Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Summary

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Abstract

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (p_rel), synaptic facilitation and depression, as well as the muscle membrane time constant (τ_m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Introduction

Studying synaptic transmission at the neuromuscular junction provides insights into the dynamic relationship between the nervous and skeletal muscular systems and is an excellent model for examining synaptic physiology. The levator auris longus (LAL) is a thin muscle, allowing for the neuromuscular junctions to be easily visualized. Previous reports have described the convenience of using the LAL to examine synaptic drugs and toxins and have characterized the skeletal muscle fiber type characteristics of the LAL^1,2. Numerous studies have used the LAL to examine neuromuscular physiology

Protocol

All animal procedures were performed in accordance with the Animal Care and Use Committee of Wright State University.

1. Mouse Euthanasia

1. In a fume hood, place the mouse in an airtight glass anesthetizing chamber.
2. Expose the mouse via inhalation to a lethal dose of isoflurane (saturating, or ~25%). Leave the mouse in the chamber until no breathing can be observed.
3. Remove the mouse from the chamber and perform a cervical dislocation as a secondary method of euthanasia.

2. Removal of Hair from the Dorsal Surface of Head, Neck, and Back

1. Use an electric shaver to rem....

Results

Figure 8 shows an example of the current pulses (Figure 8A) and the voltage responses (Figure 8B) from one LAL fiber under current-clamp from a 12-week-old wild type R6/2 mouse. The presence of mEPPs indicates that these records were taken from the motor endplate. The records were obtained in normal physiological saline solution. These current-clamp records can be analyzed to determine the R_in

Discussion

Described here is the preparation and use of the mouse LAL muscle for the measurement of neuromuscular transmission under current- or voltage-clamp conditions. There are several important points to consider for dissecting out the LAL. Cleaning excess connective tissue from the muscle aids in electrode impalement, as the electrodes can snag the connective tissue when positioning them for impalement. However, only remove connective tissue that can be taken away easily to limit the chances of damaging the muscle. The isolat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Olympus Compound Microscope	Olympus	BX51WI
10x Objective	Olympus	UMPLFLN10XW
40x Objective	Olympus	LUMPLFLN40XW
Borosilicate Glass	Sutter Instruments	BF150-86-7.5
CCD Camera	Santa Barbara Instruments Group	ST-7XMEI
Mater-9 Pulse Generator	AMPI
Iso-flex Stimulus Isolator	AMPI
pCLAMP 10 Data Acquisition and Analysis Software	Molecular Devices	1-2500-0180
Concentric Bipolar Electrode	FHC	CBDSH75
Ball-joint Manipulator	Narishige
Non-metalic Syringes 34 Gauge	World Precision Instruments	MF34G-5
Nikon Stereomicroscope	Nikon	SMZ800N
No. 5 Forceps	Fine Science Tools
Spring Scissors	Fine Science Tools	15006-09
No. 2 Forceps	Roboz	RS-5Q41
Microdissecting Scissors	Roboz	RS-5912SC
Sylgard 184 Silicone Elastomer Kit	Dow Corning	2404019862
Hair Removal Cream	Nair
Grass SD9 Stimulator	Grass Medical
Model P-1000 Micropipette Puller	Sutter Instruments	P-1000
Axon Digidata 1550 Low-noise Data Acuisition System	Molecular Devices
Low Pass Bessell Filter	Warner Instrument Corp.	LPF-8
Left-handed Micromanipulator	Siskiyou Corp.	MX1641/45DL
Right-handed Micromanipulator	Siskiyou Corp.	MX1641/45DR
Single Motion Controler	Siskiyou Corp.	MC100e
Crossed Roller Micromanipulator	Siskiyou Corp.	MX1641R	This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller
All chemicals were orded from Fisher except,
BTS	Toronto Research Chemicals	B315190
CTX	Alomone Labs	C-270
4-Di-2-Asp	Molecular Probes		Molecular probes is no longer a company. Now ordered through Fisher