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Vocal fold (VF) scar and sulcus cause severe vocal problems, but optimal methods have not been established. Total replacement of the mucosa is required particularly for cases in which the whole lamina propria is occupied by severe fibrosis and vibratory function is totally lost. The amniotic membrane (AM) has been proven to have regenerative potential, as it contains stem cells and growth factors. The current study investigated the biocompatibility and effects of AM for regeneration of the VF mucosa.In vitro and in vivo studies.Vocal fold fibroblasts (VFFs) from 13 Sprague-Dawley rats were seeded on AM and subjected to histology and immunohistochemistry, and gene expressions in the VFFs on AM were examined in in vitro study. Twelve New Zealand White rabbits were used in in vivo study. VFs were stripped down and were reconstructed with AM. The regenerative effects were examined 3 months later by histological examination.In vitro study indicated VFFs survived on AM and stained positively for Ki67, vimentin, and fibronectin. Gene expressions of Has1, Has2, and Hgf were significantly increased in the VFFs on AM compared with the other groups. The in vivo study indicated AM-transplanted VFs showed a significantly higher density of hyaluronic acid and lower density of collagen compared with sham VFs.The current preliminary study suggests biocompatibility and possible regenerative effects of AM for VFs.NA Laryngoscope, 132:2017-2025, 2022.

OBJECTIVES/HYPOTHESIS Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs). STUDY DESIGN In vitro. METHODS VFFs were isolated from 10-week-old, male Sprague-Dawley rats, and estrogen receptor alpha (ERα) and G protein-coupled receptor 30 (GPR30) were examined via immunostaining and quantitative polymerase chain reaction (qPCR). VFFs were treated with estradiol (E2, 10-7 , 10-8 or 10-9 M) ± transforming growth factor beta 1 (TGF-β1, 10 ng/mL). ICI 182,780 (ICI, 10-7 M) or G36 (10-7 M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis. RESULTS ERα and GPR30 were expressed in VFFs at both the protein and the mRNA levels. E2 (10-7 M) did not alter Smad3, Smad7, Acta2 mRNA, or extracellular matrix related genes. However, the combination of E2 (10-8 M) and TGF-β1 significantly increased Smad7 (P = .03) and decreased Col1a1 (P = .04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P = .009). CONCLUSIONS E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis. LEVEL OF EVIDENCE NA Laryngoscope, 131:2285-2291, 2021.

OBJECTIVES: We aimed to examine the effect of unilateral inhibition of the medullary dorsal swallowing networks on the activities of swallowing-related cranial motor nerves and swallowing interneurons. METHODS: In 25 juvenile rats, we recorded bilateral vagal nerve activity (VNA) as well as unilateral phrenic and hypoglossal activity (HNA) during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve during control and following microinjection of the GABA agonist muscimol into the caudal dorsal medulla oblongata in a perfused brainstem preparation. In 20 animals, swallowing interneurons contralateral to the muscimol injection side were simultaneously recorded extracellularly and their firing rates were analyzed during swallowing. RESULTS: Integrated VNA and HNA to the injection side decreased to 49.0 ± 16.6% and 32.3 ± 17.9%, respectively. However, the VNA on the uninjected side showed little change after muscimol injection. Following local inhibition, 11 out of 20 contralateral swallowing interneurons showed either increased or decreased of their respective firing discharge during evoked-swallowing, while no significant changes in activity were observed in the remaining nine neurons. CONCLUSION: The neuronal networks underlying the swallowing pattern generation in the dorsal medulla mediate the ipsilateral motor outputs and modulate the contralateral activity of swallowing interneurons, suggesting that the bilateral coordination of the swallowing central pattern generator regulates the spatiotemporal organization of pharyngeal swallowing movements. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2187-2198, 2021.

Oropharyngeal swallowing is centrally mediated by a swallowing central pattern generator (Sw-CPG) in the medulla oblongata. The activity of the Sw-CPG depends on the sensory inputs determined by physical and chemical bolus properties. Here we investigate the sensory-motor integration during swallowing arising from different sensory sources. To do so we electrically stimulated the superior laryngeal nerve and we triggered swallowing with oral injections of distilled water or capsaicin solution and extracellularly recorded from swallowing interneurons in arterially perfused brainstem preparations of rats. We recorded the activities of 40 neurons, while monitoring the motor activities of the phrenic, vagal and hypoglossal nerves. Eighteen neurons responded to electrical stimulation of the ipsilateral superior laryngeal nerve, and 6 neurons were excited by oral fluid injection, while 16 non-respiratory neurons did not receive afferent inputs to either electrical or physiological stimuli. The cellular activities displayed by swallowing interneurons during electrical and physiological stimulation of pharyngeal and laryngeal afferent input reveal complex adaptations of the timing of firing patterns and frequencies. The modulation of neuronal activity is likely to contribute to the coordination of efficient bolus transfer during the pharyngeal stage of swallowing.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein 1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

We recorded membrane potentialp changes in 45 pharyngeal motoneurons (PMs) including 33 expiratory modulated and 12 nonrespiratory neurons during breathing, swallowing, and coughing in decerebrate paralyzed cats. Four types of membrane potential changes were observed during swallowing: 1) depolarization during swallowing (n = 27), 2) depolarization preceded by a brief (≤ 0.1 s) hyperpolarization (n = 4), 3) longer term (> 0.3 s) hyperpolarization followed by depolarization (n = 11), and 4) hyperpolarization during the latter period of swallowing (n = 3). During coughing, PMs showed two types of membrane potential changes (n = 10). Nine neurons exhibited a ramp-like depolarization during the expiratory phase of coughing with the potential peak at the end of expiratory phase. This depolarization was interrupted by a transient repolarization just before the potential peak. The membrane potential of the remaining neuron abruptly depolarized at the onset of the expiratory phase and then gradually decreased even after the end of the expiratory phase. Single-shock stimulation of the superior laryngeal nerve (SLN) induced inhibitory postsynaptic potentials in 19 of 21 PMs. Two motoneurons exhibited an SLN-induced excitatory postsynaptic potential. The present study revealed that PMs receive the central drive, consisting of a combination of excitation and inhibition, from the pattern generator circuitry of breathing, swallowing, and coughing, which changes the properties of their membrane potential to generate these motor behaviors of the pharynx. Our data will provide the basis of studies of pharyngeal activity and its control from the medullary neuronal circuitry responsible for the upper airway motor activity.NEW & NOTEWORTHY We have provided the first demonstration of the multifunctional activity of the pharyngeal motoneurons at the level of membrane potential during respiration, swallowing, and coughing.

Summary Objectives Medialization procedures, such as type I thyroplasty, arytenoid adduction, and vocal fold injection, are popular treatments for dysphonia due to unilateral vocal fold paralysis (UVFP). However, dysphonia occasionally persists after medialization procedures owing to tension imbalance. This tension imbalance causes diplophonia, asymmetry and aperiodic vibrational flutter in travelling wave motion. Currently, there is no established treatment for tension imbalance. We herein report two cases with residual dysphonia due to tension imbalance following medialization for chronic UVFP, and another case presenting with dysphonia due to tension imbalance following chronic unilateral vocal fold paresis. Methods Three patients underwent voice therapy using flow phonation to facilitate increased airflow management in speech as well as forward oral resonance by focusing on balanced airflow. Phonatory outcomes were evaluated using stroboscopic findings, aerodynamic and acoustic measures, as well as self-rating. Results Aerodynamic assessments, acoustic findings and self-ratings improved in all three cases after voice therapy. Stroboscopic findings prior to voice therapy showed asymmetric vibration with glottic gap, which was improved after voice therapy. Fundamental frequency (F0) also increased post-therapy. Conclusions In a previous canine study, it was shown that enhanced breath support with expiratory airflow resulted in increased F0, suggesting that enhanced breath support could increase vocal fold tension. The increased F0 achieved in the present cases following voice therapy may increase vocal fold tension with breath support. Thus, voice therapy using flow phonation may be effective for supporting vocal fold tension and improving dysphonia due to tension imbalance following UVFP and paresis.

Summary Objectives Vocal fold atrophy following unilateral vocal fold paralysis is caused by atrophy of the thyroarytenoid (TA) muscle and remains a challenge. Medialization procedures are popular treatment options; however, hoarseness often remains due to the reduction in mass or tension of the TA muscle. Therefore, in addition to medialization procedures, TA muscle reinnervation is desirable. In vivo studies have shown the potential for basic fibroblast growth factor (bFGF) to affect muscular and nerve regeneration. The present study aimed to examine the regenerative effects of bFGF on restoration of TA muscle atrophy caused by recurrent laryngeal nerve transection. Study design Prospective animal experiments with controls. Methods TA muscle atrophy was induced by unilateral transection of the recurrent laryngeal nerve. One month after transection, different doses (200 ng, 100 ng, 10 ng) of bFGF in 50 µL were repeatedly injected into the TA muscle four times with an interval of 1 week between injections. Saline only was injected in the sham group. Larynges were harvested for histologic and immunohistochemical examination 4 weeks after the final injection. Results The cross-sectional TA muscle area was significantly larger in the bFGF-treated groups compared with the sham-treated groups. Immunohistochemistry indicated that bFGF significantly increases the number of neuromuscular junctions and satellite cells in the TA muscle. Conclusions These results suggest that local application of bFGF to the TA muscle may improve TA muscle atrophy caused by recurrent laryngeal nerve paralysis. Furthermore, bFGF may have regenerative effects on both nerves and muscles.

OBJECTIVES The purpose of this study was to investigate the influence of laryngeal afferent inputs on brainstem circuits that mediate and transmit swallowing activity to the orofacial musculature. METHODS Experiments were performed on 19 arterially perfused juvenile rats. The activities of swallowing interneurons in relation to their respective motor outputs in the hypoglossal and vagus nerves were assessed during fictive swallowing with or without concurrent laryngeal sensory stimulation at intensities of 20, 40, and 60 μA. RESULTS The hypoglossal nerve activity was gradually enhanced with increasing intensity of the sensory stimulation, while the vagus nerve activity was not altered. The activities of various interneurons were modulated by the laryngeal stimulation, but more than 50% of the recorded neurons were inhibited by the stimulation. Some interneurons demonstrated no obvious change in their discharge rates with laryngeal sensory stimulation during fictive swallowing. CONCLUSION Laryngeal afferent inputs partially modulated the swallowing motor activity via enhanced or suppressed activities of the swallowing interneurons, while the essential motor pattern underlying the pharyngeal stage of swallowing remained basically unchanged. Thus, the output patterns of the complex sequential movements of swallowing could be basically predetermined and further adjusted according to sensory information related to the properties of the ingested food by a swallowing central pattern generator. LEVEL OF EVIDENCE NA Laryngoscope, 130: 1885-1893, 2020.

Pharyngeal swallowing is controlled by synaptic interactions within a swallowing central pattern generator (sw-CPG) that is composed of a dorsal and a ventral swallowing group (VSG). Here, we used electrical stimulation (10 s) of the superior laryngeal nerve (SLN; 20 Hz; pulse width: 100 μs) to explore the role of the VSG in an arterially-perfused brainstem preparation of rats. To investigate the effects of pharmacological lesion (local microinjection of an GABA(A)-R agonist) of the nucleus retroambiguus (NRA), a designated component of the VSG, we recorded phrenic (PNA) and vagal nerve (VNA) activities. Control SLN stimulation with stepwise increasing stimulus intensities (from 20 μA to 160 μA) elicited robust suppression of PNA and evoked sequential swallowing activity in the VNA. Lesioning of the NRA had no effect on the pattern of pharyngeal swallowing, but significantly increased the sensory gating of SLN inputs. We conclude that the NRA is not part of the VSG, but appears to have important roles for the central gating of swallowing.

We aimed to investigate and validate the cellular activity patterns and the potential topographical organization of neurons of the medullary swallowing pattern generator (Sw-CPG). We used the perfused brainstem preparation as an innovative experimental model that allows for stable neuronal recording in the brainstem.Animal model.Experiments were conducted in 14 juvenile Wistar rats. The activities of the phrenic, vagus, and hypoglossal nerves were recorded at baseline, and fictive swallowing was elicited by stimulation of the superior laryngeal nerve. Extracellular action potentials of 72 swallowing-related neurons were recorded in the Sw-CPG of the dorsal medulla oblongata.Neurons could be classified into three types: sensory relay, and neurons that were excited or inhibited during fictive swallowing. Approximately one-third of the neurons likely received monosynaptic input from the laryngeal afferents. One-third of neurons recorded showed respiratory-related activity, most of which exhibited inspiratory modulation. The neurons were widely distributed in the nucleus tractus solitarius and reticular formation.The perfused brainstem preparation of rat fully preserves the Sw-CPG. The recorded cellular activities and general topographical organization of swallowing neurons are in accordance with previous in vivo studies. Thus, the perfused brainstem preparation is an ideal experimental model to advance the understanding of neuronal mechanisms underlying swallowing.NA Laryngoscope, 129:E72-E79, 2019.

The retrotrapezoid/parafacial respiratory group (RTN/pFRG) located ventral to the facial nucleus plays a key role in regulating breathing, especially enhanced expiratory activity during hypercapnic conditions. To clarify the roles of the RTN/pFRG region in evoking coughing, during which reflexive enhanced expiration is produced, and in swallowing, during which the expiratory activity is consistently halted, we recorded extracellular activity from RTN/pFRG neurons during these fictive behaviors in decerebrate, paralyzed, and artificially ventilated guinea pigs. The activity of the majority of recorded respiratory neurons was changed in synchrony with coughing and swallowing. To further evaluate the contribution of RTN/pFRG neurons to these nonrespiratory behaviors, the motor output patterns during breathing, coughing, and swallowing were compared before and after brain stem transection at the caudal margin of RTN/pFRG region. In addition, the effects of transection at its rostral margin were also investigated to evaluate pontine contribution to these behaviors. During respiration, transection at the rostral margin attenuated the postinspiratory activity of the recurrent laryngeal nerve. Meanwhile, the late expiratory activity of the abdominal nerve was abolished after caudal transection. The caudal transection also decreased the amplitude of the coughing-related abdominal nerve discharge but did not abolish the activity. Swallowing could be elicited even after the caudal end transection. These findings raise the prospect that the RTN/pFRG contributes to expiratory regulation during normal respiration, although this region is not an essential element of the neuronal networks involved in coughing and swallowing.

Sensory-motor control of the pharyngeal swallow requires sensory afferent inputs from the pharynx and larynx evoked by introducing bolus into the pharynx. Patients with reduced sensitivity of the pharynx and larynx are likely to have a swallowing impairment, such as pre-swallow aspiration due to delayed swallow triggering. Interferential current stimulation applied to the neck is thought to improve the swallowing function of dysphagic patients, although the mechanism underlying the facilitatory effect of such stimulation remains unknown. In the present study, we examined the changes in the elicitability of swallowing due to the stimulation and the responses of the swallowing-related neurons in the nucleus tractus solitarius and in the area adjacent to the stimulation in decerebrate and paralyzed guinea pigs. The swallowing delay time was shortened by the stimulation, whereas the facilitatory effect of eliciting swallowing was attenuated by kainic acid injection into the nucleus tractus solitarius. Approximately half of the swallowing-related neurons responded to the stimulation. These data suggest that the interferential current stimulation applied to the neck could enhance the sensory afferent pathway of the pharynx and larynx, subserving excitatory inputs to the neurons of the swallowing pattern generator, thereby facilitating the swallowing reflex.

Stimulation of vestibular receptors elicits distinct changes in blood flow to the forelimb and hindlimb, showing that the nervous system has the capacity to produce changes in sympathetic outflow which are specific for a particular region of the body. However, it is unclear whether the rostral ventrolateral medulla (RVLM), the primary region of the brainstem that regulates sympathetic outflow to vascular smooth muscle, has the appropriate connectivity with sympathetic preganglionic neurons to generate anatomically patterned responses. To make this determination, the retrograde fluorescent tracer Fast Blue was injected into the T(4) spinal cord segment of cats, which regulates upper body blood flow, whereas Fluoro-Ruby was injected into the T(10) segment to label projections to a region of the spinal cord that regulates lower body blood flow. More neurons were single-labeled by a particular tracer (92 %) than were double labeled by both tracers (8 %), supporting the notion that the RVLM can regulate sympathetic outflow from a limited number of spinal cord segments. Since a large fraction of RVLM neurons that control sympathetic outflow in rodents contain epinephrine, we additionally determined whether the tracer-labeled cells were immunopositive for the enzyme tyrosine hydroxylase (TH), which participates in the synthesis of catecholamines. Double labeling by the two tracers injected into the spinal cord was more common for TH-immunopositive neurons than for the general population of RVLM neurons: 19 % of the TH-positive cells contained both Fast Blue and Fluoro-Ruby, 30 % contained one of the tracers, and 51 % were not labeled by either tracer. Furthermore, many spinally projecting neurons in close proximity to the RVLM catecholaminergic neurons (41 % of the population) were not immunopositive for TH, suggesting that feline RVLM is neurochemically heterogeneous.

The parabrachial and adjacent Kölliker-Fuse (PBN/KF) nuclei play a key role in relaying visceral afferent inputs to the hypothalamus and limbic system and are, thus, believed to participate in generating nausea and affective responses elicited by gastrointestinal (GI) signals. In addition, the PBN/KF region receives inputs from the vestibular system and likely mediates the malaise associated with motion sickness. However, previous studies have not considered whether GI and vestibular inputs converge on the same PBN/KF neurons, and if so, whether the GI signals alter the responses of the cells to body motion. The present study, conducted in decerebrate cats, tested the hypothesis that intragastric injection of copper sulfate, which elicits emesis by irritating the stomach lining, modifies the sensitivity of PBN/KF neurons to vertical plane rotations that activate vestibular receptors. Intragastric copper sulfate produced a 70% median change in the gain of responses to vertical plane rotations of PBN/KF units, whose firing rate was modified by the administration of the compound; the response gains for 16 units increased and those for 17 units decreased. The effects were often dramatic: out of 51 neurons tested, 13 responded to the rotations only after copper sulfate was injected, whereas 10 others responded only before drug delivery. These data show that a subset of PBN/KF neurons, whose activity is altered by a nauseogenic stimulus also respond to body motion and that irritation of the stomach lining can either cause an amplification or reduction in the sensitivity of the units to vestibular inputs. The findings imply that nausea and affective responses to vestibular stimuli may be modified by the presence of emetic signals from the GI system.

Anatomical studies have demonstrated that the vestibular nuclei project to nucleus tractus solitarius (NTS), but little is known about the effects of vestibular inputs on NTS neuronal activity. Furthermore, lesions of NTS abolish vomiting elicited by a variety of different triggering mechanisms, including vestibular stimulation, suggesting that emetic inputs may converge on the same NTS neurons. As such, an emetic stimulus that activates gastrointestinal (GI) receptors could alter the responses of NTS neurons to vestibular inputs. In the present study, we examined in decerebrate cats the responses of NTS neurons to rotations of the body in vertical planes before and after the intragastric administration of the emetic compound copper sulfate. The activity of more than one-third of NTS neurons was modulated by vertical vestibular stimulation, with most of the responsive cells having their firing rate altered by rotations in the head-up or head-down directions. These responses were aligned with head position in space, as opposed to the velocity of head movements. The activity of NTS neurons with baroreceptor, pulmonary, and GI inputs could be modulated by vertical plane rotations. However, injection of copper sulfate into the stomach did not alter the responses to vestibular stimulation of NTS neurons that received GI inputs, suggesting that the stimuli did not have additive effects. These findings show that the detection and processing of visceral inputs by NTS neurons can be altered in accordance with the direction of ongoing movements.

There is accumulating evidence that fibroblasts are target cells for steroids such as sex hormones and corticoids. The characteristics of fibroblasts vary among tissues and organs. Our aim in this study is to examine differences in responses to steroid hormones among fibroblasts from different cervicothoracic regions. We compared the actions of steroid hormones on cultured fibroblasts from the vocal folds, which are considered to be the primary target of steroid hormones, and the trachea and esophagus in adult male rats. Expression of steroid hormone receptors (androgen receptor, estrogen receptor α, and glucocorticoid receptor) was identified by immunofluorescence histochemistry. Androgen receptor was much more frequently expressed in fibroblasts from the vocal fold than in those from the trachea and esophagus. Cell proliferation analysis showed that administration of testosterone, estradiol, or corticosterone suppressed growth of all 3 types of fibroblasts. However, mRNA expression for extracellular matrix–associated genes, including procollagen I and III and elastin, and hyaluronic acid synthase I was elevated only by addition of testosterone to fibroblasts from the vocal fold. These results indicate that each steroid hormone exerts region-specific effects on cervicothoracic fibroblasts with different properties through binding to specific receptors.

To examine the relationship between the neuronal networks underlying respiration and non-respiratory behaviors such as vocalization and airway defensive reflexes, we compared the activity of respiratory neurons in the ventrolateral medulla during breathing with that during non-respiratory behaviors including vocalization, swallowing, and coughing in guinea pigs. During fictive vocalization the activity of augmenting expiratory neurons ceased, whereas the other types of expiratory neurons did not show a consistent tendency of increasing or decreasing activity. All inspiratory neurons discharged in synchrony with the phrenic nerve activity. Most of the phase-spanning neurons were activated throughout the vocal phase. During fictive swallowing, many expiratory and inspiratory neurons were silent, whereas many phase-spanning neurons were activated. During fictive coughing, many expiratory neurons were activated during the expiratory phase of coughing. Most inspiratory neurons discharged in parallel with the phrenic nerve activity during coughing. Many phase-spanning neurons were activated during the expiratory phase of coughing. These findings indicate that the medullary respiratory neurons help shape respiratory muscle nerve activity not only during breathing but also during these non-respiratory behaviors, and thus suggest that at least some of the respiratory neurons are shared among the neuronal circuits underlying the generation of breathing and non-respiratory behaviors.

Although it is well established that bulbospinal neurons located in the rostral ventrolateral medulla (RVLM) play a pivotal role in regulating sympathetic nerve activity and blood pressure, virtually all neurophysiological studies of this region have been conducted in anesthetized or decerebrate animals. In the present study, we used time- and frequency-domain analyses to characterize the naturally occurring discharges of RVLM neurons in conscious cats. Specifically, we compared their activity to fluctuations in carotid artery blood flow to identify neurons with cardiac-related (CR) activity; we then considered whether neurons with CR activity also had a higher-frequency rhythmic firing pattern. In addition, we ascertained whether the surgical removal of vestibular inputs altered the rhythmic discharge properties of RVLM neurons. Less than 10% of RVLM neurons expressed CR activity, although the likelihood of observing a neuron with CR activity in the RVLM varied between recording sessions, even when tracking occurred in a very limited area and was higher after vestibular inputs were surgically removed. Either a 10-Hz or a 20- to 30-Hz rhythmic discharge pattern coexisted with the CR discharges in some of the RVLM neurons. Additionally, the firing rate of RVLM neurons, including those with CR activity, decreased after vestibular lesions. These findings raise the prospect that RVLM neurons may or may not express rhythmic firing patterns at a particular time due to a variety of influences, including descending projections from higher brain centers and sensory inputs, such as those from the vestibular system.

A central pattern generator (CPG) for swallowing in the medulla oblongata generates spatially and temporally coordinated movements of the upper airway and alimentary tract. To reveal the medullary neuronal network of the swallowing CPG, we examined the cytoarchitecture of the swallowing CPG and axonal projections of its individual neurons by extracellular recording and juxtacellular labeling of swallowing-related neurons (SRNs) in the medulla in urethane-anesthetized and paralyzed guinea pigs. Three major types of neuronal discharge patterns were identified during fictive swallowing induced by stimulation of the superior laryngeal nerve: early (burst-like activation during the pharyngeal stage), late (activation after the pharyngeal stage), and inhibited (inhibition during the pharyngeal stage) types. Sixteen neurons were successfully labeled in the nucleus tractus solitarii (NTS) and in the medullary reticular formation (RF). No motoneuron was labeled. The SRNs in the NTS had axons projecting to the NTS, RF, nucleus ambiguus, nucleus hypoglossus, and dorsal motor nucleus of the vagus on the ipsilateral side. Some NTS SRNs projected only within the NTS. The axons of SRNs in the RF projected also to the NTS, RF, motor nuclei on the ipsilateral side, and to the other side RF. These findings show anatomic substrates for the neuronal network of the CPG for swallowing, which consists of complex neuronal connections among SRNs in the NTS, RF, and motor nuclei.

Research on animal models as well as human subjects has demonstrated that the vestibular system contributes to regulating the distribution of blood in the body through effects on the sympathetic nervous system. Elimination of vestibular inputs results in increased blood flow to the hindlimbs during vestibular stimulation, because it attenuates the increase in vascular resistance that ordinarily occurs in the lower body during head-up tilts. Additionally, the changes in vascular resistance produced by vestibular stimulation differ between body regions. Electrical stimulation of vestibular afferents produces an inhibition of most hindlimb vasoconstrictor fibers and a decrease in hindlimb vascular resistance, but an initial excitation of most upper body vasoconstrictor fibers accompanied by an increase in upper body vascular resistance. The present study tested the hypothesis that neurons in the principal vasomotor region of the brainstem, the rostral ventrolateral medulla (RVLM), whose projections extended past the T10 segment, to spinal levels containing sympathetic preganglionic neurons regulating lower body blood flow, respond differently to electrical stimulation of the vestibular nerve than RVLM neurons whose axons terminate rostral to T10. Contrary to our hypothesis, the majority of RVLM neurons were excited by vestibular stimulation, despite their level of projection in the spinal cord. These findings indicate that the RVLM is not solely responsible for establishing the patterning of vestibular-sympathetic responses. This patterning apparently requires the integration by spinal circuitry of labyrinthine signals transmitted from the brainstem, likely from regions in addition to the RVLM.

The purpose of the present study was to determine whether murines could be substituted for traditional experimental mammals to study the brainstem mechanism of vocalization. We conducted systematic electrical and chemical stimulation of the brainstem in guinea pigs to identify the similarities in the call sites between murines and other mammals. We further examined whether or not fictive vocalization could be induced in paralyzed guinea pigs, an experimental model which facilitates neuronal recording in the brainstem. The sites where electrical stimulation evoked vocalization were distributed continuously from the periaqueductal grey (PAG) to the lower brainstem. This call area usually ended at the most caudal part of the inferior olive and thus did not continuously extend to the nucleus retroambiguus. Microinjections of d,l-homocysteic acid and bicuculline induced vocalization at the PAG, parabrachial nucleus, and the most dorsal part of the pontine reticular formation. The brainstem call areas and vocal motor patterns induced from these areas were approximately consistent with those in other mammals. Fictive vocalization induced by PAG stimulation could be identified from activities of the phrenic, abdominal, and superior laryngeal nerves in paralyzed guinea pigs. We thus concluded that guinea pigs can be utilized in studies of brainstem vocal mechanism.