This study was financially supported mainly by the two grants from Lundbeck Foundation (Grant number R191-2015-931 and Grant number R290-2018-751). Additionally, the study was financially supported by Novo Nordisk Foundation Challenge Programme (Grant number NNF14OC0011633) as part of the International Diabetic Neuropathy Consortium.

All subjects must provide written consent prior to examination, and the protocol must be approved by the appropriate local ethical review board. All methods described here were approved by the Regional Scientific Ethical Committee and Danish Data Protection Agency.
1. Preparation of the subject
<ol>
	<li>Assess subjects&#39; medical histories to ensure that they do not have any previous nervous system disorders other than the disease group that will be investigated.</li>
	<li>Inform the subject in detail about the examinations and request to obtain written consent.
	<ol>
		<li>Inform the subject about the insertion of two needles in a leg muscle and that the muscle fibers will be stimulated with weak current.</li>
		<li>Explain that the sensation may feel slightly unpleasant.</li>
		<li>Inform the subject that the stimulation can be turned off immediately at any moment during the recording in case of any discomfort.</li>
	</ol>
	</li>
	<li>Clean the subject&#39;s lower leg with alcohol.</li>
	<li>Insert the stimulating monopolar needle electrode (25 mm x 26 G) over the anterior tibial muscle and adhesive surface electrode as the anode 1 cm distal to the monopolar needle (Figure 1).</li>
	<li>Place a ground electrode distal to the anode.</li>
	<li>Insert the recording concentric needle electrode (25 mm x 30 G) about 2cm proximal to the stimulating monopolar needle electrode along the muscle fibers (Figure 1).</li>
	<li>Connect the recording concentric needle and ground electrodes to the preamplifier.</li>
	<li>Ask the subject to remain silent and avoid movement during the examination.</li>
	<li>Zero the output of the stimulator and connect the stimulating electrodes to the stimulator (Figure 1).</li>
	<li>Maintain the skin temperature between 32&#8211;36 &#176;C using a warming lamp.</li>
</ol>
2. Recording of the MVRCs
<ol>
	<li>Start the semi-automated recording software using the muscle excitability recording protocol and turn on the stimulator. Stimulations will start at 2.5 mA with 1 Hz.</li>
	<li>Increase the stimulus intensity manually by hitting the Insert key until a response is recorded (max = 10 mA).
	<ol>
		<li>Adjust the stimulating and recording needles if necessary, until recording an acceptable response with a stimulus intensity of less than 10 mA. The shape of the muscle action potential should be triphasic, if possible, and stable. Avoid large twitches of the whole muscle.</li>
		<li>Invert the muscle action potential by hitting the minus key (-) if the potential appears upside down. 
		NOTE: A magenta horizontal line appears on the screen indicating the width of the action potential.</li>
	</ol>
	</li>
	<li>Adjust the position and length of the magenta line by dragging the line with the mouse. The green horizontal line represents the baseline.</li>
	<li>Click OK to start recording the MVRCs.</li>
	<li>Select a stimulus response relationship from the main options.</li>
	<li>Increase stimulus intensity by hitting the Insert key to a max of 10 mA or tolerable.</li>
	<li>Click OK to start descending the stimulus response curve.</li>
	<li>Click OK when the test stimulus reaches zero.</li>
	<li>Set the stimulus intensity to level for stable latency.</li>
	<li>Click OK to return to the main menu.</li>
	<li>Select the option 1/2/5 conditioning stims for RC.</li>
	<li>Select a protocol from recovery cycle options (e.g., start quick recovery cycle [skip alternate delays]), which is the default. 
	NOTE: The recording continues automatically for 34 steps with decreasing inter-stimulus intervals (ISIs).</li>
	<li>Make sure that muscle action potential is stable during the recording and that the needle has not moved. The screen changes automatically to main options when the 34 steps have completed.</li>
	<li>Click on Finish recording | Close file | OK, unless a ramp-up frequency or 20 Hz s recordings is being performed.</li>
	<li>Finish the recording and save the data by clicking on the Close file and save data button.</li>
</ol>
3. MVRC analyses
<ol>
	<li>Start the analyzing software program to perform the analysis offline.</li>
	<li>Select the recording that will be analysed and click on the OK button.</li>
	<li>Click on Load parameters from the Files menu.</li>
	<li>Select MANAL9 option for the analysis. If this is not present on the list, click on Browse to find this file. Click OK to continue.</li>
	<li>When a description of MAnal9 muscle excitability analysis appears, click OK to continue.
	<ol>
		<li>Invert the muscle action potential by typing MM-1 if the potential appears upside down.</li>
		<li>Right-click the mouse to make the magenta line visible. Set the window to the base of the peak response and with a width corresponding roughly to the width of the action potential at that height. Drag with the mouse to adjust the window. The window determines the latencies within which the height and latency are measured, as indicated by the pale blue lines, and green line indicates the baseline. Click OK to continue.</li>
	</ol>
	</li>
	<li>Click OK to remeasure the latencies and peaks. This will be done automatically. 
	NOTE: In the display of the remeasured latencies, the latencies are measured to shorter delays than original ones. This is because the responses to conditioning stimuli alone were subtracted from responses to the conditioning plus the test. This ensures that conditioning stimuli do not interfere with latency measurements. As is indicated in the prompt box, single bad points can be eliminated by positioning the cursor (vertical red line) over the point and hitting the ~ key. The bad point is replaced with mean of values on either side in same channel. If there are no bad points, set DE (display end) to just after the last latency required.</li>
	<li>Click OK to create an RMC file.</li>
	<li>Ignore most of the options appearing in the &#34;Create RCC or RMC&#34; form, since these are concerned with measurements of C-fiber rather than MVRCs. Click Save and Exit to continue. After saving the RMC file, the prompt box provides different options</li>
	<li>If frequency ramp and/or repetitive stimulation data have been recorded, follow the instructions to analyse these. Otherwise, select Go straight to create MEM file option to create a MEM file. Click OK to continue.</li>
	<li>Click Save and Exit to continue.</li>
	<li>Click OK to add the RMC data to MEM file.</li>
	<li>Click Add from Input RMC file to add this data to the MEM file, then change the directory to save the composite MEM file. Then, click Save and Exit to save it.</li>
	<li>Click OK to save the remeasured QZD file to allow differentiation from the original QZD file using a # sign.</li>
</ol>

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	<li>Tankisi, H., 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=Pathophysiology+inferred+from+electrodiagnostic+nerve+tests+and+classification+of+polyneuropathies.+Suggested+guidelines.">Pathophysiology inferred from electrodiagnostic nerve tests and classification of polyneuropathies. Suggested guidelines.</a> Clinical Neurophysiology. 116 (7), 1571-1580 (2005).</li><li>Gregorio, C. C., Hudecki, M. S., Pollina, C. M., Repasky, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+denervation+on+spectrin+concentration+in+avian+skeletal+muscle.">Effects of denervation on spectrin concentration in avian skeletal muscle.</a> Muscle and Nerve. 11 (4), 372-379 (1988).</li><li>Kotsias, B. A., Venosa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+sodium+and+potassium+permeabilities+in+the+depolarization+of+denervated+rat+muscle+fibers.">Role of sodium and potassium permeabilities in the depolarization of denervated rat muscle fibers.</a> Journal of Physiology. 392, 301-313 (1987).</li><li>Kirsch, G. E., Anderson, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+multi-fiber+muscle+velocity+recovery+cycles+recorded+at+a+single+site+using+submaximal+stimuli.">Validity of multi-fiber muscle velocity recovery cycles recorded at a single site using submaximal stimuli.</a> Clinical Neurophysiology. 123 (11), 2296-2305 (2012).</li><li>Z'Graggen, W. J., Troller, R., Ackermann, K. A., Humm, A. M., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+recovery+cycles+of+human+muscle+action+potentials:+repeatability+and+variability.">Velocity recovery cycles of human muscle action potentials: repeatability and variability.</a> Clinical Neurophysiology. 122 (11), 2294-2299 (2011).</li><li>Lee, J. H. F., Boland-Freitas, R., Ng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+excitability+changes+in+normal+human+aging.">Sarcolemmal excitability changes in normal human aging.</a> Muscle and Nerve. 57 (6), 981-988 (2018).</li><li>Lee, J. H. F., Boland-Freitas, R., Ng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+differences+in+sarcolemmal+excitability+between+human+muscles.">Physiological differences in sarcolemmal excitability between human muscles.</a> Muscle and Nerve. 60 (4), 433-436 (2019).</li><li>Humm, A. M., Bostock, H., Troller, R., Z'Graggen, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+ischaemia+in+patients+with+orthostatic+hypotension+assessed+by+velocity+recovery+cycles.">Muscle ischaemia in patients with orthostatic hypotension assessed by velocity recovery cycles.</a> Journal of Neurology, Neurosurgery and Psychiatry. 82 (12), 1394-1398 (2011).</li><li>Z'Graggen, W. J., 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=Velocity+recovery+cycles+of+human+muscle+action+potentials+in+chronic+renal+failure.">Velocity recovery cycles of human muscle action potentials in chronic renal failure.</a> Clinical Neurophysiology. 121 (6), 874-881 (2010).</li><li>Z'Graggen, W. J., 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=Muscle+membrane+dysfunction+in+critical+illness+myopathy+assessed+by+velocity+recovery+cycles.">Muscle membrane dysfunction in critical illness myopathy assessed by velocity recovery cycles.</a> Clinical Neurophysiology. 122 (4), 834-841 (2011).</li><li>Lee, J. H., Boland-Freitas, R., Liang, C., Ng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+depolarization+in+sporadic+inclusion+body+myositis+assessed+with+muscle+velocity+recovery+cycles.">Sarcolemmal depolarization in sporadic inclusion body myositis assessed with muscle velocity recovery cycles.</a> Clinical Neurophysiology. 19 (31205-2), 1388 (2019).</li><li>Tan, S. V., Z'Graggen, W. J., Hanna, M. G., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+muscle+membrane+properties+in+the+sodium+channel+myotonias.">In vivo assessment of muscle membrane properties in the sodium channel myotonias.</a> Muscle and Nerve. 57 (4), 586-594 (2018).</li><li>Tan, S. V., 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=In+vivo+assessment+of+muscle+membrane+properties+in+myotonic+dystrophy.">In vivo assessment of muscle membrane properties in myotonic dystrophy.</a> Muscle and Nerve. 54 (2), 249-257 (2016).</li><li>Tan, S. V., 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=Membrane+dysfunction+in+Andersen-Tawil+syndrome+assessed+by+velocity+recovery+cycles.">Membrane dysfunction in Andersen-Tawil syndrome assessed by velocity recovery cycles.</a> Muscle and Nerve. 46 (2), 193-203 (2012).</li><li>Tan, S. V., 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=Chloride+channels+in+myotonia+congenita+assessed+by+velocity+recovery+cycles.">Chloride channels in myotonia congenita assessed by velocity recovery cycles.</a> Muscle and Nerve. 49 (6), 845-857 (2014).</li><li>Boland-Freitas, 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=Sarcolemmal+excitability+in+the+myotonic+dystrophies.">Sarcolemmal excitability in the myotonic dystrophies.</a> Muscle and Nerve. 57 (4), 595-602 (2018).</li><li>Stalberg, E., 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=Standards+for+quantification+of+EMG+and+neurography.">Standards for quantification of EMG and neurography.</a> Clinical Neurophysiology. 130 (9), 1688-1729 (2019).</li><li>Witt, A., 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=Muscle+velocity+recovery+cycles+in+neurogenic+muscles.">Muscle velocity recovery cycles in neurogenic muscles.</a> Clinical Neurophysiology. 130 (9), 1520-1527 (2019).</li><li>Kristensen, R. 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H.B. receives royalties from UCL for sales of his Qtrac software used in this study. The other authors have no potential conflicts of interest. All authors have approved the final article.

MVRCs, as programmed in the recording software, is a highly automated procedure, but care is needed to obtain reliable results. In the recording stage, while adjusting the needles, it is important to avoid stimulating the end-plate zone or nerve. This usually leads to large twitches of the whole muscle, which increases the risk of displacement of the stimulation and/or recording needle during recording MVRCs. To date, the method has been applied to several muscles that have better described end-plate zone; however, the e...

Nerve conduction studies (NCS) and electromyography (EMG) are the conventional electrophysiological methods used for the diagnosis of neuromuscular disorders. NCS enables detection of axonal loss and demyelination in the nerves1, while EMG can differentiate whether myopathy or neurogenic changes are present in the muscle due to nerve damage. However, NCS or EMG provide limited information about muscle fiber membrane properties and underlying disease mechanisms. This information can be achieved using intracellular electrodes in isolated muscles from muscle biopsies2,3,4. However, it is of clinical importance to use methodologies using recordings from intact muscles in patients.
The velocity of a second muscle fiber action potential changes as a function of the delay after the first5, and this velocity recovery function (or recovery cycle) has been shown to change in dystrophic or denervated muscles. The yield of such recordings from single muscle fibers was, however, too low to be of use as a clinical tool6. However, Z&#39;Graggen and Bostock later found that multi-fiber recordings, obtained by direct stimulation and recording from the same bundle of muscle fibers, provide a fast and simple method of obtaining such recordings in vivo7. A sequence of paired pulse electrical stimuli with varying interstimulus intervals (ISIs) is used in this method7,8,9,10,11.
The evaluated MVRC parameters include the following: 1) muscle relative refractory period (MRRP), which is the duration after a muscle action potential until the next action potential can be elicited; 2) early supernormality (ESN); and 3) late supernormality (LSN). ESN and LSN are the periods after the refractory period in which the action potentials are conducted along the muscle membrane faster than normal. The depolarizing afterpotential, and potassium accumulation in the t-tubules of the muscle respectively, are hypothesized as the main causes for the two periods of supernormality.
The wide applicability of MVRCs to muscle disorders has been shown in detecting membrane depolarization in ischemia7,10,12 and renal failure13, as well as providing information about muscle membrane abnormalities in critical illness myopathy14 and inclusion body myositis15. Frequency ramp and intermittent 15 Hz and 20 Hz simulation protocols have since been introduced. MVRCs, together with these additional protocols, have demonstrated the different effects on muscle membrane excitability related to loss-of-function or gain-of-function mutations in various muscle ion channels in the inherited muscle ion channelopathies (i.e., sodium channel myotonia, paramyotonia congenita16, myotonic dystrophy17, Andersen-Tawil syndrome18, and myotonia congenita19,20).
In a recent study, the applicability of MVRCs to neurogenic muscles was shown for the first time. The term &#34;neurogenic muscle&#34; refers to the secondary changes in skeletal muscles that develop as denervation and reinnervation after any injury to the anterior horn cells or motor axons. Denervation is characterized in EMG as spontaneous activity (i.e., fibrillations [fibs] and positive sharp waves [psws]), while large motor unit potentials with prolonged duration and increased amplitude present reinnervation21. EMG changes are evident in denervated muscles, but the underlying cellular changes in muscle fiber membrane potentials have only been demonstrated in experimental studies on isolated muscle tissue2,3,4. MVRCs provide further insight into in vivo human muscle membrane properties regarding the denervation process.
This paper describes the methodology of MVRCs in detail. It also summarizes the changes in neurogenic muscles in a subgroup of patients from a previously reported study22 and healthy control subjects that enables determination of whether the method is appropriate for a planned study.
The recordings are performing using a recording protocol that is part of a software program. Other equipment used is an isolated linear bipolar constant current stimulator, 50 Hz noise eliminator, isolated electromyography amplifier, and analogue-to-digital converter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 Hz Noise Eliminator</td>
			<td>Digitimer Ltd</td>
			<td></td>
			<td>Humbug</td>
		</tr>
		<tr>
			<td>Analogue-to-Digital Converter</td>
			<td>National Instruments</td>
			<td></td>
			<td>NI-6221</td>
		</tr>
		<tr>
			<td>Analysing software program</td>
			<td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td>
			<td></td>
			<td>QtracP, MANAL9</td>
		</tr>
		<tr>
			<td>Disposable concentric needle electrode, 25 mm x 30G</td>
			<td>Natus</td>
			<td></td>
			<td>Dantec DCN</td>
		</tr>
		<tr>
			<td>Disposable monopolar needle electrode, 25 mm x 26G</td>
			<td>Natus</td>
			<td></td>
			<td>TECA elite</td>
		</tr>
		<tr>
			<td>Isolated EMG amplifier</td>
			<td>Digitimer Ltd</td>
			<td></td>
			<td>D440</td>
		</tr>
		<tr>
			<td>Isolated linear bipolar constant-current stimulator</td>
			<td>Digitimer Ltd</td>
			<td></td>
			<td>DS5</td>
		</tr>
		<tr>
			<td>Software and recording protocol</td>
			<td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td>
			<td></td>
			<td>QtracW software, M3REC3 recording protocol written by Hugh Bostock, Istitute of Neurology, London, UK)</td>
		</tr>
	</tbody>
</table>

muscle velocity recovery cycles to examine muscle membrane properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.

The following results were obtained in a subgroup of patients from a recent study22, in which there were fibs/psws in all sites showing profuse denervation activity. The results showed that changes in muscle fibers after denervation were assessed in vivo using the MVRC technique described in this protocol. MVRCs showed changes consistent with depolarization of the resting membrane potential in the neurogenic muscle fibers.
Fourteen patients were compared with 29 healthy...

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Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...

muscle-velocity-recovery-cycles-to-examine-muscle-membrane-properties

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14.0K Views.  Aarhus University Hospital. Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Video: Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Fabrication and Surgical Implantation of Electromyographic Electrodes in Mice

Recording Forelimb Muscle Activity in Head&#45;Fixed Mice with Chronically Implanted EMG Electrodes

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with unprecedented precision in head-fixed mice performing a variety of tasks. The small size of the mouse makes the measurement of motor output difficult, as the traditional method of electromyographic (EMG) recording of muscle activity was designed for larger animals like cats and primates. Pending commercially available EMG electrodes for mice, the current gold-standard method for recording muscle activity in mice is to make electrode sets in-house. This article describes a refinement of established procedures for hand fabrication of an electrode set, implantation of electrodes in the same surgery as headplate implantation, fixation of a connector on the headplate, and post-operative recovery care. Following recovery, millisecond-resolution EMG recordings can be obtained during head-fixed behavior for several weeks without noticeable changes in signal quality. These recordings enable precise measurement of forelimb muscle activity alongside in vivo neural recording and/or perturbation to probe mechanisms of motor control in mice.

Author Spotlight: Investigating Mouse Motor Cortex Interactions from Muscle Activity to Neural Dynamics

This protocol describes the hand fabrication and surgical implantation of electromyographic (EMG) electrodes in the forelimb muscles of mice to record muscle activity during head-fixed behavior experiments.

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with ...

Standardized Evaluation of DIAm EMG Post-C2SH in Rats to Minimize Variability

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection (C2SH) model of cSCI that disrupts eupneic ipsilateral diaphragm (iDIAm) electromyographic (EMG) activity during breathing in rats. To evaluate recovery of DIAm motor control, the extent of deficit due to C2SH must first be clearly established. By verifying a complete initial loss of iDIAm EMG during breathing, subsequent recovery can be classified as either absent or present, and the extent of recovery can be estimated using the EMG amplitude. Additionally, by measuring the continued absence of iDIAm EMG activity during breathing after the acute spinal shock period following C2SH, the success of the initial C2SH may be validated. Measuring contralateral diaphragm (cDIAm) EMG activity can provide information about the compensatory effects of C2SH, which also reflects neuroplasticity. Moreover, DIAm EMG recordings from awake animals can provide vital physiological information about the motor control of the DIAm after C2SH. This article describes a method for a rigorous, reproducible, and reliable C2SH model of cSCI in rats, which is an excellent platform for studying respiratory neuroplasticity, compensatory cDIAm activity, and therapeutic strategies and pharmaceuticals.

Author Spotlight: Neuromotor Control and Recovery of Diaphragm Function Following Cervical Spinal Hemisection in Rats

Respiratory complications are the leading cause of death in individuals with cervical spinal cord injury (cSCI). Animal models of cSCI are essential for mechanistic evaluations and pre-clinical studies. Here, we introduce a reproducible method to assess functional recovery of diaphragm muscle (DIAm) activity following unilateral C2 spinal hemisection (C2SH) in rats.

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection ...