Name: levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions
Uploaded: 2018-05-05T14:00:00
Description: Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

We thank Dr. Mark M. Rich and Daniel Miranda for editorial comments, Ahmad Khedraki for helping establish this technique, and Wright State University for financial support (startup fund to A.A.V.).

All animal procedures were performed in accordance with the Animal Care and Use Committee of Wright State University.
1. Mouse Euthanasia
<ol>
	<li>In a fume hood, place the mouse in an airtight glass anesthetizing chamber.</li>
	<li>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.</li>
	<li>Remove the mouse from the chamber and perform a cervical dislocation as a secondary method of eutha...

<ol>
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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...

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 LAL1,2. Numerous studies have used the LAL to examine neuromuscular physiology3,4,5,6,7,8. For electrophysiology, the ability to easily observe LAL neuromuscular junctions allows for the accurate placement of microelectrodes at the motor endplate and greatly reduces space clamp issues in recording synaptic transmission. Current-clamp recordings of the muscle membrane properties, such as the membrane time constant (&#964;m) and input resistance (Rin) are readily obtained. Furthermore, these properties can be measured from the same muscle fibers used to record neuromuscular transmission, allowing for a direct comparison of synaptic function to the muscle membrane properties. Analysis of these data can provide key insights into the physical mechanisms of many neuromuscular diseases and states of altered activity.
A key aspect of the technique described here is the use of voltage-clamp&#160;for synaptic recordings, which are not subject to the non-linearities encountered in current-clamp&#160;and are independent of the muscle membrane properties. Advantages of using voltage-clamp as opposed to current-clamp to examine neuromuscular transmission were established by pioneering efforts in the 1950s9. Under current-clamp, EPPs that exceed 10-15 mV in amplitude are not a linear product of the mEPP amplitude9. For example, if the average mEPP is 1 mV, an EPP of 5 mV can be assumed to be the product of 5 mEPPs (QC of 5); whereas, an EPP of 40 mV will be the product of more than 40 mEPPs. This non-linearity at larger EPPs occurs because the driving force for the EPP, which is the difference between the membrane potential and equilibrium potential for the acetylcholine receptor (~-10 mV), substantially decreases during large EPPs. This issue is avoided in voltage-clamp experiments because the muscle membrane potential does not change during voltage-clamp experiments. A drawback is that voltage-clamp experiments are technically more difficult to complete than current-clamp recording. With this in mind, McLachlan and Martin developed a straightforward mathematical correction that accounts for non-linearities in current-clamp recordings of EPPs10. The corrections work well11,12,13, but importantly, assume that the muscle membrane properties have not been disrupted.
The muscle membrane properties are especially important to consider if studying conditions or disease states that disrupt the muscle. For example, skeletal muscle from the R6/2 transgenic model of Huntington&#39;s disease is hyperexcitable due to a progressive reduction in the resting chloride and potassium currents14,15. As a consequence, mEPPs and EPPs are amplified in the R6/2 skeletal muscle. Certainly, additional factors can alter mEPPs and EPPs. Work with a different model of Huntington&#39;s disease mice (R6/1) found changes in EPPs that seemed to be related to SNARE-proteins8. To assess the mechanisms causing altered neuromuscular transmission, it would be beneficial to eliminate the effects of altered muscle membrane properties by using a voltage-clamp. In a recent study, the R6/2 neuromuscular transmission was studied under both current- and voltage-clamp conditions using the technique described herein. The entirety of the motor endplates were voltage-clamped with less than 1% error by placing two microelectrodes within the length constant of the endplate16. It was shown that voltage-clamp and corrected current-clamp records yielded contrasting measurements of neuromuscular transmission in R6/2 muscle. This highlights that it may be difficult to correct EPPs for non-linearities if the muscle membrane properties have been altered and shows the benefits of obtaining voltage-clamp records that are independent of the muscle membrane properties. The protocol presented herein is ideal for examining conditions or disease states that affect synaptic transmission and the postsynaptic membrane properties.

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus Compound Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:288px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">UMPLFLN10XW</td>
			<td style="width:288px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">LUMPLFLN40XW</td>
			<td style="width:288px;"></td>
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			<td height="24" style="height:25px;width:216px;">Borosilicate Glass</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">BF150-86-7.5</td>
			<td style="width:288px;"></td>
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			<td height="40" style="height:40px;width:216px;">CCD Camera</td>
			<td style="width:183px;">Santa Barbara Instruments Group</td>
			<td style="width:119px;">ST-7XMEI</td>
			<td style="width:288px;"></td>
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			<td height="29" style="height:29px;width:216px;">Axoclamp 900A Amplifier</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;">2500&#8208;0179&#160;&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Mater-9 Pulse Generator</td>
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			<td height="21" style="height:21px;width:216px;">Iso-flex Stimulus Isolator</td>
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			<td height="42" style="height:42px;width:216px;">pCLAMP 10 Data Acquisition and Analysis Software</td>
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			<td style="width:119px;">1-2500-0180</td>
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			<td height="21" style="height:21px;width:216px;">Concentric Bipolar Electrode</td>
			<td style="width:183px;">FHC</td>
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			<td height="21" style="height:21px;width:216px;">Ball-joint Manipulator</td>
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			<td height="52" style="height:52px;width:216px;">Non-metalic Syringes 34 Gauge</td>
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			<td height="21" style="height:21px;width:216px;">Nikon Stereomicroscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
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			<td height="21" style="height:21px;width:216px;">No. 5 Forceps</td>
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			<td style="width:119px;">15006-09</td>
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			<td height="21" style="height:21px;width:216px;">No. 2 Forceps</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5Q41</td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;width:216px;">Microdissecting Scissors</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5912SC</td>
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			<td height="42" style="height:42px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
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			<td align="right" style="width:119px;">2404019862</td>
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			<td height="21" style="height:21px;width:216px;">Hair Removal Cream</td>
			<td style="width:183px;">Nair</td>
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			<td height="27" style="height:27px;width:216px;">Grass SD9 Stimulator</td>
			<td style="width:183px;">Grass Medical</td>
			<td style="width:119px;"></td>
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			<td height="46" style="height:46px;width:216px;">Model P-1000 Micropipette Puller</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
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			<td height="37" style="height:37px;width:216px;">Axon Digidata 1550 Low-noise Data Acuisition System</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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			<td style="width:183px;">Warner Instrument Corp.</td>
			<td style="width:119px;">LPF-8</td>
			<td style="width:288px;"></td>
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			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DL</td>
			<td style="width:288px;"></td>
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			<td height="44" style="height:44px;width:216px;">Right-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DR</td>
			<td style="width:288px;"></td>
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			<td height="44" style="height:44px;width:216px;">Single Motion Controler</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MC100e</td>
			<td style="width:288px;"></td>
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			<td height="161" style="height:161px;width:216px;">Crossed Roller Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641R</td>
			<td style="width:288px;">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</td>
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			<td colspan="3" height="21" style="height:21px;width:517px;">All chemicals were orded from Fisher except,</td>
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			<td height="42" style="height:42px;width:216px;">BTS</td>
			<td style="width:183px;">Toronto Research Chemicals</td>
			<td style="width:119px;">B315190</td>
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			<td height="21" style="height:21px;width:216px;">CTX</td>
			<td style="width:183px;">Alomone Labs</td>
			<td style="width:119px;">C-270</td>
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			<td height="42" style="height:42px;width:216px;">4-Di-2-Asp</td>
			<td style="width:183px;">Molecular Probes</td>
			<td style="width:119px;"></td>
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levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions

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 (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;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.

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 Rin</...

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

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.

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

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The overall goal of this procedure is to isolate the levator auris longus muscle and its nerve in order to record synaptic currents at the neural muscular junction. This method can help answer key questions in synaptic physiology such as the end plate current size, quantal content, probability of release and relationships between neurotransmission and muscle excitability. The main advantage of this technique is the detailed electrophysiological recordings from a single synapse can easily be combined with live cell optical experiments.Though this method can provide insight into synaptic function and feedback communication between nerve and muscle, it can also be applied to other systems ranging from drosophila to mammals. Generally, individuals new to this method will struggle because the mammalian nerve muscle prep is fragile, particularly the nerve. Carefully acquiring and interpreting the subsequent voltage clamp data can also be challenging.Once mastered, this technique can be done in one hour if it is performed properly. Demonstrating the procedure will be Steve Burke from my laboratory. To begin this procedure, under a stereo-dissecting microscope, make a small incision in the skin on the back of the mouse at the level of the scapulae.Use a pair of microdissection scissors. Cut the skin over the head and the back. Next, pull up the skin along the incision near the scapulae.Cut the muscles that are inferior to the LAL by starting at the right scapula. Then, gently lift the cut tissue immediately to the right of the midline. Make a cut toward the left ear with the blades of the scissors pressed against the cranium to remove several layers of muscle that are inferior to the left LAL.Avoid cutting the nerve that innervates the LAL which wraps around the ear canal and enter the muscles on the medial side of the ear. Continue cutting through the ear canal keeping as much of the nerve attached as possible. Then, cut the fatty tissue behind the ear canal along the ventrolateral portion of the ear.Cut along the left scapula similar to the procedures done on the right side to remove the muscle completely from the mouse. Next, cut off the pinna of the ear along the base leaving the cartilaginous part of the ear attached to the LAL. Flip the muscle so that the inferior side is facing up.To isolate LAL muscle, place the LAL and the surrounding tissue into a Petri dish with a silicon elastomer bottom and pin the dissected tissue to the bottom. Wash the tissue frequently in a physiological saline solution. Then, place an insect pin through the ear canal to hold the prep in place.Using smaller pins, pin the remaining tissue on the opposite side of the midline of the LAL. Use a pair of forceps. Gently pull the skin up on the lateral portion of the ear to stretch the muscle out and place a small pin through the skin.Repeat this step until the tissue is well-secured to the dish. Remove the muscles that are covering the LAL and those bound to the LAL via connective tissue and are especially tight near the midline. Subsequently, pull up the overlying muscle layer and cut the connective tissue with the blades oriented toward the muscle layer being pulled.Cut toward the midline until about 3/4 of the way to the midline and keep removing muscle layers until only the LAL remains. During the cleaning process, ensure that the nerves are not damaged. Following that, remove some of the remaining connective tissue that is covering the LAL to aid in impalement of the electrodes.Only remove tissue that can be done easily without risk of damaging the LAL in the process. To identify the nerve that innervates the LAL, using a nerve stimulator, touch the nerves with the nerve stimulator. When the muscle contracts, the correct nerve has been identified.Then, carefully grab the tissue near the nerve and use spring scissors to separate the nerve from the tissue surrounding the ear. To minimize damage, keep most of the nerve embedded in some surrounding tissue which will be used later to secure the nerve to the recording dish. At this point, the investigator can take an hour break as long as the LAL is bathed in 20 milliliters or more of a physiological saline solution.Next, unpin and transfer the muscle to a stage insert under the dissection microscope for electrophysiology experiments. Pin the muscle at the ends and along its edge. Position the nerve perpendicular to the muscle fibers and pin it to the bottom of the dish through some excess tissue that was left intact at the end of the nerve.Keep the tissue bathed in a physiological saline solution at all times. For electrophysiology experiment, secure the perfusion chamber with the LAL to the microscope stage. Place the reference electrode into a cup filled with three-molar potassium chloride which is connected to the recording chamber via an agar bridge.At this point, position the nerve-stimulating electrode on the nerve. Expose the LAL prep to 4-Di-2-Asp for 10 minutes to achieve adequate florescence for neuromuscular junction visualization. After 10 minutes, exchange the 4-Di-2-Asp solution with the normal calcium solution.In the meantime, fill the glass capillaries with the appropriate solutions for the voltage-sensing and current-passing electrodes. Gently tap the capillary to remove any air bubbles and secure the filled capillary into the electrode holder on the headstage. Using an upright microscope with standard bright field and florescence illumination, look for a bright band of fluorescent green neuromuscular junctions running perpendicular to the muscle fibers along the prep with a low-magnification water-emersion objective.Then, switch to a higher magnification water-emersion objective to identify a neuromuscular junction on the top layer of muscle in order to examine with electrophysiology. Using primarily bright field, position the electrode above the muscle membrane within 100 microns of the identified neuromuscular junction. This figure shows an example of the current pulses and the voltage responses from one LAL fiber under current clamp from a 12-week-old wild-type R62 mouse.The records were obtained in normal physiological saline solution and the presence of mEPPs indicates that these records were taken from the motor end plate. Here is a representative recording of an EPC and two mEPCs obtained under voltage clamp condition. This figure shows the superimposed EPCs and mEPCs from a representative fiber.Once mastered, this technique can be done in one hour if it is performed properly. Following this procedure, other methods like live-cell imaging with membrane dyes such as FM143 or histology can be performed in order to answer questions related to vesicle release uptake or morphological changes. After its development, this technique paved the way for researchers in the field of physiology to explore synaptic transmission and nerve-cell communication in organisms ranging from drosophila to mammals.After watching this video, you should have a good understanding of how to isolate the innervated levator auris longus muscle in order to record synaptic currents at the neuromuscular junction. While attempting this procedure, it's important to remember to take great care in removing the muscle tissue adjoined to the LAL and isolating the nerve.

Research

JoVE Journal

Neuroscience

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