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Laryngeal muscles are specialized for fine control of voice, speech and swallowing, and may differ from limb muscles in many aspects. Since muscles and their controlling motor neurons communicate via neuromuscular junctions (NMJ), we hypothesized that NMJs in laryngeal muscles have specialized characteristics different from limb muscles.
In vivo study.
Single muscle fibers from 12 Sprague-Dawley rats (6 male, 6 female) were used to analyze the postsynaptic side of NMJs from laryngeal thyroarytenoid (TA), cricothyroid (CT), posterior cricoarytenoid (PCA), limb soleus (SOL), and extensor digitorum longus (EDL) muscles. NMJs were labeled with rhodamine-conjugated-α-bungarotoxin. With confocal microscopy, we counted cluster fragments, and measured the NMJ area, both absolute and normalized (corrected by muscle fiber diameter) for at least 10 single fibers from each muscle of each animal. Differences between genders were also compared.
Cluster fragments of postsynaptic NMJs were more numerous in PCA and TA compared to CT, SOL and EDL muscles (P <.01) in both male and female rats. NMJ cluster fragments were more numerous in female than in male rats only in the TA muscle (P <.01). The absolute area covered by the NMJs showed SOL>EDL>PCA>CT>TA (P <.01), however, with normalization, the SOL=EDL=PCA>CT=TA.
Differences found in NMJ surface and organization between laryngeal and limb muscle fibers may relate to specialized laryngeal muscle functions. Differences in NMJs between male and female rats were found only in the TA muscle, suggesting an underlying mechanism for some gender-specific laryngeal disorders related to abnormal TA muscle activity.
The intrinsic laryngeal muscles control the shape and tension of the vocal folds which mediate fine control of phonation, vocalization, respiration, swallowing and airway protection in mammals. Laryngeal muscles have rapid shortening speed, high active tension and high fatigue resistance 1–3. Muscle activity is directly modulated by nerve impulses from motor neurons via axons to muscle fibers to induce muscle contractions. The junction between a motor neuron and a muscle fiber is referred to as the neuromuscular junction (NMJ). Postsynaptic components of NMJs include acetylcholine receptors (AChRs) which accept acetylcholine released from the nerves, thus evoking a muscle impulse. Each adult single muscle fiber maintains an NMJ of the specific appropriate size, efficiency and spatial distribution to maintain its functional properties.
Important functional and structural differences exist between limb and cranial muscles. For instance, cranial muscles have higher ratios of end-plate size to muscle fiber volume than the limb muscles 4. In addition, intrinsic laryngeal muscles were recently reported to be spared or less affected in some degenerative neuromuscular diseases 5–9 with increased fragmentation of NMJs. Considering that fine structure of the NMJ is closely related to its function 4, the specialized functional regulation of the laryngeal muscles may be due to their specific NMJ morphology. Such differences may, in turn, make it less likely for neuromuscular diseases to injure laryngeal muscles.
Comparing the NMJs of limb and laryngeal muscles could increase our understanding of the mechanisms underlying these reported differences. Fine structural comparisons between limb and cranial muscle NMJs are lacking. Most studies have focused on either limb or laryngeal muscles with an emphasis on the effect of aging and degenerative disorders. In addition, most of these studies have investigated morphological changes in the NMJ with aging in whole laryngeal muscles 10,11. A more detailed structural comparison between limb and cranial muscle NMJs is therefore needed.
Recent evidence suggests a possible difference in laryngeal muscle NMJ structure and function between genders. For example, females are much more likely (~80% predominance) to be affected by spasmodic dysphonia (SD) 12–14, a disease characterized by the involuntary movements and inconsistent contractions of the intrinsic laryngeal muscles. Intramuscular botulinum toxin injections are used to inhibit acetylcholine release 15 from nerve endings and to decrease the amount of ACh binding to AChR, therefore reducing muscle contractions. Some reports indicate that the amount of botulinum toxin required to treat SD may be lower in men, and that the side effects of breathiness (loss of voice) due to denervation are greater in men 16. The mechanisms underlying these gender differences in prevalence and treatment are rarely studied and remain unknown. Possible NMJ structural differences in laryngeal muscles between genders could be one of many underlying mechanisms.
Most studies of NMJ morphologies and structures are performed on whole muscle tissue sections and use alpha-bungarotoxin, a drug that binds to the AChR and thus labels the postsynaptic site of the NMJ. In the present study, we use the same label but we examined the postsynaptic NMJ morphology of single muscle fibers isolated from laryngeal muscles of the Sprague-Dawley rat. We studied three intrinsic laryngeal muscles, i.e. the posterior cricoarytenoid (PCA), cricothyroid (CT) and thyroarytenoid (TA); and two limb muscles, the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL). We hypothesized that 1) NMJs of laryngeal muscles would show a different morphology from that of the limb muscles; 2) NMJs would show different morphologies among individual laryngeal muscles; and, 3) laryngeal NMJ morphology differences may exist between genders.
Twelve young adult Sprague-Dawley rats (Harlan, Indianapolis, IN) including 6 males and 6 females, were used for this experiment. All rats were maintained on a 12-hour light/dark cycle and given ad libitum access to food and water. All procedures were carried out in accordance with National Institutes of Health guidelines on the care and use of laboratory animals. The study protocol was approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee.
Rats were euthanized with an intraperitoneal injection of sodium pentobarbital (190 mg/kg body weight) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS) followed by a freshly prepared cold 4% paraformaldehyde (PFA) solution in 0.1 M PBS. Under an operating microscope (Zeiss, LR66238C, Germany), the PCA, CT, TA, SOL and EDL muscles from the right side of each animal were isolated. All the muscles were fixed in 4% PFA at room temperature for two hours and then moved into 0.1 M PBS.
Muscles were longitudinally cut into small pieces and the mid-portion from the same area in each muscle was selected for fiber separation. Single muscle fibers in each muscle were identified, separated apart and stained for acetylcholine receptors (AChR, postsynaptic NMJ) following the procedure described below. Muscle fibers were incubated in 4 % bovine serum albumin (BSA) blocking solution for 1 hour, then moved to tetramethylrhodamine α-bungarotoxin (BTX, 1:500; Invitrogen, CA, USA) diluted in 0.1M PBS for 1 hour at 4°C to label AChR. After 3 washes with 0.1M PBS, muscle fibers were incubated in Hoechst 33342 (1:500, Sigma) for 15 minutes to label the nuclei, then washed in PBS three times, mounted in Vectashield mounting medium (Vector Labs, Burlingame, CA), and sealed.
NMJ images were acquired on a Zeiss LSM 510 confocal microscope with a 40x N.A. 1.4 objective lens as a series of optical sections separated by 1 μm increments. Series were recorded regardless of the orientation of the NMJ in relation to the microscope focus plane. A three-dimensional (3D) reconstituted image of each labeled postsynaptic NMJ was obtained by doing 64 maximum projections of the data, covering 360 degrees around the Y axis. A 3D movie of each NMJ was created (see supplementary material). The 3D movie was used, for each NMJ, to determine the projection that best reflected the total size of the NMJ. This 2D projection was then used for the measurements. The muscle fiber diameter was determined from the background Hoechst staining. At least 125 images of muscle fibers with AChR staining were taken from each muscle and from 12 animals.
Morphology/size of the postsynaptic NMJs was analyzed using ImageJ software (developed by Wayne Rasband at the National Institutes of Health and freely available at rsbweb.nih.gov). The discontinuous fragments of each postsynaptic NMJ were counted and the muscle fiber area covered by each NMJ was measured. Muscle fiber diameter was measured to calculate the normalized postsynaptic NMJ area (postsynaptic NMJ area/muscle fiber diameter). The largest diameter adjacent to the postsynaptic NMJ on 2D stack images was measured. After developing the measurement methods, a pilot study for the first ten images of NMJs was performed by two independent investigators (XF and TZ) and their results were compared. As no differences were found, all of the measures were made by one investigator while blinded to the muscle group identity.
The images were analyzed by an investigator blinded to the muscle groups. All values were expressed as the mean ± standard deviation. Data were analyzed by two-way ANOVA with gender (male, female) and different muscles (PCA, CT, TA, SOL and EDL) as grouping variables. Post-hoc analysis was performed using a t-test with the Bonferroni adjustment method. Differences were considered significant at P < 0.05.
Figure 1 shows representative 2D images of NMJs in five different skeletal muscles. The soleus and EDL muscles show the classical appearance of NMJs, sometimes referred to as “pretzel-shaped”. In contrast, AChRs in PCA and TA muscles were grouped into multiple distinct small, rounded clusters, whereas the NMJ in CT muscle looked more similar to the EDL muscle.
Quantification confirmed that in both male and female rats, postsynaptic NMJs of the PCA and TA had more fragments compared to the SOL and EDL muscles (P < .05), whereas CT muscle showed similar amounts of cluster fragments compared to the SOL and EDL muscles (P > .05) (Figs. 2A, 2B). SOL had more NMJ cluster fragments than the EDL (P < .01). No difference was found between PCA and TA muscles (P > .05). A significant gender difference was found in the TA muscle, where the females showed more cluster fragments than the males (P < .01, Fig. 2C). In the PCA muscle, there was a tendency toward more cluster fragments in the female rats than the male rats; the difference was not statistically significant, which may be due to a larger standard deviation (P > .05).
Occasionally, multiple NMJs were observed in a single muscle fiber (Fig. 3). These types of muscle fiber were only found in PCA muscle (3 among 125 fibers from 12 rats) in this study.
Table I lists the resulting measurements of NMJs from these experiments. Because no gender differences were found in these measures, data from both male and female rats were combined and the comparison was made between different muscles. Limb muscles showed the biggest NMJ area (SOL>EDL>PCA>CT>TA, P < .05) and muscle fiber diameter (SOL>EDL>PCA>CT>TA, P < .05), but normalized NMJ area was similar to the PCA (P > .05) (SOL=EDL=PCA>CT=TA). For all three laryngeal muscles analyzed, PCA showed the biggest normalized NMJ area compared to the CT and TA muscles (P < .05).
The purpose of this study was to compare the morphology of neuromuscular junctions (NMJs) at the single muscle fiber level in young adult Sprague-Dawley rats in laryngeal and limb muscles. The advantage of studying the NMJ in single muscle fibers is the ability to view the whole NMJ regardless of its orientation and to match the morphology of each NMJ precisely to its muscle fiber. A recently published paper suggested that 3-D measurements are recommended for NMJ morphology testing 17, but measurements of 2-D projections appear ideal to identify the changes of NMJ in morphology and size 15.
Taking advantage of confocal microscopy, we combined the 3-D image collection and 2-D projection so that the full surface area of each NMJ could be measured regardless of its orientation and matched to the fiber diameter accurately. The major findings were: 1) the PCA and TA muscles had more fragmented NMJs than the CT, SOL and EDL muscles; 2) only the TA muscle showed male-female differences (more fragmented NMJ cluster fragments in the female than in the male rats); 3) multiple NMJs were found in some fibers of the PCA muscle; and 4) PCA, SOL and EDL muscles had larger normalized NMJ areas than the TA and CT muscles. In the rat the laryngeal muscles are used for precise patterns of ultrasonic vocalization 18 and in the human for speech, which both require precise muscle tension control. Rats may provide a good model for studying the NMJ structure and distribution in single muscle fibers of laryngeal muscles due to their size and easy accessibility for tissue collection. Large numbers of whole fibers can be collected, ensuring that NMJs will be present in each fiber, and large numbers of animals can be collected, improving the reliability of the results. In addition, the fiber types in the selected limb and laryngeal muscles are well known. For example, among laryngeal muscles in rats, the TA, an adductor muscle, expresses more fast fibers (100%) than PCA (80%), the abductor, and CT (70%), the vocal fold tensor 19. TA and PCA showed more fragments compared to the CT muscle, suggesting that these two muscles may express similar muscle twitch patterns during the modulation of laryngeal adduction and abduction. CT muscle was the most similar in morphology and fragmentation to the EDL muscle, which is composed of 90% fast-twitch fibers. However, the limb muscle of the rat, SOL, a representative muscle with mostly slow-twitch muscle fibers (90%) showed more fragments compared to the EDL. It seems that the limb muscles do not follow the rule of intrinsic laryngeal muscles - the higher the proportion of fast-twitch fibers, the higher the number of NMJ cluster fragments. A possible explanation for these differences is that the shape and size of NMJs could change significantly depending on muscle fiber types and physiological status in a single muscle 20–23. NMJ development, maturation and maintenance are determined by the signals between the nerve terminal and the muscle fiber, which play distinct roles in postsynaptic differentiation of the neuromuscular synapse 24,25. Specifically, development of intrinsic laryngeal muscles and their innervation differs from that in limb muscles. For example, human laryngeal innervation shows delayed maturation compared to other skeletal muscles 26 which may suggest that the development and maturation of laryngeal muscle innervation could be further modulated by their specialized functions after birth.
In addition to the differences between laryngeal and limb muscles, there are also marked differences in NMJ cluster morphology among different intrinsic laryngeal muscles. These differences may underlie the distinct sensitivities of laryngeal muscles to muscle diseases. For example, intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy (AChRs are fragmented in the affected muscles) 5–8, except the CT muscle. Interestingly, another neuromuscular disease, myasthenia gravis, also shows more severe electrophysiological pathology in CT muscle compared to other intrinsic laryngeal muscles 9. The highly specialized morphology of NMJs may predispose some laryngeal muscles to be more sensitive or resistant to some neuromuscular diseases.
However, in some laryngeal diseases with increased muscle tension, like spasmodic dysphonia (SD), the PCA and TA muscles show higher levels of activity 27. This may reflect possible correlates of higher synapse strength with the specialized NMJ clusters. In support of this concept, synapse strength of laryngeal muscles is higher in females and can be increased by the female hormone, estrogen 28,29. In our study, the TA muscle in female rats had more AChR clusters than in male rats, which strongly suggests a correlation between larger numbers of AChR clusters and higher synapse strength. The PCA muscle also had the largest NMJ area compared to the other intrinsic laryngeal muscles.
Our findings could provide a valuable model for future testing of the possible link between NMJ organization and synapse strength in humans, and might provide useful information for the development of NMJ targeting strategies for treating laryngeal muscle abnormalities. For example, the PCA muscle may play a role in abductor spasmodic dysphonia (ABSD) where botulinum toxin injection has not consistently been therapeutically helpful 30. If it were shown that our results could be extrapolated to human PCA NMJs then it might be investigated whether higher numbers of synapses may require t higher doses of botulinum toxin for injection.
In addition to the fine structural differences, we found a few single muscle fibers displaying multiple NMJs, consistent with reports of human laryngeal muscles innervated at multiple NMJs by a single axon 26,31. We did not stain the neural axon in single muscle fibers in the present study. Perhaps multiple NMJs in rats might be related with vocalization and might be correlated with the fine control of vocal fold movement required for speech in humans, similar to the multi-innervation reported in extraocular muscles 32. The similar observations of multiple NMJs in laryngeal muscles suggest homology between rats and humans in laryngeal muscle innervation. However, there are differences between rat and human laryngeal muscle physiology and muscle composition. For example, laryngeal muscles in the rat comprise more fast-twitch fibers than those in humans 17. Therefore, more work is needed to substantiate some of the differences found here in the rat between limb and laryngeal muscles, in humans. The present study did not test the fiber type (fast or slow twitch fiber) or the single muscle fiber function for each NMJ analyzed. Furthermore, human samples are irreplaceable when it comes to the study of speech and voice disorders. The rat model remains valuable, however, for further studies on the development of innervation and of physiological functioning in the laryngeal muscles. Ultimately, linking NMJ morphology to single fiber type/function could help provide a better understanding of laryngeal muscles.
Rat intrinsic laryngeal muscles, especially the PCA and TA muscles, present specialized post-synaptic NMJs compared to the limb muscles. Further study of these NMJ functions will improve our understanding of laryngeal muscles specifically affected or spared in neurological disorders. The NMJ gender differences in the TA muscle may suggest an underlying mechanism to examine in future research for the study of gender-related differences in some laryngeal disorders related to abnormal TA muscle activity.
Animation of 64 maximum projections of tetramethylrhodamine α-bungarotoxin staining of a neuromuscular junction in the laryngeal thyroarytenoid muscle of the rat.
This work was supported by the Intramural Programs of the National Institute of Neurological Disorders and Stroke, and National Institute of Arthritis and Musculoskeletal and Skin Diseases, and by funds from the Department of Otolaryngology, Wake Forest School of Medicine. We thank Dr. Iris Leng, Department of Biostatistics, Wake Forest University School of Medicine, for her assistance with the statistical analyses. We also thank Karen Potvin Klein, MA, ELS (Research Support Core, Wake Forest University Health Sciences) for her editorial contributions to this manuscript.
Dr. Ludlow is a consultant for Passy Muir Inc and Alfred Mann Foundation and receives support from U54 NS065701.
Part of the data were presented at the 40th Annual Meeting of the Society of Neuroscience, San Diego, California, U.S.A., November 13–17, 2010.
The authors have no other funding, financial information, or conflicts of interest to report.