Tara G. Bautista

Ponto-medullary nuclei involved in the generation of sequential pharyngeal swallowing and concomitant protective laryngeal adduction in situ

Laryngeal adduction is a major mechanism in sealing off entry to the trachea to prevent aspiration during swallowing. An experimental protocol to reliably elicit sequential swallowing by oral injection of small volumes of water was developed in the in situ perfused brainstem preparation of juvenile rats. The sequential swallowing motor pattern consists of two distinct components: (i) phasic swallowing, indicated by rhythmic sequential vagal nerve bursting, and (ii) protective laryngeal adduction, indicated by background tonic vagal discharge. Pharmacological manipulation of the Kölliker–Fuse nucleus revealed that it specifically mediates the protective tonic laryngeal adduction, while GABAergic neurotransmission is needed in the nucleus of the solitary tract for the generation of the sequential swallowing motor pattern. We conclude that sequential swallow motor patterning, including effective airway protection, requires balanced excitatory–inhibitory synaptic interaction within the nucleus of the solitary tract and the Kölliker–Fuse nucleus, as well as between the two nuclei.

The temporal relationship between non‐respiratory burst activity of expiratory laryngeal motoneurons and phrenic apnoea during stimulation of the superior laryngeal nerve in rat

Non‐technical summary  Nerve fibres in the larynx detect foreign substances and elicit a stereotypical airway protective response that can be simulated by electrical stimulation of the superior laryngeal nerve (SLN). In humans the response includes cough, swallowing and a cessation of breathing (apnoea). It is still unknown precisely how the central nervous system coordinates swallowing and breathing, and at which point the two vital systems converge and diverge in the brain. Here we report a temporal, sequential relationship between excitation of expiratory laryngeal motoneurons that close the larynx during swallowing, and inhibition of breathing, during stimulation of the SLN in rat. The two phenomena can be dissociated by inactivating different brain areas. This work therefore has implications for diseases such as sudden infant death syndrome and Parkinsons disease, in which incoordination of breathing and protective behaviours may result in aspiration of irritants and subsequent death or aspiration pneumonia.

Inhibition of the pontine Kölliker-Fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat

The pontine Kölliker-Fuse nucleus (KF) has established functions in the regulation of inspiratory-expiratory phase transition and the regulation of upper airway patency via laryngeal valving mechanisms. Here we studied the role of the KF in the gating and modulation of eupneic hypoglossal motor activity (HNA) using the in situ perfused brainstem preparation, which displays robust inspiratory HNA. Microinjection of glutamate into the KF area triggered complex and often biphasic modulation (excitation/inhibition or inhibition/excitation) of HNA. Subsequent transient pharmacological inhibition of KF by unilateral microinjection of GABA-A receptor agonist isoguvacine reduced HNA and while bilateral microinjections completely abolished HNA. Our results indicate that mixed and overlapping KF pre-motor neurons provide eupneic drive for inspiratory HNA and postinspiratory vagal nerve activity. Both motor activities have important functions in the regulation of upper airway patency during eupnea but also during various oro-pharyngeal behaviors. These results have potential implications in the contribution of state-dependent modulation of KF hypoglossal pre-motor neurons during sleep-wake cycle to obstructive sleep apnea.

Acute intermittent hypoxia induced neural plasticity in respiratory motor control

Respiratory neural networks can adapt to rapid environmental change or be altered over the long term by various inputs. The mechanisms that underlie the plasticity necessary for adaptive changes in breathing remain unclear. Acute intermittent hypoxia (AIH)‐induced respiratory long‐term facilitation (LTF) is one of the most extensively studied types of respiratory plasticity. Acute intermittent hypoxia‐induced LTF is present in several respiratory motor outputs, innervating both pump muscles (i.e. diaphragm) and valve muscles (i.e. tongue, pharynx and larynx). Long‐term facilitation is present in various species, including humans, and the expression of LTF is influenced by gender, age and genetics. Serotonin plays a key role in initiating and modulating plasticity at the level of respiratory motor neurons. Recently, multiple intracellular pathways have been elucidated that are capable of giving rise to respiratory LTF. These mainly activate the metabolic receptors coupled to Gq (‘Q’ pathway) and Gs (‘S’ pathway) proteins. Herein, we discuss AIH‐induced respiratory LTF in animals and humans, as well as recent advances in our understanding of the synaptic and intracellular pathways underlying this form of plasticity. We also discuss the potential to use intermittent hypoxia to induce functional recovery following cervical spinal injury.

The Physiological significance of postinspiration in respiratory control

The term postinspiration is commonly used in the scientific literature concerned with neural generation and the control of breathing movements. Because postinspiration belongs functionally to the mechanical act of expiration, the physiological significance of postinspiration as a distinct phase of the breathing cycle is often underappreciated. The present review will give an overview of the physiological significance of postinspiratory motor activity in laryngeal adductor (constrictor) muscles and the crural diaphragm. The functional importance of postinspiratory motor activity is discussed for the eupneic respiratory cycle, and for various protective respiratory reflex mediations (e.g., sneeze, cough, and breath-hold). Also, the implications of recruited postinspiratory activity during nonventilatory behaviors such as vocalization, swallowing, or vomiting are underpinned. Finally, we describe the impact of absence or malfunction of postinspiratory motor function in neurological diseases.

Cholinergic inputs to laryngeal motoneurons functionally identified in vivo in rat: a combined electrophysiological and microscopic study.

The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviors such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus in the modulation of laryngeal motoneuron (LMN) activity. The present study investigated the anatomical nature of cholinergic input to inspiratory‐ (ILM) and expiratory‐modulated (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus. Using combined in vivo intracellular recording, dye filling, and immunohistochemistry, we demonstrate that LMNs identified in Sprague–Dawley rat receive several close appositions from vesicular acetylcholine transporter‐immunoreactive (VAChT‐ir) boutons. ELMs receive a significantly greater number of close appositions (mean ± standard deviation [SD]: 47 ± 11; n = 5) than ILMs (32 ± 9; n = 8; t‐test P < 0.05). For both LMN types, more close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two‐way analysis of variance [ANOVA], P < 0.0001). Using fluorescence confocal microscopy, almost 90% of VAChT‐ir close appositions (n = 45 boutons on n = 4 ELMs) were colocalized with the synaptic marker synaptophysin. These results support a strong influence of cholinergic input on LMNs and may have implications in the differential modulation of laryngeal muscle activity. J. Comp. Neurol. 518:4903–4916, 2010.

Neuronal Mechanisms Underlying the Laryngeal Adductor Reflex

Objectives: Electromyographic studies of the laryngeal adductor reflex, glottal closure occurring in response to laryngeal stimulation, have demonstrated an early ipsilateral response (R1) and a late bilateral response (R2). To better define the physiologic properties of these responses, we recorded responses from expiratory laryngeal motoneurons (ELMs) in rats during stimulation of the superior laryngeal nerve (SLN). Methods: Single unit extracellular recordings were obtained from 5 ELMs, identified by their antidromic responses to recurrent laryngeal nerve stimulation and postinspiratory firing pattern, in 4 Sprague-Dawley rats. Results: Unilateral stimulation of the SLN (at 20 Hz) stopped both phrenic nerve inspiratory activity and ELM postinspiratory activity. However, the ELMs displayed robust tonic firing, consisting of non-respiratory burst activity and single action potentials. The single action potentials were identified as short-latency ones (5 to 10 ms) activated by ipsilateral SLN stimulation, with an occurrence rate of 90%, and long-latency ones (20 to 50 ms) activated by bilateral SLN stimulation, with occurrence rates of 47% on the ipsilateral side and 58% on the contralateral side. Conclusions: The R1 response appears to be the result of the short-latency action potentials, orthodromically activated by ipsilateral stimulation of the SLN. The R2 response is likely to be a result of the long-latency action potentials that can be recorded from ELMs on both sides.

Learning to breathe: Habituation of Hering–Breuer inflation reflex emerges with postnatal brainstem maturation

The Hering-Breuer (HBR) reflex is considered a major regulatory feedback for the generation and patterning of respiratory activity. While HBR is important in neonates, its significance in adults is controversial. Previous experiments that investigated the plasticity of entrainment of the respiratory rhythm by vagal input demonstrated postnatal changes in HBR plasticity. Here we analyzed postnatal changes in the plasticity of HBR by mimicking the classic lung inflation tests with repetitive tonic vagal stimulation across different postnatal stages in an in situ perfused brainstem preparation of rat. The study shows that neonates stereotypically exhibit HBR stimulus-dependent prolongation of expiration while juvenile preparations (>postnatal day 16) showed significant habituation of HBR following repetitive stimulation. Subsequent experiments employing physiological lung inflation tests in situ confirmed HBR habituation in juveniles. We conclude that postnatal emergence of HBR habituation explains the weak contribution and high activation threshold of HBR in the regulation of eupnea.

The midbrain periaqueductal grey has no role in the generation of the respiratory motor pattern, but provides command function for the modulation of respiratory activity

It has previously been shown that stimulation of cell-columns in the periaqueductal grey (PAG) triggers site-specific cardiorespiratory effects. These are believed to facilitate changes in behaviour through coordinated changes in autonomic outflow. Here, we investigated whether PAG-evoked respiratory commands can be studied in situ using the decerebrate perfused brainstem preparation. Phrenic, vagus and abdominal iliohypogastric nerves were recorded before and after microinjection of L-glutamate (30-50 nl, 10 mM) or isoguvacine (GABA-receptor agonist, 30-50 nl, 10 mM) into the PAG. L-glutamate microinjection triggered a range of site-specific respiratory modulations (n = 17 preparations). Subsequent microinjection of isoguvacine into the same PAG sites had no effect on the baseline respiratory motor pattern or rhythm. We conclude that while the PAG has no function in respiratory pattern generation, PAG-evoked respiratory modulations can be evoked in situ in the absence of higher brain centres and while homeostatic parameters that may affect respiratory drive are held static.

Expiratory-modulated laryngeal motoneurons exhibit a hyperpolarization preceding depolarization during superior laryngeal nerve stimulation in the in vivo adult rat

Swallowing requires the sequential activation of tongue, pharyngeal and esophageal muscles to propel the food bolus towards the stomach. Aspiration during swallow is prevented by adduction of the vocal cords during the oropharyngeal phase. Expiratory-modulated laryngeal motoneurons (ELM) exhibit a burst of action potentials during swallows elicited by electrical stimulation of the superior laryngeal nerve (SLN). Here we sought to investigate changes in membrane potential in ELM during superior laryngeal nerve stimulation in the anaesthetised, in vivo adult rat preparation. Intracellular recordings of ELM in the caudal nucleus ambiguus (identified by antidromic activation from the recurrent laryngeal nerve) demonstrated that ELM bursting activity following SLN stimulation is associated with a depolarization that is preceded by a small hyperpolarization. During spontaneous ELM bursts, the preceding hyperpolarization separated the bursting activity from its usual post-inspiratory activity. These findings demonstrate that the in vivo adult rat preparation is suitable for the study of swallow-related activity in laryngeal motoneurons.