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 ...

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

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F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

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The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

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Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

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Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...