Voltage-gated calcium channels (VGCCs) are critical for presynaptic calcium influx during regnlated neurotransmitter release (Catterall, 2011). unc-2 encodes the a! subunit of the C. elegans PIQ type VGCC. UNC-2 localizes to presynaptic terminals, and promotes calcium influx and neurotransmitter release (Richmond et a!., 200 I; Saheki and Bargmann, 2009). unc-2 has been reported to be exp ressed in the DVB neuron, and to regnlate the Exp step (Mathews et a!., 2003). Consist ently, we observed the Exp step in only about 40% of the defecation cycles in unc-2( /jl) null mutants (Fignre 3.6 A). In unc-2 mutants expressing PKA[CA], the Exp step was observed in about 60% of cycles (Fignre 3.6A). The incomplete restoration of the Exp step by PKA[CA] is consistent with the idea that UNC-2 functions downstream of or in parallel to PKA to regnlate Exp. 76 To test whether the rhythmic calcium transients are mediated by UNC-2, we examined GCaMP3 fluorescence in synaptic regions of DVB neurons in unc-2 mutants . Surprisin gly, fluorescent transients were observed in all cycles including the normal cycles that had the Exp steps and the incomplete cycles without the Exp steps (Figure 3.6B and 3.6C and S.7 and S3.8). However, there was a sig uificant reduction in the average amplitude of fluorescent transients in the incomplete cycles (that lacked Exp, n�JS) compared to the fluorescent transients in the normal cycles (those with Exp, n�22) (Figure 3.6B, 3.6C and 3.6D). To determine if the reduction in fluorescence amplitude in incomplete cycles was a secondary effect of compromised Exp in these mutants, we examined fluorescence amplitudes in mutants lacking unc-25/GAD. We found no difference in the average amplitude between normal and incomplete cycles in unc-25/GAD mutants (n�JO and 31 cycles, respec tively, and Figure 3.6 D). These results indicate that UNC-2 is not absol utely required for the generation of calcium transients but rather regulates the size of calcium transients , possibly allowing them to reach the threshold required for triggering Exp. 3.4.6 Other non-voltage-gated calcium channels may also mediate calcium influx in the DVB neuron Because unc-2 does not appear be to the only channel to mediate the calcium transients in DVB neurons for the Exp step, we next sought to identify other calcium channels that might function in this process. The C. elegans genome encodes two additional VGCC a! subuni ts: egl-19, the L-type a! subunit, and cca-1, the T-type a! subunit (Lee et a!., 1997; Steger et a!., 2005). We found that loss- of -function cca-1NGCC mutants had normal Exp steps, whereas 77 A B 3 0 2 u.. -... u.. <l 1 0 -1 Q) 1.0 * � 0.8 (.) (U 0.6 0.. 0.. 0.4 >< w 0.2 0.0 � o " o " � � 0 0 0 0 � rj (j rj (j (f (f � � � � �' � � � unc-2, normal cycle (22 out of 40 cycles) ,. Exp 5 Time(s) 10 D 0 !:!:: X "' E u. <I c 3 0 2 u.. -... u.. <l 1 0 15 -1 • normal cycle 2.5 • incomplete cycle *** 2 1.5 1 0.5 0 unc-2NG CC a1 unc-25/GAD unc-2, incom plete cycle (18 out of 40 cycles) 5 Time(s) 10 15 Figure 3.6 unc-2NGCC functions dowstream of PKA to mediate calcium influx in the GABAergic neurons. (A) Quantification of the Exp step of young adult wonns with the indicated genoty pes. vjlsl 03 was used for the PKA[CA] st rain. (B) and (C) Representative trace of GCa:tvfP3 flu orescence in the synaptic region of DVB neurons in unc-2 mutants in nonnal cycles, with the Exp step (B) and incomplete cycles, without the Exp step (C). (D) Comparison of the maximal change (L1Fmax1Fa) of GCa:tvfP3 flu orescence in the synaptic region of DVB neurons between the normal cycles and the incomplete cycles in unc-2 mutants and unc-25 mutants. vjls58, the transgenic strain with GCa:tvfP3 exp ressed in the DVB neuron, was used. Normal cycles: n=22 in unc-2(! jl) mutants; n=lO in unc-25(e156 ) mutan ts. Incomplete cycles: n=l8 in unc-2(/jl) mutants ; n=31 in unc-25(e156 ) mutants. Means and standard errors are shown. Asterisks indicate significant differe nces: * P < 0.05, *** P < 0.0005 in Student's t-test. 78 loss- of -function egl-19NGCC mutants displayed a modest reduction in the number of cycles with Exp (Figure 3.7A). Interestingly, in double mutants lacking both unc-2 and egl-19, the Exp step was nearly eliminated (Figure 3.7B). Furt hermore, PKA[CA}; egl-19; unc-2 triple mutants had the same, severely reduced Exp fr equency as the egl-19; unc-2 double mutants (Figure 3.7B). Thus, two VGCCs, egl-19 and unc-2, function downstream ofPKA to control the Exp step. To directly test whether egl-19NGCC mediates calcium influx in DVB neurons, we next examined the effects of egl-19 mutations on GCaMP3 fluorescence in DVB neu rons. We were unable to analyze calcium influx in egl-19 mutants due to an unexpected genetic interaction between egl-19 and unc-13(s69) (used to paralyze animals for imaging without stopping the defecation cycle) that supp ressed the Exp defects of egl-19 mutants (see Materials and Methods). Instead, we examined calcium influx in egl-19; unc-2 double mutants (which were sufficiently paralyzed for imaging without the unc-13(s69) mutation). We found that the fraction of incomplete cycles increased to 80% (from 40% in unc-2 mutan ts, Figure 3.7C and Video S3.9). Finally, no calcium influx was observed at all in 35% of the incomplete cycles, a defect that was not observed in the unc-2 mutants alone (Figure 3.7C and Video S3. 10). In egl-19; unc-2 double mutants expressing PKA[CA] trans genes, these defects in calcium influx were as severe as those observed in egl-19; unc-2 mutants (Figure 3.7C), suggesting that egl-19 and unc-2 function downstream of PKA to promote calcium influx into DVB. egl-19 and unc-2 mutations were not able to block all effe cts of PKA[CA] trans genes, including the increased frequency of ectopic calcium transients and the increa sed duration of normal and ectopic calcium transients (Figures 3.7D and S4 and Video S3.11 and S3. 12). In 79 A 1.0 <ll 0.8 u � 0.6 .... <ll a. 0.4 a. X w 0.2 c 1.0 rJl <ll 0.8 u >. (.) 0.6 - 0 c <ll e 0.4 <ll a... 0.2 0.0 ••• B 1.0 <ll 0.8 u � 0.6 .... <ll � 0.4 X w 0.2 n.s. _j f:J. F/F 0 3s D E 6 � rJl j :::1 <ll ar� 2.5 .... a. - rJl 2 o E 0 :::1 � Q 1.5 "' ro L1 (.) 1 <l ••• ••• n.s. Figure 3. 7 Other non-voltage-gated calcium channels are required from calcium influx in the GABAergic neurons. (A) and (B) Quantifi cation of the Exp step of young adult worms with the indicated genotype types. (C) Classification of different patterns of pBoc, fluorescent spikes in DVB and Exp in worm s with the indicated genotyp es. unc-2: 40 cycles in 21 anim als, egl-19; unc-2: 19 cycles in 11 animals, PKA[CA}; egl-19; unc-2: 24 cycles in 12 animals, PKA[CA] : 23 cycles in 12 animals. (D) and (E) Quantification of the duration and amplitude of regular DVB calcium spikes in worms with indicated genoty pes. PKA[CA] represents transgenic worms with constitutively active PKA specifically expressed in GABAergic neurons (v jls1 03 in (B) and vj ls1 02 in (C, D)). Note that in (C), (D) and (E), unc-2 and PKA[CA] strains, but not the egl-19; unc-2 and the PKA[CA] ; egl-19; unc-2 strains contain the unc-13( s69) mutation for immobilization, as egl-19;unc-2 alone were almost completely paralyzed. vj 1s58, the transgenic strain with GCa:tv1P3 exp ressed in the DVB neuron, was used for the unc-2 strain; while vj ls64 was used for calcium imaging in other genoty pes. Means and standard errors are shown. Asterisks indicate significant difference between indicated group and significant difference from wild type in (A), PKA[CA] in (B) and egl-19; unc-2 mutants in (D) and (E): * p< 0.05; ***P < 0.005 in Student's t-test. "n.s." indicates no significant difference between indicated grou ps. 80 addition, the amplitude of regular calcium spikes of the incomplete cycles in egl-19; unc- 2mutants expressmg PKA[CA] transgenic was similar to those in PKA[CA] tran sgenic animals, but significantly larger than those observe in egl-19; unc-2 double mutants (Figure 3.7E). Together these results suggest that the eff ects of egl-19 and unc-2 mutations on Exp can in part be attributed to their requirement for calcium influx in DVB neurons and that additional calcium channels must act downstream of PKA to mediate calcium influx in DVB. 3.5 Discussion In this study, we identify PKA as the maJ or downstream target of cAMP in the NLP-40-AEX-2/GPCR peptidergic signaling pathway that functions in the GABAergic neurons to regulate the Exp step during the defecation motor program in C. elegans. PKA controls rhythmic activation of the GABAergic neurons by promoting presynaptic calcium influx in these neu rons. We find that the mechanism by which PKA functions to promote rhyth mic calcium influx is partially dependent on the P/Q-type VGCC, UNC-2 and the L-type VGCC, EGL- 19. These results suggest that PKA promotes rhythmic neurotransmitter release by controlling calcium influx in neurons during a rhythmic behavior. 3.5.1 PKA is essential for presynaptic calcium influx in DVB neurons Our results suggest that PKA functions as an essential cue for calcium influx in neurons for their activation to control rhythmic behaviors, because both calcium spikes in DVB neurons and Exp steps are abolished in most of the defecation cycles in animals expressing PKA[DN] 81 transgenes (Figure 3.2 B, 3.2 D, 3.4C and 3.4 D). To our knowledge, this is the first in vivo example showing that PKA in neurons is absolutely required for the execution of a rhythmic behavior. In many preparati ons, PKA has been shown to modulate biological processes by regulating intracellular calcium concentration. One classic example is that PKA mediates the enhancement of calcium influx in cardiac myocytes to modulate the rate of heart beating in response to norepinephrine from the sympathetic nervous system (Hell, 2010). In neurons, PKA also has a modulatory role in regulation of synaptic transmission and synaptic plasticity by phosphorylating several synaptic vesicle proteins that fun ction downstream of calcium influx (Seino and Shibasaki, 2005). 3.5.2 UNC-2 and EGL-19 mediate part of PKA-dependent calcium influx in DVB neurons How does PKA control calcium influx in the GABAergic neurons? Our results suggest that PKA acts at least partially through UNC-2, the P/Q-type VGCC, and EGL- 19, the L-type VGCC. unc-2 has been reported to be exp ressed in the DVB neuron and egl-19 is expressed in many neurons as well (Arellano-Carba jal et a!., 20 11; Lee et a!., 1997; Mathews et a!., 2003). Our behavioral and calcium imaging analys is of egl-19; unc-2 double mutants expr essing PKA[CA] trans genes suggests that one or both of these channels must also function in enteric muscles to promote Exp since PKA[CA] can restore normal calcium spike amplitudes but it fails to restore the Exp step in egl-19; unc-2 double mutants (Figure 3.7B, 3.7C and 3.7E). Consistent with this idea, egl-19 has been reported to be exp ressed in muscles, including some of the enteric muscles (Lee et a!., 1997). Our data indicates that other non voltage-gated calcium channels must be also 82 required for calcium influx in DVB neurons for the Exp step, since egl-19 and unc-2 mutations together could not completely block the calcium influx in DVB neurons or the effects of PKA[CA] transgenes on calcium influx (Figure 3.7C, 3.7D and 3.7E). The identification of these channels will further our understand ing of the mechanism underlying PKA-dependent calcium influx in these neuron s. PKA may regulate calcium influx either by direct phosphorylation of UNC-2 and/or EGL- 19 or by an indirect mechanism involving, for example, the regulation of membrane potential. Indeed, both mechanisms have been reported. The fight-or-flight response is contro lled by PKA-dependent phosphorylation of the L-type calcium channels Cavl.1 and Cav l.2 which mediate calcium influx in skeletal and cardiac muscles, respec tively, to enhance their contraction (Emrick et a!., 2010; Hell, 2010). EG L-19 does not have the homologous phosphorylation site. In pancreatic beta cells, PKA has been reported to phosphorylate ATP-sensi tive potassi um channels to regulate the membrane potential (Light et a!., 2002). Interestingly, the egi-36 /Shaw type potassi um channel functions in DVB to regulate the Exp step (Johnstone et a!., 1997), raising the possibility that it may be a target for PKA. Our results also show that PKA[CA] trans genes increases the duration of calcium spikes in DVB neurons in both wild type and egl-19; unc-2 mutan ts, suggesting that PKA regulates calcium spike dynamics independently of egl-19 and unc-2. PKA might regulate the open time or the inactivation of other calcium channels or reduce the rate of calcium clearance from the synaptic region in DVB neu rons. Consistent with our observation, in jection of the PKA catalytic subunits in cells has been reported to increase the duration of calcium currents (Castellucci et a!., 83 1980; Kaczmarek et a!., 1980; Osterrieder et a!., 1982). 3.5.3 The activity ofPKAin the GABAergic neurons during the def ecation cycle Our previous work indicates that NLP-40 functions as the timing signal from the intestine and it delivers the timing information to the GABAergic neurons by instructing their rhytlunic activation (Wang et a!., 2013). In this study, we show that the downstream effector PKA is also instructive for the activation of the GABAergic neurons, because the PKA[CA] trans genes can elicit ectopic calcium spikes in between cycles. The observation that ectopic calcium spikes in DVB neurons do not occur in wild type animals suggests that endogenous PKA activity in these neurons must somehow be turned down rapidly following each Exp step. Thus, we propose a model in which rhytlunic activation of PKA by the NLP-40 -AEX-2/GPCR peptidergic pathway stimulates rhythmic calcium influx in the GABAergic neurons to drive the Exp step. It will be interesting to directly determine whether cAMP levels and/or PKA activity oscillate in DVB neurons and whether these oscillations correspond with the calcium oscillations in vivo. Several negative feedback mechanisms following PKA activation that have been reported in other preparations may also help establish rhytlunic PKA activity in DVB neu rons. These include the activation of by phosphodiesterases (PDE s) that break down cAMP, calcium-mediated inhibition of adenylyl cyclases and activation of specific phosphatases that counteract PKA activity (Gancedo, 2013; Sasso ne-Corsi, 2012; Wong and Scott, 2004). PKA activity has been reported to be essential for the initiation of the cAMP-PKA-Ca 2 + oscillation circuit in insulin-secreting MIN6 beta cells (Ni et a!., 20 10). Thus, the interp lay between cAMP, PKA and calcium may be a 84 general mechanism by which PKA generates oscilla tory signaling circuits in neurons to control rhythmic behavi ors. Rhyt hmic PKA activation may be essential for the reliability of the Exp step during the defecation cycle, because PKA[CA] trans genes cannot fully restore Exp to animals that lack the NLP-40-AEX-2/GPCR peptidergic signaling pathway components (Fignre 3.3B). Similarly, cons titutive acy-J/a denylyl cyclase exp ression in the GABAergic neurons also only partially rescues the Exp defects of aex-2 mutants (Mahoney et a!., 2008). Interestingly, gsa-] (gf) /Gas mutations ahnost fully rescue the Exp defects in aex-2 /GPCR mutants (Mahoney et a!., 2008). Since GSA-1/Ga.s functions upstream of both adenylyl cyclase and PKA, it is possi ble that gsa-] (gf) mutants retain the proper negative feedback mechanisms that allow PKA activity to remain rhythmic. Rhyt hmic PKA activation may work together with other mechanisms to ensure the proper execution of the Exp step. Indeed, it has been postulated that a permissive signal may control the refractory period of enteric muscles by allowing Exp to occur only within a small window of time following the beginning of each cycle (the pBoc step) (Mahoney et a!., 2008; Wang et a!., 2013). We speculate that this permissive signal may be entrained to the pacemaker activity in the intestine, because most of the Exp steps observed in the aex-2 mutants expressing PKA[CA] did not happen at random times, but rather occurred a few seconds after the pBoc step. The absence of this permissive signal in between cycles may also explain why PKA[CA] elicits ectopic calcium spikes in DVB neurons more frequently than ectopic Exp steps. The identification of this permissive signal or the nature of the refractory period of enteric muscles would provide more 85 comprehensive understan ding on how the rhyt lunic Exp is reliably generated. 3.5.4 Using genetically-encoded PKA transgenes to dissect the fu nction ofPKA in vivo In mamma ls, several genes encode different PKA regulatory and catalytic subunits, and each of these genes may have several different isoforms due to alternative splicing (Skalhegg and Tasken, 2000). Although many PKA isoforms have been knocked out and knocked in in mice to dissect the role of PKA in vivo, compensation by remaining isoforms has complicated the interpretation of these studies (Brandon et a!., 1997; Niswender et a!., 2005; Willis et a!., 2011). Unlike mamma ls, C. elegans has a single gene (kin-2) for the PKAregulatory subunit and a single gene (kin- I) for the catalytic subunit, both of which share high similarities with their counterparts in higher mamma ls. Thus, C. elegans has the potential to be a good genetic model to dissect the physiological roles of PKA. However, studies on the contribution of PKA sigualing in C. elegans have been limited since null mutants of either kin-] /catalytic subunit or kin-2/ regulatory subunit are lethal. Various strategies using weak alleles of kin-2, pharmacological treatments with PKA inhibitors, and RNAi-mediated knockdown, have revealed roles for PKA in regulating neurotransmitter release, axon regeneration and behaviors (Ghosh-Roy et a!., 20 10; Murray et a!., 2008; Schade et a!., 2005). However, non-specific effects of phar macological PKA inhibitors and spreading of RNAi limit the utility of these approaches (May and Plasterk, 2005; Murray, 2008). The ability to manipulate PKA activity in a tissue specific manner using the PKA variants deve loped in this study represents a powerful approach for probing the function of PKA sigualing lll Vl VO. 86 Mouse PKA Ria 1 KI N-2a 1 Mouse PKA Ria 63 KIN-2a 55 Mouse PKA Ria 125 KIN-2a 110 Mouse PKA Ria 187 KI N-2a 172 Mouse PKA Ria 249 KIN-2a 2 3 4 Mouse PKA Ria 3 11 KIN-2a 296 RQI I C.KT I I RTD s � s�PIIN PWKGRRII -YTIII A A slvi . Y K I --- RII EGI NPDAA � r E•K--R-SG� I KIII -rEI KI oAI • Mouse PKA Ria KIN-2a 3 7 3 I Fi slsv 381 358 YI KI MT 366 124 109 186 171 248 233 310 295 372 357 Figure S3.1 Alignment of C. elegans PKA regulatory subunit (KIN-2a) with mouse PKA regulatory subunit (Ria). KIN-2a is well conserved. Pink color indicates identity and green color represents similarity. The positions for site A and site B, where cAMP binds, are indicated by black rectan gles. The asterisk "*" indicates the Glycine residue in KIN-2a (G31 0), which was substitute d by Aspartic acid (G31 0D) to make dominant negative PKA (PKA[DN] ). 87 Mouse PKA Ca KIN-1a Mouse PKA Ca KIN-1a Mouse PKA Ca KIN-1a Mouse PKA Ca KIN-1a Mouse PKA Ca KIN-1a Mouse PKA Ca KIN-1a 1 1 55 63 117 125 179 187 241 249 303 311 * 351 359 116 124 178 186 240 248 302 310 Figure S3.2 Alignment of C. elegans PKA catalytic subunit (KIN-la) with mouse PKA catalytic subunit (Ca). KIN- la is well conserved. Pink color indicates identity and green color represents simila rity. The two asterisks "*" represent the Histidine and the Tryptophan residues in KIN -la (H96, W205), which were substituted by Glutamine (H96Q) and Arginine (W205R), respectively, to make constitutively active PKA(PKA[CA]). 88 1.0 Q) () >. 0.8 () ,_ Q) 0.6 0.. 0.. X 0.4 w 0.2 0.0 ;!,�� �� Figure S3.3 Constitutively active PKA specifically in GABAergic neurons mimics kin-2( lj) mutants. Quantification of the Exp step of young adult worm s with the indicated genot ypes. kin-2(l f} denotes the loss-f unction allele of PKA regulatory subunit, kin-2( ce17 9) . PKA[CA] denotes PKA constitutively active transgenic worms (v jlsl 02 and vjlsl 03) expressing the mutated catalytic subunit kin-1 a(Ji96Q, W205R) in GABAergic neurons using the unc-47 full length promoter. The null mutants, nl p-40(tm4 085), were used. Means and standard errors are shown. Asterisks indicate significant differences from nl p-40 mutants : * P < 0.05, ** P < 0.01 in Student's t-test. 89 E u -� Q) (.) (/) roo (.)l{) coN > ..... 0� (.) (/) · a.� 0 · -a. � (/) A 3.5 3 2.5 2 1.5 1 0.5 0 *** * B 6 u ..-... 5 ·a. � 0 (/) 4 �� 0 � 3 § E :.;:::; -� 2 ro u 1... - :J ro 0 (.) 1 0 *** c (.) ·a. (/) 0 Q) -� (.) ·- Q) a. ..... (/) o E 0 :J u.. · -- (.) X- CII ro tfU <J 3.5 3 2.5 2 1.5 1 0.5 0 Figure S3.4 EGL-19 and UNC-2 do not completely block ectopic calcium spikes in DVB neurons induced by constitutively active PKA. (A) Av erage frequency of ectopic calcium spikes in DVB neurons during a 250-second imaging period in egl-19; unc-2 (0.0±0.00 events per 250 seconds, n=ll anim als), PKA[CA}; egl-19; unc-2 (1.3 ±0.55 events per 250seconds, n=l2 animals) and PKA[CA} (2.5±0.57 events per 250 seconds, n=l2 animals). (B) and (C) Quantifi cation of the duration and amplitude of ectopic DVB calcium spikes in worms with indicated genot ypes. vj 1s64, a transgenic strain with GCa:MP3 exp ressed in DVB neuron was used for calcium imaging. PKA[CA] represents trans genic worms with constitutively active PKA specifically expressed in GABAergic neurons (vjls102). Means and standard errors are shown. Asterisks indicate significant difference from egl-19; unc-2 mutants in (A) and between indicated groups in (C): *, P<0.05; ***, p < 0.005 in Student's t-test. "n.s." indicates no significant difference between indicated grou ps. 90 Chapter 4 Conclusions and Perspectives 4.1 Signif icance Timing information originating from pacemakers needs to be reliably transmitted to downstream neurons in the output pathways to ensure the proper execution of rhyth mic behav iors. However, the mechanism underlying the time delivery process remains elusive. In my dissertati on, I identify a neuropeptide (NLP-40) as the timing messenger for rhyth mic enteric muscle contractions (Exp) during the defecation motor program in C. elegans and reveal the underlying molecular mechanism by which it carries the timing information and delivers it to downstream neu rons. The intestine (the pacemaker) relays the timing information encoded by calcium oscillations to the downstream GABAergic neurons via SNT -2/synaptotagrnin-dependent NLP-40 release. Once released, NLP-40 instructs the rhythmic activation of these neurons by binding to its receptor AEX-2/GPCR. The activation of this GPCR signaling pathway is coupled to calcium influx through the Gas-AC-cAMP-PKA-VGCCs signaling cascade in the GABAergic neuron s. Although neuropeptides have been implicated in regulating rhyth mic behaviors (Mertens et a!., 2007; Taghert, 2001; Vosko et a!., 2007), the molecular mechanisms by which neuropeptides function as timing messengers to mediate the communication between pacemakers and downstream neurons and how neuropeptides regulate the physiology of neurons in circuits for rhyth mic behaviors remain unclear. My dissertation work advances current understanding of neuropeptide signaling and rhyth mic behaviors in several aspects: first, I show that neuropeptides 91 could directly mediate ultradian rhythmic behaviors with a period less than 24 hours; second, my work suggests that synaptotagrnin-dependent rhythmic release of neuropeptide may represent a novel and conserved way to relay timing information from pacemaker to downstream neu rons; third, in contrast to the classic modulatory role of neurope ptides, I demonstrate that NLP-40 functions more like a neurotransmitter, since it is both instructive and essential for the activation of the GABAergic neur ons; fourth, PKA can act downstream of the timing messenger from the pacemaker to mediate the activation of the motor neurons in a rhyth mic behavior circuit. My results support the idea that rhythmic release of neuropeptides from pacemakers may represent a general mechanism to convey timing information to control rhythmic behavioral outputs . However, in order to directly prove this implication, future experiments are still needed to address remaining questions about the relationship between the dynamics of the NLP-40 signaling and Exp . In addition, it is also very interesting to further study how Exp is coordinated with aBoc and pBoc during the defecation motor program. I will discuss some of these future directions as follows. 4.2 Directly testing rhythmic release of NLP -40 fr om the intestine My results demonstrate that NLP-40 is the timing signal from the pacemaker (intestine) for the Exp step during the defecation process in C. elegans and I propose that calcium oscillations in the intestine may drive rhythmic NLP-40 release through SNT -2/synaptotagrnin, the putative calcium sensor on DCVs containing NLP-40. However, direct evidence for this 92 speculation is still lacking. It will be interesting to directly examine whether NLP-40 is rhythmic released. One possi ble approach is to use !otal internal reflection fluorescence (TIRF) microscopy to image pHluorin-tagged NLP-40 during the defecation cycle. pHluorin is a pH sensitive variant of green fluorescent 12rotein (GFP) and has served as a very useful tool to study neurotransmitter and neuropeptide release (Levitan, 2004; Miesenbock et a!., 1998). The acidic enviro rnn ent of lumenal side of synaptic vesicles and dense core vesicles quenches the fluorescence of pHluorin; when these vesicles fuse with plasma membrane and release their contents, pHluorin would be exposed to the enviro rnn ent that is more neutral and its fluorescence will be enhanced (Miesenbock et a!., 1998). However, the cuticle prevents directly applying TIRF microscopy to examine NLP-40 release in intact animals, because TIRF microscopy only allows selec tive visualization of a very tiny depth of the sample ( � lOOnm from the glass interf ace). Additionally, the movement of worms may also impose another barrier for TIRF imaging in vivo . Recent observations that the dissected C. elegans intestine preparation is able to preserve normal calcium oscillations may resolve both problems (Espelt et a!., 2005; Teramoto and Iwasaki, 2006). In addition, the development of red geneti cally-encoded calcium indicator (R -GECO 1) makes it possible to detect both calcium oscillations and the release of pHluorin-tagged NLP-40 simultaneously (Zhao et a!., 20 11). The model that I propose in the disse rtation predicts that each calcium oscillation in the intestine would be accompanied by synchronous release of NLP-40 in wild type animals but not snt-2 mutants . 93 Another intriguing idea is to generate a "synthetic" reporter system that responses to the released NLP-40 in vivo. One promising candidate is to use the FMRFamide-peptide gated sodium channel (FaNaCh) isolated from the snail Helix aspersa and its ligand (Lingueglia et a!., 1995), which have been succes sfully used to study neuropeptide secretion in culture cells (Whim, 2002; Whim and Moss, 200 1). This "FMRFamide tagging technique" is performed as follows: first fuse the neuropeptide of interest with the FMRFamide peptide (the ligand), and then express this fusion protein together with the FaNaCh channel in the same cell. The principle is that once the neuropeptide of interest is released, the FMRFamide peptide from the same dense core vesicles would also be released and it would bind to and open the FaNaCh chann els, which could be easily detected by whole-cell voltage clamp technique to monitor the membrane potential (Whim and Moss, 200 I). This technique holds the potential to examine NLP-40 release from the intestine in vivo. However, it is technically impossi ble to directly patch on the intestinal cells in intact ani mals, because these cells are not accessible. One possi ble way to overcome this technical problem is to express the FaNaCh channel in a different cell and use calcium indictors, such as GCaMP3, to detect the opening of FaNaCh channel. For example, the FaNaCh channel could be expressed in the DV A neuron, a neuron that is very close to the intestine. If the rhyth mic release model is correct, I speculate that the DV A neuron with FaNaCh would undergo rhythmic activation, like the DVB neuron does, because once the FMRFamide-tagged NLP-40 is released fr om the intestine, the FMRFamide would activate FaNaCh and induce sodium influx, leading to the activation of the DVA neuron, which would be could detected with GCaMP3 . As the first trial, I attempted to express the FMRFamide-tagged NLP-40 in the intestine and the FaNaCh channel 94 in GABAergic neurons in aex-2 mutants . If the "FMRFamide tagging technique" works, the FMRFamide, released together with NLP-40 from the intestine, is predicted to open the FaNaCh channels and activate GABAergic neuro ns, which should suppress the Exp defects in aex-2 mutan ts. However, I did not observe this prediction in the transgenic worms. Reasons for this include the incorrect processing and/or modification of FMRFamide-tagged NLP-40 in the intestine and the relative low affmity of FMRFamine and FaNaCh compared to NLP-40 and AEX-2 (Lingueglia et a!., 1995; Wang et a!., 2013). Results of such experiments would provide direct evidence showing rhyth mic release of neuropeptide from the pacemaker is the timing delivering mechanism in C. elegans and may help to understand how neuropeptides mediate rhyth mic behaviors in other organi sms. 4.3 Mechanisms of the termination of NLP-40 signaling The peptide in jection experiment shows that NLP-40 is instructive for the activation of DVB neurons (Figure 2.6). During the defecation motor program, DVB neurons in wild type animals undergo rhyth mic activation and no calcium spikes between cycles are observed (Figure 3.4). These data suggest that the NLP-40 signaling pathway must be rapidly turned off to preserve the rhythmicity established by SNT -2-dependent release of NLP-40 fr om the inte stine. The mechanisms to terminate the effect of NLP-40 signaling may occur at multiple leve ls. One possibility is that NLP-40 peptide itself can be degraded by peptidases in the pseudocoelom after it activates the receptor. Desensi tization and/or inte rnalization of AEX-2/GPCR after its activation by NLP-40 peptide may represent another way to stop this peptidergic signaling. 95 Additionally, activation of PDEs, which would break down cAMP, and other negative feedback mechanisms may also contribute to the down regulation of the PKA sigualing in the GABAergic neurons to prevent the GABAergic neurons to become activated at wrong time . Thus, it would be very interes ting to test whether mutants defective in peptidas es, factors that are required for the GPCR desens itization and PDEs have multiple calcium spikes in DVB neurons during each cycle, similar to what is observed in animals expr essing PKA[CA] trans genes . 4.4 Directly examining cAMP levels and PKA activity in the DVB neuron My results suggest that in the DVB neuron, rhyth mic oscillations of cAMP and PKA drive rhyth mic calcium influx during each defecation cycle. It would be very interesting to directly observe the cAMP levels and PKA activity in the DVB neuron in vivo using currently available gene tically-encoded cAMP and PKA indicators, most of which are based on the fluorescence resonance energy transfer (FRET) between YFP and CFP when cAMP binds (Berrera et a!., 2008; Nagai et a!., 2000; Zhang et a!., 200 1). Although some of these indicators have been tested in vivo to examine the cAMP sigualing on regulating circadian rhyth mic behavior (Shafer et a!., 2008), it is not clear whether the dynamics of these indicators is fast enough to reflect the relatively quick change of cAMP concentration in the DVB neuron, since cAMP should oscillate every 50 seconds, as predicted. If these indicators indeed work to reveal the cAMP levels in the DVB neuron, it would be equally important to further determine whether the cAMP or PKA activity oscillations are correlated with the rhythmic calcium influx. 96 I also found that the calcium spikes in the DVB neuron generally start at the synaptic region and propagate along the axon to the cell body, suggesting that cAMP levels and/or PKA activity may be inc reased locally at the synaptic region. Visualization of cAMP and PKA activity with geneti cally-encoded indicators in the DVB neuron in vivo would provide clear answer to this speculation. However, this speculation is not very surprising, as compartmentation of cAMP and PKA signaling has been documented in other systems (Steinberg and Brunton, 200 I; Wong and Scott, 2004). For example, a-kinase anchoring proteins (AKAPs) are a large family of sca ffold proteins that specify the local events of cAMP and PKA signaling. Each of the AKAPs may have different localization signals and different docking sites for many other signaling molecules, including PDEs and phosphat ases, which help to establish local signaling (Wong and Scott, 2004). However, only one gene (aka- 1) that encodes AKAP is identified in the C. elegans genome. In addition, C. elega ns only has a single gene for the PKA catalytic subunit (kin- I) and a single gene for the PKA regulatory subunit (kin-2). How could compartmen talized PKA signaling be achieved in the DVB neuron? Both kin-] and kin-2 have been reported to generate many different isoforms due to alternative splicing (Pastok et a!., 2012; Tabish et a!., 1999). It is possi ble that each of the isoforrns of KIN-I and/or KIN-2 may specifically localize in a particular sub-domain within in the cell, which could help to establish local cAMP and/or PKA signaling in the DVB neuron during defecation cycle. 4.5 What are other calcium channels that mediate PKA-dependent calcium influx in the DVB neuron? 97 I have shown that PKA is absol utely required for the calcium influx in the DVB neuron and that constitutively active PKA is able to trigger ectopic calcium spikes. In addition, I identify two voltage-gated calcium channels (UNC-2 and EGL-19) that partially mediate PKA-dependent calcium influx in the DVB neuron. What are the non-voltage-gated calcium channels that function downstream of PKA in the DVB neuron? One way to identify these channels is to first examine mutants lacking other calcium channel genes for defects in the Exp step and then perform calcium imaging on the DVB neuron in those mutants that have Exp def ects . NCA- 1 and NCA-2 are a1-like subunits of voltage-insensi tive cation leak channels that regulate neuronal activity in C. elegans (Yeh et a!., 2008). However, the null mutants lacking both nea-l and nca-2 have normal Exp steps (H. W. and D.S., unp ublished data), suggesting NCA- 1 and NCA-2 are not likely to regulate calcium influx in the DVB neuron. The C. elegans genome also encodes several transient receptor potential (TRP) chan nels, which have been implicated in olfaction, mechanosenstaion and osmosensation and can be regulated by GPCR sigualing (Kahn-Kirby and Bargmann, 2006). It would be interesting to examine whether mutants that are defective in TRP channels would display abnormal Exp. unc-68, which encodes the ryanodine receptor in C. elegans, does not seem to be a promising candidate, as the unc-68(r 1162 ) mutants have gro ssly normal Exp steps (H.W. and D.S., unpub lished data). Another candidate is itr-1, which encodes the IP3 receptor on endoplasmic reticulum and has been shown to be essential for the generation of calcium oscillations in the intestine (Dal Santo et a!., 1999). However, its function in the DVB neuron on the Exp step has not been determ ined. Direct examination of the loss-of -function itr-1 mutants may not reveal the cell-autonomous contribution of itr-1 on the calcium influx in the 98 DVB neuron, as the defects in the intestinal calcium oscillations in itr-1 mutants may affect the SNT -2-dependent release of NLP-40, which would impact the calcium influx in the DVB neuron in a non-cell-autonomous manner. Thus, it would be valuable to see whether knockdown itr-1 specifically in the GABAergic neurons (for example by tissue-specific hairpin RNAi) would leads to the Exp defects. 4.6 What is the penni ssive signal for the Exp step? It has been suggested that enteric muscles have a refractory period, which prevents ectopic Exp in between cycles (Mahoney et a!., 2008; Wang et a!., 20 13). Activating the AVL and DVB neurons in between cycles either by optogenetics or by NLP-40 P3-l peptide in jection fails to cause out-of-phase Exp step. Consist ently, muscimol, the GABA receptor agonist, cannot cause enteric muscle contractions at random time but only allows enteric muscles to contract within a small time window (from a couple of seconds before pBoc to a few seconds after pBoc, personal communication with Erik Jorgensen). The refractory period of enteric muscles may reflect an intrinsic physiological property of enteric muscles following contraction. After mus cle excitation, potassium channels allow K + to flow out of cell and reduce the membrane potential. One tempting hypothesis to explain the ref ractory period of enteric muscles is that potassium channels may function in enteric muscles to negatively regulate muscle excitability by hyperpolarizing them in between cycle. Consist ently, several potassium channels, including exp-2, egl-2, unc-103 and egl-36 are reported to be expressed in the enteric muscles and the single gain-of -function mutants for each of these potassium channels have defects in the Exp step (Davis 99 et a!., 1999; Elkes et a!., 1997; Reiner et a!., 2006; Weinshenker et a!., 1999). In contrast, the single loss- of-function mutants for these potassium channels display gro ssly normal Exp (Davis et a!., 1999; Johnstone et a!., 1997; Reiner et a!., 1999; Weinshe nker et a!., 1999). This suggests a functional redundancy of enteric muscle potassium channel s. One way to directly test this hypothesis is to determine whether muscimol or activating AVL and DVB neurons by optogenetics would induce enteric muscle contraction at random time m mutants carrymg loss -function alleles of all these potassium channe ls. The refractory period of enteric muscle can explain the observation that enteric muscles could not contract in between cycles, but it imposes a very intriguing question: what is the sigual that removes this refractory period at the right time, so enteric muscles would become responsive to GABA that is released from AVL and DVB neurons during each defecation cycle? One possible mechanism is that the intestine may release a permissive sigual acting on enteric muscles to transiently remove the refectory period and that this sigual is somehow coupled to the calcium oscillations in the intestine. Extensive genetic screens have been performed to identify genes that are esse ntial for the defecation motor program (Thomas, 1990). Although it is possible that these screens might not be saturated, the mutants collected so far do not seem to provide a hint for another permissive sigual from the intestine that directly acts on enteric muscles. Another possibility is that the int rinsic property of the enteric muscles may dictate its ability to contract at a certain time interval, which is approximately 50 seconds. During each defecation cycle, the activation of enteric muscles by GABA from AVL and DVB neurons would lead to the opening of several voltage-gated potassium channels, such as exp-2 and egl-36, which would reploarize 100 enteric muscles. The slow inactivation of these potassium channels would eventually hyperpolarize these muscles in between cycles, and the membrane potential would gradually return to the resting membrane potential in about 50 seconds. Biophysical analys is of the inactivation kinetics of these potassium channels would help to test this possi bility. However, these two hypotheses are not mutually exclusive. It is equally possible that these two mechanisms may both exist and they cooperate to ensure the ability of enteric muscle to contract within a small time window. 4.7 How are the three steps in defecation motor program coordinated? The calcium oscillation in the intestine peaks and initiates the defecation cycle every 50 seconds: the posterior body wall muscles first contract (pBoc ); about 3-4 seconds later, anterior body wall muscles contract ( aBoc) and the enteric mus cle contraction (Exp) follows immediately. How is the relative timing of these three mus cle contractions (pBoc, aBoc and Exp) in each defecation cycle is precisely controlled by the single clock (calcium oscillation) in the intestine? The answer probably lies in the different circuitry structures and different signaling molecules for each of the three mus cle contracti ons. The pBoc step is achieved by a single node circuit between the intestine and posterior body wall muscles and no neurons are required (Beg et a!., 2008). During each defecation cycle, the calcium oscillation originates from the posterior intes tine. It immediately triggers pBoc through the release proton from the intestine into the posterior pseudocoelom; protons then bind 101 to the fast proton-gated ion channels in the posterior body wall muscles to cause contraction (Figure 1.3 and (Beget a!., 2008; Ffeiffer et a!., 2008)). Unlike pBoc, the Exp step is contro lled by two-step sigualing pathway across three different tissues: the intestine first relays the timing information to the GABAergic neurons (AVL and DVB), which in tum control enteric muscle cont raction. The intestine releases the neuropeptide NLP-40 into the pseudocoelom, which activates AVL and DVB through a relatively slower GPCR sigualing cascade, and these two neurons release fast neurotransmitter GABA to cause enteric mus cle contraction (Figure 1.3 and (Beg and Jorgensen, 2003; Wang et a!., 2013)). How do these different circuits and siguals explain the 3-to-4-second delay between pBoc and Exp? Both AVL and DVB are activated to trigger the Exp step (Mcintire et a!., 1993). But unlike DVB, the AVL neuron is located in the anterior part of C. elegans. Thus, NLP-40 released from the intestine needs to diffuse across a relatively long distance within the pseudocoelom to activate AEX-2/GPCR on the AVL neuron. Indeed, I observe a 3.5- second delay between NLP-40 P3-l peptide in jection at the posterior peudoceolom and calcium response in DVB neuron, suggesting that the diffusion may account for most of the delay between pBoc and Exp . It has been reported that the calcium oscillations originating in the posterior intestine will propagate to the anterior intestine in about 3 seconds (at the speed of �340 J11llls) and that this posterior-to-anterior calcium wave is essential for both aBoc and Exp steps (Peters et a!., 2007; Teramoto and Iwasaki, 2006). I speculate that this posterior-to-anterior calcium wave may drive the NLP-40 release from the intestine in a similar manner, which facilitates the diffusion of NLP-40 to the anterior end to activate the A VL neuron. In addition, unlike protons which act as fast transmitters to directly 102 open ion chann els, NLP-40 is a relatively slow transmitter that acts through a GPCR signaling cascade. It has been estimated that it takes about 0.5 seconds for Gas activation following GPCR stimulation (Lohse et a!., 2008). The circuit for aBoc includes three tissues as well: the intestine signals to the A VL neuron, which triggers the contraction of anterior body wall muscles. However, the signals from the intestine and the AVL neuron for the aBoc step are still unknown (Fignre 1.3). Although aBoc and Exp are contro lled by similar but distinct circuits, genetic evidence suggests that some signals for the aBoc and Exp steps might be shared (Mahoney et a!., 2008; Mcintire et a!., 1993; Thomas, 1990). The genetic screen by Thomas identified a class of mutants (aex) which displayed defects in both aBoc and Exp, but not pBoc (Thomas, 1990). I find that both nlp-40 mutants and snt-2 mutants showed mild defects in aBoc frequency and timing (Figure S2.2 and data not shown), similar to those observed in ae.x-2 mutants (Mahoney et a!., 2008; Thomas, 1990), suggesting that in addition to mediating the Exp step, the NLP-40-AEX-2 signaling may also be involved in the regulation of the aBoc step. However, GAB A, the neurotransmitter from AVL and DVB neurons for the Exp step, is not required for the aBoc step, as unc-25 mutants have normal aBoc (Mahoney et a!., 2008; Mcintire et a!., 1993). Nonet heless, the relatively similar timing of aBoc and Exp during the defecation motor program could be attributed to the similar two-node circuits and similar signal molecules. More experiments are needed to identify the genes for the aBoc step. It is interesting to note that mutants lacking UNC-3 1/CAPS, an essential protein for dense core vesicle exocytosis, appears to have the aBoc step in only about 50% of cycles, suggesting that intestine may secrete another peptide to control aBoc. In addition, the order of aBoc and Exp is 103 reversed in about 75% of the cycles in unc-31 mutants where both aBoc and Exp are present (Speese et a!., 2007). Further analysis of unc-31 mutants may help to shed light on how the aBoc step is cont rolled. 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Zhao, Y., Araki, S., Wu, J, Teramoto, T., Chang, Y.F., Nakano, M., Abdelf attah, AS., Fuj iwara, M., Ishihara, T., Nagai, T., et al. (2011). An expanded palette of genetically encoded Ca(2)(+) indicators. Science 333, 1888-1891. 115 Appendix Al Publications 1. Wang H, Sieburth D. (20 13) PKA controls calcium influx into motor neurons during a rhythmic behavior. PLoS Genetics, in press. 2. Chan JP, Staab TA, Wang H, Mazzasette C, Butte Z, Sieburth D. (2013) Extrasynaptic Muscarinic Acety lcholine Receptors on Neuronal Cell Bodies Regulate Presynaptic Function in Caenorhabditis elegans. Journal of Neuroscience, 33( 35):141 46-14159. 3. Wang H, Girskis K, Janssen T, Chan JP, Dasgupta K, Knowles JA, Scho ofs L, Sieburth D. (2013) Neuropeptide secreted from a pacemaker activates neurons to control a rhyth mic behavior. Current Biology 23(9):746-754 (Highlighted by: Zhao B, Sch afer WR. (20 13) Neuropeptide Signa ling: From the Gut. Current Biology 23(11): R481-R483) The chapter 2 in this dissertation is a reprint of the publication 3; the chapter 3 in this dissertation is a reprint of the publication 1. 116 A2 Legends of videos # Video S2.1 Real time calcium imaging in the DVB neuron of wild type. unc-13(s69);v ;Js58 was used, since unc-13(s69) ahnost paralyzed but still have normal Exp steps. One represen tative defecation cycle was shown. A calcium spike in the synaptic region of the DVB neuron was observed immediately before the Exp step. Video S2.2 Real time calcium imaging in the DVB neuron of nl p-40( tm4085 ) mutants. nlp-40( tm4085)unc-13(s69);v;Js58 was used. One representative defecation cycle was shown. No calcium spikes in the synaptic region of the DVB neuron were observed. Video S2.3 Real time calcium imaging in the DVB neuron of nlp-40( tm40 85) mutants with nl p-40 eDNA specifically expressed in the intestine. v;Ex368;nlp-40(tm4085)unc-13(s69);v ;Js 58 was used. One representative defecation cycle was shown. A calcium spike in the synaptic region of the DVB neuron was observed immediately before the Exp step. Video S2.4 Real time calcium imaging in the DVB neuron of aex-2( sa3) mutants. unc-13(s69);v ;Js 58;aex-2(sa3) was used. One representative defecation cycle was shown. No 117 calcium spikes iu the synaptic region of the DVB neuron were observed. Video S2.5 Real time calcium imaging in the DVB neuron of glued nlp-40( tm40 85) mutants during P3-1 peptide injection. nlp-40(tm4085);v ;Js58 was in jected with lOf!M NLP-40 P3-l peptide. A siugle calcium spike iu cell body of DVB was observed about 4 seconds after in jection. Video S2.6 Real time calcium imaging in the DVB neuron of glued nlp-40( tm40 85) mutants during Pl peptide injection. nlp-40(tm4085);v ;Js58 was iu jected with lOf!M NLP-40 Pl peptide. No calcium spike iu cell body of the DVB neuron was observed after iu jection. Video S2.7 Real time calcium imaging in the DVB neuron of glued nlp-40( tm40 85 ); aex-2( sa3) mutants during P3-1 peptide injection. nlp-40(tm4085);v ;Js 58;aex-2(sa3) was in jected with lOf!M NLP-40 P3-l peptide. No calcium spike in cell body of DVB was observed after iu jection. Video S3. 1. Real time calcium imaging in the DVB neuron of wild type. The straiu unc-13(s69); v;Js58 was used as wild type, since unc-13(s69) mutants are almost paralyzed but still have normal Exp steps. v;Js58 is an integrated trans gene iu which GCaMP3 is exp ressed in DVB neu rons. One representative defecation cycle is shown. A calcium spike in the synaptic region of the DVB neuron was observed immediately before the Exp step. Video S3.2. Real time calcium imaging in the DVB neuron of aex-2 mutants. unc-13(s69); v;Js 58; aex-2(sa3) was used. One represen tative defecation cycle is shown. No calcium spike in the synaptic region of the DVB neuron and no Exp were observed following the pBoc. 118 Video S3.3. Real time calcium imaging in the DVB neuron of unc-25 mutants. The strain unc-13(s69); unc-25(el56); v;Js 58 was used. One representative defecation cycle is shown. A calcium spike in the synaptic region of the DVB neuron, but no Exp was observed following the pBoc. Video S3.4. Real time calcium imaging in the DVB neuron of PKA[DN] transgenic animals. The strain unc-13(s69); v;Js58; v;Js 76 was used. v;Js 76 is an integrated trans gene with PKA[DN] specifically expressed in GABAergic neu rons. One representative defecation cycle is shown. No calcium spike in the synaptic region of the DVB neuron and no Exp were observed following the pBoc. Video S3.5. Regular calcium spike in the DVB neuron of PKA[CA] transgenic animals. The strain unc-13(s69); v;Js58; v;Jsl02 was used. v;Jsl02 is an integrated tran sgene with PKA[CA] specifically exp ressed in GABAergic neu rons. One representative defecation cycle is shown. A calcium spike in the synaptic region of the DVB neuron was observed immediately before the Exp step. The duration of the calcium influx was increased. Video S3.6. Ectopic calcium spike in DVB neuron of PKA[CA] transgenic animals. The strain unc-13(s69); v;Js58; v;Jsl02 was used. v;Jsl02 is an integrated tran sgene with PKA[CA] specifically expressed in GABAergic neu rons. One representative ectopic calcium spike is shown. An ectopic calcium spike in the synaptic region of the DVB neuron was observed in between cycles. Generally, the ectopic calcium spikes did not produce ectopic Exp. The duration of the 119 calcium influx was increased. Video S3.7. Real time calcium imaging in the DVB neuron of unc-2 mutants in normal cycles. The strain unc-13(s69); v;Js58; unc-2( /jl) was used. One representative normal def ecation cycle is shown. A calcium spike in the synaptic region of the DVB neuron was observed immediately before the Exp step. Video S3.8. Real time calcium imaging in the DVB neuron of unc-2 mutants in incomplete cycles. The strain unc-13(s69); v;Js 58; unc-2( /j1) was used. One represen tative incomplete defecation cycle is shown. A weak calcium spike in the synaptic region of the DVB neuron, but no Exp was observed. Video S3.9. Real time calcium imaging in the DVB neuron of egl -19; unc-2 mutants in incomplete cycles. The strain v;Js64; egl-19( n582); unc-2(/j1) was used. v;Js64 is an integrated transgene with GCaMP3 expres sed in DVB neu rons. One representative incomplete defecation cycle is shown. A weak calcium spike in the synaptic region of the DVB neuron but no Exp was observed following pBoc. Video S3.10. Real time calcium imaging in the DVB neuron of egl-19; unc-2 mutants in incomplete cycles. The strain v;Js64; egl-19(n5 82); unc-2(/j1) was used. One representative incomplete defecation cycle without calcium spike is shown. No calcium spike in the synaptic region of the DVB neuron and no Exp were observed following pBoc. Video S3.11. Regular calcium spike in the DVB neuron of PKA[CA]; egl -19;unc-2 animals. 120 The strain v;Js 64; egl-19( n582); v;Jsl02; unc-2( /jl ) was used. v;Jsl02 is an integrated tran sgene with PKA[CA] specifically expr essed in GABAergic neu rons. One representative incomplete defecation cycle was shown. A calcium spike in the synaptic region of the DVB neuron but no Exp was observed following pBoc. The duration of the calcium spike inc reased. Video S3.12. Ectopic calcium spike in the DVB neuron of PKA [CA]; egl -19;unc-2 animals. The strain v;Js 64; egl-19( n582); v;Jsl02; unc-2( 1]1) was used. v;Jsl02 is an integrated tran sgene with PKA[CA] specifically expressed in GABAergic neu rons. One representative ectopic calcium spike was shown. An ectopic calcium spike in the synaptic region of the DVB neuron was observed in between cycles. Generally, the ectopic calcium spikes did not produce ectopic Exp. The duration of the calcium spike inc reased. # These videos are in the separate digital files accompanying this dissertati on. They can also be found in the supplemental files in my two papers (publication 1 and publication 3 in Appendix Al, Page 116). 121
Abstract (if available)
Abstract
Rhythmic behaviors are those behaviors that occur at regular timing intervals and they are widely observed in animal kingdom. The time intervals are determined by pacemakers and the rhythmic behavioral outputs are generally performed by different tissues, including downstream neurons and muscles. However, how the timing information from the pacemaker is delivered to the downstream effectors remains unclear. ❧ To address this question, I study a simple rhythmic behavior in C. elegans: the enteric muscle contraction (Exp) in the defecation motor program. The pacemaker for the defecation motor program is the intestine and the timing is encoded by intestinal calcium oscillations, which drive rhythmical enteric muscle contraction about every 50 seconds. Using an in vivo calcium imaging approach, I find that the downstream GABAergic neurons (AVL and DVB) that innervate enteric muscles undergo rhythmic activation that happens immediately before Exp during each defecation cycle, suggesting the timing information from the intestine is transmitted step by step within the circuit controlling enteric muscle contraction. ❧ It has been hypothesized that an unknown neuropeptide signal may be released from the intestine to activate the downstream GABAergic neurons to cause rhythmic enteric muscle contraction. By combing forward genetic screens and whole genome sequencing, I identify a neuropeptide-like protein (NLP-40) as the timing signal from the intestine. First, nlp-40 is essential for enteric muscle contraction. Second, nlp-40 is exclusively expressed in the intestine. Third, NLP-40 is released from the intestine, which is mediated by SNT-2/synaptotagmin, the putative calcium sensor in the dense core vesicles (DCVs), suggesting calcium oscillations in the intestine may trigger rhythmic release of NLP-40. Fourth, using optogenetics and in vivo calcium imaging, I show that NLP-40 does not impact the integrity or development of the circuit that controls enteric muscle contraction. Instead, NLP-40 is both necessary and sufficient for the rhythmic calcium influx in the downstream GABAergic neurons. Fifth, I further demonstrate that NLP-40 triggers calcium influx in the GABAergic neurons by activating its receptor AEX/GPCR. ❧ I further delineate the molecular mechanism by which the NLP-40 signaling controls the activation of the GABAergic neurons. Previous studies show that NLP-40 signaling is dependent on cAMP. Here, I develop a genetic technique to either increase or inhibit PKA activity in a tissue specific manner in C. elegans. I demonstrate that PKA is the major target of cAMP in the NLP-40 signaling pathway in the GABAergic neurons to regulate calcium influx. Furthermore, I identify two voltage-gated calcium channels (VGCCs), UNC-2 and EGL-19 that partially mediate PKA-dependent calcium influx in the GABAergic neurons. ❧ In conclusion, I present evidence to uncover a mechanism by which neuropeptides could function as timing messengers to couple pacemakers to downstream neurons to coordinate the proper execution of rhythmic behaviors. Neuropeptides may encode timing information via synaptotagmin-dependent rhythmic release from pacemakers and they activate downstream neurons through binding to their receptors (GPCRs) which leads to PKA signaling cascade to trigger the calcium influx.
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Defining the circuits and mechanisms mediating a pacemaker-controlled behavior in C. elegans
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inx-1 is a negative regulator of the expulsion step of the defecation motor program in C. elegans
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Wang, Han
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Genetic and molecular analysis of a rhythmic behavior in C. elegans: how neuropeptide signaling conveys temporal information
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Keck School of Medicine
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Doctor of Philosophy
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Genetic, Molecular and Cellular Biology
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10/11/2013
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C. elegans,calcium imaging,cAMP-dependent protein kinase(PKA),cyclic adenosine monophosphate (cAMP),defecation motor program,G protein-coupled receptor (GPCR),GABAergic neurons,neuropeptide,OAI-PMH Harvest,rhythmic behavior,synaptotagmin,voltage-gated calcium channel (VGCC)
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C. elegans
calcium imaging
cAMP-dependent protein kinase(PKA)
cyclic adenosine monophosphate (cAMP)
defecation motor program
G protein-coupled receptor (GPCR)
GABAergic neurons
neuropeptide
rhythmic behavior
synaptotagmin
voltage-gated calcium channel (VGCC)