Excitatory and inhibitory synaptic inputs shape the discharge pattern of pump neurons of the nucleus tractus solitarii in the rat

The second-order relay neurons of the slowly-adapting pulmonary stretch receptors (SARs) are called pump neurons (P cells) and are located in the nucleus tractus solitarii (NTS). We have shown recently that P cells do not act merely as simple relay neurons of SAR afferents but also receive rhythmic inputs from the central respiratory system. This study aimed to analyze two aspects of the respiratory inputs to P cells: (1) suppression of P cell firing at early inspiration (eI suppression) and (2) facilitation of P cell firing at around the period from late inspiration to early expiration (IE facilitation). This study employed extracellular recordings combined with iontophoretic applications of neuroactive drugs to single P cells, in Nembutal-anesthetized, paralyzed, and artificially ventilated rats. The results showed that several excitatory and inhibitory neurotransmitters were involved in these synaptic events. First, the glycine antagonist strychnine and the GABA_A antagonist bicuculline were applied to identify the neurotransmitters acting in eI suppression. Strychnine greatly diminished eI suppression, but bicuculline had little effect. This suggested that eI suppression was elicited by inspiratory neurons that were glycinergic and had a decrementing firing pattern. Second, on the other hand bicuculline markedly enhanced IE facilitation as well as the baseline frequency of P cell firing. The enhancement of IE facilitation was distinctive even when the effects of increased baseline firing on this enhancement were taken into account. Third, IE facilitation was diminished by applications of the NMDA glutamate receptor antagonists D-2-amino-5-phosphonovaleric acid (APV) and dizocilpine (MK-801). These results suggested that glutamatergic synapses on P cells from some unidentified respiratory neurons form excitatory inputs for IE facilitation and GABA_A receptor-mediated processes control the strength of IE facilitation, possibly at the presynaptic level. Finally, iontophoretic application of the non-NMDA glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione disodium (CNQX), almost completely abolished P cell firing in response to both lung inflation and electrical stimulation of the vagus nerve. This confirmed the previous report that glutamate is the primary neurotransmitter at the synapses between SAR afferents and P cells. We concluded that complicated synaptic inputs involving glycinergic and GABAergic inhibitions, and non-NMDA and NMDA glutamate receptor-mediated excitations form the basic pattern of P cell firing. Received: 31 March 1999 / Accepted: 8 June 1999     

Functional morphology and physiology of slowly adapting pulmonary stretch receptors

Since the original work by Hering and Breuer (1868) on slowly adapting pulmonary stretch receptors (SARs), numerous studies have demonstrated that these receptors are the lung vagal afferents responsible for eliciting the reflexes evoked by moderate lung inflation. SARs play a role in controlling breathing pattern, airway smooth muscle tone, systemic vascular resistance, and heart rate. Both anatomical and physiological studies support the contention that SARs, by their close association with airway smooth muscle, continuously sense the tension within the myoelastic components of the airways caused by lung inflation, smooth muscle contraction, and/or tethering of small intrapulmonary airways to the lung parenchyma. As a result, the receptor field location within the tracheobronchial tree of a SAR plays an important role in its discharge pattern, with variations in airway transluminal pressure and airway smooth muscle orientation being important modulating factors. The disruption of airway myoelastic components in various pulmonary diseases would be expected to alter the discharge pattern of SARs, and contribute to changes in breathing pattern and airway smooth muscle tone.     

Electrophysiological and pharmacological analysis of synaptic inputs to pulmonary rapidly adapting receptor relay neurons in the rat

The information from pulmonary rapidly adapting stretch receptors (RARs) to the central nervous system (CNS) is relayed in the nucleus tractus solitarii (NTS). The second-order neurons in the NTS referred to as RAR cells have recently been shown to receive rhythmic inputs from the central respiratory system in addition to the main inputs from RAR afferents. The present study analyzed these synaptic inputs by intracellular recordings from RAR cells, and by extracellular recordings combined with local applications of neuroactive drugs to RAR cells, in Nembutal-anesthetized, paralyzed, and artificially ventilated rats. The intracellular analysis identified both excitatory postsynaptic potentials (EPSPs) elicited presumably by RAR afferents and inhibitory postsynaptic potentials (IPSPs) synchronous with central inspiratory activity. This inhibitory input, called I suppression, was the origin of respiratory modulation of RAR cell firing, and its time course suggested that some unidentified inspiratory neurons with an augmenting firing pattern were the source of the inhibition. The pharmacological analysis suggested the types of neurotransmitters used in these synaptic events. First, glutamate was shown to be the primary neurotransmitter at the synapse between RAR afferents and RAR cells. Iontophoretic applications of the non-NMDA glutamate antagonist, CNQX, abolished RAR cell firing almost completely in response to lung inflation and deflation and to electrical stimulation of the vagus nerve. Second, glycinergic inputs which inhibited RAR cells in the inspiratory phase were revealed by applications of the glycine antagonist, strychnine. That is, the I suppression was greatly diminished by applications of strychnine. Third, although applications of the GABA_A receptor antagonist, bicuculline, had little effect on I suppression, bicuculline markedly increased the baseline firing of RAR cells. These results imply that the information path from RARs to the CNS is regulated at the level of RAR cells by phasically-acting glycinergic inhibition in the inspiratory phase and tonically-acting GABAergic inhibition; the results also provide new insights into the neuronal mechanisms of RAR-induced reflexes. Received: 3 February 1999 / Accepted: 12 May 1999     

Pacemaker phase shift in the absence of neural activity in guinea-pig stomach: a microelectrode array study

The nucleus tractus solitarii (NTS) is the first site of integration for primary baroreceptor afferents, which release glutamate to excite second-order neurones through ionotropic receptors. In vitro studies indicate that glutamate may also activate metabotropic receptors (mGluRs) to modulate the excitability of NTS neurones at pre- and postsynaptic loci. We examined the functional role of metabotropic glutamate receptors (mGluRs) in modulating the baroreceptor reflex in the rat NTS. Using the working heart–brainstem preparation, the baroreflex was activated using brief pressor stimuli and the consequent cardiac (heart rate change) and non-cardiac sympathetic (T8–10 chain) baroreflex gains were obtained. Microinjections of glutamate antagonists were made bilaterally into the NTS at the site of termination of baroreceptor afferents. NTS microinjection of kynurenate (ionotropic antagonist) inhibited both the cardiac and sympathetic baroreflex gains (16 ± 5% and 59 ± 11% of control, respectively). The non-selective mGluR antagonist MCPG produced a dose-dependent inhibition of the cardiac gain (30 ± 3% of control) but not the sympathetic gain. Selective inhibitions of the cardiac gain were also seen with LY341495 and EGLU suggesting the response was mediated by group II mGluRs. This effect on cardiac gain involves attenuation of the parasympathetic baroreflex as it persists in the presence of atenolol. Prior NTS microinjection of bicuculline (GABA_A antagonist) prevented the mGluR-mediated attenuation of the cardiac gain. These results are consistent with the reported presynaptic inhibition of GABAergic transmission by group II mGluRs in the NTS and constitute a plausible mechanism allowing selective feed-forward disinhibition to increase the gain of the cardiac limb of the baroreflex without changing the sympathoinhibitory component.     

Presynaptic depolarization in myelinated vagal afferent fibres terminating in the nucleus of the tractus solitarius in the cat

The presynaptic influences that act on terminals of slowly adapting lung stretch receptor afferents and aortic baroreceptor afferents within the nucleus of the solitary tract were assessed using intracellular recording and antidromic stimulation techniques.Central respiratory influences on the axcitability of lung stretch receptor terminals were observed in 29% (4 of 14) of measurements. These were confirmed in intracellular recordings where membrane depolarizations in synchrony with phrenic nerve discharge were seen in 17% (4 of 24) of fibres. In three cases membrane depolarization also occurred synchronously with artificial lung inflation.Neither tests of excitability nor intracellular recording revealed any evidence for equivalent presynaptic influences on 16 myelinated aortic baroreceptor terminals.Stimulation of the superior laryngeal nerve evoked depolarizations in 50% (7 of 14) of lung stretch receptor terminals. These took the form of complex waves of depolarization with both short (3–8 ms) and long latency (27–35 ms) components. The amplitude of the long latency response increased during the period of phrenic nerve discharge, i.e. during central inspiration.These effects are discussed in relation to the central respiratory influences on both respiratory and cardiovascular reflexes.     

GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors

The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are inhibited by activation of slowly adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second-order neurons (pump cells). Pump cells (n = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane-anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end-expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these nine cells, eight contained glutamic acid decarboxylase 67 (GAD67) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin-B, we confirmed that neurons located in or close to iNTS innervate RTN (two rats). Many such neurons contained GAD67 mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract tracing with biotinylated dextranamide (four rats) applied to iNTS also confirmed that this region innervates RTN by a predominantly GABAergic projection. This work confirms that many rat NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.     

Evidence for a monosynaptic connection between slowly adapting pulmonary stretch receptor afferents and inspiratory beta neurones

The synaptic connection between slowly adapting pulmonary stretch receptor afferents and inspiratory neurones within a region ventral to the tractus solitarius was determined using intracellular recording and spike triggered averaging techniques.When the vagus nerve was stimulated at intensities eliciting a Hering-Breuer reflex, the difference in mean latency between centrally recorded action potentials of slowly adapting pulmonary stretch receptor afferents and e.p.s.p.s of inspiratory beta neurones was 0.2 ms. This difference is indicative of a monosynaptic connection.Extracellular single unit spikes of slowly adapting pulmonary stretch receptors recorded from the nodose ganglion were used to trigger the averaging of synaptic noise recorded from inspiratory neurones. A prominent wave of synaptic depolarization was observed in all inspiratory beta neurones even when a small number of sweeps were averaged. This depolarization was absent from inspiratory alpha neurones.The shape indices of these depolarizations are consistent with a monosynaptic connection between slowly adapting pulmonary stretch receptor afferents and inspiratory beta neurones. In addition, the data raise the possibility that this connection is multiple and distributed.     

Regulation of cough by secondary sensory inputs

We have reviewed the role of afferent inputs and blood chemical changes to the central nervous system, and the way in which they modify the cough and expiration reflexes (CR and ER). Slowly adapting pulmonary stretch receptors (SARs) augment the CR, insofar as when their activity is abolished the CRs from the tracheobronchial (TB) tree and larynx are abolished or weakened. However, stimulation of SARs by lung inflation has an inconsistent effect on the CR. Activation of SARs strongly potentiates the ER from the vocal folds, by a reflex mechanism, and inhibition of SARs weakens the ER. Bronchopulmonary C-fibre receptors inhibit the CR, as do capsaicin-sensitive afferents from the heart and splanchnic bed, cutaneous cold receptors and those that respond to chest wall vibration. Nasal receptors responsive to the irritant agent capsaicin potentiate the reflex. Acute hypoxia also augments the CR, and the reflex is down-regulated by carotid body resection. On the other hand, the CR is inhibited by prolonged hypoxia and hyperoxia, and by hypercapnia. Thus different inputs to the cough-controlling mechanism in the brainstem have very varied effects on the CR. We conclude that the sensitivities of the CR and ER can be modified in a large variety of physiological and clinical conditions, and that there is no clear relationship between the reflexes and changes in breathing caused by the interventions.     

Influences of lung mechanoreceptors and carotid chemoreceptors on the response of respiratory muscle activity to tracheal occlusion

We examined the responses of respiratory muscle electromyograms (EMGs) from internal (IIC) and external intercostal (EIC) muscles and diaphragm (DIAP) to three successive occluded breaths in anesthetized spontaneously breathing rabbits. Both inspiratory and expiratory muscle EMGs progressively increased in the course of tracheal occlusion. An increase in these muscle EMGs was still observed after release of tracheal occlusion, but those effects were short-lasting. In a separate series of experiments, for assessment of possible reflex effects involved, the responses of slowly adapting pulmonary stretch receptor (SAR), rapidly adapting pulmonary stretch receptor (RAR), and carotid chemoreceptor activities to tracheal occlusion lasting for three respiratory efforts were also examined. The inspiratory discharge of SARs decreased but the expiratory discharge of SARs increased during tracheal occlusion. Although carotid chemoreceptors increased their activity in the latency of 3-6s after the onset of tracheal occlusion, the activity of RARs was greatly reduced throughout the period of tracheal occlusion. A transient increase in both carotid chemoreceptors and RARs was still observed after release of tracheal occlusion. These results suggest that alterations of inspiratory and expiratory muscle EMGs produced by tracheal occlusion would appear to be mediated by the afferent inputs from lung mechanoreceptors and carotid chemoreceptors.     

Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst2A receptor

Axon terminals synapsing on neurones in the nucleus tractus solitarii (NTS) that originate from the central nucleus of the amygdala (CeA) have been shown to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here we investigated whether such terminals also contain somatostatin (SOM), a neuropeptide found in axons distributed throughout the NTS and in somata in the CeA, and known to modulate cardiovascular reflexes when microinjected into the NTS. With fluorescence microscopy, SOM immunoreactivity was seen in the varicosities of some axons throughout the NTS that were anterogradely labelled with biotin dextran amine injected into the CeA. Such varicosities were frequently observed in close proximity to dendrites of NTS neurones that were immunoreactive for the SOM receptor sst(2A) subtype, and in many cases also for catecholamine synthesising enzymes. In the caudal, cardioregulatory zone of NTS, SOM immunoreactivity was localised by electron microscopic pre-embedding gold labelling to boutons containing dense-cored and clear pleomorphic vesicles and forming symmetrical synapses, mostly onto dendrites. Additional post-embedding gold labelling for GABA suggested that a subpopulation (29%) of GABAergic terminals sampled in this area of NTS contained SOM. Almost all boutons anterogradely labelled from the amygdala were GABA-immunoreactive (-IR) and 21% of these were SOM-IR. A similar proportion of these boutons (22%) formed synapses onto dendrites containing immunoreactivity for the SOM receptor sst(2A) subtype. These observations provide evidence that some of the GABAergic projection neurones in the CeA that inhibit baroreceptor reflex responses in the NTS in response to fear or emotional stimuli could release SOM, which might modulate the activity of NTS neurones via an action on sst(2A) receptors.     

The Hering-Breuer reflexes in the bronchial asthma attack

The correlation between pulmonary stretch receptor activity and inspiration-expiration duration quotient as well as respiratory frequency (Hering-Breuer reflexes) has been established before and during an asthma attack in the guinea-pig. The Hering-Breuer reflexes subserving the self-regulation of breathing during uninfluenced spontaneous breathing no longer prevail after induction of a bronchial asthma attack. Increased stretch receptor activity following increased lung volume does not lead to inhibition of inspiratory activity (lung inflation reflex), but to an enhancement of the asthmatic tachypnoea. The latter is assumed to result from the expiratory self-compression of the lungs (lung deflation reflex). The enhancement of the deflation reflex by increasing lung volume during the asthma attack is discussed with regard to the uneven ventilation and the conditions in lung mechanics underlying the excitation of the lung deflation or collapse endings.     

Ventilatory response to lung inflation after induced bronchospasm in healthy conscious subjects (author's transl)]

In 10 healthy conscious subjects we have studied the effect of a sustained bronchospasm induced by aerosol of carbachol on the pattern of breathing and the ventilatory response to lung inflation which reflects the Breuer Hering reflex. Bronchospasm was assessed by measuring airway resistance and functional residual capacity by plethysmography. After bronchospasm the ventilatory response to inflation was unchanged and the role played by afferents related to such a reflex in controlling pattern of breathing was preserved. Moreover the ration between the threshold volume of the reflex and tidal volume (VT) remained constant. These data suggest that this ration could represent a central set point for VT control. Variable changes in the ventilatory pattern were also observed and are discussed in relation to the previous data.     

Pulmonary stretch receptor spike time precision increases with lung inflation amplitude and airway smooth muscle tension

Slowly adapting pulmonary stretch receptors (SARs) respond to different lung inflation volumes with distinct spike counts and patterns, conveying information regarding the rate and depth of breathing to the cardiovascular and respiratory control systems. Previous studies demonstrated that SARs respond to repetitions of the same lung inflation faithfully, suggesting the possibility of modeling an SAR's discrete response pattern to a stimulus using a statistically based method. Urethane-anesthetized rabbit SAR spike trains were recorded in response to repeated 9-, 12-, and 15-ml lung inflations, and used to construct model spike trains using K-means clustering. Analysis of the statistics of the responses to different lung inflation volumes revealed that SARs fire with more temporal precision in response to larger lung inflations, because the standard deviations of real spikes clustered around the modeled spike times of responses to 15-ml stimuli were smaller than those produced by 12 or 9 ml, even at the same absolute firing frequencies. This implied that the mechanical coupling of SAR endings with pulmonary tissue is critical in determining spike time reliability. To test this, we collected SAR responses during bronchial constriction, compared them with those produced by the same SARs under normal airway resistance, and found that their firing reliability improved during bronchial constriction. These results suggest that airway distension and mechanical coupling of the receptor endings with the airway wall (partially determined by smooth muscle tone) are important determinants of SAR spike time reliability.     

Modulation of the Hering-Breuer reflex by raphe pallidus in rabbits

Activation of the pulmonary stretch receptors by lung inflation or vagal stimulation evokes Hering-Breuer (HB) reflex, which is characterized by inspiratory inhibition and expiratory prolongation. In this work, whether the HB reflex could be modulated by the serotonergic raphe pallidus (RP) was studied by comparing the strength of this reflex before and after electrical or chemical stimulation of the RP. Experiments were performed on urethane anesthetized adult rabbits. The HB reflex was simulated with electrical stimulation of the central end of cervical vagus nerve. The RP was stimulated electrically or chemically by microinjection of glutamate. We found that after either electrical stimulation or chemical stimulation of the RP, the inspiratory inhibition and expiratory prolongation of the HB reflex were significantly attenuated. This attenuation showed post-stimulation time dependency or short-term memory, as well as RP stimulation intensity dependency. Results of the present study suggested that the serotonergic RP could exert its respiratory effects by modulating the strength of HB reflex.     

The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents

Painful stimuli can evoke dramatic responses in the cardiovascular and respiratory systems. We have assessed the role of both the sympathetic and parasympathetic nervous system in mediating the reflex tachycardia that accompanies somatic nociception. We describe a major role for the nucleus tractus solitarii (NTS) as a site for integrating nociceptive and cardiorespiratory afferents. Since cardiorespiratory and nociceptive afferents terminate in the NTS, this nucleus offers a powerful opportunity for central modulation. We show that the NTS plays a major role in mediating the reflex tachycardia evoked by somatic noxious stimulation. Similar noxious stimulation attenuates the cardiac component of the peripheral chemoreceptor reflex and inhibits the peripheral chemoreceptor-evoked excitatory synaptic response of some NTS neurones. The functional interpretation we propose is that by depressing homeostatic reflexes at the NTS, noxious stimulation-evoked cardiorespiratory changes can be expressed and maintained, which may be essential for the survival of the animal.     

Vagal control of the heart: central serotonergic (5-HT) mechanisms

Cardiac vagal preganglionic neurones (CVPNs) are located within the dorsal vagal nucleus (DVN) and the nucleus ambiguus (nA). In mammals, CVPNs within the nA have small myelinated axons and mediate major chronotropic effects, those in the DVN have non-myelinated axons and mediate smaller chronotropic, dromotropic and inotropic effects. Numerous studies demonstrate important influences of serotonin (5-HT) at multiple sites controlling autonomic outflows including the nucleus tractus solitarius (NTS) where cardiorespiratory afferent fibres terminate, and the CVPNs and rostral ventrolateral medulla (RVLM), the location of sympathetic premotor neurones. We have demonstrated roles for some of the numerous 5-HT receptor subtypes (5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5-HT7) in brainstem regions involved in cardiac control. Intracisternal application of selective ligands was used to study the effect of 5-HT receptors on heart rate and its reflex control. Further electrophysiological studies were also carried out to delineate their location and the mechanisms of action of these ligands. Blocking 5-HT1A receptors attenuated bradycardias evoked by stimulating baroreceptor and cardiopulmonary afferents but not arterial chemoreceptors, whereas antagonizing 5-HT7 receptors markedly attenuated all these reflex bradycardias. Within the DVN, nA and NTS, activation of 5-HT1A receptors could excite or inhibit neurones. In the NTS 5-HT2 receptors also had variable effects; 5-HT2B receptors excite and 5-HT2C receptors inhibit. Antagonism of 5-HT3 receptors attenuated upper airway and cardiopulmonary reflex bradycardias; this is compatible with data showing that 5-HT3 receptors excite DVN and NTS neurones by a glutamate-dependent mechanism. The origin of the glutamate (neuronal or glial) remains unresolved but glia are a possibility as baroreceptor-sensitive NTS neurones receive few direct 5-HT-containing synaptic contacts. Thus, 5-HT plays a critical role in the control of vagal outflow to the heart; however, why so many different receptors are involved, and their relative functional roles, remains unresolved.     

Substance P presynaptically depresses the transmission of sensory input to bronchopulmonary neurons in the guinea pig nucleus tractus solitarii

Substance P modulates the reflex regulation of respiratory function by its actions both peripherally and in the CNS, particularly in the nucleus tractus solitarii (NTS), the first central site for synaptic contact of the lung and airway afferent fibres. There is considerable evidence that the actions of substance P in the NTS augment respiratory reflex output, but the precise effects on synaptic transmission have not yet been determined. Therefore, we determined the effects of substance P on synaptic transmission at the first central synapses by using whole-cell voltage clamping in an NTS slice preparation. Studies were performed on second-order neurons in the slice anatomically identified as receiving monosynaptic input from sensory nerves in the lungs and airways. This was done by the fluorescent labelling of terminal boutons after 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindocarbo-cyanine perchlorate (DiI) was applied via tracheal instillation. Substance P (1.0, 0.3 and 0.1 μM) significantly decreased the amplitude of excitatory postsynaptic currents (eEPSCs) evoked by stimulation of the tractus solitarius, in a concentration-dependent manner. The decrease was accompanied by an increase in the paired-pulse ratio of two consecutive eEPSCs, and a decrease in the frequency, but not the amplitude, of spontaneous EPSCs and miniature EPSCs, findings consistent with a presynaptic site of action. The effects were consistently and significantly attenuated by a neurokinin-1 (NK1) receptor antagonist (SR140333, 3 μM). The data suggest a new site of action for substance P in the NTS (NK1 receptors on the central terminals of sensory fibres) and a new mechanism (depression of synaptic transmission) for regulating respiratory reflex function.     

NMDA and non-NMDA receptors may play distinct roles in timing mechanisms and transmission in the feline respiratory network.

1. Activation of N-methyl-D-aspartate (NMDA) glutamate receptors in the brainstem network of respiratory neurones is required to terminate inspiration in the absence of lung afferents, but it is not required in the inspiratory motor act of lung inflation. In the present study we examined the involvement of non-NMDA ionotropic glutamate receptors in these two mechanisms in the adult mammal. 2. Adult cats were either decerebrated or anaesthetized with sodium pentobarbitone, paralysed and ventilated. Inspiratory motor output was recorded from the phrenic nerve and central respiratory activity from neurones in the bulbar ventral respiratory group. 3. In decerebrate vagotomized cats, ionophoretic application of 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)quinoxaline (NBQX) onto single respiratory neurones decreased their spontaneous discharge rate and abolished the excitatory effect of exogenously applied (RS) alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) but not NMDA. 4. In these animals, intravenous infusion (12 mg kg-1) of the non-NMDA receptor blockers GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-5-H-2,3-benzodi aze pine) or NBQX: (1) decreased (in 10/15 cats) or abolished (in 5/15 cats) the inspiratory-related discharge of the phrenic nerve; (2) did not prolong the inspiratory phase; (3) reduced or abolished the spontaneous discharge of respiratory neurones; and (4) profoundly decreased the excitatory effects of AMPA but not NMDA ionophoresed onto these neurones. When both the phrenic nerve and the recorded respiratory neurone were silenced, neuronal excitation by ionophoretic application of NMDA first revealed a subthreshold respiratory modulation without lengthening of the inspiratory phase, then respiratory modulation became undetectable. 5. Additional blockade of NMDA receptors by a small dose (0.15 mg kg-1) of dizocilpine (MK-801), abolished the phrenic nerve activity which persisted after NBQX (apnoea), but the discharge or the subthreshold modulation of the bulbar respiratory neurones showed a lengthening of the inspiratory phase (apneusis). 6. Elevation of FA,CO2 increased or re-established phrenic nerve discharges after blockade of non-NMDA receptors or of both NMDA and non-NMDA receptors. 7. Small doses of NBQX or GYKI 52466 induced apnoea in five of five cats anaesthetized with sodium pentobarbitone. 8. In decerebrate animals with intact vagi, GYKI 52466 and NBQX depressed the Hering-Breuer expiratory-lengthening reflex. 9. The results suggest that: (1) there is a specialization of different classes of glutamate receptors participating in timing mechanisms and transmission within the mammalian respiratory network. Neural transmission predominantly involves activation of non-NMDA receptors, acting in synergy with NMDA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.