This research was supported by grants F31DC017053-01A1 (Lenell, PI) and K23DC014517 (Johnson, PI) from the National Institute on Deafness and other Communication Disorders of the National Institutes of Health.

This study was performed in compliance with the Institutional Animal Care and Use Committee of New York University School of Medicine.
1. Dissect rat larynx
<ol>
	<li>Euthanize rat according to the institutionally approved protocol. Shave the ventral neck from the mandible to manubrium and swab with alcohol to prevent fur contamination in the tissue specimens.</li>
	<li>Under a dissecting scope with 10x magnification excise the entire larynx by creating a midline neck incision with a scalpel...

<ol>
	<li>Connor, N. P., Suzuki, T., Lee, K., Sewall, G. K., Heisey, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+junction+changes+in+aged+rat+thyroarytenoid+muscle.">Neuromuscular junction changes in aged rat thyroarytenoid muscle.</a> Annals of Otology, Rhinology and Laryngology. 111 (7), 579-586 (2002).</li><li>Suzuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Alterations+in+Myosin+Heavy+Chaing+Isoforms+in+Rat+Intrinsic+Laryngeal+Muscles.">Age-Related Alterations in Myosin Heavy Chaing Isoforms in Rat Intrinsic Laryngeal Muscles.</a> Annals of Otology, Rhinology and Laryngology. 111 (11), 962 (2002).</li><li>Johnson, A. M., Grant, L. M., Schallert, T., Ciucci, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multiple+call+categories+within+the+rich+repertoire+of+adult+rat+50-kHz+ultrasonic+vocalizations:+effects+of+amphetamine+and+social+context.">Identification of multiple call categories within the rich repertoire of adult rat 50-kHz ultrasonic vocalizations: effects of amphetamine and social context.</a> Psychopharmacology. 211 (1), 1-13 (2010).</li><li>Bowers, J. M., Perez-Pouchoulen, M., Edwards, N. S., McCarthy, M. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+dopamine+synaptic+activity:+degradation+of+50-kHz+ultrasonic+vocalization+in+rats.">Reduction of dopamine synaptic activity: degradation of 50-kHz ultrasonic vocalization in rats.</a> Behavioral Neuroscience. 123 (2), 328-336 (2009).</li><li>Ciucci, M. R., Vinney, L., Wahoske, E. J., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+translational+approach+to+vocalization+deficits+and+neural+recovery+after+behavioral+treatment+in+Parkinson+disease.">A translational approach to vocalization deficits and neural recovery after behavioral treatment in Parkinson disease.</a> Journal of Communication Disorders. 43 (4), 319-326 (2010).</li><li>Nagai, H., Ota, F., Konopacki, R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discoordination+of+laryngeal+and+respiratory+movements+in+aged+rats.">Discoordination of laryngeal and respiratory movements in aged rats.</a> American Journal of Otolaryngology. 26 (6), 377-382 (2005).</li><li>Ma, S. T., Maier, E. Y., Ahrens, A. M., Schallert, T., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+intravenous+cocaine+experience:+development+and+escalation+of+pre-drug+anticipatory+50-kHz+ultrasonic+vocalizations+in+rats.">Repeated intravenous cocaine experience: development and escalation of pre-drug anticipatory 50-kHz ultrasonic vocalizations in rats.</a> Behavioural Brain Research. 212 (1), 109-114 (2010).</li><li>Inagi, K., Schultz, E., Ford, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anatomic+study+of+the+rat+larynx:+establishing+the+rat+model+for+neuromuscular+function.">An anatomic study of the rat larynx: establishing the rat model for neuromuscular function.</a> Otolaryngology and Head and Neck Surgery. 118 (1), 74-81 (1998).</li><li>Lenell, C., Newkirk, B., Johnson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laryngeal+Neuromuscular+Response+to+Short-+and+Long-Term+Vocalization+Training+in+Young+Male+Rats.">Laryngeal Neuromuscular Response to Short- and Long-Term Vocalization Training in Young Male Rats.</a> Journal of Speech, Language, and Hearing Research. 62 (2), 247-256 (2019).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> Journal of Visualized Experiments. (99), e52793 (2015).</li><li>McMullen, C. A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+evidence+of+age-related+denervation+in+rat+laryngeal+muscles.">Functional and morphological evidence of age-related denervation in rat laryngeal muscles.</a> Journals of Gerontology. Series A: Biological Sciences and Medical Sciences. 64 (4), 435-442 (2009).</li><li>McMullen, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+stimulation-induced+changes+in+the+rodent+thyroarytenoid+muscle.">Chronic stimulation-induced changes in the rodent thyroarytenoid muscle.</a> Journal of Speech, Language, and Hearing Research. 54 (3), 845-853 (2011).</li><li>Lenell, C., Johnson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+laryngeal+muscle+fibers+and+ultrasonic+vocalizations+in+the+adult+rat.">Sexual dimorphism in laryngeal muscle fibers and ultrasonic vocalizations in the adult rat.</a> Laryngoscope. 127 (8), 270-276 (2017).</li><li>Johnson, A. M., Ciucci, M. R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocal+training+mitigates+age-related+changes+within+the+vocal+mechanism+in+old+rats.">Vocal training mitigates age-related changes within the vocal mechanism in old rats.</a> Journals of Gerontology. Series A: Biological Sciences and Medical Sciences. 68 (12), 1458-1468 (2013).</li><li>Feng, X., Zhang, T., Ralston, E., Ludlow, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+neuromuscular+junctions+of+laryngeal+and+limb+muscles+in+rats.">Differences in neuromuscular junctions of laryngeal and limb muscles in rats.</a> Laryngoscope. 122 (5), 1093-1098 (2012).</li></ol>

The authors have nothing to disclose.

Preparing rat vocal folds for neuromuscular analysis can present with various challenges. Not only are laryngeal muscles small and surrounded by cartilage, thereby making it difficult to directly extract target muscle, high variability was also found between animals in the depth of laryngeal anatomical landmarks. For muscle the cross-section plane protocol, complete vocal fold sections appeared between 21&#8722;85 sections (10 &#181;m per section) after the initial appearance of the ventral thyroid cartilage, which is qu...

The rat larynx is a well-established model to investigate structural and functional neuromuscular laryngeal adaptations to development, aging, disease, and pharmacological agents1,2,3,4,5. Consistency of histological methods is critical to this line of work, as there are multiple intricacies involved in muscle preparation and analysis as well as challenges associated with laryngeal size, shape, and topography of the muscles encapsulated within the laryngeal cartilages1,6,7,8,9,10,11. Due to the small size of the rat intrinsic laryngeal muscles, systematic embedding, freezing, and cryosectioning are critical to achieve consistent and accurate results. For example, when sectioning the rat vocal fold in the coronal plane, the neuromuscular junctions (NMJs) of four of the intrinsic laryngeal muscles are located within less than 1.8 mm of tissue depth11. Therefore, precise monitoring of laryngeal muscle anatomy during cryosectioning is imperative to accurately identify the section(s) of interest and prevent oversectioning of tissue. Oversectioning of the target muscle can result in inaccurate identification of number and topography of NMJs11 or can result in overall reductions in sample size if the target muscle is discarded due to landmark orientation confusion12. As novel models for the study of laryngeal muscle and their respective adaptations are developed, standard operating procedures are essential to ensure results are precise, reliable, and reproducible across studies.
The objective of this article is to detail preparation of the rat vocal fold for optimal longitudinal and cross-sectional analysis. Detailed methods used regularly in our laboratory are described to identify target muscle landmarks during cryosectioning. Although similar methods are used in several laboratories, greater detail is provided here than in the literature to ensure reliable and accurate replication when implemented by novice investigators. The goal of this tutorial is to provide a standard methodology for immunohistochemical (IHC) evaluation of the rat vocal fold to improve consistency across laboratories and investigations.

<table>
	<tbody>
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			<td>2-Methylbutane Certified</td>
			<td>Fisher Chemical</td>
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			<td>Aluminum Foil</td>
			<td>Fisherbrand</td>
			<td>1213101</td>
			<td></td>
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			<td>Cryo Tongs SS</td>
			<td>Thermo Scientific</td>
			<td>11679123</td>
			<td></td>
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		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat blades</td>
			<td>C.L. Sturkey D554X50</td>
			<td>22-210-045</td>
			<td></td>
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		<tr>
			<td>Disposable Base Molds 15mm x 15mm</td>
			<td>Thermo Scientific</td>
			<td>41-741</td>
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			<td>Disposable Underpads</td>
			<td>Medline</td>
			<td>23-666-062</td>
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		<tr>
			<td>Dissection kit</td>
			<td>Thermo Scientific</td>
			<td>9996969</td>
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			<td>DPBS - Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190136</td>
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			<td>Frozen Section Medium</td>
			<td>Fisher Healthcare</td>
			<td>23-730-571</td>
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			<td>Ice Bucket</td>
			<td>Bel-Art</td>
			<td>11999054</td>
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			<td>Immunostain Moisture Chamber</td>
			<td>Ted Pella Inc</td>
			<td>NC9425474</td>
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			<td>Needle holders</td>
			<td>Assi</td>
			<td>ASSI.B148</td>
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			<td>Non-Woven Sponges, 4 Ply</td>
			<td>Quick Medical</td>
			<td>9023</td>
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			<td>Orbital shaker</td>
			<td>Troemner</td>
			<td>02-217-987</td>
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			<td>Pap pen</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td>Alfa Aesar</td>
			<td>50-00-0</td>
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		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>Fisherbrand</td>
			<td>5408129</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Storage Bags</td>
			<td>Fisherbrand</td>
			<td>19240093</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Graduated Measure 32 oz/100 mL</td>
			<td>Polar Ware</td>
			<td>114231B</td>
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		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
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			<td>Task wiper</td>
			<td>Kimberly-Clark Professional&#8482; 34155</td>
			<td>06666A</td>
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			<td>Timer</td>
			<td>Fisherbrand</td>
			<td>2261840</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Pattern Scissors</td>
			<td>Assi</td>
			<td>ASSI.SAS15RV</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">NOTE: For all supplies, these are examples of equipment to purchase. The exact model is not necessary to complete our methods.</td>
		</tr>
	</tbody>
</table>

preparation of the rat vocal fold for neuromuscular analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

The representative results were part of an ongoing investigation of the effects of vocal exercise on the laryngeal neuromuscular system. Twenty-nine male Fischer 344/brown Norway rats (12 9-month-old, 17 24-month-old) were weighed and euthanized with CO2 inhalation followed by a bilateral thoracotomy.
The procedures followed the outlined protocol to label NMJs and fiber size of the lateral and medial TA muscles. The distance between laryngeal landmarks was tracked in both longitudin...

Watch this Scientific Journal Video about Preparation of the Rat Vocal Fold for Neuromuscular Analyses at JoVE.com

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

This protocol facilitates the preparation of the rat and vocal fold for optimal longitudinal and cross-sectional analysis. The protocol utilizes laryngeal tracking during cryosectioning to ensure an accurate identification of the intrinsic laryngeal muscles and to reduce the chance of tissue loss. The rant larynx is a well-established model for investigating structural and functional neuromuscular laryngeal adaptations in response to development, aging, disease, and pharmacological agents.Although this demonstration focuses on the thyroarytenoid muscle of the vocal fold, the methodology is also applicable to other intrinsic laryngeal muscles. For dissection of the rat larynx, shave the ventral neck from the mandible to the manubrium and swab the exposed area with alcohol to prevent fur contamination of the tissue specimens. Place the rat under a dissecting microscope with a 10 times magnification and use a scalpel to make a midline neck incision until the trachea is exposed.Use forceps and a sharp instrument to separate the ventral extrinsic laryngeal muscles at the midline and sever the trachea caudal to the third tracheal ring. To excise the larynx, use dissecting scissors to make an incision caudal to the third tracheal ring and rostral to the hyoid bone. Use tweezers, pins, and microscissors to remove the esophagus, thyroid gland, and extrinsic laryngeal muscles from the larynx.Using the midline between the posterior cricoarytenoid muscles as a landmark, use the microscissors to bisect the larynx dorsally between the arytenoids. Pin the lateral walls of the larynx to expose the vocal folds and use the microscissors to bisect the tissue ventrally through the midline of the thyroid cartilage between the anterior commissure of the vocal folds. Then rinse each hemilarynx in PBS for 10 seconds before delicately drying the tissue with a task wiper to reduce ice crystal formation during freezing.To fix the hemilarynx tissues, place the samples in a centrifuge tube containing 4%formaldehyde and PBS for one hour at room temperature on an orbital shaker at 70 revolutions per minute. At the end of the incubation, transfer the tissues into a new centrifuge tube and rinse the samples three times for 20 minutes in fresh PBS per wash. After the last wash, transfer the tissues into a new centrifuge tube containing a 20%sucrose 5%glycerol solution at four degrees Celsius for up to 18 hours.When the tissue has sunk to the bottom of the tube, place the hemilarynges into a cryomold filled with optimal cutting temperature medium with the medial surface of the vocal fold facing the bottom of the cryomold in the longitudinal aspect of the vocal fold parallel to the lower edge of the cryomold opening. Flash freeze the submerged tissues in isopentane chilled in a steel beaker surrounded by liquid nitrogen and wrap each mold in pre-labeled foil for minus 80 degrees Celsius storage. To acquire cross-sectional plane sections of the frozen hemilarynx tissue samples, set the cryostat chamber to minus 20 degrees Celsius and set the section thickness to 10 microns.Transfer the tissue samples to the cryostat chamber and use additional optimal temperature cutting medium to attach one embedded tissue sample to the cryostat specimen disk with the ventral thyroid cartilage facing the cryostat blade and the arytenoid cartilage facing the specimen disk. To trim the cutting medium from the frozen tissue block, advance the specimen head by 100 microns until the ventral portion of the thyroid cartilage appears. Then trim and track 30 micrometer sections from the onset of the thyroid cartilage until the lamina propria, medial thyroid arytenoid muscles, and lateral thyroarytenoid muscle can be observed by light microscopy.An accurate identification of the transparent tissues during cryosectioning is the most difficult and critical component of this protocol. Once the target muscle has been reached, collect 10 micron sections of the tissue on positively charged slides and store the sections at four degrees Celsius in PBS until they are ready to be stained. To obtain longitudinal vocal fold sections for neuromuscular junction analysis of the thyroarytenoid muscle, affix the specimens to the disks so that the epiglottitis is oriented toward the cryostat blade and the tracheal lumen faces down toward the specimen disk and trim the cutting medium as just demonstrated.Trim and track 30 micrometer sections from the onset of the thyroid until the lamina propria, and medial and lateral divisions of the thyroarytenoid muscle can be observed by light microscopy. Once the target muscle has been reached, collect 30 micrometer sections onto positively charged slides and store the sections at four degrees Celsius in PBS until they are ready to be stained. In this analysis, the distance between laryngeal landmarks was tracked in both longitudinal and cross-sectional planes using laryngeal muscles in the surrounding cartilages to determine their progression during cryosectioning.Here, the appearance of laryngeal landmarks during cross-sectional cryosectioning in temporal order with the thyroid can be observed, appearing prior to the medial thyroarytenoid muscle and the lamina propria. Here, the appearance of laryngeal landmarks during longitudinal cryosectioning in temporal order with the alar muscle appearing prior to the medial thyroarytenoid muscle and the lamina propria. Weight and laryngeal landmark appearances had weak to moderate correlations for young rats and weak correlations for aged rats.The distances among landmarks within each plane were moderately to strongly correlated for both age groups, but weakly correlated between the two dissection planes. The most important thing to remember is that small variations in the tissue embedding and mounting will result in large variations during cryosectioning. These detailed procedures allow reliable neuromuscular histological investigations of the rat vocal fold.

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2.5K Views.  New York University School of Medicine. This protocol facilitates the preparation of the rat and vocal fold for optimal longitudinal and cross-sectional analysis. The protocol utilizes laryngeal tracking during cryosectioning to ensure an accurate identification of the intrinsic laryngeal muscles and to reduce the chance of tissue loss. The rant larynx is a well-established model for investigating structural and functional neuromuscular laryngeal adaptations in response to development, aging, disease, and pharmacological agents.