Name: a molecular readout of long-term olfactory adaptation in c. elegans
Uploaded: 2012-12-22T14:00:00
Description: Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

Vi vil gjerne takke Scott Hamilton, og medlemmer av O&#39;Halloran lab for grundig lesning av dette manuskriptet. Vi vil også takke våre anonym anmelder for gode forslag og innsiktsfulle kommentarer.

1. Bygging av GFP Tagged EGL-4 Uttrykke Dyr <ol><li> Klone translasjonsforskning fusion GFP :: EGL-4 under arrangøren for ODR-3-genet (bruk 2678 bp direkte oppstrøms for startkodon): (p) ODR-3 :: GFP :: EGL-4. Den ODR-3 promoter stasjoner uttrykk i amphid Nevron parene: AWA, AWB, AWC, og svakt i ASH. </li><li> Injisere plasmid (p) ODR-3 :: GFP :: EGL-4 i wildtype (N2) dyrene på 50 ng / ul med co-injeksjon markører ved hjelp av standard bakterie-line transformasjon teknikker <sup...

<ol>
	<li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14 (4), 803 (1995).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+modulate+olfactory+acuity,+discrimination,+and+memory+in+Caenorhabditis+elegans.">Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.</a> Learning and Memory. 4 (2), 179 (1997).</li><li>Bargmann, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Neuron. 74 (3), 515 (1993).</li><li>L'Etoile, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cyclic+GMP-dependent+protein+kinase+EGL-4+regulates+olfactory+adaptation+in+C.+elegans.">The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans.</a> Neuron. 36, 1079-10 (2002).</li><li>Lee, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+entry+of+a+cGMP-dependent+kinase+converts+transient+into+long-lasting+olfactory+adaptation.">Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.</a> PNAS. 107 (13), 6016 (2010).</li><li>O'Halloran, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+AWC-mediated+olfactory+plasticity+in+Caenorhabditis+elegans.">Regulators of AWC-mediated olfactory plasticity in Caenorhabditis elegans.</a> PLoS Genetics. 5 (12), e1000761 (2009).</li><li>O'Halloran, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+cGMP+levels+affect+the+localization+of+EGL-4+in+AWC+in+Caenorhabditis+elegans.">Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans.</a> PLoS ONE. 7 (2), e31614 (2012).</li><li>Mello, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO Journal. 10, 3959-39 (1991).</li><li>Miyabayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+members+of+a+divergent+nuclear+receptor+family+in+Caenorhabditis+elegans.">Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans.</a> Dev. Biol. 215, 314 (1999).</li><li>L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+and+odor+discrimination+are+mediated+by+the+C.+elegans+guanylyl+cyclase+ODR-1.">Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1.</a> Neuron. 25 (3), 575 (2000).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451 (1995).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> Wormbook. , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71 (1974).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+Dopamine+Neuron+Degeneration+Assay+for+the+Validation+of+Potential+Parkinson's+Disease+Genes.">Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes.</a> J. Vis. Exp. (17), e835 (2008).</li><li>Pittenger, C., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+general+mechanisms+for+long-lasting+plasticity:+Aplysia+and+the+hippocampus.">In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358 (1432), 757 (2003).</li></ol>

Lukt-indusert kjernefysiske oppføring av en GFP-merket EGL-4 molekyl som er beskrevet her gir en robust molekylær avlesning av olfactory tilpasning i C. elegans. De lukt-indusert kjernefysiske translokasjon analysene er grei og krever bare noen få dager med forberedelser tid. Den pyIs500 dyr som vi har bygget for disse analysene, uttrykker en markør som lyser opp AWC nervecellen samt uttrykker GFP-merket EGL-4 protein. Dermed føler vi at en eksperimentator med liten eller ingen erfaring med denne ...

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a molecular readout of long-term olfactory adaptation in c. elegans

Under vedvarende stimulering mest sensoriske nevroner vil tilpasse deres respons ved å redusere deres følsomhet til signalet. Tilpasning respons hjelper form oppmerksomhet og også beskytter cellene fra overstimulering. Omstilling i olfactory krets av C. elegans ble først beskrevet av Colbert og Bargmann 1,2. Her forfatterne definerte parametere av olfactory tilpasning paradigme, som de brukte til å designe en genetisk skjermen for å isolere mutanter defekt i sin evne til å tilpasse seg flyktige lukt avfølt av Amphid Wing celler type C (AWC) sensoriske nerveceller. Når wildtype C. elegans dyr er utsatt for en attraktiv AWC-avfølt lukt 3 i 30 min vil tilpasse sin reaksjonsevne til lukt og vil da ignorere tilpasse lukt i en kjemotaksis atferdsdata analysen for ~ 1 time. Når wildtype C. elegans dyr er utsatt for en attraktiv AWC-ante lukt for ~ 1 hr de vil da ignorere tilpasse lukt i achemotaxis atferdsmessige analyse for ~ 3 timer. Disse to fasene av olfactory tilpasning i C. elegans ble beskrevet som kortsiktig olfactory tilpasning (indusert etter 30 min lukt eksponering), og langsiktig olfactory tilpasning (indusert etter 60 min lukt eksponering). Senere arbeidet fra L&#39;Etoile et al., Avdekket fire en protein kinase G (PKG) kalt EGL-4 som er nødvendig for både den kortsiktige og langsiktige olfactory tilpasning i AWC nerveceller. EGL-4 protein inneholder en kjernefysisk lokalisering sekvens som er nødvendig for langsiktig olfactory tilpasning svar, men unnværlig for kortsiktige olfactory tilpasning svarene i AWC 4. Etter merking EGL-4 med et grønt fluorescerende protein, var det mulig å visualisere lokalisering av EGL-4 i AWC under langvarig lukt eksponering. Ved hjelp av dette fullt funksjonell GFP-merket EGL-4 (GFP :: EGL-4) molekylet vi har vært i stand til å utvikle en molekylær avlesning av langsiktig olfactory tilpasning i AWC 5. Ved hjelp av denne molecular avlesning av olfactory tilpasning vi har vært i stand til å utføre både fremover og bakover genetiske skjermer for å identifisere muterte dyr som viser defekte subcellulære lokalisering mønstre av GFP :: EGL-4 i AWC 6,7. Her beskriver vi: 1) bygging av GFP :: EGL-4 uttrykker dyr, 2) protokollen for dyrking av dyr for langsiktig lukt-indusert kjernefysiske translokasjon analyser og 3) scoring av den langsiktige lukt-indusert kjernefysisk translokasjon hendelsen og gjenvinning (re-allergi) fra kjernekraftverket GFP :: EGL-4 tilstand.

Et eksempel på lokalisering mønster av GFP :: EGL-4in den AWC før og etter langvarig lukt eksponering er vist i figur 2. Før langvarig lukt eksponering, GFP :: EGL-4 er lokalisert til cytosol av AWC (figur 2B), og etter 80 min lukt eksponering GFP :: EGL-4 er lokalisert til kjernen av AWC (figur 2D). På atferdsmessige nivå, er dyr med cytosolic GFP :: EGL-4 i AWC tiltrukket til et punkt kilde lukt (Figur 2C plotter representative resultater fra ch...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Her beskriver vi en molekylær avlesning av langsiktig olfactory tilpasning i<em&gt; Caenorhabditis elegans</em&gt;. Proteinkinase G, EGL-4, som er nødvendig for stabile tilpasningstiltak responser i primær sensorisk neuron pair kalt AWC. Under langvarig lukt eksponering EGL-4 translocates fra cytosol til kjernen av AWC.

En molekylær Avlesning av Long-term olfactory Tilpasning i<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

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