The intrinsic laryngeal muscles of rats have myomyous junctions

1. The intrinsic laryngeal muscles of rats have myomyous junctions T Yamagata, M Hyodo, A Okada and J Desaki* Dept of Otolaryngology and Anatomy*, Ehime University School of Medicine, Japan

2. Muscle spindle in the ILM Innervation pattern of the ILM (bilateral innervation of the arytenoid muscle) Ultrastructure of the neuromuscular junctions (NMJ) of the ILM (denervation & regeneration, development, aging) Ultrastructure of the muscle fiber of the ILM (myotendonious junction;MTJ, myomyous junction;MMJ) Morphological studies about the intrinsic laryngeal muscles (ILM) at our department

3. My previous study Ultrastructure of the NMJ of the ILMs Acta Otolaryngol 120:766-770, 2000., Arch Histol Cytol 63:249-254, 2000. To observe the NMJ on the muscle fiber by a scanning electron microscope (SEM), the connective tissues must be removed. Desaki reported the method using HCl hydrolysis to observe the NMJ on the muscle fiber by SEM (1981). Using this method, we can also investigate the ultrastructure of the muscle fiber, such as NMJ and MMJ.

4. NMJ on the muscle fiber 10mm 10mm Cricothyroid muscle (ILM) Extensor digitorum longus (hindlimb)

5. MMJ on the muscle fiber Cricothyroid muscle (CT) 30mm

6. Material & method Wistar rat (13 weeks after birth), n=6 Heavy anesthesia with ketamine and sodium pentobarbiturate (i.m.) Whole larynx was excised The CT and PCA muscles were dissected (CT: cricothyroid muscle, PCA: posterior cricoarytenoid muscle)

7. Material & method 2 Fixation with 0.1M phosphate buffer + 4% glutaraldehyde (2hr) Post-fixation with 2% unbuffered osmium tetroxide (30min) HCl-hydrolysis method（8N-HCl, 60oC, 30min) (Desaki 1981) Dehydration, freeze drying and sputter coating Investigation by SEM

8. MMJ CT 10mm

9. 10mm CT

10. CT 5 mm

11. PCA 10mm

12. Myomyous Junction (MMJ) extra ocular muscles in rat, sheep and bird (Floyd; 1970, Mayr et al; 1975) regenerated muscle fiber in rat (Bormioli et al; 1977) common skeletal muscle in mouse (Torigoe et al.; 1987) (rectus abdominis, external oblique, gastrocnemius) function of MMJ integrate the fine and precise muscular contraction by making “motor units”

13. Type of MMJ Extra ocular muscle (Mayr et al.) End-to-end type Branch type

14. Result & Discussion In the CT, two types of the MMJ were identified; end-to-end and branch types. And the ends of the branch type of the MMJ were divided into two groups; finger-like tips and tiny round tips. end-to-end MMJ:finger-like tips branch MMJ:finger-like tips, branch MMJ:tiny round tips In the PCA, all the MMJ observed were branch type. And all the end of the branch type of the MMJ seemed tiny round tips. branch MMJ:tiny round tips

15. Result & Discussion 2 The previous comparative study of the CT and PCA muscles about the NMJ and MTJ reported that the PCA is primitive and that the CT has an intermediate nature between the PCA and common skeletal muscles. We suggested that the PCA has primitive MMJs and the CT has both primitive and evolutionary MMJs for the phonation.

16. Next step • Observation of the MMJs in the thyroarytenoid muscle • Correlation of the MMJs and the muscle fiber types

17. Conclusion 1.We investigated the muscle fibers of the ILM (CT and PCA) using HCl hydrolysis method for removal of the connective tissues. 2.The MMJs, connected between muscle fibers, were observed in the CT and PCA muscles. This is the first report about the MMJ of the ILM. 3.The MMJs consisted of two types; end-to-end and branch types. And the end of the MMJ divided into two groups; finger-like and tiny round tips.

18. Conclusion-2 • 4.The MMJ of the PCA muscle seems to be a primitive style, while the CT muscle has both primitive and evolutionary MMJs. • 5.Taken together, we conclude that the CT muscle of the rat gains the evolutionary structure to control the pitch of voice on phonation.