REM sleep-like episodes of motoneuronal depression and respiratory rate increase are triggered by pontine carbachol microinjections in in situ perfused rat brainstem preparation.

Hypoglossal nerve activity (HNA) controls the position and movements of the tongue. In persons with compromised upper airway anatomy, sleep-related hypotonia of the tongue and other pharyngeal muscles causes increased upper airway resistance, or total upper airway obstructions, thus disrupting both sleep and breathing. Hypoglossal nerve activity reaches its nadir, and obstructive episodes are longest and most severe, during rapid eye movement stage of sleep (REMS). Microinjections of a cholinergic agonist, carbachol, into the pons have been used in vivo to investigate the mechanisms of respiratory control during REMS. Here, we recorded inspiratory-modulated phrenic nerve activity and HNA and microinjected carbachol (25-50 nl, 10 mm) into the pons in an in situ perfused working heart-brainstem rat preparation (WHBP), an ex vivo model previously validated for studies of the chemical and reflex control of breathing. Carbachol microinjections were made into 40 sites in 33 juvenile rat preparations and, at 24 sites, they triggered depression of HNA with increased respiratory rate and little change of phrenic nerve activity, a pattern akin to that during natural REMS in vivo. The REMS-like episodes started 151 ± 73 s (SD) following microinjections, lasted 20.3 ± 4.5 min, were elicited most effectively from the dorsal part of the rostral nucleus pontis oralis, and were prevented by perfusion of the preparation with atropine. The WHBP offers a novel model with which to investigate cellular and neurochemical mechanisms of REMS-related upper airway hypotonia in situ without anaesthesia and with full control over the cellular environment.

Learning to breathe: control of the inspiratory-expiratory phase transition shifts from sensory- to central-dominated during postnatal development in rats.

The hallmark of the dynamic regulation of the transitions between inspiration and expiration is the timing of the inspiratory off-switch (IOS) mechanisms. IOS is mediated by pulmonary vagal afferent feedback (Breuer-Hering reflex) and by central interactions involving the Kölliker-Fuse nuclei (KFn). We hypothesized that the balance between these two mechanisms controlling IOS may change during postnatal development. We tested this hypothesis by comparing neural responses to repetitive rhythmic vagal stimulation, at a stimulation frequency that paces baseline breathing, using in situ perfused brainstem preparations of rats at different postnatal ages. At ages < P15 (P, postnatal days), phrenic nerve activity (PNA) was immediately paced and entrained to the afferent input and this pattern remained unchanged by repetitive stimulations, indicating that vagal input stereotypically dominated the control of IOS. In contrast, PNA entrainment at > P15 was initially insignificant, but increased after repetitive vagal stimulation or lung inflation. This progressive adaption of PNA to the pattern of the sensory input was accompanied by the emergence of anticipatory centrally mediated IOS preceding the stimulus trains. The anticipatory IOS was blocked by bilateral microinjections of NMDA receptor antagonists into the KFn and PNA was immediately paced and entrained, as it was seen at ages < P15. We conclude that as postnatal maturation advances, synaptic mechanisms involving NMDA receptors in the KFn can override the vagally evoked IOS after 'training' using repetitive stimulation trials. The anticipatory IOS may imply a hitherto undescribed form of pattern learning and recall in convergent sensory and central synaptic pathways that mediate IOS.

Postnatal emergence of synaptic plasticity associated with dynamic adaptation of the respiratory motor pattern.

The shape of the three-phase respiratory motor pattern (inspiration, postinspiration, late expiration) is controlled by a central pattern generator (CPG) located in the ponto-medullary brainstem. Synaptic interactions between and within specific sub-compartments of the CPG are subject of intensive research. This review addresses the neural control of postinspiratory activity as the essential determinant of inspiratory/expiratory phase duration. The generation of the postinspiratory phase depends on synaptic interaction between neurones of the nucleus tractus solitarii (NTS), which relay afferent inputs from pulmonary stretch receptors, and the pontine Kölliker-Fuse nucleus (KF) as integral parts of the CPG. Both regions undergo significant changes during the first three postnatal weeks in rodents. Developmental changes in glutamatergic synaptic functions and its modulation by brain-derived neurotrophic factor may have implications in synaptic plasticity within the NTS/KF axis. We propose that dependent on these developmental changes, the CPG becomes permissive for short- and long-term plasticity associated with environmental, metabolic and behavioural adaptation of the breathing pattern.

Activation of Orexin B receptors in the pontine Kölliker-Fuse nucleus modulates pre-inspiratory hypoglossal motor activity in rat.

Orexins (splice variants A and B) are hypothalamic neuropeptides that have essential functions in control of arousal and nutrition. Lack of Orexins is strongly associated with narcolepsy and sleep disordered breathing. However, the role of Orexins and particularly that of Orexin-B (OXB), in respiratory centres controlling upper-airway patency are less defined. In the present study we performed microinjections of OXB into the pontine Kölliker-Fuse nucleus (KF) of the dorsolateral pons, since this nucleus is particularly involved in the pre-motor control of upper airway muscles. The OXB mediated effects on heart, phrenic (PNA) and hypoglossal (XII-A) nerve activities were analysed in an in situ perfused brainstem preparation. Injection of OXB into the KF evoked significant augmentation of the respiratory frequency. Importantly, OXB provoked particularly prolonged pre-inspiratory discharge of the XII nerve, while no cardiovascular response was observed after KF microinjections. In summary, OXB in the KF exerts an excitatory effect on XII pre-motoneurones. Since pre-inspiratory activity of the XII is important for the decrease in upper airway resistance during inspiration, we conclude that OXB release in the KF has strong implications in the state-dependent control of upper airway patency under physiological and pathophysiological conditions.

Development of adaptive behaviour of the respiratory network: implications for the pontine Kolliker-Fuse nucleus.

Breathing is constantly modulated by afferent sensory inputs in order to adapt to changes in behaviour and environment. The pontine respiratory group, in particular the Kolliker-Fuse nucleus, might be a key structure for adaptive behaviours of the respiratory network. Here, we review the anatomical connectivity of the Kolliker-Fuse nucleus with primary sensory structures and with the medullary respiratory centres and focus on the importance of pontine and medullary postinspiratory neurones in the mediation of respiratory reflexes. Furthermore, we will summarise recent findings from our group regarding ontogenetic changes of respiratory reflexes (e.g., the diving response) and provide evidence that immaturity of the Kolliker-Fuse nucleus might account in neonates for a lack of plasticity in sensory evoked modulations of respiratory activity. We propose that a subpopulation of neurones within the Kolliker-Fuse nucleus represent command neurones for sensory processing which are capable of initiating adaptive behaviour in the respiratory network. Recent data from our laboratory suggest that these command neurones undergo substantial postnatal maturation.

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.

Central respiratory effects on motor nerve activities after organophosphate exposure in a working heart brainstem preparation of the rat.

The impact of organophosphorus compound (OP) intoxication on the activity of central respiratory circuitry, causing acetylcholinesterase (AChE) inhibition and accumulation of acetylcholine in the respiratory brainstem circuits, is not understood. We investigated the central effect of the OP Crotylsarin (CRS) on respiratory network activity using the working heart brainstem preparation, which specifically allows for the analysis of central drug effects without changes in brainstem oxygenation possibly caused by drug effects on peripheral cardio-respiratory activity. Respiratory network activity was determined from phrenic and hypoglossal or vagal nerve activities (PNA, HNA, VNA). To investigate combined central and peripheral CRS effects hypo-perfusion was used mimicking additional peripheral cardiovascular collapse. Systemic CRS application induced a brief central apnea and complete AChE-inhibition in the brainstem. Subsequently, respiration was characterised by highly significant reduced PNA minute activity, while HNA showed expiratory related extra bursting indicative for activation of un-specified oro-pharyngeal behaviour. During hypo-perfusion CRS induced significantly prolonged apnoea. In all experiments respiratory activity fully recovered after 1h. We conclude that CRS mediated AChE inhibition causes only transient central breathing disturbance. Apparently intrinsic brainstem mechanisms can compensate for cholinergic over activation. Nevertheless, combination of hypo-perfusion and CRS exposure evoke the characteristic breathing arrests associated with OP poisoning.

Pontine respiratory activity involved in inspiratory/expiratory phase transition.

Control of the timing of the inspiratory/expiratory (IE) phase transition is a hallmark of respiratory pattern formation. In principle, sensory feedback from pulmonary stretch receptors (Breuer-Hering reflex, BHR) is seen as the major controller for the IE phase transition, while pontine-based control of IE phase transition by both the pontine Kölliker-Fuse nucleus (KF) and parabrachial complex is seen as a secondary or backup mechanism. However, previous studies have shown that the BHR can habituate in vivo. Thus, habituation reduces sensory feedback, so the role of the pons, and specifically the KF, for IE phase transition may increase dramatically. Pontine-mediated control of the IE phase transition is not completely understood. In the present review, we discuss existing models for ponto-medullary interaction that may be involved in the control of inspiratory duration and IE transition. We also present intracellular recordings of pontine respiratory units derived from an in situ intra-arterially perfused brainstem preparation of rats. With the absence of lung inflation, this preparation generates a normal respiratory pattern and many of the recorded pontine units demonstrated phasic respiratory-related activity. The analysis of changes in membrane potentials of pontine respiratory neurons has allowed us to propose a number of pontine-medullary interactions not considered before. The involvement of these putative interactions in pontine-mediated control of IE phase transitions is discussed.

Serotonin targets inhibitory synapses to induce modulation of network functions.

The cellular effects of serotonin (5-HT), a neuromodulator with widespread influences in the central nervous system, have been investigated. Despite detailed knowledge about the molecular biology of cellular signalling, it is not possible to anticipate the responses of neuronal networks to a global action of 5-HT. Heterogeneous expression of various subtypes of serotonin receptors (5-HTR) in a variety of neurons differently equipped with cell-specific transmitter receptors and ion channel assemblies can provoke diverse cellular reactions resulting in various forms of network adjustment and, hence, motor behaviour. Using the respiratory network as a model for reciprocal synaptic inhibition, we demonstrate that 5-HT(1A)R modulation primarily affects inhibition through glycinergic synapses. Potentiation of glycinergic inhibition of both excitatory and inhibitory neurons induces a functional reorganization of the network leading to a characteristic change of motor output. The changes in network operation are robust and help to overcome opiate-induced respiratory depression. Hence, 5-HT(1A)R activation stabilizes the rhythmicity of breathing during opiate medication of pain.

Developmental changes in brain-derived neurotrophic factor-mediated modulations of synaptic activities in the pontine Kölliker-Fuse nucleus of the rat.

The Kölliker-Fuse nucleus (KF), part of the respiratory network, is involved in the modulation of respiratory phase durations in response to peripheral and central afferent inputs. The KF is immature at birth. Developmental changes in its physiological and anatomical properties have yet to be investigated. Since brain-derived neurotrophic factor (BDNF) is of major importance for the maturation of neuronal networks, we investigated its effects on developmental changes in the KF on different postnatal days (neonatal, P1-5; intermediate, P6-13; juvenile, P14-21) by analysing single neurones in the in vitro slice preparation and network activities in the perfused brainstem preparation in situ. The BDNF had only weak effects on the frequency of mixed excitatory and inhibitory spontaneous postsynaptic currents (sPSCs) in neonatal slice preparations. Postnatally, in the intermediate and juvenile age groups, a significant augmentation of the sPSC frequency was observed in the presence of 100 pm BDNF (+23.5+/-12.6 and +76.7+/-28.4%, respectively). Subsequent analyses of BDNF effects on evoked excitatory postsynaptic currents (eEPSCs) revealed significant enhancement of eEPSC amplitude of +20.8+/-7.0% only in juvenile stages (intermediates, -13.2+/-4.8%). On the network level, significant modulation of phrenic nerve activity following BDNF microinjection into the KF was also observed only in juveniles. The data suggest that KF neurones are subject to BDNF-mediated fast synaptic modulation after completion of postnatal maturation. After maturation, BDNF contributes to modulation of fast excitatory neurotransmission in respiratory-related KF neurones. This may be important for network plasticity associated with the processing of afferent information.