Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster
成年黑腹果蝇食窦肌肉9（CM9）的电生理记录
发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2401 浏览次数: 7920
评审: Jihyun KimAlexandros C. KokotosAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Feb 2014
Abstract
The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous postsynaptic excitatory potentials (mEPSPs) at a mature glutamatergic synapse. Combined with CM9-specific genetic technologies, the CM9 NMJ provides a powerful experimental system to better understand the regulation of neurotransmitter release at a mature synapse.
Keywords: Drosophila (果蝇)
Aging (衰老)
Neuromuscular junction (神经肌肉接头)
Neurotransmission (神经传递)
Background
A significant hurdle in defining changes in presynaptic function during aging has been due to the lack of a simple model system for performing the electrophysiological recordings necessary to thoroughly characterize the release of neurotransmitter from the presynaptic nerve terminal. Existing rodent models suffer from the significant cost issues associated with aging studies and the technical difficulty of using electrophysiological recordings on single defined nerve terminals with consistent release parameters. To overcome these obstacles, we have pioneered a model synaptic system in the adult Drosophila for analyzing the effects of age on presynaptic function during neurotransmission, the CM9 NMJ located on the fly proboscis (Rawson et al., 2012; Mahoney et al., 2014; Mahoney et al., 2016) (Figure 4A). Briefly, the presynaptic arbor of the CM9 motor neuron (MN) converges upon the 15 muscle fibres of the CM9 muscle to form 35 individual distinct innervations (Rawson et al., 2012). The CM9 MN has been shown to be necessary for the contraction of the CM9 muscle and is the only source of glutamatergic input for the CM9 muscle (Kimura et al., 1986; Gordon and Scott, 2009). Given the highly-conserved nature of the mechanisms underlying synaptic vesicle (SV) release between flies and mammals, and the resemblance to the central synapses found in the mammalian CNS, this makes the CM9 NMJ a powerful model for investigating presynaptic function.
Materials and Reagents
- Sterile disposable filter (0.22 μm pore size, aPES membrane 19.6 cm2 CA membrane) (such as Corning® 250 ml Vacuum Filter/Storage Bottle System, Corning, catalog number: 430767 )
- Borosilicate glass capillary with filament (OD 1.50 mm, ID 0.86 mm) (Sutter Instrument, catalog number: BF150-86-10 )
- Borosilicate glass capillary without filament (OD 1.50 mm, ID 0.86 mm) (Sutter Instrument, catalog number: B150-86-10 )
- Silver wire (A-M Systems, catalog number: 782000 )
- Diamond coated bench stone (such as DMT 8 in Dia-Sharp bench stone)
- Drawn out P200 tip
- 10 ml syringe (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S7510-10 )
- Minutien pins (Fine Science Tools, catalog number: 26002-10 )
- Fine paint brush
- Flies of desired genotype and age
Note: UAS constructs can be driven within the CM9 motor neuron via the use of the E49-Gal4 driver (E49-Gal4 from Ulrike Heberlein’s Gal4 collection). - 50% Bleach
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-250G )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 21115-250ML )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 63069-500ML )
- Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761-500G )
- Trehalose (Sigma-Aldrich, catalog number: T9531-25G )
- HEPES (Sigma-Aldrich, catalog number: H3375-25G )
- Sucrose (Sigma-Aldrich, catalog number: 84097-250G )
- Modified hemolymph like solution (HL3.1) (see Recipes)
Equipment
- Vannas spring scissors–2 mm cutting edge (Fine Science Tools, catalog number: 15000-03 )
- Tungsten Dissecting needle, 125 mm, Ultra fine (Roboz Surgical Instrument, catalog number: RS-6063 )
- Micro Dissecting needle holder, 5 ¼” (Roboz Surgical Instrument, catalog number: RS-6060 )
- Fine forceps (such as Dumont #5CO, Fine Science Tools, catalog number: 11295-20 and Dumont #3, Fine Science Tools, catalog number: 11231-30 )
- Dissecting stereoscopic zoom microscope (such as ZEISS, model: SteREO Discovery.V8 )
- Narrow Format Manipulator Systems (such as Sutter Instrument, model: ROE-200 )
- Horizontal Micropipette puller (such as programmable Flaming/Brown type micropipette puller, Sutter Instrument, model: P-97 ) with platinum filament
- Micro Forge electrode polisher (such as NARISHIGE, model: MF-900 )
- Upright confocal microscope (such as Olympus, model: BXS1WI ) with 10x and 40x water objectives
- Power lab 4/30 digital converter (ADInstruments, model: ML866 )
- Neuroprobe amplifier 1600 (A-M Systems, model: Model 1600 , catalog number: 680100)
- Stimulator (Digitimer, model: DS2A )
- Labchart 7 (ADInstruments)
Software
- Prism 6 (GraphPad software)
- Mini Analysis (comparable version 6.0.3, Synaptosoft Inc.)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Eaton, B. A. and Mahoney, R. E. (2017). Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster. Bio-protocol 7(14): e2401. DOI: 10.21769/BioProtoc.2401.
- Mahoney, R. E., Rawson, J. M. and Eaton, B. A. (2014). An age-dependent change in the set point of synaptic homeostasis. J Neurosci 34(6): 2111-2119.
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分类
神经科学 > 细胞机理 > 突触生理学
细胞生物学 > 细胞信号传导 > 胞内信号传导
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