लंबी अवधि में घ्राण अनुकूलन के एक आणविक Readout<em> सी. एलिगेंस</em
Summary
यहाँ हम लंबी अवधि में घ्राण अनुकूलन के एक आणविक readout का वर्णन<em> Caenorhabditis एलिगेंस</em>. प्रोटीन kinase जी, EGL-4, प्राथमिक संवेदी न्यूरॉन AWC बुलाया जोड़ी में स्थिर अनुकूलन प्रतिक्रियाओं के लिए आवश्यक है. लंबे समय तक गंध जोखिम EGL 4 cytosol से AWC के नाभिक को translocates के दौरान.
Abstract
During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L’Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.
Protocol
Representative Results
Discussion
गंध प्रेरित एक GFP टैग EGL चार अणु परमाणु प्रविष्टि यहाँ वर्णित सी. में घ्राण अनुकूलन का एक मजबूत आणविक readout प्रदान करता है एलिगेंस. गंध से प्रेरित परमाणु स्थानान्तरण assays सीधा कर रहे हैं और तैयारी के सम?…
Declarações
The authors have nothing to disclose.
Acknowledgements
हम इस पांडुलिपि के सावधान पढ़ने के लिए स्कॉट हैमिल्टन, और O'Halloran प्रयोगशाला के सदस्यों का शुक्रिया अदा करना चाहते हैं. हम भी हमारे गुमनाम समीक्षक उत्कृष्ट सुझाव और insightful टिप्पणियों के लिए धन्यवाद.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Bacto Agar | Difco | DF0140-07-4 | NGM plates |
| Sodium Chloride | Fisher Chemical | S671-10 | NGM plates |
| Bacto Peptone | Difco | DF0118-07-2 | NGM plates |
| Potassium Phosphate Dibasic | Fisher Chemical | S375-500 | S-Basal buffer and NGM plates |
| Potassium Phosphate Monobasic | Fisher Chemical | P285-500 | S-Basal buffer and NGM plates |
| Kimwipes – Small | Kimberly-Clark | LS2770 | |
| Ethanol 100% | Gold Shield Chemical Co. | 43196-115 | diluting odors for chemotaxis assays |
| Calcium Chloride | Sigma-Aldrich | C8106-500G | NGM plates |
| Magnesium Sulphate | MP Biomedicals | 150136-500G | NGM plates |
| Sodium Azide 99% | Fisher Scientific | ICN10289180 | Anesthetic |
| Agarose – UltraPure | Invitrogen | 16500-500 | Agarose pads |
| Benzaldehyde | Sigma-Aldrich | B1334-100G | AWC odor |
| Butanone, ACS Grade | Sigma-Aldrich | 360473-500ML | AWC odor |
| Microcentrifuge Tubes – 1.5 ml Colored | Denville | LS8147 | |
| Pasteur Pipet Disposable Glass 5-3/4″ | Fisher Scientific | 13-678-20B | |
| Stratalinker | Stratagene | Stratalinker 2400 | UV integration |
| Filter Vacuum Bottle – 500 ml | Nalgene | 09-740-25B |
Referências
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