We thank the highly collaborative and stimulating environment of the NeSt Lab members for useful discussion and comments on this work. Work at the NeSt Lab is currently funded by FONDECYT 1221213. The scheme in Figure 1A was created with BioRender.com.

All experimental procedures are approved by the Bioethics Committee at Universidad Austral de Chile, Chile (protocol Nr. 503/2023), and followed the norms imposed by the Bioethics Committee of the National Research and Development Agency, Chile (ANID), as well as the guidelines of the European Council Directive for the Care of Laboratory Animals. The present study used adult (3-6 months old) CF-1 mice of both sexes (30-45 g). Euthanasia was accomplished by inhalant anesthetic overdose followed by exsanguination. The reagents and equipment used in the study are listed in the Table of Materials.
1. In vivo LAL muscle electroporation
<ol>
	<li>Preparation
	<ol>
		<li>Gently sedate the mouse in an acrylic induction chamber under inhalation of 2.5% isofluorane with 0.8 L/min oxygen (following institutionally approved protocols). After that, maintain mouse sedation using a nose tubing mask connected to an inhalation anesthesia machine under a dissecting stereomicroscope. 
		&#8203;NOTE: Ensure that the mouse is asleep before starting the procedure; test the touch reflex of the mouse by gently squeezing one foot with a forceps (do this step every time after inducing anesthesia).</li>
		<li>Disinfect the cervical area at the level of the sagittal suture of the skull with the antiseptic chlorhexidine gluconate 2% and inject subcutaneously 20 &#181;L of 2 mg/mL hyaluronidase in PBS using a Hamilton syringe to facilitate the DNA plasmid access to the muscle surface.</li>
		<li>Turn off the isofluorane flow and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Weigh&#160;the mouse to calculate the appropriate Meloxicam&#160;dose (see step 1.3.2.).</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the animal to&#160;normal housing for 30 min16.</li>
	</ol>
	</li>
	<li>Surgery procedure
	<ol>
		<li>Re-anesthetize the mouse by the inhalation of 2.5% isofluorane with a 0.8 L/min oxygen mixture (following institutionally approved protocols) and keep it sedated throughout the surgical procedure using an inhalation anesthesia machine, under a dissecting stereomicroscope.</li>
		<li>Shave the cervical area using a double-edged razor blade and disinfect with chlorhexidine and 70% ethanol 3 times.</li>
		<li>Make an 8 mm incision over the sagittal suture of the skull and expose the LAL muscle by gently removing the fat and connective tissue located just under the skin using scissors/forceps. 
		NOTE: The LAL muscle is the most superficial muscle in the cranial region after skin incision. It is easily recognized by the orientation of muscle fibers that run diagonally from the middle skull towards each pinna8,16.</li>
		<li>Using a Hamilton syringe, inject 10 &#181;L of the commercially obtained DNA (0.8-1 &#181;g/&#181;L) in PBS into the fascia that is distributed throughout the muscle, forming a bubble.</li>
		<li>Place two gold needle electrodes 5 mm apart, parallel, and over muscle fibers, occupying the entire length of the LAL muscle (Figure 1A).</li>
		<li>Using an electroporator, generate five pulses of 100 V/cm of 20 ms duration each, at 1 Hz frequency.</li>
		<li>Repeat the procedure in the contralateral LAL muscle (repeating steps 1.2.4-1.2.6).</li>
	</ol>
	</li>
	<li>Suture and recovery
	<ol>
		<li>Suture (5/0 strain size, HR15 needle) the skin with absorbable polyglycolic acid thread.</li>
		<li>Inject subcutaneously the analgesic Meloxicam in doses of 1-2 mg/kg of weight to reduce post-surgery pain.</li>
		<li>Turn off the isofluorane flow and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the animal to its regular housing and monitor it for 2 days after surgery or until its recovery. Monitor pain or stress signs, allocating scores for each of the different parameters considering the humane endpoint criteria previously described19. 
		NOTE: Position the mouse in a heated pad covered with a surgical pad to prevent its body temperature from dropping during surgery. Try not to take more than 30 min for the surgical procedure, thus preventing the mouse from dying due to excess anesthesia. If the expressed protein yields low fluorescence levels, it is recommended to co-electroporate with a tracer (e.g., tdTomato or GFP) in a 1:5 ratio (tracer: DNA of interest).</li>
	</ol>
	</li>
</ol>
2. Crush nerve injury-induced denervation
NOTE: This protocol is a modification of a previously described protocol17. Perform this procedure with a difference of at least 3 days from the electroporation. Depending on the research questions, this procedure can be performed before or after electroporation.
<ol>
	<li>Anesthesia and animal positioning
	<ol>
		<li>Perform the animal sedation in an acrylic induction chamber under inhalation of 2.5% isofluorane with 0.8 L/min oxygen (following institutionally approved protocols).</li>
		<li>Weight the mouse to calculate the Meloxicam&#160;dose (see step 2.3.2.).</li>
		<li>Maintain mouse sedation with a nose tubing mask connected to an inhalation anesthesia machine under a dissecting stereomicroscope.</li>
		<li>Position the mouse on its left side (to denervate the right LAL muscle). Tape the right ear towards the nose to expose the posterior area of the pinna.</li>
	</ol>
	</li>
	<li>Surgery procedure
	<ol>
		<li>Shave the posterior area of the pinna using a double-edged razor blade and disinfect with chlorhexidine and 70% ethanol 3 times.</li>
		<li>Locate the posterior auricular vein and make an incision in the skin, 2 mm away caudally from it, of approximately 5 mm with spring scissors. Avoid cutting blood vessels and/or muscle tissue.</li>
		<li>Cut the fat and connective tissues with forceps and scissors. Find the spinal accessory nerve; continue until finding the belly of the digastric muscle and the cartilaginous auditory canal (semi-transparent whitish tissue) (Figure 2A).</li>
		<li>Locate the beginning of the facial nerve branching just below the digastric muscle.</li>
		<li>Follow the posterior auricular branch (the most dorsal branch of the facial nerve), and gently remove the connective tissue surrounding it, carefully avoiding direct nerve manipulation.</li>
		<li>Crush the posterior auricular branch of the facial nerve for 30 s, applying constant pressure on the nerve with a forceps with a 45&#176; angled tip.</li>
	</ol>
	</li>
	<li>Suture and recovery
	<ol>
		<li>Suture (5/0 strain size, HR15 needle) the skin with absorbable polyglycolic acid thread.</li>
		<li>Inject subcutaneously the analgesic Meloxicam in doses of 1-2 mg/kg of weight to reduce post-surgery pain.</li>
		<li>Remove the tape from the ear, turn off the isofluorane flow, and allow the mouse to breathe pure oxygen for 1 min.</li>
		<li>Place the animal in an empty cage (without any bedding) to recover from anesthesia.</li>
		<li>Return the mouse to its regular housing and monitor it for 2 days after surgery or until its recovery.</li>
		<li>After recovery, examine the mouse for failures in the movement of the affected ear as a sign of efficient denervation. 
		NOTE: In Sham control animals, make an incision in the skin, expose the facial nerve without touching it directly, and subsequently suture.</li>
	</ol>
	</li>
</ol>

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

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The authors have nothing to disclose.

The combination of in vivo muscle electroporation and denervation is a valuable experimental approach to investigating a regenerative niche at the NMJ4. Since the LAL muscle is superficially exposed, these combined procedures can be readily complemented with the delivery of probes or drugs that could affect protein expression or activity12. In addition, electroporation of reporter genes into the LAL muscle could provide a valuable tool to follow the activation or i...

The neuromuscular junction (NMJ) is the peripheral synapse that controls muscle contraction and, indirectly, the coordinated movement of organisms1. It is formed by a presynaptic motor axon terminal, a muscle postsynaptic domain enriched in acetylcholine receptors (AChRs), and non-myelinic terminal Schwann cells covering the axon terminal2,3,4. Upon NMJ denervation due to traumatic peripheral nerve injury, disease, or pharmacologic intervention, contractile muscle activity is lost5,6. As certain permissive microenvironments allow timely and efficient functional recovery, the search for proteins that could help the regeneration process is a permanent necessity. Here, two procedures have been combined to specifically evaluate the potential role of manipulating muscle protein expression in the short-term regeneration of the NMJ after nerve injury.
The levator auris longus (LAL) muscle, located on the dorsal surface of the skull, is a thin and flat muscle that controls the movement of the pinna. The LAL muscle consists of rostral and caudal bands, each containing two or three layers of muscle fibers7,8. The posterior auricular branch of the facial nerve innervates the LAL muscle, generating a well-described innervation pattern consisting of five rostral (R1-R5) and two caudal (C1-C2) innervation regions8,9,10. The LAL is a superficially exposed and easily accessible muscle, so its use requires minimally invasive procedures. This allows visualization of the NMJ using real-time microscopy, drug delivery, as well as the intervention of complete muscle preparations both in vivo and ex vivo11,12. Altogether, these features make the LAL muscle an excellent model to study NMJ behavior and function10,13.
In vivo electroporation is a gene-transfer technique that allows the incorporation of exogenous DNA into the tissue through the transient permeabilization of cell membranes and the mobility of DNA inside the cells, both generated after inducing an electric field14,15. This procedure is local and can be performed at any stage of the animal&#39;s development. Considering the unique features of the LAL muscle, its in vivo electroporation represents a minimally invasive, highly efficient, and quick procedure16, making it possible to observe robust protein expression two or three days after electroporation.
The facial nerve crush procedure has been widely employed in research as it offers several advantages5,17,18. This protocol has been previously modified to specifically target the posterior auricular branch of the facial nerve that innervates the cranial muscles, including the LAL muscle, to avoid facial paralysis; consequently, mice do not require special care after surgery (e.g., lubricating eye ointment for the loss of blink reflex). As NMJ reinnervation at the LAL muscle begins between 7-9 days after injury6, it is a good short-term reinnervation model compared to the commonly used nerve crush injury model of the sciatic nerve to denervate hindlimb muscles, where reinnervation occurs around three weeks post nerve injury18.
The combination of in vivo electroporation and facial nerve injury at the LAL muscle will enable researchers to evaluate the behavior of synaptic components against muscle-derived protein modulation at different times of NMJ regeneration, including short-term and long-term morphological effects6,16, through a wide variety of techniques including immunohistochemical, histological, and functional assays.

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			<td>Chlorhexidine gluconate 2%</td>
			<td>DifenPharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light dissecting stereomicroscope</td>
			<td>Motic</td>
			<td>Model SMZ-171</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Dilute 1 : 100</td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools&#160;</td>
			<td>11252-00</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Dumont Mini Forceps &#8211;Style 5</td>
			<td>Fine Science Tools&#160;</td>
			<td>11200-14</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>ECM 830 electroporator</td>
			<td>BTX Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gold needle-type electrodes&#160;</td>
			<td>Genetrodes, BTX</td>
			<td>45-0114</td>
			<td>10 mm straight</td>
		</tr>
		<tr>
			<td>Hamilton syringe&#160;</td>
			<td>Hamilton</td>
			<td>80400</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3884</td>
			<td></td>
		</tr>
		<tr>
			<td>Inhalation anesthesia machine</td>
			<td>SciVerma Scientific</td>
			<td>M3000 Table Top</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>218-082</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Instituto de Salud Publica - Chile</td>
			<td></td>
			<td>Adult (3-6 months old) CF-1 mice, of both sexes (30-45 g)&#160;</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Schick</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Glicosorb 5/0</td>
			<td>TAGUM</td>
			<td>GS0812.J</td>
			<td>Absorbable polyglycolic acid thread&#160;</td>
		</tr>
		<tr>
			<td>tdTomato plasmid</td>
			<td>Addgene</td>
			<td>54642</td>
			<td>tdTomato plasmid under the control of the CMV promoter</td>
		</tr>
		<tr>
			<td>Tramadol hydrochloride 5% (Triamcol)</td>
			<td>Drag Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td>91500-09</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>WGA Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>W7024</td>
			<td>Dilute</td>
		</tr>
		<tr>
			<td>&#945;-BTX Alexa Fluor 488&#160;</td>
			<td>Invitrogen</td>
			<td>B13422</td>
			<td>Dilute 1 : 500</td>
		</tr>
	</tbody>
</table>

combined in vivo electroporation and short&#45;term reinnervation of the cranial levator auris longus skeletal muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

Author Spotlight: Regenerative Roles of Muscle Proteins at Neuromuscular Junction Post-Nerve Injury

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

Our research aims to understand the mechanisms underlying the reiteration of the neuromuscular synapse and to evaluate the impact of inhibiting or overexpressing specific muscle-derived proteins on this regenerative process. Through muscle in vivo electroporation, we have observed the impact of muscle-derived proteins on the organization of postsynaptic acetylcholine receptors. Additionally, our denervation protocol has enabled us to identify various morphologies of postsynaptic domains and correlate them with their stability.This technique serves as an initial experimental approach to evaluate the impact of a specific muscle protein's generation, paving the way for more intricate gene-editing techniques. Moreover, these protocols are minimally invasive. This combination of experimental methods has been employed to investigate the role of Wnt signaling or NMJ regeneration.By overexpressing agonists or inhibitors of the Wnt pathway, we analyzed nerve damage and the subsequent regeneration processes. Our future research will examine the impact of muscle-derived proteins on the organization and functionality of the neuromuscular synapse in various contexts, including aging and certain pathologies, such as ALS.

The high fluorescence quantum yield of the tdTomato protein20 makes it an appropriate tracer for electroporation efficiency. Therefore, the expression of tdTomato protein in LAL muscles by in vivo electroporation was evaluated (Figure 1). LAL muscles were dissected 21 days after electroporation. Whole-mount preparations show the thin caudal band (cLAL, cyan polygon) of the LAL muscle with its two innervation regions, and the thick rostral band (rLAL, yellow p...

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

combined-vivo-electroporation-short-term-reinnervation-cranial

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JoVE Journal

Neuroscience

In Vivo Levator Auris Longus Skeletal Muscle Electroporation to Study NMJ Regeneration in Mice

To begin, make sure all dissection tools and the working space have been autoclaved and are clean. Place an anesthetized CF-1 mouse on the surgical table. Use a Hamilton syringe to subcutaneously inject 20 microliters of 2 milligrams per milliliter hyaluronidase in PBS.Weigh the mouse to determine the approximate meloxicam dose and place the animal in an empty cage for a 30-minute recovery. After re-anesthetizing the mouse, make an 8-millimeter incision over the sagittal suture of the skull. Using scissors and forceps, gently remove the fat and connective tissue beneath the skin to expose the levator auris longus or LAL muscle.With a Hamilton syringe, inject 10 microliters of commercially-sourced DNA into the fascia of the muscle. Position two gold needle electrodes 5 millimeters apart in parallel alignment over the muscle fibers, covering the full length of the LAL muscle. Now, use an electro operator to apply five electrical pulses at 100 volts per centimeter for 20 milliseconds each with a frequency of 1 hertz.Microscopic examination showed that a high proportion of muscle fibers from the rostral LAL portion were positive for the expression of tandem dimer tomato, confirming efficient in vivo electroporation-mediated gene transfer of LAL muscles.

Crush Nerve Injury&#45;Induced Denervation in Mouse Levator Auris Longus Muscle to Assess NMJ Reinnervation and Maintenance

To begin, position an anesthetized mouse on its left side. To denervate the right levator auris longus, or LAL, muscle, secure the right ear towards the nose to reveal the posterior area of the pinna. After shaving the skin and locating the posterior auricular vein, make a five millimeter incision in the skin, two millimeters caudally away from it.With forceps and scissors, remove fat and connective tissues to expose the spinal accessory nerve, the digastric muscle, and the cartilaginous auditory canal. Gently remove the surrounding connective tissue to expose the posterior auricular branch of the facial nerve. Using forceps with a 45-degree angled tip, apply a 30-second crush to the nerve with constant pressure.Finally, using a 5/0 string size and an HR15 needle, close the incision with absorbable polyglycolic acid sutures. After recovery and meloxicam treatment, examine the mouse's LAL microscopically for denervation. Confocal microscopy imaging of LAL muscles showed complete neuromuscular junction denervation five days after nerve injury.

203 Views.  Universidad Austral de Chile.  Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

Summary