This work was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant (KAW), a grant from Pfizer (WS753098, SH), and an NIH MARC fellowship (#2T34GM008253-21, JAF). The authors would like to thank Michael Sawchuk, Anusha Allawala, Nina Bubalo, Remie Mandawe and Peter Nguyen for their technical support.

Appropriate national and institutional ethics should be obtained before performing animal experiments.
1. Removal of EDL Muscle and Nerve
<ol>
	<li>Weigh and deeply anesthetize an adult mouse with inhaled isofluorane using a vaporizer with 5% isofluorane plus a 1.5 L/min oxygen flow rate or a bell jar with isofluorane soaked cotton on the bottom.</li>
	<li>Ensure that the mouse is deeply anesthetized and does not respond to a toe pinch. Quickly decapitate using large, sharpened scissors or a guillotine.</li>
	<li>Make a ventral midline cut through the ribs using scissors and remove the internal organs.</li>
	<li>Skin the animal by grasping the skin from the neck area and pulling it past the feet.</li>
	<li>Remove the legs by cutting above the hips and place the skinned legs into a dish with chilled (4 &#176;C), carbogenated (95% O2, 5% CO2) low calcium, high magnesium bicarbonate buffered saline solution containing in mM: 128 NaCl, 1.9 KCl, 1.2 KH2PO4, 26 NaHCO3, 0.85 CaCl2, 6.5 MgSO4, and 10 glucose (pH of 7.4&#177;0.05)16. The low calcium, high magnesium solution inhibits synaptic transmission during the dissection.</li>
	<li>Place the legs dorsal side up in the dish and pin the legs and hips down using needles or insect pins so that the knee and ankle joints are at a 90&#176;&#160;angle. Place a pin on each end of the feet and one pin on each of the anterior and posterior thighs to hold the tissue in place.</li>
	<li>Using Castroviejo spring scissors, lift up the top layer of muscle on the thighs and make a midline cut to expose the sciatic nerve directly below. The sciatic nerve is located just below the top muscle layer and runs above the femur from its exit near the hips until it branches into the common peroneal and tibial nerve just before the knee joint.</li>
	<li>Remove the muscle above the sciatic nerve until the point at which the deep peroneal nerve branch dives into the flexor hallucis longus (FHL) muscle is visible.</li>
	<li>Using #55 forceps, dissect the connective tissue around the peroneal branch to free it from the gastrocnemius and soleus muscles, which are on the medial side of the tibia. Cut the tendons of the gastrocnemius and soleus muscles and carefully remove them from under the nerve.</li>
	<li>Remove the superficial muscle on the lateral side of the tibia to expose three distinct tendon bundles at the ankle joint&#8212;from medial to lateral: FHL, EDL, and tibialis anterior (TA), with the EDL hidden underneath the TA.</li>
	<li>Cut the TA tendon at the ankle joint, lift the TA up and away from the EDL, and cut the muscle near the knee to remove it.</li>
	<li>Cut the FHL tendon at the ankle and lift it back to reach the area where the nerve enters the FHL. Cut just below that point and remove ~2/3 of the FHL muscle.</li>
	<li>Cut the sciatic nerve as close to the hip joint as possible and gently strip away all nerve branches except for the deep peroneal branch.</li>
	<li>Cut the EDL tendons at both the ankle and knee joints using large spring scissors.</li>
	<li>Using sharp scissors, remove the EDL, the remaining FHL and nerve from the surrounding tissue by cutting through the tibia bone at the knee and midway through the thigh. Cut away the remaining tibia bone so that just the EDL, part of the FHL and nerve remain.</li>
</ol>
2. Mounting of the EDL Muscle and Nerve into the Tissue Bath (Figure 1A)
<ol>
	<li>Before the start of the experiment, prepare the tissue bath. Constantly perfuse the bath with oxygenated (100% O2) synthetic interstitial fluid (SIF) containing (in mM) 123 NaCl, 3.5 KCl, 0.7 MgSO4, 1.7 NaH2PO4, 2.0 CaCl2, 9.5 NaC6H11O (sodium gluconate), 5.5 glucose, 7.5 sucrose, and 10 N-2-hydroxyethylpiperazine-N&#8242;-2-ethanesulfonic acid (HEPES); pH 7.4&#177;0.0517. A flow rate of 15-30 ml/min is recommended. Shown is a commercially available bath of 25 ml capacity with two stimulating electrodes fixed to the bottom of the bath, a mounted tissue post and a mount for a force and length controller (approximate bath dimensions 8.5 cm x 3 cm x 1 cm; for specifications see Table of Materials/Equipment).</li>
	<li>Use the remaining FHL tissue to handle the isolated muscle-nerve and place it into the tissue bath. Place a small piece of sylgard on the bottom of the dish and use an insect pin through the remaining FHL tissue to stabilize the muscle.</li>
	<li>Use 6-0 silk sutures to tie both tendons and affix one end to the tissue post and the other to the lever arm of the force and length controller (see Table of Materials/Equipment for specifications). Use the smallest suture length that is practical.&#160;Remove the insect pin and sylgard after you have tied the tendons. NOTE: To facilitate easy connection to the lever arm a small piece of wire can be bent into a &#8220;j&#8221; shaped hook and fixed into the lever arm with epoxy. The suture can then be tied to the wire instead of threaded into the small hole on the lever arm.</li>
	<li>Make suction electrodes from SA 16 glass by first using a glass micropipette puller (Heat = 286, Pull = 0, Velocity = 150, Time = 200); break the tip back and manually grind it on a sharpening stone until there is about a 3 mm taper. Melt the tip using a microforge to the desired tip inner diameter of between 10 - 100 &#181;m depending on the area of nerve one wishes to sample from (see Figures 1B-1C for electrode schematic and Table of Materials/Equipment for product information).</li>
	<li>Fill a premade glass suction electrode to the inner silver wire with SIF.</li>
	<li>Suction the cut end of the nerve into the electrode and connect to the positive port of a differential amplifier. Wrap the electrode with a chlorided silver wire that connects to the negative port of the headstage. Ground the SIF bath by running a second chlorided silver wire from the bath to the headstage&#8217;s ground port. Also ground the perfusion tubing to the Faraday cage at multiple points to mitigate electrical noise introduced via the perfusion pumps.</li>
	<li>Stimulate the muscle via the electrodes mounted on either side of the muscle in the tissue bath to induce a twitch contraction. Alternatively place a stimulating electrode on the cut end of the nerve. Increase the stimulating voltage until a peak contractile force is observed and then increase the voltage by an additional 15% to reach supramaximal voltage (0.5 msec pulse width). Continue the twitch contractions at the supramaximal voltage, with a 10 sec rest in-between, but vary the length of the muscle until a peak contractile force is reached to find the optimal length (Lo) of the muscle. All length ramps and vibrations will start with the muscle at this length.</li>
	<li>Allow the muscle-nerve preparation to remain in the bath for at least 1 hr before subsequent data collection to allow the tissue to reach the bath temperature and for normal synaptic transmission to recover following dissection in a low calcium solution.</li>
	<li>To collect data at a temperature other than room temperature, place a temperature probe into the tissue bath near the muscle. Slowly bring the bath up to temperature by pumping heated water through the tissue bath base plate. Wrap clay microwavable heating pads around the SIF reservoir to help maintain a steady temperature.</li>
</ol>
3. Data Collection
<ol>
	<li>To identify an afferent as a spindle afferent, record the neuronal activity during repeated twitch contractions produced by a 0.5 msec supramaximal voltage stimulus delivered once every second. NOTE: Muscle spindle afferents should pause during the twitch contraction18,19 (Figure 2).</li>
	<li>Use data acquisition software to apply length changes at different speeds and to different lengths. Use a custom script to automate this task (see supplemental information for a screenshot of the script and directions on how to customize the stretches given). Apply ramp-and-hold stretches of 4 sec at stretch lengths of 2.5%, 5%, and 7.5% Lo and stretch speeds of 20, 40, or 60% Lo/sec 15. NOTE: See user manual for specific force and length controller for necessary voltage-to-millimeter conversion factor.</li>
	<li>At the end of the experiment, determine muscle health at 24 &#176;C using maximal isometric tetanic contractions (500 msec train, 120 Hz train frequency, 0.5 msec pulse width, supramaximal voltage). Compare the peak contractile force to previously reported values (~24 N/cm2 9,20).</li>
</ol>

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The open access charges for this manuscript have been paid by Aurora Scientific, Inc., the company that makes the force and length controller and in vitro bath plate used in this manuscript.

The goal of this article was to describe a method for recording from muscle spindle afferents in an isolated mouse muscle-nerve preparation. We have found that mouse spindle afferents respond similarly to stretch as those from rats, cats and humans15, and other laboratories have used mice as model organisms to study sensory neurons in both the muscle and the skin in vitro (for example3,13) .
Muscle sensory afferents can be recorded for at least 6-8 hr at both 24 &#176;C and 34 &#176;C. To minimize handling the EDL and nerve, use the remaining portion of the FHL to handle the tissue whenever possible. Following the dissection, wait at least 1 hour to start data collection to allow the tissue to equilibrate to bath temperature and for normal synaptic transmission to recover. Previously, muscle health was verified on a subset of muscles used in this preparation by determining that the maximal tetanic contractile force generated by the muscles is similar to that reported by others at the beginning and end of the experiment15.
Muscle spindle afferents were identified functionally in this preparation by looking for a characteristic pause in firing in response to twitch contraction (Figure 2) as well as the expected instantaneous frequency increases in response to length changes (Figure 3). In our experience, most afferents that fire in control conditions are muscle spindle afferents. The length of the nerve retained in this preparation (~7 mm maximum) is not long enough to use afferent conduction velocity differences to identify muscle afferent subtypes13. Additionally, unlike in cats and humans, measures of dynamic sensitivity were not able to clearly differentiate Group Ia and II afferents in mice (for further discussion see Wilkinson&#160;et al.15). Other subtypes of afferents (i.e., Group III and IV) can be identified using additional functional tests in this preparation, for instance by adding substances like capsaicin, ATP, or bradykinin, decreasing bath pH, exposing the muscle to ischemia, etc.3,13,21-23 Using suction electrodes allows the activity from multiple sensory neurons to be recorded at once, which increases the amount of data that can be collected from a single muscle. This preparation can be used to gauge the overall effect of a perturbation on sensory neuron population responses or the responses of identified afferents. If the spike shapes are unique enough, up to 4 sensory neurons can be discriminated by software (both Spike2 (Cambridge Electronic Design) and LabChart Pro (AD Instruments) have performed similarly). In cases where neurons cannot be discriminated, changes in electrode placement or tip diameter can easily be implemented.
In summary, the mouse muscle-nerve in vitro preparation is a simple experimental approach that can be used to investigate the response properties of muscle sensory afferents to various physicochemical perturbations, injury and disease models. Additionally, this preparation is ideally suited to take advantage of the powerful genetic tools available in mice, including transgenic animals and optogenetic tools.

Skeletal muscles are innervated by sensory neurons that provide the central nervous system with important information about the muscle environment. Muscle spindles are highly specialized encapsulated structures composed of intrafusal muscle fibers that are located in parallel with extrafusal muscle fibers. Spindles are innervated by Group Ia and II afferents that encode changes in muscle length and intrafusal fiber tone is dynamically regulated by innervating gamma motor neurons1. Group Ib afferents are positioned between muscle fibers and their tendon insertions and are sensitive to changes in muscle force, for instance during muscle contraction2. The Group Ia, Ib and II afferents provide proprioceptive feedback that aids in appropriate motor control. Additional populations of muscle afferents located throughout the muscle (Group III and IV) serve to signal metabolite buildup, the presence of nociceptive stimuli, and muscle temperature3. This muscle sensory information is critical to maintaining homeostasis, preventing muscle damage and modulating movement. Muscle afferents can alter their firing patterns based on prior experience4. Activation of nociceptors can lead to the induction of chronic pain states5 and aberrant signaling from the muscle proprioceptors can lead to problems with balance or movement6. We use an isolated muscle-nerve in vitro preparation to study the response of muscle sensory neuron receptor endings from adult mice of both sexes (shown are responses from 2-4 month old C57Bl/6 mice). The mouse is the model genetic species for studies in mammals. This preparation requires the isolation of the deep peroneal branch of the sciatic nerve and the extensor digitorum longus (EDL) muscle, a fast twitch muscle of the peroneal group found in the lateral part of the lower leg. The EDL is often used to study muscle contractile properties7-9, has tendons that are easy to isolate and is small enough to allow adequate diffusive oxygen supply at rest and with reasonable contraction duty cycles10. Other muscles in this area (for instance soleus and tibialis anterior) are of similar size and this preparation could be easily modified to record from afferents from these muscles. A suction electrode is placed onto the cut end of the nerve to record muscle sensory afferent firing. Individual neurons can be identified and analyzed based on their spike shape using spike sorting software. Stimulating electrodes in the bath or on the nerve can be used to evoke muscle contraction. Muscle length and force can be controlled and measured using commercially available systems. Similar in vitro preparations have been used in rodents to study Group III and IV muscle afferents in the rat EDL11, Group Ia and II spindle afferents in the rat fourth lumbrical toe muscle12 and Group III and IV muscle afferents in the mouse plantar muscle13.
An in vitro system has the advantages of pharmacological accessibility and direct control of perfusate and physiochemical variables like temperature and pH. An in vitro approach eliminates the potential in vivo confounds of anesthesia and muscle perfusion status. While the muscle-nerve preparation allows for the study of the direct response of a perturbation on the afferents, the ability to study gamma efferent modulation of spindle sensitivity and other integrated responses that is possible with in vivo14 or ex vivo3 preparations is sacrificed as there are no neuronal cell bodies in this preparation.
This preparation was previously used to characterize the response of mouse muscle spindle afferents to a battery of ramp and hold stretches and vibrations and it was determined that mouse spindle afferent responses were similar to those reported in other species such as rats, cats, and humans. Mouse spindle afferent responses were found to be similar at both 24 &#176;C and 34 &#176;C bath temperatures, although at 34 &#176;C absolute firing rates were faster and the afferents were more able to respond to faster length changes15. We describe below how this preparation can be used to identify and study the muscle spindle afferents. Moreover, this preparation can easily be modified to study the response of other muscle afferent subtypes13, to compare the properties of sensory afferents in response to a drug or disease state or assorted other variables (e.g., age, sex, gene knockout).

<table><tbody><tr><td>Force and Length Controller &amp;amp; Tissue Bath</td><td>Aurora Scientific, Inc.</td><td>300C-LR &amp;amp; 809B-IV&amp;nbsp;</td><td></td></tr><tr><td>Masterflex L/S Digital Drive Perfusion Pump &amp;amp; Easy Load II Pump Head</td><td>Cole Parmer</td><td>EW-07523-80 &amp;amp; EW-77200-60</td><td></td></tr><tr><td>2 Channel Microelectrode AC Differential Amplifier &amp;amp; Headstage Amplifier</td><td>A-M Systems</td><td>Model 1800; 700000 &amp;amp; 700500</td><td></td></tr><tr><td>Simulator</td><td>Grass&lt;br/&gt; OR&lt;br/&gt; Aurora Scientific, Inc.</td><td>S88&lt;br/&gt; OR&lt;br/&gt; 701 C</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Swift</td><td>892070</td><td></td></tr><tr><td>Fiber Optic Light Source</td><td>Fiberlite</td><td>Model 190</td><td></td></tr><tr><td>Analog to Digital Board</td><td>AD Instruments</td><td>PL3508/P (PowerLab 8/35)</td><td></td></tr><tr><td>Data Acquisition &amp;amp; Analysis Software</td><td>AD Instruments</td><td>LabChart Pro 7</td><td></td></tr><tr><td>Electrode Holder</td><td>A-M Systems</td><td>672445</td><td></td></tr><tr><td>Electrode Glass</td><td>Dagan</td><td>SA16</td><td></td></tr><tr><td>Electrode Puller</td><td>Sutter Instrument Co.</td><td>P-80/PC</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-9</td><td></td></tr><tr><td>Isoflourane</td><td>MWI Veterinary Supply</td><td>18627</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Surgical Tools&lt;/strong&gt;</td></tr><tr><td>Large Dissecting Scissors</td><td>Fine Science Tools</td><td>14014-17</td><td></td></tr><tr><td>Large Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Large Spring Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>#5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>#55 Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Sharp Scissors</td><td>Fine Science Tools</td><td>14059-11</td><td></td></tr><tr><td>6-0 Silk Suture</td><td>Fine Science Tools</td><td>18020-50</td><td></td></tr><tr><td>Chemicals for Low Calcium- High Magnesium aCSF and SIF&lt;br/&gt; (Low Calcium solution is equilibriated with 95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt; gas and pH of 7.4 &amp;plusmn; 0.05; SIF is equilibriated with 100% O2 gas and pH of 7.4 &amp;plusmn; 0.05)</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888-1KG</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>230391-500G</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>223506-500G</td><td></td></tr><tr><td>Sodium gluconate</td><td>Sigma</td><td>S2054-500G</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S8282-500G</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767-500G</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S5016-500G</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-250G</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P0662-500G</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P3911-500G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6014-500G</td><td></td></tr></tbody></table>

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.

The response of muscle afferents can be recorded following a variety of perturbations, depending on which afferent subtype is being studied. Representative responses of muscle spindle afferents to muscle contraction and ramp and hold stretch are shown here. To identify an afferent as a spindle afferent, twitch contractions are given once every second (0.5 msec pulse width) to see if there is a pause in firing during contraction18,19. Figure 2 shows a representative trace of neuronal activity and muscle tension during these twitch contractions. No neuronal activity is observed during twitch contractions, as expected for spindle afferents. If the recorded afferent was a Group Ib Golgi tendon organ afferent, an increase in firing rate during the contraction would be expected.
Under control conditions most afferents with a regular firing pattern (~12 impulses/sec at 24 &#176;C and ~32 impulses/sec at 34 &#176;C) are muscle spindle afferents. A subset of spindle afferents will only fire during stretch (in our hands ~11% of spindle afferents)15. Figure 3A shows a representative raw trace of two muscle spindle afferents responding to a ramp and hold stretch produced by the force and length controller. The Spike Histogram feature of LabChart is used to identify and analyze the instantaneous firing frequency of the two individual neurons separately (Figures 3B-3C).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig1highres.jpg" width="500" /> 
Figure 1.&#160;Isolated Muscle Preparation A)&#160;The extensor digitorum longus (EDL) and innervating sciatic nerve are mounted in an isolated tissue bath perfused with oxygenated synthetic interstitial fluid (SIF). An extracellular amplifier connected to a suction electrode records neural activity. A dual force and length controller&#8212;with appropriate software&#8212;controls and measures muscle force and length. The tissue bath electrodes deliver stimuli to produce twitch or tetanic contractions. Pumping heated water through internal bath plate channels controls the tissue bath temperature. B)&#160;Pulled glass suction electrode with ~3 mm tapered tip. C)&#160;Magnified depiction of the ideal tip shape for the suction electrode that is produced using a microforge. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51948/51948fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig2highres.jpg" width="500" /> 
Figure 2.&#160;Spindle Afferent Response to Twitch Contraction. Neuronal activity (top trace) and muscle tension (bottom trace) from 30 twitch contractions are superimposed with top overlapping trace in black. Afferent activity pauses during contraction-induced tension increase, which is a characteristic response of muscle spindle afferents. Bracket and arrow above the top trace denote the time during contraction when activity is paused.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51948/51948fig3highres.jpg" width="600" /> 
Figure 3.&#160;Spindle Afferent Response to Ramp and Hold Stretch A)&#160;Raw neural activity of two afferents (top trace) during a ramp and hold stretch applied to the EDL (muscle length shown in bottom trace). B)&#160;Instantaneous firing frequency (Inst. Fr., impulses per sec) of the unit exhibiting activity at Lo from A (shown in blue). C)&#160;Instantaneous firing frequency of the smaller unit that only fires during the stretch (shown in orange). Both units exhibit the spike frequency adaptation during stretch that is characteristic of muscle spindle afferents1.

Watch this Scientific Journal Video about An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents at JoVE.com

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.

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 ...

an-vitro-adult-mouse-muscle-nerve-preparation-for-studying-firing

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.0K Views.  San José State University.  Muscle sensorische neuronen betrokken zijn bij proprioceptor signaleren en rapporteren over metabole toestand en verwondingen gerelateerde gebeurtenissen. We beschrijven een volwassen muis in vitro spier-zenuw voorbereiding op studies over-stretch geactiveerde spieren afferenten. 

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

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Medicine

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

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

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

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

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