Name: functional isolation of single motor units of rat medial gastrocnemius muscle
Uploaded: 2020-12-26T13:00:00.000+00:00
Duration: 6 min 54 s
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 of 60 mg&#183;kg-1).</li>
	<li>After approximately 5 min, check the depth of anesthesia by pinching the rat&#8217;s ear or forelimb with blunt forceps. Go to the next steps of the protocol only when no reflex action is observed.</li>
	<li>During the surgery, check the animal for reflex actions every 10-15 minutes and supplement anesthesia if the animal responds to a pinch with movement (usually, 10 mg&#183;kg-1&#183;h-1 sodium pentobarbital, IP).</li>
</ol>
2. Surgery
<ol>
	<li>Prepare the animal for the surgical procedure by shaving the fur over the left hindlimb from the heel to the hip (the first segment, muscle and nerve isolation), the right hindlimb from the heel to the hip (the second segment, ground electrode), and the backside from the tail to the thoracic segments (the third segment, laminectomy). Antiseptic is not necessary because of the acute nature of the experiment.
	<ol>
		<li>Place the rat on its abdomen on a heating pad (37 &#176;C &#177; 1 &#176;C.)</li>
	</ol>
	</li>
	<li>Laminectomy
	<ol>
		<li>Using sharp-blunt scissors, cut the skin along the spinal column from the sacrum up to the thoracic vertebrae.</li>
		<li>Separate the skin from the underlying muscles.</li>
		<li>Using blunt tip scissors, cut out the longissimus muscle on both sides of the sacrum and lumbar spinous processes.</li>
		<li>Identify the S1 vertebra as the lowest segment. Using sharp-blunt scissors, cut and remove the spinous processes from L6 to L2 vertebrae. Next, using fine rongeurs, remove the transverse processes L6-L2 and perform a laminectomy over L6 &#8211; L2 segments (first transverse processes, then lamina, begin with L6 vertebral segment) to expose the lumbar segments of the spinal cord covered by the dura mater. Be careful not to cut the sacral bone and L1 spinous process, which will be used as a fixation point for animal immobilization.</li>
		<li>Using sharp scissors, cut the spinal cord (its caudal fragment) and the dorsal and ventral roots at L2 vertebrae segment level, at the upper border of laminectomy. Place small pieces of dried gel foam to stop bleeding. Next, place a thin, saline-soaked cotton wool over the exposed spinal cord segments.</li>
	</ol>
	</li>
	<li>Isolation of medial gastrocnemius muscle and its nerve
	<ol>
		<li>Using sharp-blunt scissors, make a longitudinal cut on the posterior side of the left hind limb, from the Achilles tendon to the hip.</li>
		<li>Grasp the skin with the forceps and separate the skin from the underlying muscles on both sides of the incision.</li>
		<li>Locate the popliteal fossa at the back of the knee joint, which is covered by the biceps femoris muscle. Using scissors, make a cut between the anterior and posterior part of this muscle.</li>
		<li>Moving upwards, cut the two heads of the biceps femoris all the way to the hip to expose the sciatic nerve.</li>
		<li>Using blunt forceps and scissors, separate the lateral from the medial head of the gastrocnemius muscle and cut the distal insertion (Achilles tendon) of the medial gastrocnemius muscle. Preserve the fragment of Achilles tendon as long as possible in order to use it to connect to the force transducer.</li>
		<li>Identify the medial gastrocnemius (MG) nerve. Using the forceps and scissors, cut all remaining collaterals of the sciatic nerve, including collaterals to posterior biceps and semitendinosus. Leave the supply blood vessels to the medial gastrocnemius intact.</li>
		<li>Thread a non-elastic ligature through the Achilles tendon and make three knots.</li>
		<li>Place a saline-soaked piece of cotton wool under the exposed nerve and muscle.</li>
		<li>Using serrated forceps, close the skin over the operated area.</li>
		<li>Using sharp-blunt scissors, make a 2 cm incision in the skin and underlying connective tissue along the anterior side of the left hind limb for immobilization with a metal clamp (3.1.6.).</li>
	</ol>
	</li>
</ol>
3. Preparation for the recording and stimulation
<ol>
	<li>Vertebral column and leg fixation and muscle arrangement
	<ol>
		<li>Using a steel clamp, fix the left hind limb by putting the clamp on the tibia.</li>
		<li>Place the rat in the custom-made adjustable frame (isolated copper wire, 1 mm), pull up the skin flaps around the laminectomy using four ligatures and suture the skin to the frame in order to form a pool for paraffin oil (size approximately 50 mm x 50 mm) over the exposed spinal cord.</li>
		<li>Using a Dumont #55 forceps, lift the dura mater at the intersection of the spinal cord, cut it caudally up to the sacral bone and retract it.</li>
		<li>Using a blunt glass rod, separate left and right dorsal and ventral roots at successive levels, taking care not to damage them.</li>
		<li>Fill the pool over the spinal cord with warm (37 &#176;C) paraffin oil, covering the exposed ventral and dorsal roots.</li>
		<li>Place the rat on the custom-made aluminum plate (length 260 mm, width 120 mm, height 80 mm) with a pool (length 135 mm, width 100 mm, depth 45 mm) for his hindlimbs connected to the closed-loop heating system. The plate is a place where the animal will be immobilized and the experiment will be performed.</li>
		<li>Fix the clamp put on the left hindlimb with the metal bar to immobilize the hindimb.</li>
		<li>Fix the vertebral column by putting steel clamps at the sacral bone and the L1 vertebra to immobilize the animal body and eliminate the artefacts in force recordings related to respiratory movements.</li>
		<li>Connect the left medial gastrocnemius muscle with the non-elastic ligature to the force transducer (with compliance of 50 &#956;m/250 mN, measurement range 0-1000 mN) via the Achilles tendon.</li>
		<li>Fill the chamber for hindlimbs with warm (37 &#176;C) paraffin oil to cover the medial gastrocnemius muscle and maintain the oil temperature at 37 &#176;C &#177; 1 &#176;C using the temperature probe and the automatic system.</li>
	</ol>
	</li>
	<li>Placement of electrodes for action potentials recording and stimulation
	<ol>
		<li>Insert a bipolar silver-wire electrode through the middle part of the medial gastrocnemius muscle, perpendicular to its long axis. Maintain about 5 mm distance between the two electrodes located along a long axis of the muscle. These electrodes will be used to record motor unit action potentials (MUAPs). Connect the electrodes to the low-noise amplifier.</li>
		<li>Stretch the operated muscle to a passive tension of 100 mN, controlled by the force transducer. For rat medial gastrocnemius at this stretch the MUs of three types develop the highest twitch force15.</li>
		<li>Using sharp-blunt scissors, make a 2 cm incision in the skin of the right hind limb and insert a silver-wire electrode to be used as a reference electrode.</li>
		<li>Place and fix a custom-made insulated metal plate (size 30 mm x 13 mm) above the exposed spinal roots. Put left pairs of ventral and dorsal roots (L4, L5 and L6) on the plate.</li>
		<li>Add saline to the pool formed by the skin around the laminectomy. The saline level should be below the insulated plate.</li>
		<li>Place a silver wire stimulating electrode (two silver wires, 0.5 mm diameter, length 50 mm) over the exposed spinal roots, place a positive pole 3 mm above the plate in oil, whereas the negative pole in the saline (added to the pool, below the plate) and connect to the stimulator.</li>
	</ol>
	</li>
</ol>
4. Motor unit recordings
<ol>
	<li>Stimulating with electrical rectangular pulses (0.1 ms duration, amplitude up to 0.5 V), select the ventral roots (L4, L5 and L6); ventral root stimulation evokes contraction of muscles whereas there is no such effect for dorsal roots. Eliminate dorsal roots from the plate. For the medial gastrocnemius, most axons are in the L5 ventral root.</li>
	<li>Using a pair of Dumont #55 forceps and magnifying glasses, split L5 or L4 ventral roots into very &#64257;ne bundles of axons (grasp the cut end of the ventral rootlet with both forceps and peel the rootlet apart); place one of these bundles on a silver wire electrode and stimulate (0.1 ms rectangular pulses of amplitude up to 0.5 V) to achieve activity of a single MU. A solid support (metal bar) is very useful for manipulating thin bundles of axons, which can be used as a hand support for using forceps. Note also that an additional source of light is necessary.</li>
	<li>By progressively increasing the intensity of the stimulus, identify a single MU on the basis of the evoked &#8220;all-or-none&#8221; character of the twitch contraction and action potential stimulus. Carefully test the evoked activity at a stimulation around the threshold.
	<ol>
		<li>When more than one MU is contracting in the studied muscle and increasing level of the force as well as increasing amplitude or changing shape of the action potential are visible, go back to step 4.2 and split the bundle of axons again. Note that the strongest MUs in rat medial gastrocnemius have about 70 times larger twitch force than the weakest ones and when very strong MU is twitching the second, weak MU may be not evident. Note also that some MUs have their muscle fibers located out of the recording area of the electrode and are not visible in electromyogram; in such a case changes in the stimulus amplitude may have effect in the force but not in action potential.</li>
	</ol>
	</li>
	<li>Stimulate a motor unit with a stimulation protocol necessary for the aim of experiment. For a basic stimulation protocol necessary to calculate all basic motor unit contractile and action potentials properties, include the following.
	<ol>
		<li>Include 5 stimuli at 1 Hz (5 single twitches recorded and averaged; averaging is eliminating noise, which is especially important for the weakest MUs).</li>
		<li>Include a series of stimuli at 10, 20, 30, 40, 50, 60, 75, 100 and 150 Hz frequencies with a duration of 500 ms (these recordings enable calculation of the force-frequency relationship, the maximum tetanic force at 150 Hz as well as the sag at 20-40 Hz stimulation).</li>
		<li>Include the fatigue test (tetani evoked by trains of 14 stimuli at 40 Hz frequency, repeated every second for 4 minutes).</li>
		<li>Include at least 10 s time intervals between all the elements of the protocol above.</li>
		<li>Repeat the process with successive isolated motor units.</li>
	</ol>
	</li>
	<li>Terminate the experiment and euthanize the animal using intraperitoneal administration of a lethal dose of pentobarbital sodium (180 mg&#183;kg-1).</li>
</ol>
5. Electronic apparatus
NOTE: The custom-made computer program controls the stimulator, providing the possibility to create variable patterns of stimulation, including those indicated in step 4.4. The program cooperates with the analog-to-digital converter (at least 10 kHz for the MUAP and force recordings).
<ol>
	<li>Connect the AC amplifier to the computer by the analog-to-digital converter and in parallel to the oscilloscope.</li>
	<li>Connect the force transducer to the computer by the analog-to-digital converter and in parallel to the oscilloscope. Use the force transducer to control the passive muscle force during experiment. Note that during experiment, the passive force may decrease; therefore, it is necessary to increase the muscle length to keep the passive muscle force constant.</li>
</ol>

<ol>
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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. 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Authors have no conflict of interest to disclose.

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...

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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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

5.2K Views.  Poznań University of Physical Education. 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.

Video: Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

Optimized Isolation of Skeletal Muscle-Derived EVs for Research and Therapeutics

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles (SkM-EVs) from rodent muscle tissues through mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It demonstrates consistency and reliability. The isolated SkM-EVs provide insights into muscle homeostasis and diseases, with potential applications as diagnostic biomarkers or therapeutic vehicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

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Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

Procedures for Rat in situ Skeletal Muscle Contractile Properties

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles