This protocol describes the procedures for dissection of Drosophila larva and intracellular recording in the Drosophila larval muscle. |
Category | Glycosyltransferases & related proteins |
Protocol Name | Electrophysiological recording in the Drosophila larval muscle |
Authors
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Itoh, Kazuyoshi
Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University
Komatsu, Akira
*
Department of Judo Therapy, Teikyo University
Nishihara, Shoko
*
Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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Drosophila Ringer solutions
0 Ca2+, 4Mg2+-HL3 solution (zero-Ca++ saline) (pH 7.1) in mM
70 NaCl, 5 KCl, 4 MgCl2, 10 NaHCO3, 5 Trehalose, 115 Sucrose, 5 HEPES or BES |
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4Mg2+-HL3 solution (pH 7.1) in mM
70 NaCl, 5 KCl, 1.5 CaCl2, 4 MgCl2, 10 NaHCO3, 5 Trehalose, 115 Sucrose, 5 HEPES or BES |
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Instruments
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a metallic cage for minimizing electric noise |
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a vibration-free table using N2 gas |
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an amplifier for intracellular recording (Duo 773, or Electro 705: World Precision Instruments Inc., Sarasota, FL) |
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an electric stimulator and an isolator (SEN-3401 and SS-501J: Nihon Kohden Corp., Tokyo, Japan) |
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a data acquisition system (PowerLab, PC, Lab Chart software: AD Instruments, Dunedin, New Zealand) |
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a glass pipette puller for glass micro electrodes and suction electrodes (Sutter Instrument: P-97 Flaming/Brown Micropipette Puller) |
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a micro forge for glass suction electrodes (MF-830: Narishige Co., Ltd., Tokyo, Japan) |
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manipulators (a micromanipulator MM-3 and a water hydraulic micromanipulator MHW-4: Narishige Co., Ltd.) |
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a stereomicroscope and a dark-field illuminator connected to light source with glass fibers |
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dissecting apparatus: a dissecting magnetic chamber, insect pins (#0), carbon razors and its holder, Vannas scissors for ophthalmologists |
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Methods |
1. |
Dissection of Drosophila larva
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1) |
Wash a wandering third-instar larva in distillated water and place a larva over a drop of Ca++ -free saline in the dissecting magnetic chamber (Fig. 1. B-1). |
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Pin the position between posterior spiracles and the pharyngeal apparatus of the larva using the center pins with the dorsal side of the larva facing up (Fig. 1. B-2). |
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Stretch a larva using the center pins and fill the magnetic chamber with zero-Ca++ saline. |
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Use a razor’s edge to make a small hole at the dorsal midline of the larva. |
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5) |
Cut the larva at the dorsal midline using a pair of Vannas scissors from the hole to the anterior end and then from the hole to the posterior end (Fig. 1. B-3). |
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6) |
Use tweezers to clean out internal organs other than the muscular system and tracheae (Fig. 1. B-4). |
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7) |
Wash intensively with drops of saline to remove organ debris as much as possible. |
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8) |
Use the scissors to cut the segmental nerve and then remove the central nervous system using tweezers (Fig. 1. B-5). |
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9) |
Stretch gently the posterior and anterior sides of the larva using the corner pins. |
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2. |
Electrophysiological recording of excitatory junction potentials (EJPs) in the Drosophila larval muscle
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1) |
Change the saline in the chamber with 4Mg2+-HL3 solution and then place the chamber on the stage plate of dark-field stereomicroscope on vibration-free table in metallic cage. |
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2) |
Make ‘glass suction electrodes’ and ‘glass micro electrodes’ from the borosilicate glass with a glass pipette puller. Blunt ‘glass suction electrodes’ are forged to have an inner diameter of 7-8 μm. |
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3) |
Fill in the ‘glass micro electrode’ with 3M KCl using 1 mL syringe with a long needle. |
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4) |
On the vibration-free table, load a ‘glass suction electrode’ to a manipulator connecting the isolator of the electric stimulator and a ‘glass micro electrode’ to another manipulator connecting an amplifier. |
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5) |
Draw the free cut end of the lateral nerve into the glass suction electrode by gentle sucking a syringe connected to it by a tigon tube (Fig. 2B). |
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6) |
Insert the ‘glass micro electrode’ into muscle cell #6 at abdominal segment 2-6 by a gentle tapping of the manipulator (Fig. 2B). |
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7) |
Stimulate the nerve with a stimulator at 1 Hz with suprathreshold stimulating pulse. |
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At the same time, record the EJPs at room temperature using ‘Lab Chart’ software (Fig. 3). |
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Analyze only the recordings with resting membrane potentials below -60 mV. |
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Discussion | The amplitude of EJPs in wild-type larvae and the mutant larvae of a glycosyltransferase were recorded and compared (Fig. 3). There is no significant difference between these amplitudes. It is suggested that the synaptic transmission at neuromuscular junction is normal in the mutant larvae. |
Figure & Legends |
Figure & Legends
Fig. 1. Dissection of a larval muscle on magnetic chamber.
(A) Magnetic chamber for dissection. (B) Dorsal view of a larva and dissection procedure (1-5).
Copyright holder is Cold Spring Harbor Laboratory Press.
Fig. 2. Pattern diagram of Drosophila larval muscle and electrophysiological experiment.
(A) Body wall muscle of a third instar larva stained with FITC-phalloidin. Each muscle is numbered (1-31). Modified from Figure 11.1., page 176 in reference 1. (B) Motor nerve at abdominal segment 2-6 is drawn into ‘glass suction electrode’ and stimulated with a stimulator at 1 Hz. Intracellular recording is performed by ‘glass micro electrode’ which is inserted into muscle cell #6.
Copyright holder for Figure 2.(A) is Cold Spring Harbor Laboratory Press.
Fig. 3. Intracellular recordings.
Traces showing excitatory junction potentials (EJPs) evoked by 1 Hz stimulation, recorded from muscle cell #6 in third instar larvae from wild-type larvae (upper record) and from the mutant larvae of the glycosyltransferase (lower record). Loss of the glycosyltransferase activity has no effect on the amplitude. |
Copyrights |
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This work is released underCreative Commons licenses
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Date of registration:2016-07-08 14:04:18 |
- Bellen, H.J., and Budnik, V. The neuromuscular junction. In: Sullivan W, Hawley RS, Ashburner M, editors. Drosophila protocols. Cold Spring Harbor Laboratory; 2000. pp. 175–199.
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Komatsu, Akira,
Nishihara, Shoko,
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Komatsu, Akira,
Nishihara, Shoko,
(2016).
Electrophysiological recording in the Drosophila larval muscle.
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Komatsu, Akira,
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(2016).
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