बनती है और का उपयोग यौगिकों की bioactivity के लिए इलैक्ट्रोफिजियोलॉजी स्क्रीन करने के लिए परख Nanoinjection ड्रोसोफिला मेलानोगास्टर विशालकाय फाइबर सिस्टम
Summary
एक तेजी से<em> Vivo में</em> Neuromodulatory विशालकाय फाइबर प्रणाली (GFS) का उपयोग यौगिकों के लिए परीक्षण परख<em> ड्रोसोफिला मेलानोगास्टर</em> वर्णित है. पशु के सिर में GFS का electrophysiological रिकॉर्डिंग के साथ nanoinjections न्यूरॉन्स या मांसपेशियों पर यौगिकों की bioactivity प्रकट कर सकते हैं.
Abstract
Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled “Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster ” from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.
Protocol
Discussion
यहाँ प्रस्तुत / nanoinjection इलेक्ट्रोफिजियोलॉजी bioassay फल मक्खी के तंत्रिका तंत्र में यौगिकों की एक तेजी से जांच के लिए अनुमति देता है. यह vivo में तकनीक है कि परिसर के छोटे मात्रा की आवश्यकता के लिए एक अच्छी तरह…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम विशेष Aline Yonezawa में मारी प्रयोगशाला और Godenschwege के प्रयोगशाला, के सदस्यों को स्वीकार करना और टिप्पणी के लिए, इस प्रोटोकॉल के साथ मदद की. इस काम के मस्तिष्क संबंधी विकार के लिए राष्ट्रीय संस्थान और एफएम और टैग करने के लिए स्ट्रोक R21NS06637 अनुदान द्वारा वित्त पोषित किया गया था, अटल बिहारी राष्ट्रीय विज्ञान फाउंडेशन पुरस्कार 082,925 संख्या, URM द्वारा वित्त पोषित किया गया था: भविष्य के शोधकर्ताओं के लिए एकीकृत जीवविज्ञान.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Recording glass electrodes: borosilicate glass capillaries | World Precision Instruments | 1B100F-4 | 1.0mm OD, 0.58mm ID |
| Stimulator | Grass Instruments | Model S48 | |
| Amplifier | Getting Instruments Inc. | Model 5A | |
| Data acquisition Software: Digidata | Molecular Devices | Model 1440A | |
| Data collection software: pCLAMP | Molecular Devices | Version 10 | |
| Stereomicroscope with fiber optic microscope ring illuminator | AmScope | SM-4T Model HL250-AR |
|
| Dissecting scope for mounting | AmScope | SM-2TZ | |
| Kite Manual Micromanipulator & Tilting Base | World Precision Instruments | Model # M3301 Kite: Model # KITE-M3-L |
|
| Drosophila melanogaster Wild 10E genotype (wild type strain) | Bloomington Stock center | Stock # 3892 | |
| Vertical pipette puller | David Kopf Instruments | Model 700c | |
| Injection glass micropipettes: Borosilicate glass capillaries | World Precision Instruments | Catalogue # 4878 | 1.14mm OD, 0.5mm ID |
| Silicon oil | Fisher | Catalogue # S159-500 | |
| Beveler | Sutter Instrument Co. | K.T. Brown Type Model # BV-10 |
|
| Nanoliter2000 | World Precision Instruments | Catalogue # B203XVY | |
| Blue food coloring | McCormick | N/A | Ingredients: Water, Propylene Glycol, FD&C Blue 1, and 0.1% Propylparaben (preservative). |
| Methyllycaconitine citrate (MLA) | Tocris Bioscience | Catalogue # 1029 | |
| Plastic wax sticks | Hygenic Corporation (Akron Ohio USA) |
References
- Koehn, F. E., Carter, G. T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 4, 206-220 (2005).
- Miljanich, G. P. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr. Med. Chem. 11, 3029-3040 (2004).
- Layer, R. T., Wagstaff, J. D., White, H. S. Conantokins: peptide antagonists of NMDA receptors. Curr. Med. Chem. 11, 3073-3084 (2004).
- Lewis, R. J. Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters. IUBMB Life. 56, 89-93 (2004).
- Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. Making an escape: development and function of the Drosophila giant fibre system. Semin. Cell Dev. Biol. 17, 31-41 (2006).
- Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R., Bellen, H. J. The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS biology. 4, e63 (2006).
- Jan, L. Y., Jan, Y. N. L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction. The Journal of physiology. 262, 215-236 (1976).
- Usherwood, P. N., Machili, P., Leaf, G. L-Glutamate at insect excitatory nerve-muscle synapses. Nature. 219, 1169-1172 (1968).
- Marrus, S. B., Portman, S. L., Allen, M. J., Moffat, K. G., DiAntonio, A. Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction. The Journal of neuroscience : the official journal of the Society for Neuroscience. 24, 1406-1415 (2004).
- Petersen, S. A., Fetter, R. D., Noordermeer, J. N., Goodman, C. S., DiAntonio, A. Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron. 19, 1237-1248 (1997).
- Qin, G. Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila. The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 3209-3218 (2005).
- Schuster, C. M. Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle. Science. 254, 112-114 (1991).
- Tanouye, M. A., Wyman, R. J. Motor outputs of giant nerve fiber in Drosophila. Journal of. 44, 405-421 (1980).
- Augustin, H., Allen, M. J., Partridge, L. Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster. J. Vis. Exp. (47), e2412 (2011).
- Allen, M. J., Godenschwege, T., Zhang, B., Freeman, M. R., Waddell, S. . Drosophila Neurobiology. , 215-224 (2010).
- Blagburn, J. M., Alexopoulos, H., Davies, J. A., Bacon, J. P. Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study. J. Comp. Neurol. 404, 449-458 (1999).
- Thomas, J. B., Wyman, R. J. Mutations altering synaptic connectivity between identified neurons in Drosophila. J. Neurosci. 4, 530-538 (1984).
- Baird, D. H., Schalet, A. P., Wyman, R. J. The Passover locus in Drosophila melanogaster: complex complementation and different effects on the giant fiber neural pathway. Genetics. 126, 1045-1059 (1990).
- Gorczyca, M., Hall, J. C. Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis. J. Neurogenet. 1, 289-313 (1984).
- Allen, M. J., Murphey, R. K. The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter. The European journal of neuroscience. 26, 439-445 (2007).
- Mejia, M. A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit. Toxicon. 56, 1398-1407 (2010).
- Stork, T. Organization and function of the blood-brain barrier in Drosophila. J. Neurosci. 28, 587-597 (2008).
