Roles of glutamate and GABA of the Kölliker-Fuse nucleus in generating the cardiovascular chemoreflex.

The Kölliker-Fuse (KF) nucleus is a part of the parabrachial complex, located in the dorsolateral pons. It is involved in the chemoreflex-evoked cardiovascular and respiratory changes, but the role of GABA and glutamate in cardiovascular chemoreflex has not been shown yet. This study was performed to determine the role of GABA, glutamate, and their interaction in the KF, in cardiovascular chemoreflex in anesthetized rat. The antagonists were microinjected into the KF, and arterial pressure, heart rate, and single-unit responses were recorded simultaneously. The chemoreflex was evoked by i.v. injection of KCN, consisted of a short pressor followed by long bradycardia responses. Both responses were significantly attenuated by injection of a synaptic blocker (CoCl2) into the KF, confirming involvement of the KF in generating the reflex. Microinjection of AP5, an NMDA receptor antagonist, into the KF significantly attenuated the pressor and bradycardia responses, while blocking the AMPA receptors by CNQX had no significant effect. Blockade of GABAA receptors by bicuculline methiodide (BMI) potentiated both responses. Co-injection of BMI and CNQX potentiated the responses too. Co-injection of BMI and AP5 had no significant effect on the pressor response but significantly attenuated the bradycardia response. In conclusion, the KF plays a role in generating cardiovascular chemoreflex via its glutamate NMDA but not AMPA receptors. GABA inhibits both components of this reflex through GABAA receptors. There is an interaction between GABAA and NMDA receptors in regulating the bradycardia response of the reflex. Single-unit results were also presented which were correlated with and supported the homodynamic findings.

Impaired orexin receptor expression in the Kölliker-Fuse nucleus in sudden infant death syndrome: possible involvement of this nucleus in arousal pathophysiology.

OBJECTIVES: As well known, the sudden infant death syndrome (SIDS) is characterized by the sudden death of a seemingly healthy infant during sleep, frequently resulted from a deficit in arousal phase. Awakening from sleep requires a fully developed and functioning neuronal respiratory network to modulate the ventilation as needed. The pontine Kölliker-Fuse nucleus (KFN) plays a pivotal role in breathing control, thanks to its interconnections with the widespread serotonin and noradrenaline neurons in the brainstem. Numerous studies to date have focused on the implication of orexin, a neuropeptide synthesized by neurons of the lateral hypothalamus, with major projections to the brainstem raphé nuclei and locus coeruleus, in arousal, a neurobiological process closely linked to breathing modifications. The aim of our research has been to demonstrate that also the KFN is a fundamental component of the orexin system, actively involved in arousal. METHODS: We have evaluated the expression and distribution of the orexin receptors (orexin-1 and orexin-2 receptors) particularly in the rostral pons, where the KFN is located, of 25 SIDS cases and 18 controls. RESULTS: An intense orexin-1 innervation around the KF neurons has been detected in almost all the controls and only in 20% of SIDS cases. DISCUSSION: On the basis of these results, we believe that: (1) the KFN plays a leading role not only in providing a regular breathing rhythm but also in the coordination of the sleep-to-wake transition; (2) a defective orexin expression in the KFN could prevent arousal, thus assuming a crucial importance in causing SIDS.

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.

Deficiency of GABAergic synaptic inhibition in the Kölliker-Fuse area underlies respiratory dysrhythmia in a mouse model of Rett syndrome.

KEY POINTS: Life threatening breathing irregularity and central apnoeas are highly prevalent in children suffering from Rett syndrome. Abnormalities in inhibitory synaptic transmission have been associated with the physiopathology of this syndrome, and may underlie the respiratory disorder. In a mouse model of Rett syndrome, GABAergic terminal projections are markedly reduced in the Kölliker-Fuse nucleus (KF) in the dorsolateral pons, an important centre for control of respiratory rhythm regularity. Administration of a drug that augments endogenous GABA localized to this region of the pons reduced the incidence of apnoea and the respiratory irregularity of Rett female mice. Conversely, the respiratory disorder was recapitulated by blocking GABAergic transmission in the KF area of healthy rats. This study helps us understand the mechanism for generation of respiratory abnormality in Rett syndrome, pinpoints a brain site responsible and provides a clear anatomical target for the development of a translatable drug treatment. Central apnoeas and respiratory irregularity are a common feature in Rett syndrome (RTT), a neurodevelopmental disorder most often caused by mutations in the methyl-CpG-binding protein 2 gene (MECP2). We used a MECP2 deficient mouse model of RTT as a strategy to obtain insights into the neurobiology of the disease and into mechanisms essential for respiratory rhythmicity during normal breathing. Previously, we showed that, systemic administration of a GABA reuptake blocker in MECP2 deficient mice markedly reduced the occurrence of central apnoeas. Further, we found that, during central apnoeas, post-inspiratory drive (adductor motor) to the upper airways was enhanced in amplitude and duration in Mecp2 heterozygous female mice. Since the pontine Kölliker-Fuse area (KF) drives post-inspiration, suppresses inspiration, and can reset the respiratory oscillator phase, we hypothesized that synaptic inhibition in this area is essential for respiratory rhythm regularity. In this study, we found that: (i) Mecp2 heterozygous mice showed deficiency of GABA perisomatic bouton-like puncta and processes in the KF nucleus; (ii) blockade of GABA reuptake in the KF of RTT mice reduced breathing irregularity; (iii) conversely, blockade of GABAA receptors in the KF of healthy rats mimicked the RTT respiratory phenotype of recurrent central apnoeas and prolonged post-inspiratory activity. Our results show that reductions in synaptic inhibition within the KF induce rhythm irregularity whereas boosting GABA transmission reduces respiratory arrhythmia in a murine model of RTT. Our data suggest that manipulation of synaptic inhibition in KF may be a clinically important strategy for alleviating the life threatening respiratory disorders in RTT.

[Distribution of GABAergic neurons in pneumotaxic center nuclei in the early postnatal period in norm and in prenatal deficiency of serotoninergic system in rats].

The aim of this study was to determine the distribution of GABAergic neurons in pneumotaxic center structures (parabrachial complex medial subnucleus and Kölliker-Fuse nucleus) in norm and in deficiency of serotoninergic system during the prenatal period of development in Wistar rats. Reduction of endogenous serotonin levels in fetal rats was achieved by tryptophan hydroxylase inhibition with para-chlorophenylalanine (PCPA), which was administered to female rats on Day 16 of gestation. Material was obtained from the area of the pons from experimental and control (intact) rat pups at early postnatal (Days 5, 10 and 12) and juvenile (Day 20) periods. At each time point, 5-6 animals were studied from both experimental and control groups. To demonstrate GABAergic neurons, antibodies against glutamate decarboxylase (GAD-67), the enzyme involved in its synthesis, were used. The results have shown that Kölliker-Fuse nucleus contained a population of GABAergic neurons at early postnatal period, the size of which was preserved until juvenile age. In parabrachial complex medial subnucleus during the early postnatal period, a small number of GABAergic neurons was detected, which was somewhat increased by juvenile age. Serotonin deficiency in pneumotaxic center structures lead to a reduction of the numbers of GABAergic neurons, GABAergic synapses and their clusters. A reduction of serotonin levels during the prenatal period may cause the disturbances in the inhibitory afferent signaling of the pneumotaxic center nuclei and lead to the changes of local inhibitory GABAergic networks in its nuclei, resulting in the disturbances of the inhibitory processes in the center structures.

Kölliker's organ and the development of spontaneous activity in the auditory system: implications for hearing dysfunction.

Prior to the "onset of hearing," developing cochlear inner hair cells (IHCs) and primary auditory neurons undergo experience-independent activity, which is thought to be important in retaining and refining neural connections in the absence of sound. One of the major hypotheses regarding the origin of such activity involves a group of columnar epithelial supporting cells forming Kölliker's organ, which is only present during this critical period of auditory development. There is strong evidence for a purinergic signalling mechanism underlying such activity. ATP released through connexin hemichannels may activate P2 purinergic receptors in both Kölliker's organ and the adjacent IHCs, leading to generation of electrical activity throughout the auditory system. However, recent work has suggested an alternative origin, by demonstrating the ability of IHCs to generate this spontaneous activity without activation by ATP. Regardless, developmental abnormalities of Kölliker's organ may lead to congenital hearing loss, considering that mutations in ion channels (hemichannels, gap junctions, and calcium channels) involved in Kölliker's organ activity share strong links with such types of deafness.

Kölliker­Fuse neurons send collateral projections to multiple hypoxia-activated and nonactivated structures in rat brainstem and spinal cord.

The Kölliker­Fuse nucleus (KFN) in dorsolateral pons has been implicated in many physiological functions via its extensive efferent connections. Here, we combine iontophoretic anterograde tracing with posthypoxia c-Fos immunohistology to map KFN axonal terminations among hypoxia-activated/nonactivated brain stem and spinal structures in rats. Using a set of stringent inclusion/exclusion criteria to align visualized axons across multiple coronal brain sections, we were able to unequivocally trace axonal trajectories over a long rostrocaudal distance perpendicular to the coronal plane. Structures that were both richly innervated by KFN axonal projections and immunopositive to c-Fos included KFN (contralateral side), ventrolateral pontine area, areas ventral to rostral compact/subcompact ambiguus nucleus, caudal (lateral) ambiguus nucleus, nucleus retroambiguus, and commissural­medial subdivisions of solitary tract nucleus. The intertrigeminal nucleus, facial and hypoglossal nuclei, retrotrapezoid nucleus, parafacial region and spinal cord segment 5 were also richly innervated by KFN axonal projections but were only weakly (or not) immunopositive to c-Fos. The most striking finding was that some descending axons from KFN sent out branches to innervate multiple (up to seven) pontomedullary target structures including facial nucleus, trigeminal sensory nucleus, and various parts of ambiguus nucleus and its surrounding areas. The extensive axonal fan-out from single KFN neurons to multiple brainstem and spinal cord structures("one-to-many relationship"') provides anatomical evidence that KFN may coordinate diverse physiological functions including hypoxic and hypercapnic respiratory responses, respiratory pattern generation and motor output,diving reflex, modulation of upper airways patency,coughing and vomiting abdominal expiratory reflex, as well as cardiovascular regulation and cardiorespiratory coupling.