Name: functional isolation of single motor units of rat medial gastrocnemius muscle
Uploaded: 2020-12-26T13:00:00
Description: Watch this Scientific Journal Video about Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle at JoVE.com

This work was supported by Polish National Research Centre grant 2018/31/B/NZ7/01028.

All procedures need to be approved by the local ethics committee and adhered to the European Union guidelines on animal care as well as the national law on the protection of animals.
NOTE: Each experimenter involved in this procedure must be trained in basic surgical procedures and has to obtain a valid license for performing animal experiments.
1. Anesthesia
<ol>
	<li>Anesthetize the rat with an intraperitoneal injection of sodium pentobarbital (an initial dose o...

<ol>
	<li>Burke, R. E., Levine, D. N., Tsairis, P., Zajac, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+types+and+histochemical+profiles+in+motor+units+of+the+cat+gastrocnemius.">Physiological types and histochemical profiles in motor units of the cat gastrocnemius.</a> Journal of Physiology. 234, 723-748 (1973).</li><li>Celichowski, J., Drzyma&#322;a-Celichowska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+motor+units+in+the+medial+gastrocnemius+muscle+of+male+and+female+rats.">The number of motor units in the medial gastrocnemius muscle of male and female rats.</a> Journal of Physiology and Pharmacology. 58, 821-828 (2007).</li><li>Grottel, K., Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+motor+units+in+medial+gastrocnemius+muscle+of+the+rat+in+light+of+variability+of+their+principal+properties.">Division of motor units in medial gastrocnemius muscle of the rat in light of variability of their principal properties.</a> Acta Neurobiologiae Experimentalis. 50, 571-588 (1990).</li><li>Celichowski, J., Krutki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+and+plasticity+of+motor+unit+properties+in+mammalian+skeletal+muscle.">Variability and plasticity of motor unit properties in mammalian skeletal muscle.</a> Biocybernetics and Biomedical Engineering. 32 (4), 33-45 (2012).</li><li>Gardiner, P. F., Olha, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+and+electromyographic+characteristics+of+rat+plantaris+motor+unit+types+during+fatigue+in+situ.">Contractile and electromyographic characteristics of rat plantaris motor unit types during fatigue in situ.</a> Journal of Physiology. 385, 13-34 (1987).</li><li>Drzyma&#322;a-Celichowska, H., Kaczmarek, P., Krutki, P., Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summation+of+slow+motor+unit+forces+at+constant+and+variable+interpulse+intervals+in+rat+soleus+muscle.">Summation of slow motor unit forces at constant and variable interpulse intervals in rat soleus muscle.</a> Journal of Electromyography and Kinesiology. 30, 1-8 (2016).</li><li>Krutki, P., Celichowski, J., &#321;ochy&#324;ski, D., Pogrzebna, M., Mr&#243;wczy&#324;ski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+differences+of+motor+units+properties+in+the+medial+gastrocnemius+muscle+of+cat+and+rat.">Interspecies differences of motor units properties in the medial gastrocnemius muscle of cat and rat.</a> Archives Italiennes de Biologie. 144, 11-23 (2006).</li><li>Burke, R. E., Rudomin, P., Zajac, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+activation+history+on+tension+production+by+individual+muscle+units.">The effect of activation history on tension production by individual muscle units.</a> Brain Research. 109, 515-529 (1976).</li><li>Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+the+regulation+of+motor+unit+contraction+in+the+skeletal+muscle.">Mechanisms underlying the regulation of motor unit contraction in the skeletal muscle.</a> Journal of Physiology and Pharmacology. 51, 17-33 (2000).</li><li>Burke, R. E., Levine, D. N., Salcman, M., Tsairis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+units+in+cat+soleus+muscle:+physiological,+histochemical+and+morphological+characteristics.">Motor units in cat soleus muscle: physiological, histochemical and morphological characteristics.</a> Journal of Physiology. 238, 503-514 (1974).</li><li>Milner-Brown, H. S., Stein, R. B., Yemm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contractile+properties+of+human+motor+units+during+voluntary+isometric+contractions.">The contractile properties of human motor units during voluntary isometric contractions.</a> Journal of Physiology. 228, 285-306 (1973).</li><li>Taylor, A., Stephens, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+human+motor+unit+contractions+by+controlled+intramuscular+microstimulation.">Study of human motor unit contractions by controlled intramuscular microstimulation.</a> Brain Research. 117, 331-335 (1976).</li><li>Westling, G., Johansson, R. S., Thomas, C. K., Bigland-Ritchie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+contractile+and+electrical+properties+of+single+human+thenar+motor+units+in+response+to+intraneural+motor-axon+stimulation.">Measurement of contractile and electrical properties of single human thenar motor units in response to intraneural motor-axon stimulation.</a> Journal of Neurophysiology. 64, 1331-1338 (1990).</li><li>Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+units:+anatomy,+physiology+and+functional+organization.">Motor units: anatomy, physiology and functional organization.</a> APS Handbook of Physiology Series, Section 1, The Nervous System. 11, 345-422 (1981).</li><li>Celichowski, J., Grottel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dependence+of+the+twitch+course+of+medial+gastrocnemius+muscle+of+the+rat+and+its+motor+units+on+stretching+of+the+muscle.">The dependence of the twitch course of medial gastrocnemius muscle of the rat and its motor units on stretching of the muscle.</a> Archives Italiennes de Biologie. 130, 315-325 (1992).</li><li>Celichowski, J., Grottel, K., Bichler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+profile+of+unfused+tetani+of+fast+motor+units+with+respect+to+their+resistance+to+fatigue+in+the+rat+medial+gastrocnemius+muscle.">Differences in the profile of unfused tetani of fast motor units with respect to their resistance to fatigue in the rat medial gastrocnemius muscle.</a> Journal of Muscle Research and Cell Motility. 20, 681-685 (1999).</li><li>Krutki, P., 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=Division+of+motor+units+into+fast+and+slow+on+the+basis+of+profile+of+20+Hz+unfused+tetanus.">Division of motor units into fast and slow on the basis of profile of 20 Hz unfused tetanus.</a> Journal of Physiology and Pharmacology. 59, 353-363 (2008).</li><li>Drzyma&#322;a-Celichowska, H., Krutki, P., Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summation+of+motor+unit+forces+in+the+rat+medial+gastrocnemius+muscle.">Summation of motor unit forces in the rat medial gastrocnemius muscle.</a> Journal of Electromyography and Kinesiology. 20, 599-607 (2010).</li><li>Kaczmarek, P., Celichowski, J., Drzyma&#322;a-Celichowska, H., Kasi&#324;ski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+image+of+motor+unit+architecture+in+the+mechanomyographic+signal+during+single+motor+unit+contraction.+In+vivo+and+simulation+study.">The image of motor unit architecture in the mechanomyographic signal during single motor unit contraction. In vivo and simulation study.</a> Journal of Electromyography and Kinesiology. 19, 553-563 (2009).</li><li>Celichowski, J., Krutki, P., Bichler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+conduction+velocity+of+motor+units+of+rat's+medial+gastrocnemius+muscle.">Axonal conduction velocity of motor units of rat's medial gastrocnemius muscle.</a> Journal of Physiology (Paris). 90, 75-78 (1996).</li></ol>

If performed correctly by experienced scientists, the surgical component of the described protocol should be completed within approximately two hours. One should take particular care to maintain stable physiological conditions of the animal during the surgery, particularly body temperature and depth of anesthesia, which should be systematically controlled by assessing pinna and withdrawal reflexes. Following the surgery, it should be possible to maintain stable recording conditions for at least six hours.
<p class="j...

Motor units (MUs) are the smallest functional units of skeletal muscles. Therefore, understanding their function, plasticity and contractile properties, as well as the mechanisms of their force regulation, is crucial for progress in muscle physiology. The basic contractile properties of MUs and the proportions of their physiological types have been documented for numerous muscles, predominantly the hindlimb muscles in experimental animals. However, both the plasticity of MU properties and the mechanisms of MU force regulation are still not fully understood.
The principle of the described method is extensive denervation of the hindlimb muscles except the investigated one and laminectomy at the lumbar vertebrae in order to prepare thin ventral rootlets, each one containing a single &#34;functional&#34; motor axon, stimulated electrically to record the force and action potential of the MU. Using the technique described in this paper, it is possible to isolate more than half of the MUs of the medial gastrocnemius muscle in a successful experiment. The rat medial gastrocnemius is composed of on average 52 MUs (females) or 57 MUs (males) of three physiological types: S (slow), FR (fast resistant) and FF (fast fatigable)1,2, and have variable contractile properties3. For experiments comparing mean values for MUs in the control and experimental groups, isolation and recording of 10-30 MUs for each of these groups are necessary. Critically, individual MUs may be accessible for stimulation for time periods exceeding one hour. Moreover, since this technique allows for recording both MU force and action potentials, this method is suitable for studying phenomena associated with force production, assessing the effect of fatigue, and observing the relationship between the force and action potentials.
Previous studies have confirmed that MU contractile properties are plastic and may be modulated by numerous interventions. Experiments using the technique described here have been performed on rat medial gastrocnemius4 or other hindlimb muscles of the rat5,6 as well as on cat muscles7, using a similar method of single MU isolation. Another series of experiments using trains of stimuli delivered at variable inter-pulse intervals provided observations concerning motor control processes, and the results in general turn attention to the history of stimulation, including considerable effects of a shift in time scale of even one stimulus, crucial for force production8,9.
MUs may also be studied using alternative methods. First, one method is direct stimulation of motoneurons. Burke used intracellular stimulation of motoneurons in cat medial gastrocnemius and soleus with glass microelectrodes used in parallel to determine the electrophysiological properties of these neurons1,10. Other methods have been proposed to study MUs in human muscles, which require considerably lower intervention. For all these methods, the stimulating and recordings electrodes are inserted into the muscle or nerve, and force is recorded from the finger or from the foot. The first of these methods was used to study MUs in the first dorsal interosseous muscle. For this muscle, contracting with a low force, in the electromyogram recorded with the needle electrode inserted into the muscle the action potentials of only one active motor unit were identified. Then the fragments of a muscle force recorded in parallel and following each action potential were averaged (spike-triggered averaging). This method enables extraction of the force of one motor unit from the muscle force recording11. However, the methodological weakness of this procedure is that no single twitch force but rather fragments of tetanic contractions were averaged. Human MUs may also be studied using the second method of intramuscular electrical microstimulation using an electrode inserted into the muscle12, which stimulates a fragment of an axonal tree, leading to activation of a single motor unit. The third method is microstimulation with an electrode inserted into the nerve. When the electrode activates only one motor axon in the nerve, only one motor unit contracts13. These last methods have some limitations, including stability and quality of the recording, ethical restrictions and access to the experimental material. This protocol has been extensively used in cats in the 70&#39;s and 80&#39;s14.

<table>
	<tbody>
		<tr>
			<td>Force transducer</td>
			<td>custom-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11255-20</td>
			<td>Dumont #55 with extra light and fine shanks</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11150-10</td>
			<td>Extra Fine Greafe Forceps</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11026-15</td>
			<td>Special cupped pattern for superior grip</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11023-10</td>
			<td>Slim 1x2 teeth</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11251-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fine Science Tools</td>
			<td>No. 13003-10</td>
			<td>Hartman</td>
		</tr>
		<tr>
			<td>Isolation Unit</td>
			<td>Grass Instruments</td>
			<td>S1U5A</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Noise Bioamplifer</td>
			<td>World Precision Instruments</td>
			<td>Order code 74030</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holders</td>
			<td>Fine Science Tools</td>
			<td>No. 12503-15</td>
			<td>With tungsten carbide jaws</td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>No. 16021-14</td>
			<td>Friedman-Pearson</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>No. 14101-14</td>
			<td>Straight sharp/blunt with large finger loops</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>No. 14075-11</td>
			<td>Curved blunt/blunt</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>No. 14084-08</td>
			<td>Extra fine bonn</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>No. 15000-00</td>
			<td>Straight, ideal for cutting nerves</td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Grass Instruments</td>
			<td>S88</td>
			<td>Dual Output Square Pulse Stimulator</td>
		</tr>
	</tbody>
</table>

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.

Parameters of motor unit contractions and action potentials can be calculated on the basis of recordings when stable conditions of recordings are ensured. Figure 1 presents a representative recording of the single twitch of a fast MU. The upper trace shows the motor unit action potential. The delay between stimulus delivery and onset of the motor unit action potential is due to conduction time from ventral root to muscle. Figure 2 shows a representative recordin...

Watch this Scientific Journal Video about Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle at JoVE.com

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

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 ...

This method allows the functional isolation of single motor units in handling masses of experimental animals. The main advantage of this technique is recording of force and action potentials of individual motor units of identified physiological type. Helping to demonstrate the procedure will be Professor Jan Celichowski, head of the department of neurobiology.Begin by cutting the skin along the spinal column, from the sacrum up to the thoracic vertebrae, with sharp-blunt scissors. Identify the S1 vertebra as the lowest segment. Then cut and remove the longissimus muscles, and the spinous processes from L6 to L2 vertebrae.Use fine rongeurs to remove the transverse processes from L6 to L2, and perform a laminectomy over L6 to L2 segments to expose the lumbar segments of the spinal cord covered by the dura mater. Using sharp scissors, cut the spinal cord and the dorsal and ventral roots at L2 vertebrae segment, level at the upper border of the laminectomy. Place small pieces of dry gel foam to stop the bleeding.Next, use sharp-blunt scissors to make a longitudinal cuddle on the posterior side of the left hind limb, from the Achilles tendon to the hip. Locate the popliteal fossa at the back of the knee joint, which is covered by the biceps femoris muscle, and make a cup between the anterior and posterior part of this muscle. Moving upwards, cut the two heads of the biceps femoris all the way to the hip to expose the sciatic nerve.Using blunt forceps and scissors, separate the lateral from the medial head of the gastrocnemius muscle, and cut the distal insertion of the medial gastrocnemius muscle. Identify the medial gastrocnemius or MG nerve, then use forceps and scissors to cut all remaining collaterals of the sciatic nerve including collaterals to posterior biceps and semitendinosus. Bred a non elastic ligature through the Achilles tendon and make three knots.Then make a two centimeter incision in the skin and underlying connective tissue along the anterior side of the left hind limb for a mobilization with a middle clan. Fix the left hind limb by putting a steel clamp on the tibia. Place the rat in a custom made adjustable frame.Pull the skin flaps around the laminectomy with four ligatures. And suture them to the frame in order to form a pool for paraffin oil over the exposed spinal cord. Use a Dumont 55 forceps to lift the dura mater at the intersection of the spinal cord.Then cut it caudally up to the sacral bone and retract it. Separate the left and right dorsal and ventral roots at successive levels with a blunt glass rod. Fill the pool over the spinal cord with warm paraffin oil, covering the exposed ventral and dorsal roots.Place the rat on a custom made aluminum plate with a pool for its hind limbs, connected to the closed loop heating system. Fix the clamp on the left hind limb with the metal bar to immobilize it. Fix the vertebral column by putting steel clamps at the sacral bone and the L1 vertebra.Then connect the left medial gastrocnemius muscle with the non elastic ligature to the force transducer via the Achilles tendon. Insert a bipolar silver wire or electrode through the middle part of the muscle, perpendicular to its long axis, and fill the chamber for hind limbs with warm paraffin oil to cover the medial gastrocnemius muscle. Stretch the operated muscle to a passive tension of 100 millinewton, controlled by the force transducer.Then use sharp-blunt scissors to make a two centimeter incision in the skin of the right hind limb, and insert a silver wire electrode to be used as a reference electrode. Place and fix a custom made insulated metal plate above the exposed spinal roots. Put left pairs of ventral and dorsal roots on the plate.And add saline to the pool formed by the skin around the laminectomy. Place a silver wire stimulating electrode over the exposed spinal roots, then place a positive pole three millimeters above the plate in oil, and the negative pole in the saline. Stimulate the ventral roots with electrical rectangular pulses, evoking contraction of muscles.Use a pair of Dumont 55 forceps and magnifying glasses to split L5 or L4 ventral roots into very fine bundles of axons. Place one of these bundles on a silver wire electrode and stimulate it to observe activity of a single motor unit. By progressively increasing the intensity of the stimulus, identify a single motor unit on the basis of the evoked all-or-none character of the twitch contraction and action potential stimulus.Parameters of motor unit contractions and action potentials can be calculated using recordings when stable conditions of recordings are insured. A representative recording of the single twitch of a fast motor unit is shown here. The upper trays shows the motor unit action potential.The delay between stimulus delivery and onset of the motor unit action potential is due to conduction time from the ventral root to the muscle. A representative recording of the unfused tetanus force of a fast motor unit and a train of motor unit action potentials are shown here along with the time positions of the applied stimuli. Before attempting this procedure, keep in mind that the splitting of the ventral root into thin filaments needs some training and experience.

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