Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.
Before starting prepare:
HL3 Solution:
Preparation of stimulating and recording pipettes:
Part 1: Dissection of Drosophila Larvae
Figure 1. Schematic showing the steps involved in cutting the motor axons of dissected larvae. The CNS is lifted with forceps and the motor axons are cut at the base of the brain.
Part 2: Intracellular recordings from larval muscle cells.
Nerve Stimulation:
Part 3: Representative Results
Figure 3. Representative intracellular recording from muscle 6 showing the evoked EJP’s is response to electrical stimulation of the segmental nerve, and the sporadic minature endplate potentials, or mEJP’s. EJP amplitudes in muscle 6 of healthy wild-type larvae, such as Canton S, are typically around 40 mV and the mEJP amplitudes between 1-3 mV.
The methods described here provide a relatively quick and broad way to detect changes in synaptic function at the NMJ. The ability to perform electrophysiological recordings using intact animals in vivo, and perform genetic or pharmacological manipulations, make Drosophila an ideal animal model for investigating the physiological and genetic aspects of neurotransmission.
Since muscle cells are very large, some might prefer to add an additional step to this protocol for two electrode voltage clamp (TEVC) recording. This can be performed on the same larval preparation with the intracellular electrode in place, by positioning a current-passing electrode on the cell. Once the cell is sufficiently voltage clamped, the current response can be recorded.
Although muscle 6 is the most commonly used for electrophysiology recordings, muscles 7 and 12 can also be used.
The methods described here show the essentials of Drosophophila NMJ electrophysiology – techniques that were first described by Jan and Jan in 1976, and have since become the model system for researching synaptic physiology.
Material Name | Tipo | 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 |