Temporal expression of Laminin-111 in the developing rat larynx.

Laminin-111 is a basement membrane protein that participates in motor innervation and reinnervation. During axonal pathfinding, laminin-111 interacts with netrin-1 (NTN1) and changes its attractant growth cone properties into repulsion. While previous models of recurrent laryngeal nerve (RLN) transection show increased Laminin-111 and NTN1 production after injury, developmental expression in the larynx has not been defined. This study investigates the expression of laminin-111 in laryngeal muscles during primary laryngeal innervation of Sprague Dawley rats. Adult larynges and embryos were sectioned for immunohistochemistry with ßIII-Tubulin, laminin subunit α-1 (LAMA1), NTN1, and α-bungarotoxin. Sections were processed for single-molecule inexpensive RNA fluorescence in situ hybridization analysis of LAMA1 mRNA. LAMA1 expression increased in all intrinsic laryngeal muscles, except the medial thyroarytenoid (MTA), at E20.5. At E20.5 there was increased expression in the lateral thyroarytenoid (LTA) and posterior cricoarytenoid (PCA) compared to the MTA. NTN1 upregulation was limited to the LTA and lateral cricoarytenoid (LCA) at E16.5 without any increase in the MTA or PCA. LAMA1 and NTN1 expression did not strictly follow expected patterns relative to the known timing of innervation and does not appear to be acting similarly to its role following RLN injury. These differences between developmental and post-injury innervation provide targets for investigations of therapeutics after nerve injury.

Development of the rat larynx: a histological study.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the cartilaginous and muscular development of the rat larynx. STUDY DESIGN: Histologic evaluation. METHODS: The larynges of Sprague Dawley rats of embryonic day (E) 13, 15, 17, 19, 21, postnatal day 0, 14, and adult of 250 gm were collected. Four larynges of each age were harvested, cut into 15-µm serial sections, stained with hematoxylin and eosin, and evaluated under light microscopy. Representative digital images were recorded and evaluated at the preglottic (supraglottic in humans), glottic, and postglottic (subglottic in humans) levels. RESULTS: Brachial arches were observed at E13. At E17, immature structures of the larynx, including skeletal muscle, cartilage, and the lumen were identifiable. Chondrification and muscle formation were clearly seen by E19. The muscular and cartilagenous components of the larynx were well established by E21. During the span between birth and adult maturation, the size of the larynx increased from a height of 1.10 mm to 2.90 mm, and from a width of 1.80 mm to 5.40 mm, and from a length of 1.38 mm to 4.77 mm in the stained section. Although developed at E21, the laryngeal structures continued to grow by approximately 30%. CONCLUSION: Rat laryngeal development parallels that in mice and humans. In the rat, at E17 immature structures of the larynx are identifiable, they are well developed at birth and grow by approximately 30% into adulthood. Understanding the chronology and morphology of the embryogenesis of the rat laryngeal musculature is essential and will allow for further evaluation of the embryologic innervation of these muscles.

Implications of peripheral muscular and anatomical development for the acquisition of lingual control for speech production: a review.

OBJECTIVES: Normally developing children learn to produce intelligible speech during rapid, non-uniform growth of their articulators and other vocal tract structures. The purpose of this review is to focus attention on the consequences of peripheral growth and development for the acquisition of lingual control for speech production. This paper (1) reviews physiological underpinnings of tongue shaping and movements that are likely to be changing in young children; (2) estimates, from previously published studies, the net consequences of growth of multiple vocal tract structures on lingual control; (3) integrates our findings with the example of [R] production, and (4) highlights areas where further investigations would be most helpful. PATIENTS AND METHODS: The authors searched the literature, including the PubMed database, for studies of the development of muscle proteins, muscle fibers, and motor units of the tongue, and of the growth of the tongue, jaw, adenoids, soft and hard palates, oral and pharyngeal cavities, and the vocal tract as a whole. CONCLUSIONS: Substantial anatomical and muscular data sets focused on children from 1-4 years of age, and rigorous definitions of the tongue boundaries are needed.

Developing laryngeal muscle of Xenopus laevis as a model system: androgen-driven myogenesis controls fiber type transformation.

The developmental programs that contribute to myogenic stem cell proliferation and muscle fiber differentiation control fiber numbers and twitch type. In this study, we describe the use of an experimental model system-androgen-regulated laryngeal muscle of juvenile clawed frogs, Xenopus laevis-to examine the contribution of proliferation by specific populations of myogenic stem cells to expression of the larynx-specific myosin heavy chain isoform, LM. Androgen treatment of juveniles (Stage PM0) resulted in upregulation of an early (Myf-5) and a late (myogenin) myogenic regulatory factor; the time course of LM upregulation tracked that of myogenin. Myogenic stem cells stimulated to proliferate by androgen include a population that expresses Pax-7, a marker for the satellite cell myogenic stem cell population. Since androgen can switch muscle fiber types from fast to slow even in denervated larynges, we developed an ex vivo culture system to explore the relation between proliferation and LM expression. Cultured whole larynges maintain sensitivity to androgen, increasing in size and LM expression. Blockade of cell proliferation with cis-platin prevents the switch from slow to fast twitch muscle fibers as assayed by ATPase activity. Blockade of cell proliferation in vivo also resulted in inhibition of LM expression. Thus, both in vivo and ex vivo, inhibition of myogenic stem cell proliferation blocks androgen-induced LM expression and fiber type switching in juveniles.

Sexually distinct development of vocal pathways in Xenopus laevis.

Deterministic rules, rather than experience, are thought to regulate the development of simple behaviors in vertebrates and invertebrates. We revisited this issue through examination of the sexually distinct vocalizations of African clawed frogs (Xenopus laevis), a reproductive behavior used by sexually mature males and females. We discovered that, as expected for simple behavior, female vocalizations develop through deterministic rules. The rare calls of juvenile females are indistinguishable from those of adult females. The vocal pathways of juvenile females, as measured by the contractile properties of the laryngeal muscles (the vocal muscles) and the laryngeal motoneuron somata (vocal motoneurons) size, are the developmental default and do not differentiate as they mature. Male Xenopus, in contrast, produce extensive vocalizations with rudimentary acoustic structure before reaching sexual maturity. Moreover, the functional properties of the vocal central pattern generator mature before muscle fibers and motoneuron size are fully masculinized. The results suggest that neuronal activity during development may be important in organizing the contractile properties of the muscle fibers in male, but not in female Xenopus.

Preliminary evaluation of noninvasive microscopic imaging techniques for the study of vocal fold development.

Understanding pediatric voice development and laryngeal pathology is predicated on a detailed knowledge of the microanatomy of the layered structure of the vocal fold. Our current knowledge of this microanatomy and its temporal evolution is limited by the lack of pediatric specimen availability. By providing the capability to image pediatric vocal folds in vivo, a noninvasive microscopy technique could greatly expand the existing database of pediatric laryngeal microanatomy and could furthermore make longitudinal studies possible. A variety of natural-contrast optical imaging technologies, including optical frequency domain imaging (OFDI), full-field optical coherence microscopy (FF-OCM), and spectrally encoded confocal microscopy (SECM) have been recently developed for noninvasive diagnosis in adult patients. In this paper, we demonstrate the potential of these three techniques for laryngeal investigation by obtaining images of excised porcine vocal fold samples. In our study, OFDI allowed visualization of the vocal fold architecture deep within the tissue, from the superficial mucosa to the vocalis muscle. The micron-level resolution of SECM allowed investigation of cells and extracellular matrix fibrils from the superficial mucosa to the intermediate layer of the lamina propria (LP) (350 microm penetration depth). The large field of view (up to 700 microm), penetration depth (up to 500 microm), and resolution (2x2x1microm [XxYxZ]) of FF-OCM enabled comprehensive three-dimensional evaluation of the layered structure of the LP. Our results suggest that these techniques provide important and complementary cellular and structural information, which may be useful for investigating pediatric vocal fold maturation in vivo.

Age-related changes in human thyroarytenoid muscles: a histological and histochemical study.

We examined the thyroarytenoid muscles of 23 larynges in order to assess function-related characteristics and to discover age-related changes. The neonatal thyroarytenoid muscle differed from limb muscles in the slow maturation of fibre types. In adults, we examined the medial part of the thyroarytenoid muscle. It showed a larger variation in fibre size and more endomysial connective tissue than is common for limb muscles. Structural and histochemical evidence of ageing developed from approximately the 6th decade. It comprised a marked increase of endomysial connective tissue and striking myopathic changes of muscle fibres. Up to 20% of the muscle fibres showed at some places of some sections evidence of mitochondrial accumulations and increased mitochondrial enzyme activity (ragged red fibres). The rise of such ragged red fibres is commonly related to the development of mutations in mitochondrial DNA. Significant myopathic changes including mitochondrial abnormalities develop in the thyrovocalis muscle with age and may play a role in the functional deficit of the larynx in old age.

Response of the cricothyroid and thyroarytenoid muscles to stereotactic injection of substance P into the region of the nucleus tractus solitarius in developing dogs.

Substance P (SP), a putative sensory neurotransmitter, mediates reflex laryngeal adductor activity in developing dogs. Such reflex activity includes life-threatening laryngospasm induced by stimulation of distal esophageal afferent nerves. The site of SP's activity is unknown. This research was undertaken to determine whether injection of SP into the nucleus tractus solitarius (NTS) of developing beagles alters laryngeal adductor motor activity. Six animals, 57 to 78 days of age, underwent stereotactic injection of 5 to 10 microL of SP into the region of the NTS, identified by electrical stimulation of the ipsilateral superior laryngeal nerve. In 8 additional studies, SP was injected into the cerebellum (2) or brain stem (6) distant from the NTS. Cardiovascular and electromyographic (EMG) responses of the diaphragm and the cricothyroid (CT) and/or thyroarytenoid (TA) muscles were recorded in all 6 animals. Injection of SP into the region of the NTS induced a decrease in blood pressure in all animals and an increase in either ipsilateral CT or TA activity. Three of these animals experienced mixed apnea characterized by sustained EMG activity (spasm) of the ipsilateral CT or TA muscles and an absence of diaphragm EMG activity. The apnea event was fatal in 1 of these animals. In the 6 animals who underwent injections in the brain stem but outside the region of the NTS, diaphragm and laryngeal EMG activity generally did not change after injection of SP, with the exception of 1 animal who experienced a mild, short-lived increase in ipsilateral CT activity. A brief phasic increase in ipsilateral CT activity was seen in both animals who underwent injection of SP into the cerebellum. A putative sensory neurotransmitter, SP evokes ipsilateral CT and/or TA EMG activity when injected into the region of the NTS in developing beagle dogs. This research suggests that SP in the NTS may play a role in mediating life-threatening laryngeal adductor reflexes in developing mammals and may provide important information regarding therapeutic intervention.

Muscle-dependent and hormone-dependent differentiation of the vocal control premotor nucleus robustus archistriatalis and the motornucleus hypoglossus pars tracheosyringealis of the zebra finch.

Sex differences in the vertebrate brain (brain sex) are thought to develop owing to the tissue specific action of gonadal hormones similar to the development of secondary sex characteristics of the body. Small sex differences in body anatomy could, however, retrogradely control the sexual differentiation of the central nervous system. This possibility has so far been verified only for motorneuron pools, since the connectivity of sex-specific higher brain areas to the sexual dimorphic periphery is frequently not well known. Here, we tested whether somatic sex differences feed back on higher brain areas by bilateral denervation of the syringeal musculature of zebra finches before, during, and after onset of estrogen-sensitive sexual differentiation of forebrain vocal nuclei such as RA (nucleus robustus archistriatalis). In the zebra finch, the sound-producing musculature (the syrinx), the syrinx motornucleus hypoglossus pars tracheosyringealis (nXIIts), and the RA are much larger in males compared to females. Tract tracing studies revealed that the volume and neuron size distribution of the nXIIts was sexually dimorphic in intact but not in animals denervated as juveniles. In contrast, the volume of RA and size of RA neurons of denervated animals were highly sexually dimorphic. Furthermore, estrogen masculinized the RA of denervated females. Thus, sexual differentiation of the RA but not of the nXIIts appears independent of somatic sex differences. The syrinx muscles are, however, important for the soma size of those RA neurons that project to the nXIIts.

[Morphological development of the intrinsic laryngeal muscles in rat--a scanning electron microscopic and histochemical study].

The postnatal development of the subneural apparatus (SNA) and differentiation of muscle fibers of the intrinsic laryngeal muscles (ILMs) in the rat were investigated on the 1st, 5th, 15th, 30th and 90th days after birth. The cricothyroid (CT), thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles in the ILMs were examined. Furthermore, we compared the development of these muscles with that of the skeletal muscles of the hindlimb, the extensor digitorum longus (EDL) and soleus (SOL) muscles. The SNA was observed with a scanning electron microscope. The SNA was quantitatively analyzed by use of two parameters: the ratio of the length of the secondary synaptic cleft to its maximal width (L/W ratio) and the ratio of the area of the secondary synaptic clefts to that of the primary synaptic cleft(s) (ASC/APC ratio). The muscle fiber types were classified according to the density of each muscle fiber by the ATPase stain. On the 15th postnatal day, the SNAs of the CT and TA muscles had a tendency to show more advanced development than those of the PCA muscle. The SNAs of the ILMs appeared to develop earlier than those of the EDL and SOL muscles. The L/W and ASC/APC ratios of the ILMs inclined to be higher than those of the hindlimb muscles. On the 30th day, the SNAs of the PCA muscle were still immature, while those of the CT and TA muscles had become nearly mature. The EDL muscle showed completely mature development and outstripped the ILMs. In contrast, the SNA of the SOL muscle was the least mature of all muscles. The L/W ratios of the ILMs were not significantly different from each other, while the ASC/APC ratio of the PCA muscle was significantly lower than those of both the CT and TA muscles. The L/W and ASC/APC ratios of the EDL muscle were the highest of all muscles examined and those of the SOL muscle were the lowest. The muscle fiber types were not significantly different in the ILMs throughout the course. On the 30th day, the EDL muscle had reached the mature adult proportion of the muscle fiber types, while the ILMs and SOL muscle needed further development. Phylogenetically, the TA muscle appeared as a sphincter of the lower airway and then the PCA muscle, as a dilator, appeared. The CT muscle arose after separation of the thyroid and cricoid cartilages. These findings infer that the lag in development of the PCA to the TA muscle in early postnatal life was due to the importance of the protective function of the lower airway and the phylogenetical difference. Moreover, these results suggest that the ILMs, which are indispensable for sucking, develop early in life, followed by rapid development of the EDL muscle to start quadruped walking after the 15th day. Thus, different muscles need to develop as an animal grows because distinct functions of muscles are necessary for normal living. In conclusion, the ILMs may have a specific mode of postnatal development in contrast with those of the hindlimb muscles.

Androgen-directed development of the Xenopus laevis larynx: control of androgen receptor expression and tissue differentiation.

The larynx of adult male Xenopus laevis differs markedly from that of the female; masculinization of the larynx requires androgen secretion during postmetamorphic development. Early in postmetamorphic development, androgen stimulates laryngeal cell proliferation and androgen binding activity is high. Later, androgen induces laryngeal cell differentiation and binding levels decrease. Here we explore the relation between laryngeal differentiation and androgen receptor expression. In untreated females, the larynx expresses high levels of androgen receptor mRNA early in postmetamorphic life; levels decline as females mature. The highest level of androgen receptor message is found in the undifferentiated laryngeal elastic precartilage of both sexes. When juveniles are exposed to androgen, laryngeal cell proliferation is stimulated within 48 hr in both sexes. Short exposures to androgen result in a biphasic response of AR mRNA levels; a marked down-regulation (4 hr) is followed by recovery at 8 hr (males) or 48 hr (females). Following longer periods of androgen treatment (3 weeks), AR mRNA expression is down-regulated and male-typical differentiation of elastic cartilage is induced in both sexes. Thus laryngeal growth responses to androgen are closely related to expression levels of androgen receptor mRNA.

Androgen directs sexual differentiation of laryngeal innervation in developing Xenopus laevis.

In adult Xenopus laevis, innervation of the vocal organ is more robust in males than in females. This sex difference originates during tadpole development; at stage 56, when the gonads first differentiate, the number of axons entering the larynx is the same in the sexes, but by stage 62, innervation is greater in males. To determine if androgen secretion establishes sex differences in axon number, we treated tadpoles with antiandrogen or androgen beginning at stage 48 or 54 and counted laryngeal nerve axons at stage 62 using electron microscopy. When male tadpoles were treated with the antiandrogen hydroxyflutamide, axon numbers were reduced to female-typical values; axon numbers in females were unaffected by antiandrogen treatment. When female tadpoles were treated with the androgen DHT (dihydrotestosterone), axon numbers were increased to male-like values. These findings suggest that endogenous androgen secretion during late tadpole stages in males is required for the sexual differentiation of laryngeal innervation observed from stage 62 on. Because androgen treatment and laryngeal innervation affect myogenesis in postmetamorphic frogs, numbers of laryngeal dilator muscle fibers were determined for hormonally manipulated tadpoles. At stage 62, vehicle-treated males had more laryngeal axons than females; laryngeal muscle fiber numbers did not, however, differ in the sexes. Both male and female tadpoles, treated from stage 54 with DHT, had more muscle fibers at stage 62 than vehicle-treated controls. Thus, while endogenous androgen secretion during late tadpole stages is subthreshold for the establishment of masculinized muscle fiber numbers, laryngeal myogenesis is androgen sensitive at this time and can be increased by suprathreshold provision of exogenous DHT. A subgroup of tadpoles, DHT treated from stage 54 to 62, was allowed to survive, untreated, until postmetamorphic stage 2 (PM2: 5 months after metamorphosis is complete). Androgen treatment between tadpole stages 54 and 62 does not prevent the ontogenetic decrease in axon numbers characteristic of laryngeal development. In addition, the elevation in stage 62 axon numbers produced by DHT-treatment at late tadpole stages was not associated with elevated numbers of laryngeal muscle fibers at PM2. Juvenile males normally maintain elevated axon numbers (relative to final adult values) through PM2 and the presence of these additional axons may result from--rather than contribute directly to--laryngeal muscle fiber addition.

Laryngeal muscle and motor neuron plasticity in Xenopus laevis: testicular masculinization of a developing neuromuscular system.

In Xenopus laevis, the sexual differentiation of the neuromuscular system responsible for courtship song is controlled by testicular androgen secretion. To explore the sensitivity of this system to androgenic stimulation, male and female frogs were gonadectomized and given testis transplants at seven different developmental stages between the end of metamorphosis and adulthood, grown to sexual maturity, and the laryngeal muscle fibers and motor axons were counted. Muscle fiber and axon numbers in males were not affected by the testicular transplant at any stage. In females, testicular transplants at all developmental stages increased muscle fiber numbers in adulthood. Values attained were, however, significantly less than those of adult intact or testis-transplanted males. Testis transplantation increased laryngeal axon numbers in females to levels equivalent to those of intact males; this effect was obtained at every stage of postmetamorphic development including adulthood. To further explore androgen regulation in adults, males and females were gonadectomized and implanted with silicone tubes containing testosterone propionate for 1.5-3 years and laryngeal muscle fibers and axon numbers compared to those of gonadectomized or sham-operated adult controls. Neither treatment with exogenous androgen nor gonadectomy had any effect on laryngeal muscle fiber or axon number in either males or females; values did not differ from those of sham-operated controls. We conclude that testicular secretions can induce laryngeal muscle fiber and axon addition in females throughout postmetamorphic life. This degree of plasticity, exhibited after the period when adult values are normally attained, stands in contrast to the effects of administration of synthetic androgen and suggests that the degree of plasticity in adult females may be underestimated if exogenous hormones rather than testicular transplants are provided.

Developmental changes in sequential activation of laryngeal abductor muscle and diaphragm in infants.

In animals and human adults, upper airway muscle activity usually precedes inspiratory diaphragm activity. We examined the interaction of the posterior cricoarytenoid muscle (PCA), which abducts the larynx, and the diaphragm (DIA) in the control of airflow in newborn infants to assess the effect of maturation on respiratory muscle sequence. We recorded tidal volume, airflow, and DIA and PCA electromyograms (EMG) in 12 full-term, 14 premature, and 10 premature infants with apnea treated with aminophylline. In most breaths, onset of PCA EMG activity preceded onset of DIA EMG activity (lead breaths). In all subjects, we also observed breaths (range 6-61%) in which PCA EMG onset followed DIA EMG onset (lag breaths). DIA neural inspiratory duration and the neuromechanical delay between DIA EMG onset and inspiratory flow were longer in lag than in lead breaths (P < 0.05 and P < 0.01, respectively). The frequency of lag breaths was greater in the premature infants [33 +/- 4% (SE)] than in either the full-term infants (21 +/- 3%, P < 0.03) or the premature infants with apnea treated with aminophylline (16 +/- 2%, P < 0.01). We conclude that the expected sequence of onset of PCA and DIA EMG activity is frequently disrupted in newborn infants. Both maturation and respiratory stimulation with aminophylline improve the coordination of the PCA and DIA.

The vocal motor neurons of Xenopus laevis: development of sex differences in axon number.

Sex differences in the number of muscle fibers in the larynx of clawed frogs (Xenopus laevis) develop after metamorphosis. In order to examine possible contributions of neural innervation to this process, we prepared sections of the laryngeal nerve from tadpole stage 56, when the sexes can first be distinguished, through adulthood, and counted axons on electron micrographs. The adult number of axons is achieved by a sexually differentiated pattern of axonal addition and loss. Axon numbers are high at tadpole stage 56 and equal for males and females; thereafter, males have more axons. Sex differences are most pronounced at tadpole stage 62 because between stages 59 and 62 the number of laryngeal axons in males increases by an average of 119 per nerve. Ultrastructural evidence is congruent with the hypothesis that new axons are added to the laryngeal nerve between tadpole stages 56 and 62. The loss of axons from the laryngeal nerve is greater for females than for males. Between tadpole stages 56 and adulthood, overall axon number decreases by 47% in males and by 64% in females. Signs of axonal degeneration are present in both sexes before metamorphosis but are rare at juvenile or adult stages. The numbers of axons in juvenile frogs do not differ from those in adults and continue to be greater in males than in females. In contrast to the amount of axon addition and loss, the timing of axon loss and the percentage of myelinated axons is the same for males and females throughout development. Thus sex differences in the innervation of laryngeal muscle originate before metamorphosis and could contribute to the marked sex differences in muscle fiber addition that occur thereafter.