Exploring the role of the Kölliker-Fuse nucleus in breathing variability by mathematical modelling.

The Kölliker-Fuse nucleus (KF), which is part of the parabrachial complex, participates in the generation of eupnoea under resting conditions and the control of active abdominal expiration when increased ventilation is required. Moreover, dysfunctions in KF neuronal activity are believed to play a role in the emergence of respiratory abnormalities seen in Rett syndrome (RTT), a progressive neurodevelopmental disorder associated with an irregular breathing pattern and frequent apnoeas. Relatively little is known, however, about the intrinsic dynamics of neurons within the KF and how their synaptic connections affect breathing pattern control and contribute to breathing irregularities. In this study, we use a reduced computational model to consider several dynamical regimes of KF activity paired with different input sources to determine which combinations are compatible with known experimental observations. We further build on these findings to identify possible interactions between the KF and other components of the respiratory neural circuitry. Specifically, we present two models that both simulate eupnoeic as well as RTT-like breathing phenotypes. Using nullcline analysis, we identify the types of inhibitory inputs to the KF leading to RTT-like respiratory patterns and suggest possible KF local circuit organizations. When the identified properties are present, the two models also exhibit quantal acceleration of late-expiratory activity, a hallmark of active expiration featuring forced exhalation, with increasing inhibition to KF, as reported experimentally. Hence, these models instantiate plausible hypotheses about possible KF dynamics and forms of local network interactions, thus providing a general framework as well as specific predictions for future experimental testing. KEY POINTS: The Kölliker-Fuse nucleus (KF), a part of the parabrachial complex, is involved in regulating normal breathing and controlling active abdominal expiration during increased ventilation. Dysfunction in KF neuronal activity is thought to contribute to respiratory abnormalities seen in Rett syndrome (RTT). This study utilizes computational modelling to explore different dynamical regimes of KF activity and their compatibility with experimental observations. By analysing different model configurations, the study identifies inhibitory inputs to the KF that lead to RTT-like respiratory patterns and proposes potential KF local circuit organizations. Two models are presented that simulate both normal breathing and RTT-like breathing patterns. These models provide testable hypotheses and specific predictions for future experimental investigations, offering a general framework for understanding KF dynamics and potential network interactions.

Dose-dependent Respiratory Depression by Remifentanil in the Rabbit Parabrachial Nucleus/Kölliker-Fuse Complex and Pre-Bötzinger Complex.

BACKGROUND: Recent studies showed partial reversal of opioid-induced respiratory depression in the pre-Bötzinger complex and the parabrachial nucleus/Kölliker-Fuse complex. The hypothesis for this study was that opioid antagonism in the parabrachial nucleus/Kölliker-Fuse complex plus pre-Bötzinger complex completely reverses respiratory depression from clinically relevant opioid concentrations. METHODS: Experiments were performed in 48 adult, artificially ventilated, decerebrate rabbits. The authors decreased baseline respiratory rate ~50% with intravenous, "analgesic" remifentanil infusion or produced apnea with remifentanil boluses and investigated the reversal with naloxone microinjections (1 mM, 700 nl) into the Kölliker-Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex. In another group of animals, naloxone was injected only into the pre-Bötzinger complex to determine whether prior parabrachial nucleus/Kölliker-Fuse complex injection impacted the naloxone effect. Last, the µ-opioid receptor agonist [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (100 µM, 700 nl) was injected into the parabrachial nucleus/Kölliker-Fuse complex. The data are presented as medians (25 to 75%). RESULTS: Remifentanil infusion reduced the respiratory rate from 36 (31 to 40) to 16 (15 to 21) breaths/min. Naloxone microinjections into the bilateral Kölliker-Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased the rate to 17 (16 to 22, n = 19, P = 0.005), 23 (19 to 29, n = 19, P < 0.001), and 25 (22 to 28) breaths/min (n = 11, P < 0.001), respectively. Naloxone injection into the parabrachial nucleus/Kölliker-Fuse complex prevented apnea in 12 of 17 animals, increasing the respiratory rate to 10 (0 to 12) breaths/min (P < 0.001); subsequent pre-Bötzinger complex injection prevented apnea in all animals (13 [10 to 19] breaths/min, n = 12, P = 0.002). Naloxone injection into the pre-Bötzinger complex alone increased the respiratory rate to 21 (15 to 26) breaths/min during analgesic concentrations (n = 10, P = 0.008) but not during apnea (0 [0 to 0] breaths/min, n = 9, P = 0.500). [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection into the parabrachial nucleus/Kölliker-Fuse complex decreased respiratory rate to 3 (2 to 6) breaths/min. CONCLUSIONS: Opioid reversal in the parabrachial nucleus/Kölliker-Fuse complex plus pre-Bötzinger complex only partially reversed respiratory depression from analgesic and even less from "apneic" opioid doses. The lack of recovery pointed to opioid-induced depression of respiratory drive that determines the activity of these areas.

Neurochemistry of the Kölliker-Fuse nucleus from a respiratory perspective.

The Kölliker-Fuse nucleus (KF) is a functionally distinct component of the parabrachial complex, located in the dorsolateral pons of mammals. The KF has a major role in respiration and upper airway control. A comprehensive understanding of the KF and its contributions to respiratory function and dysfunction requires an appreciation for its neurochemical characteristics. The goal of this review is to summarize the diverse neurochemical composition of the KF, focusing on the neurotransmitters, neuromodulators, and neuropeptides present. We also include a description of the receptors expressed on KF neurons and transporters involved in each system, as well as their putative roles in respiratory physiology. Finally, we provide a short section reviewing the literature regarding neurochemical changes in the KF in the context of respiratory dysfunction observed in SIDS and Rett syndrome. By over-viewing the current literature on the neurochemical composition of the KF, this review will serve to aid a wide range of topics in the future research into the neural control of respiration in health and disease.

The pontine Kölliker-Fuse nucleus gates facial, hypoglossal, and vagal upper airway related motor activity.

The pontine Kölliker-Fuse nucleus (KFn) is a core nucleus of respiratory network that mediates the inspiratory-expiratory phase transition and gates eupneic motor discharges in the vagal and hypoglossal nerves. In the present study, we investigated whether the same KFn circuit may also gate motor activities that control the resistance of the nasal airway, which is of particular importance in rodents. To do so, we simultaneously recorded phrenic, facial, vagal and hypoglossal cranial nerve activity in an in situ perfused brainstem preparation before and after bilateral injection of the GABA-receptor agonist isoguvacine (50-70 nl, 10 mM) into the KFn (n = 11). Our results show that bilateral inhibition of the KFn triggers apneusis (prolonged inspiration) and abolished pre-inspiratory discharge of facial, vagal and hypoglossal nerves as well as post-inspiratory discharge in the vagus. We conclude that the KFn plays a critical role for the eupneic regulation of naso-pharyngeal airway patency and the potential functions of the KFn in regulating airway patency and orofacial behavior is discussed.

Endogenous glutamatergic inputs to the Parabrachial Nucleus/Kölliker-Fuse Complex determine respiratory rate.

The Kölliker-Fuse Nucleus (KF) has been widely investigated for its contribution to "inspiratory off-switch" while more recent studies showed that activation of the Parabrachial Nucleus (PBN) shortened expiratory duration. This study used an adult, in vivo, decerebrate rabbit model to delineate the contribution of each site to inspiratory and expiratory duration through sequential block of glutamatergic excitation with the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5). Glutamatergic disfacilitation caused large increases in inspiratory and expiratory duration and minor decrease in peak phrenic activity (PPA). Hypoxia only partially reversed respiratory rate depression but PPA was increased to >200 % of control. The contribution of PBN activity to inspiratory and expiratory duration was equal while block of the KF affected inspiratory duration more than expiratory. We conclude that in the in vivo preparation respiratory rate greatly depends on PBN/KF activity, which contributes to the "inspiratory on- "and "off-switch", but is of minor importance for the magnitude of phrenic motor output.

Excitation-inhibition balance regulates the patterning of spinal and cranial inspiratory motor outputs in rats in situ.

Spinal phrenic nerve activity (PNA) drives the diaphragm but cranial hypoglossal nerve activity (HNA) also expresses synchronous activity during inspiration. Here, we investigated the effects of local disinhibition (bilateral microinjections of bicuculline) of the nucleus tractus solitarius (NTS), the pre-Bötzinger complex and Bötzinger complex core circuit (pre-BötC/BötC) and the Kölliker-Fuse nuclei (KFn) on the synchronization of PNA and HNA in arterially-perfused brainstem preparations of rats. To quantitatively analyze the bicuculline effects on a putatively distributed inspiratory central pattern generator (i-CPG), we quantified the phase synchronization properties between PNA and HNA. The analysis revealed that bicuculline-evoked local disinhibition significantly reduced the strength of phase synchronization between PNA and HNA at any target site. However, the emergence of desynchronized HNA following disinhibition was more prevalent after NTS or pre-BötC/BötC microinjections compared to the KFn. We conclude that the primary i-CPG is located in a distributed medullary circuit whereas pontine contributions are restricted to synaptic gating of synchronous HNA and PNA.

Tauopathy in the periaqueductal gray, kölliker-fuse nucleus and nucleus retroambiguus is not predicted by ultrasonic vocalization in tau-P301L mice.

Upper airway and vocalization control areas such as the periaqueductal gray (PAG), kölliker-fuse nucleus (KF) and nucleus retroambiguus (NRA) are prone to developing tauopathy in mice expressing the mutant human tau P301L protein. Consequently, impaired ultrasonic vocalization (USV) previously identified in tau-P301L mice at the terminal disease stage of 8-9 months of age, was attributed to the presence of tauopathy in these regions. Our aim was to establish whether the onset of USV disorders manifest prior to the terminal stage, and if USV disorders are predictive of the presence of tauopathy in the PAG, KF and NRA. USVs produced by tau-P301L and wildtype mice aged 3-4, 5-6 or 8-9 months were recorded during male-female interaction. Immunohistochemistry was then performed to assess the presence or degree of tauopathy in the PAG, KF and NRA of mice displaying normal or abnormal USV patterns. Comparing various USV measurements, including the number, duration and frequency of calls, revealed no differences between tau-P301L and wildtype mice across all age groups, and linear discriminant analysis also failed to identify separate USV populations. Finally, the presence of tauopathy in the PAG, KF and NRA in individual tau-P301L mice did not reliably associate with USV disorders. Our findings that tauopathy in designated mammalian vocalization centres, such as the PAG, KF and NRA, did not associate with USV disturbances in tau-P301L mice questions whether USV phenotypes in this transgenic mouse are valid for studying tauopathy-related human voice and speech disorders.

Reduced computational modelling of Kölliker-Fuse contributions to breathing patterns in Rett syndrome.

KEY POINTS: Reduced computational models are used to test effects of loss of inhibition to the Kölliker-Fuse nucleus (KFn). Three reduced computational models that simulate eupnoeic and vagotomized respiratory rhythms are considered. All models exhibit the emergence of respiratory perturbations associated with Rett syndrome as inhibition to the KFn is diminished. Simulations suggest that application of 5-HT1A agonists can mitigate the respiratory pathology. The three models can be distinguished and tested based on their predictions about connections and dynamics within the respiratory circuit and about effects of perturbations on certain respiratory neuron populations. ABSTRACT: Rett syndrome (RTT) is a developmental disorder that can lead to respiratory disturbances featuring prolonged apnoeas of variable durations. Determining the mechanisms underlying these effects at the level of respiratory neural circuits would have significant implications for treatment efforts and would also enhance our understanding of respiratory rhythm generation and control. While experimental studies have suggested possible factors contributing to the respiratory patterns of RTT, we take a novel computational approach to the investigation of RTT, which allows for direct manipulation of selected system parameters and testing of specific hypotheses. Specifically, we present three reduced computational models, developed using an established framework, all of which successfully simulate respiratory outputs across eupnoeic and vagotomized conditions. All three models show that loss of inhibition to the Kölliker-Fuse nucleus reproduces the key respiratory alterations associated with RTT and, as suggested experimentally, that effects of 5-HT1A agonists on the respiratory neural circuit suffice to alleviate this respiratory pathology. Each of the models makes distinct predictions regarding the neuronal populations and interactions underlying these effects, suggesting natural directions for future experimental testing.

The Kölliker-Fuse nucleus orchestrates the timing of expiratory abdominal nerve bursting.

Coordination of respiratory pump and valve muscle activity is essential for normal breathing. A hallmark respiratory response to hypercapnia and hypoxia is the emergence of active exhalation, characterized by abdominal muscle pumping during the late one-third of expiration (late-E phase). Late-E abdominal activity during hypercapnia has been attributed to the activation of expiratory neurons located within the parafacial respiratory group (pFRG). However, the mechanisms that control emergence of active exhalation, and its silencing in restful breathing, are not completely understood. We hypothesized that inputs from the Kölliker-Fuse nucleus (KF) control the emergence of late-E activity during hypercapnia. Previously, we reported that reversible inhibition of the KF reduced postinspiratory (post-I) motor output to laryngeal adductor muscles and brought forward the onset of hypercapnia-induced late-E abdominal activity. Here we explored the contribution of the KF for late-E abdominal recruitment during hypercapnia by pharmacologically disinhibiting the KF in in situ decerebrate arterially perfused rat preparations. These data were combined with previous results and incorporated into a computational model of the respiratory central pattern generator. Disinhibition of the KF through local parenchymal microinjections of gabazine (GABAA receptor antagonist) prolonged vagal post-I activity and inhibited late-E abdominal output during hypercapnia. In silico, we reproduced this behavior and predicted a mechanism in which the KF provides excitatory drive to post-I inhibitory neurons, which in turn inhibit late-E neurons of the pFRG. Although the exact mechanism proposed by the model requires testing, our data confirm that the KF modulates the formation of late-E abdominal activity during hypercapnia. NEW & NOTEWORTHY The pons is essential for the formation of the three-phase respiratory pattern, controlling the inspiratory-expiratory phase transition. We provide functional evidence of a novel role for the Kölliker-Fuse nucleus (KF) controlling the emergence of abdominal expiratory bursts during active expiration. A computational model of the respiratory central pattern generator predicts a possible mechanism by which the KF interacts indirectly with the parafacial respiratory group and exerts an inhibitory effect on the expiratory conditional oscillator.

The Kölliker-Fuse nucleus acts as a timekeeper for late-expiratory abdominal activity.

While the transition from the inspiratory to the post-inspiratory (post-I) phase is dependent on the pons, little attention has been paid to understanding the role of the pontine respiratory nuclei, specifically the Kölliker-Fuse nucleus (KF), in transitioning from post-I to the late expiratory (late-E) activity seen with elevated respiratory drive. To elucidate this, we used the in situ working heart-brainstem preparation of juvenile male Holtzman rats and recorded from the vagus (cVN), phrenic (PN) and abdominal nerves (AbN) during baseline conditions and during chemoreflex activation [with potassium cyanide (KCN; n=13) or hypercapnia (8% CO2; n=10)] to recruit active expiration. Chemoreflex activation with KCN increased PN frequency and cVN post-I and AbN activities. The inhibition of KF with isoguvacine microinjections (10mM) attenuated the typical increase in PN frequency and cVN post-I activity, and amplified the AbN response. During hypercapnia, AbN late-E activity emerged in association with a significant reduction in expiratory time. KF inhibition during hypercapnia significantly decreased PN frequency and reduced the duration and amplitude of post-I cVN activity, while the onset of the AbN late-E bursts occurred significantly earlier. Our data reveal a negative relationship between KF-induced post-I and AbN late-E activities, suggesting that the KF coordinates the transition between post-I to late-E activity during conditions of elevated respiratory drive.

Kölliker-Fuse nuclei regulate respiratory rhythm variability via a gain-control mechanism.

Respiration varies from breath to breath. On the millisecond timescale of spiking, neuronal circuits exhibit variability due to the stochastic properties of ion channels and synapses. Does this fast, microscopic source of variability contribute to the slower, macroscopic variability of the respiratory period? To address this question, we modeled a stochastic oscillator with forcing; then, we tested its predictions experimentally for the respiratory rhythm generated by the in situ perfused preparation during vagal nerve stimulation (VNS). Our simulations identified a relationship among the gain of the input, entrainment strength, and rhythm variability. Specifically, at high gain, the periodic input entrained the oscillator and reduced variability, whereas at low gain, the noise interacted with the input, causing events known as "phase slips", which increased variability on a slow timescale. Experimentally, the in situ preparation behaved like the low-gain model: VNS entrained respiration but exhibited phase slips that increased rhythm variability. Next, we used bilateral muscimol microinjections in discrete respiratory compartments to identify areas involved in VNS gain control. Suppression of activity in the nucleus tractus solitarii occluded both entrainment and amplification of rhythm variability by VNS, confirming that these effects were due to the activation of the Hering-Breuer reflex. Suppressing activity of the Kölliker-Fuse nuclei (KFn) enhanced entrainment and reduced rhythm variability during VNS, consistent with the predictions of the high-gain model. Together, the model and experiments suggest that the KFn regulates respiratory rhythm variability via a gain control mechanism.

The role of the Kölliker-Fuse nuclei in the determination of abdominal motor output in a perfused brainstem preparation of juvenile rat.

The abdominal muscles are largely quiescent during normal breathing but may exhibit tonic activity or subtle respiratory modulation. The origin of baseline abdominal motor nerve activity (AbNA) if present remains uncharacterised. The contribution of the Kölliker-Fuse nucleus (KF) in the dorsolateral pons in the patterning and amplitude of AbNA was investigated using in situ perfused brainstem preparations of juvenile rats (n=12). Two types of AbNA were observed: Type I - expiratory-modulated (n=7), and Type II - weakly inspiratory/post-inspiratory-modulated (n=5). Despite this, all preparations exhibited the same bi-phasic late expiratory/postinspiratory bursts upon elicitation of the peripheral chemoreflex. Interestingly, the type of AbNA exhibited correlated with postinspiratory duration. Targeted microinjections of GABA-A receptor agonist isoguvacine (10mM; 70nl) into KF however did not significantly modify pattern or amplitude of baseline AbNA in either Type besides the selective abolition of the postinspiratory phase and, consequently, postinspiratory modulation in AbNAwhen present. In sum, the KF is not a major contributorin setting baseline abdominal motor output.

Dorsal and ventral aspects of the most caudal medullary reticular formation have differential roles in modulation and formation of the respiratory motor pattern in rat.

The respiratory pattern generator of mammals is anatomically organized in lateral respiratory columns (LRCs) within the brainstem. LRC compartments serve specific functions in respiratory pattern and rhythm generation. While the caudal medullary reticular formation (cMRF) has respiratory functions reportedly related to the mediation of expulsive respiratory reflexes, it remains unclear whether neurons of the cMRF functionally belong to the LRC. In the present study we specifically investigated the respiratory functions of the cMRF. Tract tracing shows that the cMRF has substantial connectivity with key compartments of the LRC, particularly the parafacial respiratory group and the Kölliker-Fuse nuclei. These neurons have a loose topography and are located in the ventral and dorsal cMRF. Systematic mapping of the cMRF with glutamate stimulation revealed potent respiratory modulation of the respiratory motor pattern from both dorsal and ventral injection sites. Pharmacological inhibition of the cMRF with the GABA-receptor agonist isoguvacine produced significant and robust changes to the baseline respiratory motor pattern (decreased laryngeal post-inspiratory and abdominal expiratory motor activity, delayed inspiratory off-switch and increased respiratory frequency) after dorsal cMRF injection, while ventral injections had no effect. The present data indicate that the ventral cMRF is not an integral part of the respiratory pattern generator and merely serves as a relay for sensory and/or higher command-related modulation of respiration. On the contrary, the dorsal aspect of the cMRF clearly has a functional role in respiratory pattern formation. These findings revive the largely abandoned concept of a dorsal respiratory group that contributes to the generation of the respiratory motor pattern.

The Kölliker-Fuse nucleus: a review of animal studies and the implications for cranial nerve function in humans.

To review the scientific literature on the relationship between Kölliker-Fuse nucleus (KF) and cranial nerve function in animal models, with view to evaluating the potential role of KF maturation in explaining age-related normal physiologic parameters and developmental and acquired impairment of cranial nerve function in humans. Medical databases (Medline and PubMed). Studies investigating evidence of KF activity responsible for a specific cranial nerve function that were based on manipulation of KF activity or the use of neural markers were included. Twenty studies were identified that involved the trigeminal (6 studies), vagus (9), and hypoglossal nerves (5). These pertained specifically to a role of the KF in mediating the dive reflex, laryngeal adductor control, swallowing function and upper airway tone. The KF acts as a mediator of a number of important functions that relate primarily to laryngeal closure, upper airway tone and swallowing. These areas are characterized by a variety of disorders that may present to the otolaryngologist, and hence the importance of understanding the role played by the KF in maintaining normal function.

µ opioid receptor activation hyperpolarizes respiratory-controlling Kölliker-Fuse neurons and suppresses post-inspiratory drive.

KEY POINTS: In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker-Fuse (KF) maintains upper airway patency and a normal respiratory pattern. In this study, activation of µ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats. Nerve recordings in an in situ preparation showed that activation of µ opioid receptors in the KF eliminated the post-inspiration phase of the respiratory cycle. In brain slices, µ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance. These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid-induced respiratory disturbances, particularly the impairment of upper airways. ABSTRACT: Opioid-induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of µ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi-intact rats, injection of opioid agonists DAMGO or [Met(5) ]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The µ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart-brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post-inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of µ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid-induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage-clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2 , characteristics of a G protein-coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine-mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by µ opioid agonists are likely mediators of the opioid-induced loss of post-inspiration and induction of apneusis.

Respiratory and sympathetic chemoreflex regulation by Kölliker-Fuse neurons in rats.

Chemoreceptor activation increases phrenic nerve activity (PNA) and sympathetic nerve activity (SNA). The dorsolateral pontine neurons, including the parabrachial nucleus and the Kölliker-Fuse (KF) region project to several brainstem areas involved in autonomic and respiratory regulation. Here the objective was to further test the hypothesis that the KF region could contribute to central and peripheral sympathetic chemoreflex activation. In urethane-anesthetized sino-aortic denervated or intact and vagotomized male Wistar rats (N = 7-8/group), hypercapnia (end-expiratory CO2 from 5 to 10 %) or KCN increased mean arterial pressure (MAP), splanchnic SNA, and PNA frequency and amplitude. Bilateral injection of muscimol (GABA-A agonist; 2 mM-50 nl) into the KF region increased resting PNA amplitude and reduced resting PNA frequency, without significant changes in resting MAP and SNA. Bilateral blockade of the KF region reduced the rise in MAP, sSNA, and PNA frequency and amplitude produced by hypercapnia or hypoxia. Our data suggest that the KF neurons could integrate and modulate breathing and sympathetic outflow during chemoreceptor activation.

Contributions of the pre-Bötzinger complex and the Kölliker-fuse nuclei to respiratory rhythm and pattern generation in awake and sleeping goats.

We investigated in three groups of awake and sleeping goats whether there are differences in ventilatory responses after injections of Ibotenic acid (IA, glutamate receptor agonist and neurotoxin) into the pre-Bötzinger complex (preBötC), lateral parabrachial (LPBN), medial (MPBN) parabrachial, or Kölliker-Fuse nuclei (KFN). In one group, within minutes after bilateral injection of 10µl IA (50mM) into the preBötC, there was a 10-fold increase in breathing frequency, but 1.5h later, the goats succumbed to terminal apnea. These data are consistent with findings in reduced preparations that the preBötC is critical to sustaining normal breathing. In a second group, increasing volumes (0.5-10µl) of IA injected at weekly intervals into the preBötC elicited a near-dose-dependent tachypnea and irregular breathing that lasted at least 5h. There were apneas restricted to wakefulness, but none were terminal. Postmortem histology revealed that the preBötC was 90% destroyed, but there was a 25-40% above normal number of neurons in the presumed parafacial respiratory group that may have contributed to maintenance of arterial blood gas homeostasis. In a third group, bilateral injections (1 and 10µl) of IA into the LPBN, MPBN, or KFN did not significantly increase breathing in any group, and there were no terminal apneas. However, 3-5h after the injections into the KFN, breathing frequency was decreased and the three-phase eupneic breathing pattern was eliminated. Between 10 and 15h after the injections, the eupneic breathing pattern was not consistently restored to normal, breathing frequency remained attenuated, and there were apneas during wakefulness. Our findings during wakefulness and NREM sleep warrant concluding that (a) the preBötC is a primary site of respiratory rhythm generation; (b) the preBötC and the KFN are determinants of respiratory pattern generation; (c) after IA-induced lesions, there is time-dependent plasticity within the respiratory control network; and (d) ventilatory control mechanisms are state dependent.

Bidirectional plasticity of pontine pneumotaxic postinspiratory drive: implication for a pontomedullary respiratory central pattern generator.

The "pneumotaxic center" in the rostral dorsolateral pons as delineated by Lumsden nine decades ago is known to play an important role in promoting the inspiratory off-switch (IOS) for inspiratory-expiratory phase transition as a fail-safe mechanism for preventing apneusis in the absence of vagal input. Traditionally, the pontine pneumotaxic mechanism has been thought to contribute a tonic descending input that lowers the IOS threshold in medullary respiratory central pattern generator (rCPG) circuits, but otherwise does not constitute part of the rCPG. Recent evidence indicates that descending input from the Kölliker-Fuse nucleus (KFN) within the pneumotaxic center is essential for gating the postinspiratory phase of the three-phase respiratory rhythm to control the IOS in vagotomized animals. A critical question arising is whether such a descending pneumotaxic input from KFN that drives postinspiratory activity is tonic (null hypothesis) or rhythmic with postinspiratory phase modulation (alternative hypothesis). Here, we show that multifarious evidence reported in the literature collectively indicates that the descending pneumotaxic input may exhibit NMDA receptor-dependent short-term plasticity in the form of a biphasic neural differentiator that bidirectionally and phase-selectively modulates postinspiratory phase duration in response to vagal and peripheral chemoreceptor inputs independent of the responses in inspiratory and late-expiratory activities. The phase-selectivity property of the descending pneumotaxic input implicates a population of pontine early-expiratory (postinspiratory/expiratory-decrementing) neurons as the most likely neural correlate of the pneumotaxic mechanism that drives post-I activity, suggesting that the pontine pneumotaxic mechanism may be an integral part of a pontomedullary rCPG that underlies the three-phase respiratory rhythm.

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

Both swallowing and respiration involve postinspiratory laryngeal adduction. Swallowing-related postinspiratory neurons are likely to be located in the nucleus of the solitary tract (NTS) and those involved in respiration are found in the Kölliker-Fuse nucleus (KF). The function of KF and NTS in the generation of swallowing and its coordination with respiration was investigated in perfused brainstem preparations of juvenile rats (n = 41). Orally injected water evoked sequential pharyngeal swallowing (s-PSW) seen as phasic, spindle-shaped bursting of vagal nerve activity (VNA) against tonic postinspiratory discharge. KF inhibition by microinjecting isoguvacine (GABAA receptor agonist) selectively attenuated tonic postinspiratory VNA (n = 10, P < 0.001) but had no effect on frequency or timing of s-PSW. KF disinhibition after bicuculline (GABAA receptor antagonist) microinjections caused an increase of the tonic VNA (n = 8, P < 0.01) resulting in obscured and delayed phasic s-PSW. Occurrence of spontaneous PSW significantly increased after KF inhibition (P < 0.0001) but not after KF disinhibition (P = 0.14). NTS isoguvacine microinjections attenuated the occurrence of all PSW (n = 5, P < 0.01). NTS bicuculline microinjections (n = 6) resulted in spontaneous activation of a disordered PSW pattern and long-lasting suppression of respiratory activity. Pharmacological manipulation of either KF or NTS also triggered profound changes in respiratory postinspiratory VNA. Our results indicate that the s-PSW comprises two functionally distinct components. While the primary s-PSW is generated within the NTS, a KF-mediated laryngeal adductor reflex safeguards the lower airways from aspiration. Synaptic interaction between KF and NTS is required for s-PSW coordination with respiration as well as for proper gating and timing of s-PSW.

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.