Projection targets of efferent neurons in the internal branch of the superior laryngeal nerve in rats: microdissection and glycogen depletion, immunhistochemical nerve fibre analysis, electromyographic study and three dimensional reconstruction

Tracer studies have revealed that the internal branch of the superior laryngeal nerve carries sensory but also motor neuron fibres. Our aim was to investigate the potential target(s) of these efferent fibres in vivo and ex vivo using a murine model. The regional anatomy of the rat larynx was explored and detailed using the modified Sihler's procedure. Additonally, the laryngeal muscles were subjected to glycogen depletion via electrostimulation of the internal branch of the superior laryngeal nerve or the inferior laryngeal nerve. Glycogen content was examined histologically via periodic acid-shift staining and staining for glycogen phosphorylase activity. Furthermore, EMG recordings from the posterior cricoartyenoid and cricothyroid muscles were obtained and the fibre distribution within the superior laryngeal nerve was studied immunohistochemically. All animal experiments were conducted under appropriate ethics approval. For functional considerations, computer assisted 3D-reconstructions were prepared. In comparison to the non-stimulated contralateral side, ipsilateral stimulation of the internal branch of the superior laryngeal nerve led to a significant depletion of glycogen content in the muscle fibres forming the ventrolateral part of the posterior cricoarytenoid muscle (95% confidence interval for p < 0.05). Solely the inferior laryngeal nerve innervates the dorsomedial part of this muscle. This result was further confirmed by EMG records and immunohistochemistry of the muscle nerves. The ventrolateral part of the posterior cricoarytenoid muscle, the true abductor of the vocal folds, is innervated by motor neurons from both the inferior laryngeal nerve and internal branch of the superior laryngeal nerve in rats in vivo.