ड्रोसोफिला लार्वा के NMJ से Synaptic क्षमता रिकॉर्डिंग के लिए electrophysiological तरीके
Summary
यहाँ हम ड्रोसोफिला लार्वा की neuromuscular जंक्शन पर synaptic प्रसारण को मापने के लिए electrophysiological विधियों का वर्णन. रिलीज पैदा कृत्रिम मोटर न्यूरॉन axons उत्तेजक द्वारा शुरू की है, और NMJ के माध्यम से संचरण postsynaptic मांसपेशियों में पैदा की प्रतिक्रिया से मापा जा सकता है.
Abstract
In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 – A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP’s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP’s and minature excitatory junction potentials (mEJP’s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP’s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.
Protocol
शुरू करने से पहले तैयार: भटक yhird instar ड्रोसोफिला लार्वा HL3.1 समाधान (संशोधित hemolymph तरह) Sylgard विच्छेदन (पारदर्शी सिलिकॉन रबर) प्लेटें छोटे (35 x 10 मिमी) प्लास्टिक पेट्री ब्रेंट और McCabe (2008) 3 द्वारा वर्णित ?…
Discussion
यहाँ वर्णित तरीकों एक अपेक्षाकृत जल्दी और व्यापक NMJ पर synaptic समारोह में परिवर्तन का पता लगाने के लिए रास्ता प्रदान. बरकरार पशुओं का उपयोग vivo में electrophysiological रिकॉर्डिंग प्रदर्शन, और आनुवंशिक या औषधीय जोड़तोड़ …
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Small Petri dishes (35 x 10 mm) | Becton Dickinson | 1008 | ||
| SYLGARD 182 Silicone Elastomer Kit | Dow Corning Corporation | 3097366-1004 | ||
| Dissecting microscope | Carl Zeiss | 475002-9902 | ||
| Light for microscope | Schott | KLI500 | ||
| Dissection pins | Fine Science Tools | 26002-10 | ||
| pClamp 9 software | Axon CNS, Molecular Devices | PCLAMP 9 STANDARD | ||
| Dissection scissors: 3mm Vannas Spring Scissors | Fine Science Tools | 15000-0 | ||
| Dumont SS Forceps | Fine Science Tools | 11200-33 | ||
| Dumont #5 Forceps | Fine Science Tools | 11252-20 | ||
| Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in) | World Precision Instruments, Inc. | TW100F-4 | ||
| Borosilicate glass capillaries, with filament (1.2 mm, 4 in) | World Precision Instruments, Inc. | 1B120F-4 | ||
| Sutter P-2000 Laser Based Micropipette Puller | Sutter Instruments | Model P-2000 | ||
| Pipette polisher | Narishiga | MF-83 | ||
| Axon HS-2A head stage | Axon CNS, Molecular Devices | Model HS-2A | ||
| Micromanipulators | Sutter Instruments | MP-85 | ||
| Axoclamp 2B amplifier | Axon CNS, Molecular Devices | AXOCLAMP 2B | ||
| Clampex Software | Axon CNS, Molecular Devices | v 8.2.0.235 | ||
| Mini analysis software. v 6.0.3 | Synaptosoft | |||
| Brownlee Precision Amplifier | Brownlee | Model 410 | ||
| NaCl | Baker | 4058-01 | ||
| KCl | Sigma | p-9333 | ||
| NaHCO3 | Sigma | s6297-1kg | ||
| Trelahose | Sigma | TO167 | ||
| Sucrose | Fisher | bp220-212 | ||
| HEPES | Sigma | h-3375 | ||
| MgCl-6H2O | Sigma | m2670-1kg | ||
| CaCl2 | Fisher | c79-500 | ||
| Master-8 Pulse Generator | A.M.P.I | |||
| Vibration table for electrophysiology set up | Technical manufacturing corporation | |||
| Faraday Cage |
References
- Atwood, H. L., Govind, C. K., Wu, C. F. Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J. Neurobiol. 24 (8), 1008-1024 (1993).
- Feng, Y., Ueda, A., Wu, C. F. A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae. J Neurogenet. 18 (2), 377-402 (2004).
- Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers. J Neurosci. 16, 5443-5456 (1996).
- Brent, J. R., Werner, K. M., McCabe, B. D. Protocol for dissection of Drosophila larvae. J Vis Exp. , (2008).
- Jan, L. Y., Jan, Y. N. Properties of the larval neuromuscular junction in Drosophila melanogaster. J Physiol (Lond). 262, 189-214 (1976).
- Katz, L. C., Shatz, C. J. Synaptic activity and the construction of cortical circuits. Science. 274, 1133-1138 (1996).
Tags
Electrophysiological MethodsRecording Synaptic PotentialsNMJDrosophila LarvaeNeuromuscular JunctionSynaptic TransmissionLarval Neuromuscular SystemSynaptic PhysiologyNeurotransmissionExperimental ManipulationIntracellular RecordingMuscle InnervationTransilluminationExcitatory Junction PotentialsGlutamate ReleaseIntracellular ElectrodeAction Potentials
Cite This Article
Imlach, W., McCabe, B. D. Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae. J. Vis. Exp. (24), e1109, doi:10.3791/1109 (2009).