Rauthan, Manish et al. (2009) International Worm Meeting "Role of neurotransmitters and neuropeptides in C. elegans nicotine dependent behavior."

Feng, Zhaoyang, Rauthan, Manish, Xu, X. Z. Shawn

[International Worm Meeting, 2009]

Nicotine is the main addictive drug in tobacco. In mammals, nicotine exerts its effects by binding to nicotinic acetylcholine receptors (nAChRs), which are ligand-gated ion channels and allow both mono- and divalent cations to pass through them. Nicotine acts by increasing the dopamine levels in mesolimbic dopamine system, thereby modulating the brain reward centre. Besides dopamine, other neurotrasmitters such as norepinephrine, serotonin and glutamate are involved in different aspects of nicotine dependence. Previous studies in our laboratory established C. elegans as a genetic model for the study of nicotine dependence (Feng, Z. et al. Cell 2006;127(3):621-33). Nicotine elicits various behavior responses in C. elegans such as acute response, adaptation, withdrawal and sensitization. We have found that neuropeptides are involved in nicotine-dependent behavior in worms. We have also found that various neurotransmitters such as dopamine, octopamine and tyramine are involved in modulating nicotine-dependent behavior in worms. In addition, we are identifying novel genes involved in regulating nicotine responses.

Zhaoyang Feng et al. (2003) International Worm Meeting "C. elegans, a genetically tractable model for the study of nicotinic signaling"

Anthony Kempf, William Schafer, Zhaoyang Feng, Marika Orlov

[International Worm Meeting, 2003]

Nicotine abuse is the most critical but preventable public health problem. Although nicotinic acetylcholine receptor (nAChR) is identified as the main target of nicotinic signaling, the molecular and neuronal circuits of nicotinic signaling remain unknown. Using invertebrate models such as c. elegans or Drosophila metanogaster as animal models to study drug abuse has recently attracted the attention of researchers because of their simple nervous system, and the available powerful genetic tools in these models [Schafer WR, J. Neurosciences, 2002, Wolf FW and Heberlein U, Cur. Opp. in Neuroscience, 2003]. Utilizing a quantitative c. elegans behavior analysis system (Feng Z. et al, abstract in this meeting), a c. elegans locomotory acclimation behavior is identified and defined. Using this locomotory acclimation behavior as a behavioral assay, the c. elegans animals were found to response acutely to nicotine at a wild concentration range including concentrations close to physiological concentration in human blood after a cigarette consummation. This locomotory acclimation behavior is also found can be used in the study of nicotine adaptation, withdrawal, and possibly sensitization. The distinct roles of several nicotinic receptor subunits (unc-29, unc-38, and unc-63), Go and Gq signaling circuits, and several neuronal transmitters (dopamine, serotonin, glutamate) in the locomotory acclimation behavior, and their functions in nicotinic signaling are discussed.

Rauthan, M. et al. (2017) International Worm Meeting "MicroRNA regulation of nicotine-dependent behavior in C. elegans."

Rauthan, M., Ronan, E.A., Gong, J., Xu, X.Z.S., Liu, J., Wescott, S.

[International Worm Meeting, 2017]

Tobacco smoking is the leading cause of preventable death in developed nations. Nicotine is the principle addictive substance in cigarettes. Chronic exposure to nicotine up-regulates nAChRs and is thought to play a critical role in the primary steps of nicotine dependence, but the underlying mechanisms are not well understood. We have previously developed a C. elegans model of nicotine dependent behavior, and shown that the nAChR gene acr-15 is required for acute response to nicotine (Feng et al., 2006). Here we identify a key role for microRNA in regulating nicotine-dependent behavior by modulating nAChR expression in C. elegans. Specifically, we show that chronic nicotine treatment down-regulates microRNA machinery, leading to up-regulation of another nAChR gene that is specifically required for nicotine withdrawal behavior. This effect is mediated by a microRNA that recognizes the 3'UTR of nAChR transcripts. These observations uncover an interesting phenomenon that different nAChRs mediate distinct aspects of nicotine dependence in C. elegans. Our results reveal a functional link between nicotine, microRNA, nAChRs, and nicotine-dependent behavior. Reference(s): 1. Feng, Z., Li, W., Ward, A., Piggott, B.J., Larkspur, E., Sternberg, P.W., and Xu, X.Z.S. (2006). A C. elegans model of nicotine-dependent behavior: regulation by TRP family channels. Cell 127, 621-633.

Ward, Alex et al. (2009) International Worm Meeting "Eyeless but not blind: Phototransduction in C. elegans."

Liu, Jie, Kang, Lijun, Decaluwe, Brandon, Gao, Jingwei, Xie, Zhixiong, Ma, Di, Ward, Alex, Yu, Yong, Inada, Hitoshi, Mori, Ikue, Xu, X.Z. Shawn, Nishio, Nana

[International Worm Meeting, 2009]

It has long been assumed that the nematode C. elegans lacks the sense of light, mainly because it lives in the soil and does not have eyes. However, we have recently reported the surprising observation that C. elegans in fact possesses a simple visual system and engages in phototaxis behavior that is mediated by photoreceptor cells and light-sensitive channels [1]. Here we elucidate the phototransduction cascade in C. elegans photoreceptor cells through a combination of electrophysiological and behavioral analysis. As is the case with vertebrate photoreceptor cell rods and cones, C. elegans phototransduction is also mediated by G signaling and cGMP-sensitive CNG channels. Interestingly, instead of signaling through phosphodiesterases (PDEs), light-activated G proteins appear to be coupled to guanylate cyclases that produce cGMP, thereby resulting in opening of CNG channels. Our studies identify a new sensory modality in C. elegans and suggest that animals living in dark environments (e.g. soil and caves) may not be presumed to be blind. Our data also reveal a surprising conservation in phototransduction between vertebrates and C. elegans, indicating that C. elegans represents a powerful genetic model for the study of phototransduction. [1] Ward, A.*, Liu, J.*, Feng, Z., and Xu, X.Z.S. (2008) Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nature Neuroscience 11, 916-22 *co-first authors.

Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

<div id="i4c-draggable-container" style="position: fixed; z-index: 1499; width: 0px; height: 0px;">

Zhaoyang Feng et al. (2007) International Worm Meeting "The regulatory roles of glutamate, dopamine and serotonin in nicotine dependence in C. elegans."

Zhaoyang Feng, Shawn X.Z. Xu, Li Li

[International Worm Meeting, 2007]

Nicotine dependence is a major health problem in the U.S. The primary molecular target of nicotine is nicotinic acetylcholine receptors (nAChRs). In mammals, nicotine binds to nAChRs in the ventral tegmental area which leads to the stimulation of mesolimbic dopamine system. Nicotine also modulates the expression and functionality of nAChRs. Nevertheless, the genetic networks underlying nicotine dependence remain to be elucidated C. elegans has been demonstrated as a genetic model for many human disorders/diseases. In a recent report (Feng, Z. et. al. Cell. 2006;127(3):621-33), we have established C. elegans as a genetic model for the study of nicotine dependence. We have found that the behavioral responses to nicotine in worms parallel those observed in mammals and the genes and pathways regulating nicotine dependence are conserved between worms and mammals. For example, TRPC (transient receptor potential classic), a conserved gene family that we have reported as novel regulators for nicotine dependence in worms has recently been suggested to be important for nicotine addiction in humans (Bierut L.J. et. al. Hum. Mol. Genet. 2007;16(1):24-35). Here we examined the possible roles of neurotransmissions in nicotine dependence and found glutamate, serotonin and dopamine but not tyramine and octopamine neurotransmissions are important for nicotine dependence in worms. These observations consist with the regulatory roles of mammalian glutamate, dopamine and serotonin neurotransmissions in nicotine dependence, and further highlight the value of using C. elegans as a model for the study of dependence to addictive substances, such as nicotine.

Zhaoyang Feng et al. (2003) International Worm Meeting "A relational database powered quantitative c. elegans behavioral analysis system and its possible application in systematic analysis of nicotinic signaling"

Zhaoyang Feng, William Schafer, Megan Palm, John Wittig

[International Worm Meeting, 2003]

C. elegans is particularly suitable for genetic analysis of the molecular basis of behavior because it has a simple nervous system of 302 neurons, and the synaptic connectivity, the cell line of each neuron are precisely known. Unfortunately, c. elegans behavior is one of most intractable biological phenomena: behavior changes from time to time; the behavioral differences among variants are subtle; and human observation is subjective and labor intensive. A quantitative c. elegans behavioral analysis system powered by relational database is developed in this study. Features extrapolated from machine vision analysis of c. elegans behavioral video are exported as two parts in database: the time to time behavioral data are saved in a widely available commercial spreadsheet format (Microsoft excel); and the statistical summary of time to time data is saved in a relational database. The features fall into three major catalogs: behavior (speed, reversal, turn angle, neck movement, etc), body posture (wave amplitude, wave length, etc), and morphology (body size, body length, etc). The outputted spreadsheet data is suitable for detail dissecting of several c. elegans behaviors including state-switch of locomotion. The system has been utilized in a study of the function of dopaminergic neurons in taping response, and a study of nicotinic signaling (Feng Z. et al, abstract in this meeting). Given the power of relational database, systematic analysis of c. elegans behavior is also possible. To test this possibility, the neural network feature selection, and principle component analysis (PCA) are utilized to study the effect of nicotine on N2 c. elegans, and several genetically engineered mutants. The features exported to database, and the systematic study results will be presented and discussed.

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Gilleard JS et al. (1995) International C. elegans Meeting "REGULATION OF THE CUTICULAR COLLAGEN GENE dpy-7"

Barry JD, Gilleard JS, Johnstone IL

[International C. elegans Meeting, 1995]

We are interested in identifying trans acting factors which control cuticular collagen gene expression. Sequences sufficient for regulated expression of dpy-7 appear to reside relatively close to the initiator ATG since the wild type gene only requires 310bp of 5' flanking sequence for transformation repair of phenotype. We have analysed sequences necessary and sufficient for dpy-7 expression using lac-Z fusions and we have identified a 95bp fragment, encompassing the transcritional start sites, which is sufficient to produce lac-Z expression predominantly, or possibly exclusively, in hypodermal cells. This fragment includes a region in which 65 out of 72bp are conserved in the 5' flank of the C.briggsae dpy-7 homolog and interspecies transformation rescue and lac-Z fusion experiments have shown that the dpy-7 cis regulatory elements are functionally conserved. A 15bp deletion at the 5' end of this homology which removes a AGATAA motif, also present in C.briggsae, completely ablates lac-Z expression . We are attempting to identify transcription factors which bind to this motif by South Western expression library screening. We are also searching for new C.elegans GATA factor homologs with a particular interest in any which are expressed in hypodermal cells postembryonically.

Denis V Touroutine et al. (2007) International Worm Meeting "Genetic screens for mutants with altered ACR-16 nicotinic receptor localization and function."

Janet E Richmond, Joshua Jonson, Denis V Touroutine

[International Worm Meeting, 2007]

Individual C. elegans body wall muscles express two pharmacologically distinct nicotinic receptors: a levamisole-sensitive and a levamisole-insensitive receptor class. We have identified ACR-16 as an essential subunit (Touroutine et.al. JBC) of the levamisole-insensitive receptor. This receptor contributes to synaptic transmission at the C. elegans neuromuscular junction (NMJ) and also functions in neurons to mediate nicotine-dependent behavior (Feng et al., Cell). Several components required to traffic or localize the levamisole-sensitive receptor have been identified. However, molecules required to target the ACR-16 receptor to post-synaptic sites remain to be identified, in part due to the lack of a screenable phenotype in acr-16 mutants. We developed two strategies to identify those molecules. 1. We conducted a genetic screen to identify mutants with altered ACR-16::GFP expression. 2. We generated a strain expressing an ACR-16 gain-of-function receptor which desensitizes slowly and causes lethality when expressed in worm muscles. However, worms grown on plates with RNAi against acr-16 survive and are healthy. This allows as to mutagenise acr-16 RNAi-treated worms and screen for suppressors of the lethal phenotype on OP50 plates. Utilizing the first strategy and after screening 2400 haploid genomes, we identified several candidates in which ACR-16::GFP expression is altered. Among these, we found a new allele of ric-3, a gene that is required for both levamisome-receptor and ACR-16 receptor function (Halevi et. al). Another mutant identified in this screen exhibits a dramatic reduction (only 5% of the signal remains) in the ACR-16::GFP puncta that localize adjacent to UNC-17-positive puncta in the ventral nerve cord. Double mutants of unc-63 and this mutant exhibit decreased thrashing rates (77% less) when compared to unc-63 mutants alone, suggesting they have reduced functional ACR-16 receptors at the NMJ. Mutants isolated from both screens will be mapped and characterized. Touroutine et. al JBC 2005 Jul 22;280(29):27013-21. Feng Z et. al. Cell 127:621-633. Halevi et.al EMBO J, 2002 Mar 1;21(5):1012-20.