Alves, Carla et al. (2021) International Worm Meeting "The effects of venlafaxine on behavior and central nervous system of Caenorhabditis elegans"

Vanin, Ana Paula, dos Santos, Amanda, Sutorillo, Nathalia Tafarel, Barcellos, Leonardo Jose Gill, Santini, Wallace, Alves, Carla, Tamagno, Wagner Antonio, Kaizer Perin, Rosilene Rodrigues

[International Worm Meeting, 2021]

The growing consumption of psychoactive drugs, including antidepressants and antipsychotics, along with the low levels of total metabolization of these drugs, are increasing their residues in the environment. After being used, the drugs are metabolized and excreted, sometimes still in their active form, and may or may not suffer degradation when in contact with the environment. Thus, these psychoactive drugs can reach the aquatic environment and affect non-target organisms, such as zebrafish (Danio rerio) and Caenorhabditis elegans, in their natural environments. Our main hypothesis is that venlafaxine (VEN), a drug that works with the selective inhibition of the reuptake of noradrenaline and serotonin, can exert effects on the endocrine and behavioral system of zebrafish and toxicological effects on the behavior and nervous system of C. elegans. The integrity of the natural behavioral repertoire is vital for the maintenance of the species, reinforcing the importance of studies that show the potential of drugs in altering these models. Here we evaluate acute exposure to VEN, in three concentrations followed by a behavioral assessment. The behavior of C. elegans was analyzed by the body bands, pharyngeal beat, and defecation cycle; while the biochemical analyzes of the enzyme Acetylcholinesterase to assess the toxicological effects of VEN on the nervous system. We have shown that the nematode C. elegans resists changes in behavior and the nervous system. Thus, this model response was not a clear indicator of water contamination and possible zebrafish effects, probably because of the cuticle and natural behavior of the nematode. Results with zebrafish exposure to VEN have shown changes in the novel tank test, mainly the robust anxiolytic-like pattern, suggesting that VEN may be more dangerous to species conservation, since fish presenting an anxiolytic-like behavior becomes more susceptible to predation. We are working on biomarkers to study differences in the responses to these models when exposed at VEN.

Tamagno, Wagner Antoino et al. (2021) International Worm Meeting "Protective and reparative effect of dragon fruit upon central nervous system toxicity induced by copper"

Tamagno, Wagner Antoino, Sutorillo, Nathalia Tafarel, Alves, Carla, dos Santos, Amanda, Kaizer, Rosilene Rodrigues, Barcellos, Leonardo Jose Gill, Santini, Wallace, Vanin, Ana Paula

[International Worm Meeting, 2021]

Copper is an essential metal and is important in general metabolism. However, in high concentrations, it becomes toxic to the organism. The metal-induced toxicity is linked to many neurodegenerative diseases like Parkinson's, Alzheimer's, multiple sclerosis, occurring in senile aging. To prevent neurodegenerative diseases, some studies are being developed to find ways of healthy aging. Natural compounds and diets based on fruits are increasing. Dragon fruit (Hylocereus undatus) is a tropical and Latin American fruit that is gaining more popularity due to its antioxidant properties. This fruit still has low popularity, since its planting and large-scale commercialization are recent. Here we evaluate the protective and reparative effect of different doses of dragon fruit's microencapsulated pulp extract on copper-induced toxicity. The nematode Caenorhabditis elegans was used as a model, to research the effects of pitaya extract on the Cholinergic nervous system, behavior, lipidic peroxidation, and chaperon system. We observed that Cu severally increased directly the cholinesterase, chaperone, and peroxidation rates. When pitaya is applied, even to prevent or remediate, all those enzymes' rates are normalized again. Overall, the results have shown that the pulp fruit extract can be used on dietary supplementation to prevent and repair neural damage. The extract was able to regulates the chaperone system to reduce expression of heat shock protein (16.2) and reestablished the AChE levels avoiding lipidic peroxidation. Changes in the behavior, decreasing cell death biomarkers, and lipidic peroxidation caused by copper toxicity are showing, and, based on these results, we concluded that the pitaya fruit has an important role in gerontological issues.

Benedetti MG et al. (2008) Exp Gerontol "Compounds that confer thermal stress resistance and extended lifespan."

Benedetti MG, Lithgow GJ, White MP, Sampayo JN, Foster AL, Vantipalli MC, Gill MS, Olsen A

[Exp Gerontol, 2008]

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders [Collins, J.J., Evason, K., Kornfeld, K., 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp. Gerontol.; Floyd, R.A., 2006. Nitrones as therapeutics in age-related diseases. Aging Cell 5, 51-57; Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 23-30; Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 3-8]. Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.

Gallotta I et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

D'Orta M, Vitale M, Castro S, Esposito G, Hilliard M A, Di Schiavi E, Gallotta I

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

An important aspect in the study of gene function is the possibility to dissect the role exerted by a gene of interest selectively in specific cells or groups of cells. In C. elegans the available approaches are time consuming and largely restricted to non-essential genes for which mutants are available. We have developed a simple reverse genetics approach to reduce the function of specific genes in chosen C. elegans neurons (Esposito et al., 2007). By expressing sense and antisense RNA corresponding to a gene of interest under cell-specific promoters, we obtain an efficient, heritable and cell autonomous knock-down of the targeted gene function in the specific neurons. This strategy has now been used by several other labs, with a variety of genes and on different neurons (Harris et al. 2009; Yamada et al. 2009; Jose et al. 2009; Ezak et al. 2010). To optimize the efficiency of this approach we have tested the influence of genetic backgrounds that affect RNA interference. We show that in a rrf-3(pk1426) background silencing is more efficient. Although this result indicates the involvement of RNAi pathways in the silencing mechanism we have shown that there is no spreading of the silencing effect at least among amphidial chemosensory neurons and within the motorneurons of the ventral cord. We are now targeting chemosensory and mechanosensory neurons and two classes of motorneurons (GABAergic and HSN) using a variety of different promoters. We are testing the effect of silencing several essential genes on the development, morphology and survival of these neurons as well as on the behavior that they control.

Butler RA et al. (2001) Gene "An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding."

Hahn ME, Van Beneden RJ, Powell WH, Butler RA, Kelley ML

[Gene, 2001]

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

Collins, Kevin M. et al. (2013) International Worm Meeting "Understanding how neurotransmitter signaling drives two-state activity of the C. elegans egg-laying behavior circuit."

Collins, Kevin M., Koelle, Michael R.

[International Worm Meeting, 2013]

We are interested in understanding how neurotransmitter signaling allows a neural circuit to execute distinct behavior states. C. elegans regulates egg laying by alternating between an inactive phase and a ~3 minute "active" state during which clusters of 3-6 eggs are laid. Six VC motor neurons release acetylcholine to excite the vulval muscles, and two HSN motor neurons release serotonin which signals through vulval muscle receptors to promote the active phase. Four uv1 cells release tyramine to inhibit egg laying. We are using GCaMP calcium imaging in behaving animals to understand how the signaling events in the circuit produce its two state behavior. We recently reported that the vulval muscles are rhythmically excited during each locomotor body bend (1). We found the UNC-103 K+ channel depresses muscle excitability below the threshold that drives calcium transients and contraction during the inactive phase, while still allowing signals above threshold during the active phase. To understand how the HSN, VC, and uv1 neurons regulate vulval muscle excitability, we are manipulating neurotransmitter release from these cells and using GCaMP to record changes in neuron and vulval muscle activity. Activation of serotonin release from the HSNs using Channelrhodopsin induces rhythmic vulval muscle twitching and egg-laying behavior-hallmarks of the active phase. We find that VC neuron activity increases during the active phase, and preliminary results show that egg-laying events mechanically distort the uv1 cells and trigger calcium transients. We have previously shown that TRPV channels, which can be mechanically gated, are required for uv1 to inhibit egg laying (2). We propose that successive egg-laying events increase uv1 calcium signaling, promoting release of tyramine and neuropeptides which terminate the active phase of egg laying. (1) Collins and Koelle (2013). J. Neurosci. 33, 761-775. (2) Jose et al. (2007). Genetics 175, 93-105.

Alkema M et al. (1998) East Coast Worm Meeting "Determining the roles of octopamine and CREB"

Alkema M, Horvitz HR

[East Coast Worm Meeting, 1998]

Biogenic amines play an important role in synaptic plasticity and long-term memory formation. One of the best studied examples is the sensitization of the gill-withdrawal reflex in Aplysia: repeated stimulation of the cAMP pathway by serotonin leads to the activation of the cAMP-responsive element binding protein (CREB)1. CREB is required for long-term facilitation of the gill-withdrawal reflex and is thought to be important in the induction of genes involved in the restructuring of synaptic connections, one characteristic of long-term changes in behavior. Studies in Drosophila and mice support the crucial role of biogenic amines, the cAMP pathway and CREB in behavioral plasticity and long-term memory formation2. To explore processes that might underlie behavioral plasticity in the worm, we are analyzing the roles of CREB and the biogenic amine octopamine in C. elegans. Octopamine has been identified in C. elegans extracts but has not been localized to specific cells. Exogenous octopamine inhibits egg-laying3, pharyngeal pumping3 and defecation4, supporting its role as a neuromodulator. Octopamine biosynthesis requires a tyramine b-hydroxylase (TBH) activity to convert tyramine to octopamine. We characterized a putative C. elegans tyramine b-hydroxylase gene (tbh-1). The tbh-1 gene encodes a 586 amino acid protein that has 30% identity with both Drosophila TBH and the closely related mammalian dopamine b-hydroxylases (DBH). To atempt to manipulate the levels of octopamine genetically, we identified a deletion of the tbh-1 gene using a PCR screen of a chemical deletion library. tbh-1 mutants are viable, and preliminary analysis indicates that they have no obvious abnormalities in brood size, egg-laying, locomotion, mechanosensation or dauer formation. TBH-1 is localized to the cell bodies and neuronal processes of the two AIY interneurons, suggesting that the AIY neurons use octopamine as a neurotransmitter. The AIY interneurons are post-synaptic to the AFD thermosensory neurons and the chemosensory neurons, AWA, AWC and ASE5. Cell killing experiments have shown that the AIY interneurons are essential for normal thermotaxis3. AIY axon morphology does not appear to be affected in tbh-1 mutant animals, and tbh-1 mutants display normal thermotaxis. We are further analyzing the behavior of thb-1 mutantsto determine if AIY function is normal. We have cloned a C. elegans CREB gene using the EST yk217f1 as a probe. The full-length CREB cDNA predicts a protein of 360 amino acids. The similarity of this worm CREB to mammalian and Drosophila CREB family members is particularly striking in the predicted bZIP and cAMP-dependent kinase domains. The C. elegans CREB gene has alternatively spliced isoforms, and on northern blots its expression is predominantly detected in embryos and L1s. We will attempt to isolate deletion mutants of this gene and generate transgenic lines that express different isoforms under the control of heat-shock promoters. Analysis of such CREB mutant strains may allow us to determine whether CREB plays a role in behavioral plasticity in the worm. 1) Reviewed by Bailey et al. (1996). Proc. Natl. Acad. Sci. 93, 13445-13452. 2) Reviewed by Yin and Tully (1996). Curr. Op. Neurobiol. 6, 264-268. 3) Horvitz et al. (1982). Science 216, 1012-1014. 4) Weinshenker et al. (1995). J. Neurosci. 15, 6975-6985. 5) Reviewed by Bargmann and Mori (1997). C. elegans II, CSHL Press pp. 717-768.

Shu Liu et al. (2008) European Worm Meeting "Towards the search for a thermonociceptor"

Shu Liu, Maren Hertweck, Ralf Baumeister

[European Worm Meeting, 2008]

Upon exposure to noxious temperature, Caenorhabditis elegans executes an. escape reflex (Tav response) similar to the response to body touch. We had. previously shown that sensory neurons in the head and tail, but not in the. midbody region, are involved in the preception of heat. In vertebrates, the. capsaicin-sensitive vanilloid receptor VR1 of the TRP family has been shown. to be involved in the perception to heat, capsaicin, and low pH, and. several attempts have been made in other labs to relate TRP channels to the. sensation of external stimuli. We have begun a systematic phenotypic. analysis of available TRP channel mutants. Of 11 TRP mutants tested, only. osm-9 ocr-2 double mutants exhibited a severe Tav response defect. OSM-9. and OCR-2 have previously been demonstrated to function coordinately in C.. elegans olfaction and nose-touch responses (Tobin et al., 2002), and our. data support a similar model of coordinated sensory transduction in. thermonociception. OCR-2 and OSM-9 expression overlaps only in a few head. and tail neurons (Jose et al., 2007), suggesting that these could in. principle include candidates for thermonociceptors. We next tested a. variety of mutants whose genes have been reported to be expressed in. subsets of these neurons. sem-4 and unc-86 indeed displayed a reduced Tav. response in the tail, similar to the osm-9 ocr-2 double mutants'' defect. In. addition, one receptor mutant expressed in several classes of head neurons. also resulted in a mild yet significant anterior Tav defect, similar to. that seen in eat-4 and osm-9ocr-2 (79% Tav response). Taken together, we. are narrowing down our search for (a) sensory neuron(s) involved in the. perception of thermonoxious stimuli. We plan to carry out neuron ablations. to verify our findings.

Johnson, Matthew et al. (2013) International Worm Meeting "Sexually dimorphic synaptic connectivity in the C. elegans tail."

Ryan, Deborah, Portman, Douglas, Johnson, Matthew

[International Worm Meeting, 2013]

Sex-specific wiring of neural circuits is likely to have important roles in behavior. However, little is known about how sexual cues might influence synapse formation or stability. Interestingly, the recently described connectome of the C. elegans male tail (Jarrell et al., 2012, Science 337:437) has revealed several instances of sex-specific synaptic connections between non-sex-specific ("shared") neurons. In particular, the architecture of phasmid and posterior touch sensory circuitry appears to to be significantly different in the adult male compared to the adult hermaphrodite, perhaps to enable efficient male copulatory behavior. Using GRASP and other fluorescent markers, we are asking several questions about these sexually dimorphic connections. (1) Are sex differences in connectivity established in the embryo, or do these arise later, as a re-wiring process that occurs in parallel with male-specific tail neurogenesis and circuit formation? (2) What determines the sex-specificity of these connections? Is the genetic sex of the pre- and/or post-synaptic cells important, or do non-autonomous sexual cues have a role? (3) How do these sex differences in connectivity alter neural circuit function and behavior? Our preliminary results indicate that one putative hermaphrodite-specific connection, PHB-AVA, is indeed present in larval males. Unexpectedly, we can also often detect strong PHB-AVA GRASP signal in adult males. One possibility is that PHB-AVA synapses are removed as part of male tail re-wiring in L4, but that GRASP is unable to detect (or perhaps even disrupts) this removal. We are currently using GRASP to label other putative sex-specific connections to explore this further. The unique tools available in C. elegans should offer the opportunity to understand how genetic sex regulates modulators of synapse specification and maintenance to bring about sex differences in circuit connectivity. We are grateful to S. Emmons (Albert Einstein) for sharing unpublished data on male tail connectivity and to M. VanHoven (San Jose State) for sharing the wyIs157 (PHB-AVA GRASP) transgene.

Sean M O'Rourke et al. (2003) International Worm Meeting "Exploring the centrosome with proteomics and functional genomics."

Sean M O'Rourke, Bruce Bowerman

[International Worm Meeting, 2003]

The centrosome is a large eukaryotic organelle that organizes microtubules for cellular functions including setting up the mitotic spindle. Despite the important role of the centrosome, relatively little is known about its composition and regulatory mechanisms in metazoans. SPD-5 is a component of C. elegans centrosomes and contains multiple coiled-coil domains that may interact with other proteins thereby governing centrosomal structure (1). Because SPD-5 is thought to be a central component of centrosomes, it represents an excellent handle to pursue other genes and proteins that are also involved in centrosome function. We are taking two experimental routes to uncover additional centrosomal components and regulators.First, SPD-5 antibodies are being used to isolate protein complexes from embryonic extracts with the goal of purifying SPD-5-interacting proteins. Such proteins will be identified by mass spectrometry. To date, we have prepared embryonic extracts from N2 animals and we have successfully immunoprecipitated SPD-5 protein. Interestingly, two SPD-5 isoforms are apparent in our extracts, the significance of which remains to be determined. We are currently optimizing the purification procedure to obtain larger quantities of SPD-5 and interacting proteins. The interacting proteins we identify will be initially characterized by localization and RNAi inhibition studies.In the second route to understanding centrosome function, we are employing genome interaction screens to identify genes which, when knocked-down by feeding RNAi, alter the phenotypes of temperature-sensitive mutants. Building on the work of Jose Eduardo Gomes (see Gomes and Bowerman abstract), we have begun to use multiple-well plates for culturing worms and a robot to easily and accurately dispense the dsRNA-producing E. coli. We have performed a pilot semi-automated screen using the chromosome I library (2). Current results will be presented. In addition to examining spd-5, we are planning to perform genome-wide interaction RNAi feeding screens with 20 different temperature-sensitive mutants defective in several different processes. We envision generating a genetic interaction map that will be useful for determining how many different processes require any given gene. We anticipate that our use of many different sensitized genetic backgrounds will enable us to identify gene requirements missed in previous classical and genomic screens.(1) Hamill, D. R. et al. Developmental Cell 3, 673-684 (2002).(2) Fraser, A. G. et al. Nature 408, 325-330 (2000).