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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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+phenotyping+of+group+III+and+IV+muscle+afferents+in+mouse.">Comprehensive phenotyping of group III and IV muscle afferents in mouse.</a> J Neurophysiol. 109, 2374-2381 (2013).</li><li>Nichols, T. R., Cope, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-bridge+mechanisms+underlying+the+history-dependent+properties+of+muscle+spindles+and+stretch+reflexes.">Cross-bridge mechanisms underlying the history-dependent properties of muscle spindles and stretch reflexes.</a> Can J Physiol Pharmacol. 82, 569-576 (2004).</li><li>Mense, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociception+from+skeletal+muscle+in+relation+to+clinical+muscle+pain.">Nociception from skeletal muscle in relation to clinical muscle pain.</a> Pain. 54, 241-289 (1993).</li><li>Proske, U., Gandevia, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amiloride-sensitive+channels+are+a+major+contributor+to+mechanotransduction+in+mammalian+muscle+spindles.">Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles.</a> J Physiol. 588, 171-185 (2010).</li><li>Wenk, H. N., McCleskey, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+skeletal+muscle-nerve+preparation+and+in+vitro+model+of+ischemia.">A novel mouse skeletal muscle-nerve preparation and in vitro model of ischemia.</a> J Neurosci Methods. 159, 244-251 (2007).</li><li>Bullinger, K. L., Nardelli, P., Wang, Q., Rich, M. M., Cope, T. 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(48), (2011).</li><li>Hoheisel, U., Rein&#246;hl, J., Unger, T., Mense, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidic+pH+and+capsaicin+activate+mechanosensitive+group+IV+muscle+receptors+in+the+rat.">Acidic pH and capsaicin activate mechanosensitive group IV muscle receptors in the rat.</a> Pain. 110, 149-157 (2004).</li><li>Taguchi, T., Sato, J., Mizumura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+mechanical+response+of+muscle+thin-fiber+sensory+receptors+recorded+from+rat+muscle-nerve+preparations+in+vitro+after+eccentric+contraction.">Augmented mechanical response of muscle thin-fiber sensory receptors recorded from rat muscle-nerve preparations in vitro after eccentric contraction.</a> J Neurophysiol. 94, 2822-2831 (2005).</li><li>Xu, J., Gu, H., Brennan, T. 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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Force and Length Controller &#38; Tissue Bath</td>
			<td>Aurora Scientific, Inc.</td>
			<td>300C-LR &#38; 809B-IV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive Perfusion Pump &#38; Easy Load II Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07523-80 &#38; EW-77200-60</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Channel Microelectrode AC Differential Amplifier &#38; Headstage Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 1800; 700000 &#38; 700500</td>
			<td></td>
		</tr>
		<tr>
			<td>Simulator</td>
			<td>Grass 
			OR 
			Aurora Scientific, Inc.</td>
			<td>S88 
			OR 
			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 &#38; 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">Surgical Tools</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 colspan="4">Chemicals for Low Calcium- High Magnesium aCSF and SIF 
			(Low Calcium solution is equilibriated with 95% 02/5% C02 gas and pH of 7.4 &#177; 0.05; SIF is equilibriated with 100% 02 gas and pH of 7.4 &#177; 0.05)</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/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

Research

JoVE Journal

Neuroscience

16.8K Views.  San José State University. 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.

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

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...