The project was supported by Lundbeck Foundation, the Novo Nordisk Foundation, the Danish Medical Research Council, the Ludvig and Sara Elsass Foundation, the Foundation for Research in Neurology and Jytte and Kaj Dahlboms Foundation. R.A is supported by an Early Career Post-Doctoral Fellowship of the National Health and Medical Research Council of Australia (#1091006)

All experimental procedures described here complied with the Animal Care and Ethics Committee of UNSW Sydney and were performed in accordance with the National Health and Medical Research Council (NHMRC) of Australia regulations for animal experimentation.
1. Experimental Set Up
NOTE: 12 week old, female Long-Evans rats were used in this procedure.
<ol>
	<li>Anesthetize the rat in an induction chamber using 4% isoflurane and 1 L per min O2 flow rate. Co...

<ol>
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S., Moldovan, M., Krarup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+motor+axons+of+SOD1(G127X)+mutant+mice+are+susceptible+to+activity-dependent+degeneration.">Peripheral motor axons of SOD1(G127X) mutant mice are susceptible to activity-dependent degeneration.</a> Neurosci. 241, 239-249 (2013).</li><li>Fledrich, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+neuregulin-1+modulates+disease+pathogenesis+in+rodent+models+of+Charcot-Marie-Tooth+disease+1A.">Soluble neuregulin-1 modulates disease pathogenesis in rodent models of Charcot-Marie-Tooth disease 1A.</a> Nat. 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The described procedure demonstrates a simple and reliable, minimally-invasive technique that allows assessment of the biophysical properties and membrane potential of the axon in a short period of time. Compared with other more invasive techniques, which require the exposure of the nerve, the present method of axonal excitability testing induces minimal tissue damage thus enabling in vivo assessment that preserves the physiological conditions of the nerve of interest and allows for repeated measurements.
<p...

The use of electrophysiological techniques is an essential tool for the in vivo investigation of peripheral nerve function in neurological disorders. Conventional nerve conduction methods utilize supramaximal stimuli to record motor action potential amplitude and latency. These techniques therefore provide useful information on the number of conducting fibers and on the conduction velocity of the fastest fibers. A valuable complementary tool is that of axonal excitability testing. This technique uses sophisticated electrophysiological stimulation patterns to indirectly assess the biophysical properties of peripheral nerves, such as the activity of ion channels, energy-dependent pumps, ion exchange processes and membrane potential1.
Axonal excitability testing is commonly utilized in the clinical setting to investigate pathophysiological processes and effects of therapeutic interventions on various neurological disorders. Importantly, axonal excitability measures are sensitive to therapeutic interventions that affect peripheral nerve function such as intravenous immunoglobulin (IVIg) therapy2, chemotherapy3 and calcineurin inhibitor (CNI) treatment4. Although these studies have provided important insights, clinical studies often preclude investigation of early disease traits and novel therapeutic options5. Therefore, the use of these methods in animal models of neurological disorders has recently gained traction6,7,8,9. Indeed, these methods provide an opportunity to understand the specific nerve excitability changes associated with these disorders, thus advancing translational research.
The procedure described here is a simple and reliable method to record axonal excitability measures on the ulnar nerves of the intact rat.

<table>
	<tbody>
		<tr>
			<td>QTracS Program</td>
			<td>Digitimer Ltd.</td>
			<td></td>
			<td>Axonal excitability program</td>
		</tr>
		<tr>
			<td>AM-Systems 2200, Analog Stimulus Isolator, 2200V/50Hz</td>
			<td>SDR Scientific</td>
			<td>850005</td>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>High Performance AC Amplifier Model LP511</td>
			<td>Grass Technologies</td>
			<td></td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Humbug 50/60Hz Noise eliminator</td>
			<td>Quest Scientific Instruments</td>
			<td>726310</td>
			<td>Noise eliminator</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Monopolar Subdermal Needle Electrodes</td>
			<td>Grass Technologies</td>
			<td>F-E2-24</td>
			<td>Recording electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Electroencephalography Needle Electrodes</td>
			<td>Cephalon</td>
			<td>9013L0702</td>
			<td>Stimulating electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Multifunction I/O Device Model USB-6341</td>
			<td>National Instruments</td>
			<td></td>
			<td>Multifunction input/output device</td>
		</tr>
		<tr>
			<td>Iron Base Plate IP</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Rotating X-block X-4</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Magnetic Stand GJ-8</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Micromanipulator M-3333</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurement of the rat ulnar nerve with axonal excitability testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

Electrophysiological measures of the rat ulnar nerve were obtained with the present protocol. Figure 3 demonstrates a representative recording from the left ulnar nerve of a 12 week-old female Long Evans rat. Compound muscle action potential relates to the number of conducting fibers that are simultaneously activated. The supramaximal peak response (mV) (Figure 3A) demonstrates the peak response achieved when incrementally increa...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing at JoVE.com

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

The overall goal of this technique is to determine a range of excitability properties for the ulnar nerve in living rats. This method can help answer key questions in the field of neuroscience such as, can we measure the pathological changes of nerve function prior to irreversible neurodegenerative events? The main advantage of this technique is that it allows the indirect assessment of peripheral nerve function in vivo enabling repeat measurements for the characterization of disease progression.Before beginning the procedure, confirm the appropriate level of sedation by pedal withdrawal and corneal reflex in a 12-week-old female Long-Evans rat. Place the rat on a feedback controlled heat mat and insert a rectal thermometer probe to maintain the body temperature at 37 degrees Celsius. To record the compound muscle action potential, insert the reference electrode through the dorsal aspect of the fourth digit and the recording electrode through the hypothenar muscle.Place the ground electrode through the skin on the superior aspect of the forearm between the stimulating and recording electrodes. Then insert the percutaneous stimulating needle cathode approximately four millimeters distal to the cubital tunnel at the elbow and insert the anode approximately one centimeter proximally through the skin of the axilla region. Next, use a semi-automated computer controlled axonal excitability program linked to a constant current stimulator and an amplifier to apply a one millisecond square wave pulse to the ulnar nerve with the cathode needle electrode and to record the compound muscle action potential from the hypothenar muscle.Carefully adjust the angle and/or position of the cathode until an optimal biphasic response curve with a consistent amplitude is achieved and stabilize the cathode with a repositionable electrode holder. For threshold tracking, it is critically important that there is a proportionate response to the stimulus. A successful response is indicated by a sigmoid shape in the stimulus response curve.To record a stimulus response curve, incrementally increase the stimulus intensity of a one millisecond impulse until a maximum response is obtained. Record multiple axonal excitability parameters including the threshold electrotonus, current threshold relationship, and recovery cycle. Then transfer the rat into an individual cage with monitoring until full recumbency.The inward and outward rectifying currents can be assessed by depolarizing and hyper polarizing currents in the current voltage parameter. Data regarding the internodal conductances and membrane potential can be obtained by long subthreshold depolarizing and hyper polarizing currents represented by the threshold electrotonus waveform. In this representative experiment, sequential axonal excitability testing on the left and right forelimbs within 35 minutes after the loss of the pedal withdrawal reflex revealed no significant differences between the left and right ulnar nerves in the standard nerve conduction parameters, compound muscle action potential amplitude and latency nor are differences observed in the nerve excitability variables including super excitability and threshold electrotonus hyper polarizing at 90 to 100 milliseconds.Once mastered, this technique can be completed in 20 to 30 minutes if it is performed properly. While attempting this procedure, it is important to take care to place the electrodes in their appropriate positions to ensure an accurate stimulus response curve and successful threshold tracking. After its development, this technique paved the way for researchers in the field of neuroscience to explore the biophysical properties of peripheral nerves in both the clinical setting and in animal models.After watching this video, you should have a good understanding of how to perform a nerve excitability protocol and to obtain a range of biophysical indices in the ulnar nerve.

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Neuroscience

9.0K vistas.  University of New South Wales. The overall goal of this technique is to determine a range of excitability properties for the ulnar nerve in living rats. This method can help answer key questions in the field of neuroscience such as, can we measure the pathological changes of nerve function prior to irreversible neurodegenerative events? The main advantage of this technique is that it allows the indirect assessment of peripheral nerve function in vivo enabling repeat measurements for the characterization of disease progress...