Guide Caenorhibditus Elegans Volume 48 Modern Biological Analysis of an Organism

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Combined, these findings suggest that procedures using C. In addition, the development of this technology will allow for the future investigation of the molecular mechanisms that underlie the efficacious effects of novel agents using the fully tractable C. In the current studies, we employed a modified version of this assay, in which a 6-well agar test plate was prepared with a SOA placed in a defined target region on one side of each well and the vehicle, usually water, placed in a target zone on the other side of each well.

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The current experiments build on our previous work showing that C. The current work examines preference responses to two of the most widely abused stimulants, cocaine and nicotine Lee et al. Few studies have examined the reinforcing properties of stimulants in C. It should be noted that the concentrations of treatment agents needed to produce effects in C. In the current work, animals counted in the target zone containing the stimulant either cocaine or nicotine are in contact with the SOA and thus demonstrating self-exposure to the SOA.

This is also a true choice behavior, since the current study found that the addition of the aversive compound nonanone near the SOA target zone, after the preference response has been established, caused the animals to immediately move away from the SOA target zone, inducing a measurable aversive response. These findings confirm that the SOAs tested here are not simply functioning as a simple locomotor anesthetic or paralytic agent in this procedure.

Consistencies in responses to SOAs across phyla led to the hypothesis that C. Recently, C.

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Thus, to determine the predictive validity of the model, we tested the effectiveness of naltrexone to decrease preference responses, as it is one of the very few compounds shown consistently to reduce alcohol and other SOAs intake and seeking behavior in animal models as well as humans Heilig and Egli, Using vertebrate models, naltrexone has been demonstrated to reduce cocaine intake Mello et al. Naltrexone has shown mixed effects on nicotine use in humans Aboujaoude and Salame, ; Barboza et al.


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Rodent studies indicate that naltrexone can reduce nicotine-induced locomotor sensitization Goutier et al. Also, treatment with naloxanazine a selective mu1 opioid receptor antagonist significantly reduced nicotine self-administration in rats Liu and Jernigan, However, some work suggests that naltrexone may have more consistent effects to reduce conditioned responses to nicotine Liu et al. Other opioid receptors may also be efficacious targets, with the kappa-opioid receptor antagonist nor-binaltorphimine reducing nicotine seeking behavior Grella et al.

Together, these studies support a role for opioid systems in stimulant reinforcement and use and are consistent with findings in the current screen with C. However, much additional investigation is needed to identify how the opioid system may be involved in nicotine self-administration and how agents that target these systems may reduce tobacco or cocaine use in humans.

Naltrexone pre-exposure clearly reduced SOA preference Figures 2 — 3 at concentrations that do not inhibit food consumption Table 1 , benzaldehyde chemotaxis, or locomotor activity body-bend data. These data are consistent with rodent data showing that naltrexone can inhibit intake of SOAs at doses that do not affect sucrose intake or body weight Henderson-Redmond and Czachowski, In most instances, little or no prior work has been published to determine if treatment agents used to treat stimulant addictions have effects on models of addictive responses to stimulants in C.

However, varenicline pre-exposure has been shown to reduce chemotaxis to nicotine in C. Our data are consistent with these data and show selectivity and predictive validity of varenicline in this screening model. Although it is still unclear how varenicline reduces nicotine preference in C. Other possible mechanisms such as changes in drug metabolism or subtle changes in sensory systems have yet to be investigated. Interestingly, the effect of varenicline to inhibit nicotine self-exposure in this paradigm is evident at the min time point and, although still evident at 30 min, appears to degrade over time Figure 5.

This could be a reflection of the apparent strength of the nicotine preference response, or possibly rapid clearance of varenicline. In support of this idea is the apparent greater strength of the preference response, and resistance to nonanone for nicotine compared with cocaine at the concentrations used in this study Figure 6. Although it is somewhat difficult to make direct comparisons between the cocaine and nicotine data due to the differences in systems and mechanisms, and also in concentrations used to produce the respective preference responses, there are clear differences in the response to nonanone.

One possible explanation is that animals are being paralyzed by the SOAs at these concentrations. This cannot be completely ruled out as previous work has indicated that nicotine uniformly mixed in agar to concentrations from 1 to 10 mM can induce paralysis Sobkowiak et al. However, other evidence argues against the idea that the worms are paralyzed. Secondly, both groups of animals show a significant effect of nonanone to move the animals from the SOA target zone although the magnitude of the effect was less for nicotine , indicating that they are not paralyzed.

This hypothesis is further supported by examination of time-lapse videos Supplementary Video 1 which clearly show animals continuing to move in the nicotine target zone after entering during a preference test on a plate spotted with mM nicotine, and a clear movement out of the zone after the addition of nonanone. One possible explanation for the greater effect of nonanone to displace cocaine exposing animals compared to nicotine is the somewhat stronger preference response observed with nicotine vs. The increased preference response for nicotine over cocaine suggests a greater reinforcing property of the SOA as tested and as such would confer greater aversion resistance.

Future experiments will provide additional evaluation and characterization of varenicline and other compounds to inhibit the SOA preference response. Behavioral studies of addictive SOAs in C. Additional studies with other SOAs are needed to better characterize the mechanisms that underlie addictive properties of SOAs across the many classes of SOAs and how they may be consistent or divergent across species.

In the few studies conducted thus far, several molecular targets have been identified in various behavioral paradigms across SOA classes using C. Thus far, it appears that genes involved in monoamine neurotransmission mediate at least some behaviors induced by each SOA Bettinger and McIntire, ; Lee et al. In particular, mutation of the gene coding for tyrosine hydroxylase cat-2 reduced or inhibited SOA-induced behaviors for each SOA of abuse Bettinger and McIntire, ; Musselman et al.

It is widely thought that the dopamine neurotransmitter system plays an important role in drug abuse Koob, ; Koob and Volkow, and all of the SOAs discussed here have effects on dopamine neurotransmission. Similarly, in agreement with the current data, previous work has shown that manipulations that inhibit cholinergic neurotransmission in C. Overall, the known mechanisms of action of SOAs in vertebrate animals thus far show parallel findings in C. The effects of SOAs in C. However, since most of the work in C. Although C.

Moreover, C. However, the similarities in responses to SOAs between C. Interestingly, differences in receptor systems and molecular pharmacology in C. As an example, topiramate is under investigation as a possible treatment for EtOH use disorders Johnson, Topiramate has a rich pharmacology and there are several possible molecular mechanisms for reducing EtOH drinking behavior. One suggested mechanism is activity at voltage-sensitive sodium channels Johnson, , which are not present in C.

If topiramate were to be found ineffective in C. This could be conducted across SOA classes to identify the effects of divergent molecular structure or function on the results. Overall, such investigations may help to characterize the molecular and pharmacological foundations of the effects of these compounds, whether or not the findings are consistent with the anticipated results.

Further development of the model employed here is anticipated to provide the field with a new and powerful tool to discover novel targets and treatments for addiction. This work will combine the advances in our knowledge of human addiction and insight gained through the use of vertebrate behavioral models, and apply them to invertebrate models with tremendous advantages and potential for discovery on a number of levels: a the current work contributes to the establishment of a new behavioral model in C. Such an application could improve the translational utility of the model and possibly enhance predictive validity.

Once a high-throughput system is fully established, one could conceivably screen entire potential treatment agent libraries using tiny amounts of expensive compounds relative to other animal models. Future collaborative projects will employ transgenic approaches to express human genes in this model to enhance the predictive validity of the model. EE and SK wrote the initial drafts and designed and directed the experiments.

RB and BN-B reviewed content, data, and final draft of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aboujaoude, E. Naltrexone: a pan-addiction treatment? CNS Drugs 30, — Alaimo, J. Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. Alcohol Clin. Barboza, J. Anupdate on the pharmacotherapeutic interventions for smoking cessation. Expert Opin. Bargmann, C.

Neurobiology of the Caenorhabditis elegans genome. Science , — Chemosensation in C. WormBook 1— Control of larval development by chemosensory neurons in Caenorhabditis elegans. Bell, R. Google Scholar. Bettinger, J. The role of the BK channel in ethanol response behaviors: evidence from model organism and human studies. State-dependency in C. Genes Brain Behav. Bianchi, L. Culture of embryonic C. Cheong, M. An opioid-like system regulating feeding behavior in C. Corrigall, W.

Opiate antagonists reduce cocaine but not nicotine self-administration. Psychopharmacology , — Crooks, P. Nicotinic receptor antagonists as treatments for nicotine abuse. Davies, A. A central role of the BK potassium channel in behavioral responses to ethanol in C. Cell , — Davis, S. Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in C. Edwards, S. Experimental psychiatric illness and drug abuse models: from human to animal, an overview.

Methods Protoc. Engleman, E. Caenorhabditis elegans as a model to study the molecular and genetic mechanisms of drug addiction. Epstein, H. Caenorhabditis elegans: Modern Biological Analysis of an Organism.

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Cambridge, MA: Academic Press. To study the expression of gana-1 in C. However, no GFP signal was observed by fluorescence microscopy under the standard laboratory conditions. As Western blotting showed the presence of fusion protein of the expected size data not shown , we assumed that the absence of the GFP signal was caused by a pH-dependent quenching of GFP fluorescence, which has neutral to alkaline optimum pH 5. To study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product.

Immunofluorescence detection of GFP fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression. The immunofluorescence staining protocol resulted in a significant decrease of inherent intestinal granular autofluorescence previously assigned to secondary lysosomes [ 32 ]. The decrease of autofluorescence intensity together with its poorly defined emission spectra hampered co-localization study.

A A coarsely granular cytoplasmic distribution of immunopositivity green in body-wall muscle cells arrowheads. Two non-transgenic worms are shown in the background asterisks for comparison. Nuclei are counterstained in red. B Detailed view of two body wall muscle cells with coarsely granular cytoplasmic distribution of immunopositivity arrowheads and a coelomocyte asterisk , both pictures were acquired by 3D rendering of initial confocal Z-stacks.

Note: compare with figure 6. Alkalization of transgenic worms using CON A. To confirm that the absence of the GFP signal was due to the quenching of fluorescence by low pH in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment [ 33 , 34 ] to enhance the GFP emission.

The GFP signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used. The reappearance of the GFP signal after treatment of the worms with compounds increasing the acidic compartment pH indirectly confirms lysosomal localization of the fusion protein. The GFP signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining. Limited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies.

The lysosomal localization of the fusion protein was also supported by pH sensitive fluorescence of GFP that was detectable only after alkalization of the acidic cellular compartment.


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Not suprisingly, RNAi of gana-1 yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states. Nevertheless, gana-1 RNAi resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them. It is possible that a deletion allele of gana-1 may provide more insight into the function of gana-1 and efforts are underway to isolate such alleles. Deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders.

The wild type Bristol N2 strain was used for all experiments and was handled under standard laboratory conditions as described previously [ 35 ]. Nomenclature is in agreement with available Genetic Nomenclature for Caenorhabditis elegans [ 15 ] and has been approved prior to manuscript submission. The entire coding region of R07B7. Positive clones were sequenced using the Li-Cor automated fluorescent sequencer and sequences were aligned with R07B7 reference cosmid sequence in the AlignIR software Li-Cor to evaluate splicing boundaries and overall gene organization.

Confirmed or predicted amino acid sequences of melibiase family members [ 43 ] representing plant, unicellular, and animal kingdoms were aligned using ClustalW algorithm [ 44 ] and Blosum62 matrix.

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The phylogenetic tree is based on bootstraped input alignments and was constructed by maximum likelihood method with Jones-Taylor-Thornton matrix model [ 47 ]. Sequence identities between species were calculated without signal sequence in EMBOSS by Needleman-Wunsch global alignment algorithm with Blosum62 matrix, gap penalty — 10 and gap extension penalty — 0. Signal peptides were predicted at the SignalP server [ 50 ] both by algorithms using neural networks and Hidden Markov Models.

The results were compared to known signal sequences. The differences between signal peptides predicted by the algorithms are depicted in Figure 2. The model was created using the automated homology modeling server SwissModel with structure refinement and model evaluation in the DeepView program [ 52 ].

The print quality figures Figure 4 and animations Additional file 1 were ray traced using PovRay software package [ 53 ]. Fire, Stanford University. The in-frame nature of the insert was confirmed by sequencing. Transgenic animals were screened for GFP signal. Nikon Eclipse E with C1 confocal module and nm and nm lasers and differential interference contrast DIC optics was used for specimen examination. Worms were pelleted by centrifugation max.

Worms were treated with either one of two agents NH 4 Cl, concanamycin A — CON A [ 33 , 34 ], that are known to specifically increase pH in the cellular acidic compartment. Small aliquots of worms were examined after 30 min, 2, 4, 6, 8 and 24 hours. Small aliquots of worms were examined after 1, 3, 6 and 24 hours. The fixation and immunofluorescence staining procedures were based on the approaches of Nonet et al. Worms were pelleted by centrifugation RPM, 2 min.

AbA buffer was used for antibody dilution. Worms were homogenized by sonication and the concentration of protein was measured by the Hartree method [ 55 ]. The membrane was treated according to a common Western blotting protocol with chemiluminiscence detection SuperSignal, West Pico [ 56 ]. The F 1 and early F 2 progeny was screened for morphological phenotypes. N2 worms microinjected with water and fed on HT E. Prior to all activity measurements worms were washed from culture plates and repeatedly 6 times washed and centrifuged in M9 buffer and finally pelleted by centrifugation.

Fluorescence signal of the 4-methylumbelliferone was measured on the luminiscence spectrofotometer LS 50B Perkin Elmer emission nm and excitation nm. Inhibitors N-acetyl-D-galactosamine, D-galactose and D-glucose were used in 0. All measurements were performed in doublets. Biochem Biophys Res Commun. Biochim Biophys Acta. Wang AM, Desnick RJ: Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Henrissat B, Davies G: Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. Structure Camb. J Mol Biol. J Biol Chem.

WO2000063425A2 - Compound assay using nematodes - Google Patents

J Lipid Res. Blue color represents identical residues and orange stands for non-conservative changes. The colors from cyan to green represent different degrees of conservation. The surface of one monomer unit at the interface area is rendered with colors representing electrostatic potential. N-acetyl-D-galactosamine inhibitor is placed in the active site pocket of both monomer units D-GalNAc arrowhead.

K arrowhead depicts predicted dimerisation residue. MPG 8 MB. We avoided standard sucrose flotation of worms because we could not exclude unpredictable artifacts caused by this compound, which is known to induce artificial lysosomal storage in eukaryotic cells and to alter lysosomal gene expression at concentrations significantly lower [ 24 ] than those used in flotation protocols. The model of GANA-1 predicts only one active site per monomer of the enzyme.

Nevertheless, these experiments were not conducted with the pure enzyme and thus do not provide absolute proof of this hypothesis. RNA interference assays directed against the whole coding region of gana-1 , employing combination of microinjection and feeding approaches, did not reveal any abnormal morphological phenotypes. This finding supports the specificity of gana-1 RNAi. The differences between individual experiments are not surprising due to the well-known variability in the efficiency of RNAi [ 26 ]. Both enzymatic activities were lower in RNAi-treated and control worms cultured on the bacterial strain HT [ 26 ] compared to a N2 strain cultured on the OP50 strain.

RNAi previously provided sufficient level of inhibition of structural lysosomal proteins for development of abnormal phenotypes in the worm [ 27 , 28 ]; however, it is apparently not efficient enough for lysosomal catalytic proteins. To study the expression of gana-1 in C. However, no GFP signal was observed by fluorescence microscopy under the standard laboratory conditions. As Western blotting showed the presence of fusion protein of the expected size data not shown , we assumed that the absence of the GFP signal was caused by a pH-dependent quenching of GFP fluorescence, which has neutral to alkaline optimum pH 5.

To study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product. Immunofluorescence detection of GFP fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression. The immunofluorescence staining protocol resulted in a significant decrease of inherent intestinal granular autofluorescence previously assigned to secondary lysosomes [ 32 ].

The decrease of autofluorescence intensity together with its poorly defined emission spectra hampered co-localization study. A A coarsely granular cytoplasmic distribution of immunopositivity green in body-wall muscle cells arrowheads. Two non-transgenic worms are shown in the background asterisks for comparison.

Nuclei are counterstained in red. B Detailed view of two body wall muscle cells with coarsely granular cytoplasmic distribution of immunopositivity arrowheads and a coelomocyte asterisk , both pictures were acquired by 3D rendering of initial confocal Z-stacks.


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  8. Note: compare with figure 6. Alkalization of transgenic worms using CON A. To confirm that the absence of the GFP signal was due to the quenching of fluorescence by low pH in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment [ 33 , 34 ] to enhance the GFP emission. The GFP signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used.

    Caenorhabditis elegans, Volume 48 Modern Biological Analysis of an Organism Methods in Cell Biology

    The reappearance of the GFP signal after treatment of the worms with compounds increasing the acidic compartment pH indirectly confirms lysosomal localization of the fusion protein. The GFP signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining. Limited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies. The lysosomal localization of the fusion protein was also supported by pH sensitive fluorescence of GFP that was detectable only after alkalization of the acidic cellular compartment.

    Not suprisingly, RNAi of gana-1 yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states. Nevertheless, gana-1 RNAi resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them. It is possible that a deletion allele of gana-1 may provide more insight into the function of gana-1 and efforts are underway to isolate such alleles.

    Deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders. The wild type Bristol N2 strain was used for all experiments and was handled under standard laboratory conditions as described previously [ 35 ]. Nomenclature is in agreement with available Genetic Nomenclature for Caenorhabditis elegans [ 15 ] and has been approved prior to manuscript submission.

    The entire coding region of R07B7. Positive clones were sequenced using the Li-Cor automated fluorescent sequencer and sequences were aligned with R07B7 reference cosmid sequence in the AlignIR software Li-Cor to evaluate splicing boundaries and overall gene organization. Confirmed or predicted amino acid sequences of melibiase family members [ 43 ] representing plant, unicellular, and animal kingdoms were aligned using ClustalW algorithm [ 44 ] and Blosum62 matrix. The phylogenetic tree is based on bootstraped input alignments and was constructed by maximum likelihood method with Jones-Taylor-Thornton matrix model [ 47 ].

    Sequence identities between species were calculated without signal sequence in EMBOSS by Needleman-Wunsch global alignment algorithm with Blosum62 matrix, gap penalty — 10 and gap extension penalty — 0. Signal peptides were predicted at the SignalP server [ 50 ] both by algorithms using neural networks and Hidden Markov Models.

    The results were compared to known signal sequences. The differences between signal peptides predicted by the algorithms are depicted in Figure 2. The model was created using the automated homology modeling server SwissModel with structure refinement and model evaluation in the DeepView program [ 52 ].

    The print quality figures Figure 4 and animations Additional file 1 were ray traced using PovRay software package [ 53 ]. Fire, Stanford University. The in-frame nature of the insert was confirmed by sequencing. Transgenic animals were screened for GFP signal. Nikon Eclipse E with C1 confocal module and nm and nm lasers and differential interference contrast DIC optics was used for specimen examination. Worms were pelleted by centrifugation max. Worms were treated with either one of two agents NH 4 Cl, concanamycin A — CON A [ 33 , 34 ], that are known to specifically increase pH in the cellular acidic compartment.

    Small aliquots of worms were examined after 30 min, 2, 4, 6, 8 and 24 hours. Small aliquots of worms were examined after 1, 3, 6 and 24 hours. The fixation and immunofluorescence staining procedures were based on the approaches of Nonet et al. Worms were pelleted by centrifugation RPM, 2 min. AbA buffer was used for antibody dilution. Worms were homogenized by sonication and the concentration of protein was measured by the Hartree method [ 55 ].

    The membrane was treated according to a common Western blotting protocol with chemiluminiscence detection SuperSignal, West Pico [ 56 ]. The F 1 and early F 2 progeny was screened for morphological phenotypes.