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.

Effects of pharmacological lesion of the nucleus retroambiguus region on the pharyngeal phase of swallowing.

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.

Thoracic sympathetic chain stimulation modulates and entrains the respiratory pattern.

Stimulation of thoracic sympathetic chain (TSC) afferents has been shown to slow the respiratory rhythm in dogs, monkeys and humans. However, sparse information exists about the physiological role of TSC afferents in modulating respiration or the central pathways of these afferents. Here, we sought to investigate whether the perfused preparation of juvenile rats is a suitable experimental model to study the role of TSC-afferents in the modulation of respiration. We show that tonic (30s) TSC stimulation initially triggered either prolonged post-inspiratory vagal nerve discharge, or when the stimulus onset occurred in the second half of expiration, TSC stimulation also modulated late-expiratory abdominal nerve activity. Independent of the timing of the TSC-stimulation the net effect was lengthening of the expiratory interval and subtle shortening of inspiration. TSC evoked respiratory modulation showed progressive habituation during the stimulus period. Importantly, high thoracic spinal cord transections abolished the TSC-evoked respiratory modulation, indicating that TSC afferents are likely to be relayed within the thoracic spinal cord. Next, we repeatedly applied 400 ms trains of stimuli at an inter-burst interval near that of the intrinsic respiratory rate and show that rhythmic TSC stimulation has a strong potential to entrain the central respiratory rhythm. Importantly, under the imposed rhythm, TSC stimuli became aligned with the late expiratory phase. The entrainment pattern supports the hypothesis that the TSC pathway may convey extra-pulmonary visceral mechano-sensory feedback that might be sensitive to visceral mass movements during locomotion. The latter was previously discussed to significantly contribute to the locomotor-respiratory coupling in various mammalian species.

Facts and challenges in respiratory neurobiology.

Respiratory neurobiology has been a lead discipline in the field of neuroscience for almost a century. Despite this, research studies on the fundamental synaptic and cellular processes underlying the generation and modulation of breathing movements suffered a significant decline during the last decade. We still believe that respiratory neurobiology is one of the most exciting and imperative fields of neuroscience. With the first white paper concerned with the central control of breathing, we want to celebrate the global importance of breathing research.

Blockade of dorsolateral pontine 5HT1A receptors destabilizes the respiratory rhythm in C57BL6/J wild-type mice.

The neurotransmitter serotonin (5HT) acting via 5HT1a receptors (5HT1aR) is a potent determinant of respiratory rhythm variability. Here, we address the 5HT1aR-dependent control of respiratory rhythm variability in C57BL6/J mice. Using the in situ perfused preparation, we compared the effects of systemic versus focal blockade of 5HT1aRs. Blocking 5HT1aRs in the Kölliker-Fuse nucleus (KFn) increased the occurrence of spontaneous apneas and accounted for the systemic effects of 5HT1aR antagonists. Further, 5HT1aRs of the KFn stabilized the respiratory rhythm's response to arterial chemoreflex perturbations; reducing the recovering time, e.g., the latency to return to the baseline pattern. Together, these results suggest that the KFn regulates both intrinsic and sensory determinants of respiratory rhythm variability.

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.

Early abnormalities of post-sigh breathing in a mouse model of Rett syndrome.

Rett syndrome is a neurodevelopmental disease accompanied by complex, disabling symptoms, including breathing symptoms. Because Rett syndrome is caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2), Mecp2-deficient mice have been generated as experimental model. Males of Mecp2-deficient mice (Mecp2(-/y)) breathe normally at birth but show abnormal respiratory responses to hypoxia and hypercapnia from postnatal day 25 (P25). After P30, Mecp2(-/y) mice develop breathing symptoms reminiscent of Rett syndrome, aggravating until premature death at around P60. Using plethysmography, we analyzed the sighs and the post-sigh breathing pattern of unrestrained wild type male mice (WT) and Mecp2(-/y) mice from P15 to P60. Sighs are spontaneous large inspirations known to prevent lung atelectasis and to improve alveolar oxygenation. However, Mecp2(-/y) mice show early abnormalities of post-sigh breathing, with long-lasting post-sigh apnoeas, reduced tidal volume when eupnoea resumes and lack of post-sigh bradypnoea which develop from P15, aggravate with age and possibly contribute to breathing symptoms to come.

The potency of different serotonergic agonists in counteracting opioid evoked cardiorespiratory disturbances.

Serotonin receptor (5-HTR) agonists that target 5-HT(4(a))R and 5-HT(1A)R can reverse mu-opioid receptor (mu-OR)-evoked respiratory depression. Here, we have tested whether such rescuing by serotonin agonists also applies to the cardiovascular system. In working heart-brainstem preparations in situ, we have recorded phrenic nerve activity, thoracic sympathetic chain activity (SCA), vascular resistance and heart rate (HR) and in conscious rats, diaphragmatic electromyogram, arterial blood pressure (BP) and HR via radio-telemetry. In addition, the distribution of 5-HT(4(a))R and 5-HT(1A)R in ponto-medullary cardiorespiratory networks was identified using histochemistry. Systemic administration of the mu-OR agonist fentanyl in situ decreased HR, vascular resistance, SCA and phrenic nerve activity. Subsequent application of the 5-HT(1A)R agonist 8-OH-DPAT further enhanced bradycardia, but partially compensated the decrease in vascular resistance, sympathetic activity and restored breathing. By contrast, the 5-HT(4(a))R agonist RS67333 further decreased vascular resistance, HR and sympathetic activity, but partially rescued breathing. In conscious rats, administration of remifentanyl caused severe respiratory depression, a decrease in mean BP accompanied by pronounced bradyarrhythmia. 8-OH-DPAT restored breathing and prevented the bradyarrhythmia; however, BP and HR remained below baseline. In contrast, RS67333 further suppressed cardiovascular functions in vivo and only partially recovered breathing in some cases. The better recovery of mu-OR cardiorespiratory disturbance by 5-HT(1A)R than 5-HT(4(a))R is supported by the finding that 5-HT(1A)R was more densely expressed in key brainstem nuclei for cardiorespiratory control compared with 5-HT(4(a))R. We conclude that during treatment of severe pain, 5-HT(1A)R agonists may provide a useful tool to counteract opioid-mediated cardiorespiratory disturbances.

Stimulation of the rat medullary raphe nuclei induces differential responses in respiratory muscle activity.

Neural control circuits that coordinate the motor activity of the diaphragm (DIA) and the geniohyoid muscle (GH) are potentially involved in pathological conditions such as various forms of sleep apnea. Here we investigated a differential role of the raphe magnus (RMg), pallidus (RPa) and the obscurus (ROb) nuclei in the neural control of DIA and GH muscle activity in rats under volatile anesthesia. In order to characterize a topographical organization of the raphe nuclei we analyzed changes in DIA and GH during high-frequency stimulation (HFS, 10-130 Hz, 60 micros pulse width, 40-160 microA, 30s). HFS of the RMg and the ROb induced apnea, in the latter case apnea was associated with massive tonic discharge in the GH. By contrast, HFS of the RPa induced tachypnea. At caudal stimulation sites the tachypnea was accompanied by tonic DIA activity and cessation of GH. These data suggest a differential distribution of inhibitory and excitatory drives of DIA and GH muscles within distinct raphe nuclei.

Modeling the ponto-medullary respiratory network.

The generation and shaping of the respiratory motor pattern are performed in the lower brainstem and involve neuronal interactions within the medulla and between the medulla and pons. A computational model of the ponto-medullary respiratory network has been developed by incorporating existing experimental data on the medullary neural circuits and possible interactions between the medulla and pons. The model reproduces a number of experimental findings concerning alterations of the respiratory pattern following various perturbations/stimulations applied to the pons and pulmonary afferents. The results of modeling support the concept that eupneic respiratory rhythm generation requires contribution of the pons whereas a gasping-like rhythm (and the rhythm observed in vitro) may be generated within the medulla and involve pacemaker-driven mechanisms localized within the medullary pre-Botzinger Complex. The model and experimental data described support the concept that during eupnea the respiration-related pontine structures control the medullary network mechanisms for respiratory phase transitions, suppress the intrinsic pacemaker-driven oscillations in the pre-BotC and provide inspiration-inhibitory and expiration-facilitatory reflexes which are independent of the pulmonary Hering-Breuer reflex but operate through the same medullary phase switching circuits.

Whole cell recordings from respiratory neurones in an arterially perfused in situ neonatal rat preparation.

For synaptic and cellular analyses of the mammalian respiratory network, intracellular recording and good pharmacological access to respiratory neurones is required. Using an existing arterially perfused in situ preparation of neonatal rat, we report on a method allowing stable intracellular recordings of ventrolateral medullary respiratory neurones. The in situ preparation generates a recognizable eupnoeic respiratory motor pattern similar to that reported in vivo. Using this preparation, we have developed a methodology for the use of patch pipettes to record from neurones within the ventral respiratory group. This technique in the arterially perfused neonatal rat is novel and has the advantage that neurones can be recorded from an intact and well-oxygenated brainstem in which the pontine regions are known to be viable. We describe the methods, present the first whole cell recordings of numerous types of respiratory neurones from neonatal rats in such a preparation, and demonstrate the applicability of the model for neuropharmacological experiments.

Histaminergic modulation of the intact respiratory network of adult mice.

Histaminergic modulation of neuronal activity in the respiratory network was investigated under normoxic and hypoxic conditions in the working heart-brainstem preparation of adult mice. Systemic application of histamine, as well as the H-1 and H-3 receptor agonists 6-[2-(4-imidazolyl)ethylamino]- N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT) and imetit, 0.5-10 micro M, significantly increased the frequency of respiratory burst discharges. Dimaprit, an H-2 receptor agonist, had no effect on respiratory activity. To test for ongoing histaminergic modulation we applied the histamine receptor antagonists pyrilamine (H-1); cimetidine (H-2) and thioperamide (H-3), each 0.5-10 micro M. Only the H-1 receptor antagonist had significant effects, viz. reduction of respiratory frequency and depression of burst amplitude. Underlying effects of histamine receptor activation were identified at the cellular level. Intracellular recordings showed that histamine mediated an increase in synaptic drive potentials in inspiratory neurones while augmentation of inhibitory and excitatory synaptic activity was observed in expiratory neurones. The augmented synaptic depolarisation of inspiratory neurones was blocked by the H-1 receptor antagonist. Histaminergic modulation is also involved in the hypoxic response of the respiratory network. Blockade of H-1 receptors significantly attenuated secondary depression of the biphasic hypoxic responses, while hypoxic augmentation was not affected. We conclude that histamine is a functional neuromodulator, which is tonically active in the respiratory network and is activated further during hypoxia. The data indicate that histaminergic neuromodulation acts predominantly via H-1 receptors.

Glycinergic inhibition is essential for co-ordinating cranial and spinal respiratory motor outputs in the neonatal rat.

Eupnoeic breathing in mammals is dependent on the co-ordinated activity of cranial and spinal motor outputs to both ventilate the lungs and adjust respiratory airflow, which they do by regulating upper-airway resistance. We investigated the role of central glycinergic inhibition in the co-ordination of cranial and spinal respiratory motor outflows. We developed an arterially perfused neonatal rat preparation (postnatal age 0-4 days) to assess the effects of blocking glycine receptors with systemically administered strychnine (0.5-1 microM). We recorded respiratory neurones located within the ventrolateral medulla, inspiratory phrenic nerve activity (PNA) and recurrent laryngeal nerve activity (RLNA), as well as dynamic changes in laryngeal resistance. Central recordings of postinspiratory neurones revealed an earlier onset in firing relative to the onset of inspiratory PNA after exposure to strychnine (260 +/- 38.9 vs. 129 +/- 26.8 ms). After glycine receptor blockade, postinspiratory neurones discharged during the inspiratory phase. Strychnine also evoked a decrease in PNA frequency (from 38.6 +/- 4.7 to 30.7 +/- 2.8 bursts min(-1)), but amplitude was unaffected. In control conditions, RLNA comprised inspiratory and postinspiratory discharges; the amplitude of the latter exceeded that of the former. However, after administration of strychnine, the amplitude of inspiratory-related discharge increased (+65.2 +/- 15.2 %) and exceeded postinspiratory activity. Functionally this change in RLNA caused a paradoxical, inspiratory-related glottal constriction during PNA. We conclude that during the first days of life in the rat, glycine receptors are essential for the formation of the eupnoeic-like breathing pattern as defined by the co-ordinated activity of cranial and spinal motor inspiratory and postinspiratory activities.

Influence of nasotrigeminal afferents on medullary respiratory neurones and upper airway patency in the rat.

Noxious stimulation of the nasal mucosa evokes a nasotrigeminal reflex (diving response), consisting of apnoea, bradycardia and vasoconstriction. However, the central mechanisms underlying the respiratory component remain unclear. Here, we describe the influence of nasotrigeminal stimulation (NS) on different types of ventral medullary respiratory neurones and upper airway patency in the rat. In an arterially perfused working heart-brainstem preparation (WHBP), NS was induced by either irrigation of the nasal passage with cold saline (100-200 microl) or electrical stimulation of the trigeminal ethmoidal nerve (EN5, 20 Hz, 10 s, 100 micros, 0.5-2 V). Both stimuli evoked apnoea, bradycardia, a pressor response, persistent discharge in the recurrent laryngeal nerve and marked glottal adduction (P<0.001). NS evoked a distinct pattern of response in respiratory neurones: inspiratory (I) neurones ceased firing and hyperpolarized (n=19), while postinspiratory neurones depolarized (n=10) and discharged persistently. The latency of EN5-evoked synaptic responses (excitatory to postinspiratory neurones 12.3+/-2.8 ms, n=10; inhibitory to inspiratory neurones 11.8+/-1.9 ms; n=19) was comparable. During the NS-evoked apnoea burst discharges were superimposed on a maintained level of tonic activity recorded from both the recurrent laryngeal nerve (n=4) and some postinspiratory neurones (n=7). The physiological significance of these "apnoeic rhythms" is discussed. We conclude that the cardiorespiratory components of the diving response are mediated by activation of distinct sets of postinspiratory respiratory neurones.

Respiratory activity in neonatal rats.

In neonatal animals in vitro preparations have been employed widely to study the central control of respiration. These preparations have limitations in that reflex afferent inputs and kinesiological studies cannot be performed. Here, we describe an alternative in situ experimental model for studying both peripheral and central control of the respiratory system in neonatal rats. Using technology based on adult mammals, we introduce an intra-arterially perfused working heart-brainstem preparation (WHBP) that permits studies on eupnoeic respiration in neonatal rats from within a few hours of birth. Using this preparation we demonstrate a three-phase respiratory rhythm as revealed by the activity in phrenic and recurrent laryngeal motor nerves, the respiratory modulation of laryngeal resistance and the firing patterns of respiratory neurones recorded from the ventrolateral medulla. We conclude that the neonatal rat WHBP is an in situ preparation because it produces a respiratory rhythm similar to that of adult in vivo mammal preparations but distinct from in vitro preparations.

Pontine cholinergic mechanisms enhance trigeminally evoked respiratory suppression in the anesthetized rat.

In the present study, we investigated in anesthetized rats the influences of the pontine rapid-eye-movement (REM) sleep center on trigeminally induced respiratory responses. We evoked the nasotrigeminal reflex by electrical stimulation of the ethmoidal nerve (EN5) and analyzed the EN5-evoked respiratory suppression before and after injections into the pontine reticular nuclei of the cholinergic agonist carbachol. After injections of 80-100 nl of carbachol (20 mM), we observed a decrease in respiratory rate, respiratory minute volume, and blood pressure but an increase in tidal volume. In those cases in which carbachol injections alone caused these REM sleep-like autonomic responses, we also observed that the EN5-evoked respiratory suppression was significantly potentiated. Unfortunately, carbachol injections failed to depress genioglossus electromyogram (EMG) effectively, because the EMG activity was already strongly depressed by the anesthetic alpha-chloralose. We assume that pontine carbachol injections in our anesthetized rats cause autonomic effects that largely resemble REM sleep-like respiratory and vascular responses. We therefore conclude that the observed potentiation of EN5-evoked respiratory suppression after carbachol might be due to REM sleep-associated neuronal mechanisms. We speculate that activation of sensory trigeminal afferents during REM sleep might contribute to pathological REM sleep-associated respiratory failures.

NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation.

In the present study, we examined the distribution of neurons in the parabrachial nucleus (PB), the Kölliker-Fuse nucleus (KF), the spinal trigeminal nucleus caudalis (Sp5C), the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM), which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-D-aspartate (NMDA) receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases (p < or = 0.001) of Fos-immunoreactive (-ir) neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Bötzinger/pre-Bötzinger complex, and in the ventrolateral and commissural NTS the increases were only moderately significant (p < or = 0.05). With respect to the numbers of NR1-/Fos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C (p < or = 0.001) and the midlevel KF (p < or = 0.01) and moderately significant (p < or = 0.05) in the external lateral PB, Bötzinger/pre-Bötzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses.

NMDA and GABAA receptors in the rat Kolliker-Fuse area control cardiorespiratory responses evoked by trigeminal ethmoidal nerve stimulation.

1. Electrical stimulation (10 s) of the ethmoidal nerve (EN5) evokes the nasotrigeminal reflex responses, including apnoea, bradycardia and rise in arterial blood pressure. In the present study, we examined the involvement of N-methyl-D-aspartate (NMDA), AMPA/kainate, (gamma-aminobutyric acidA (GABAA) and glycine receptors in the Kolliker-Fuse (KF) nucleus in the mediation of the nasotrigeminal reflex responses. 2. Unilateral injections (n = 6) of 50-100 nl of the NMDA receptor antagonist AP5 into the KF area led to a significant blockade of the EN5-evoked respiratory depression and bradycardia. Injections placed into the midlevel of the KF area were most effective (80-90 % blockade). The rise in arterial blood pressure remained unaffected. 3. Unilateral injections (n = 6) of the AMPA/kainate receptor antagonist CNQX into the KF area failed to block EN5-evoked autonomic responses significantly. 4. Unilateral injections (n = 5) of the GABAA receptor antagonist bicuculline enhanced the EN5-evoked respiratory depression and bradycardia. The effect persisted for up to 30 s after stimulation. Bicuculline injections into the midlevel of the KF area were most effective. The increase in arterial blood pressure remained unaffected. 5. Unilateral injections (n = 5) of the glycine receptor antagonist strychnine into the KF area did not produce any significant effects on EN5-evoked autonomic responses. 6. Our results suggest that the KF area represents a mandatory relay for the nasotrigeminally induced apnoea and bradycardia which are predominantly mediated by NMDA receptors in the KF. Furthermore, it appears that KF neurons are under a potent GABAergic inhibitory control. The EN5-evoked rise in arterial blood pressure was not altered by any of the drugs and, therefore, appears not to be mediated via the KF.

The medical nucleus of the solitary tract mediates the trigeminally evoked pressor response.

The present study examined whether the nucleus of the solitary tract (NTS) plays a role in mediating the trigeminally evoked pressor response which occurs after noxious perturbation of the nasal mucosa or electrical stimulation of the ethmoidal nerve (EN5). We recorded arterial blood pressure responses to EN5 stimulations before and after injections of the calcium channel blocker CoCl2 into the NTS. Unilateral and bilateral injections of CoCl2 into the medial NTS resulted in significant blockade (p < 0.05 and p < 0.01, respectively) of the EN5-evoked pressor responses. Recovery of the pressor responses was observed 15-25 min after the CoCl2 injections. CoCl2 injections into the caudal commissural NTS remained largely ineffective with respect to blocking the EN5-evoked pressor responses. These findings strongly suggest that the medial NTS is a crucial link for the trigeminally induced pressor response.