Reorganisation of respiratory network activity after loss of glycinergic inhibition

gamma-Aminobutyric acid (GABA)-ergic and glycinergic inhibition is believed to play a major role in the respiratory network. In the present study we tested whether specific blockade of glycinergic inhibition resulted in changes in respiratory network interaction and function. Using the working heart-brainstem preparation from adult mice, we recorded phrenic nerve activity and the activity of different types of respiratory neurones located in the ventrolateral medulla. Strychnine (0.03-0.3 microM) was given systemically to block glycine receptors (Gly-R). During exposure to strychnine, post-inspiratory (PI) neurones shifted their onset of discharge into the inspiratory phase. As a consequence, the post-inspiratory phase failed and the rhythm changed from a three-phase cycle (inspiration, post-inspiration, expiration, with a frequency of about. 0.24 Hz) to a faster, two-phased cycle (inspiration expiration, frequency about 0.41 Hz). Inspiratory and expiratory neurones altered their augmenting membrane potential pattern to a rapidly peaking pattern. Smaller voltage oscillations at approximately 10 Hz and consisting of excitatory and inhibitory postsynaptic potential sequences occurred during the expiratory interval. Due to their high frequency and low amplitude, such oscillations would be inadequate for lung ventilation. We conclude that, under physiological conditions, glycinergic inhibition does indeed play a major role in the generation of a normal respiratory rhythm in adult mice. After failure of glycinergic inhibition a faster respiratory rhythm seems to operate through reciprocal GABAergic inhibition between inspiratory and expiratory neurones, while phase switching is organised by activation of intrinsic membrane properties.     

Studies on the synaptic interconnection between bulbar respiratory neurones of cats

In cats anaesthetized with pentobarbital, medullary respiratory neurones of both dorsal and ventral populations were recorded intracellularly with 1 mol·l^–1 KCl-electrodes. The neurones were classified according to the projection of their axons to the spinal cord (bulbospinal neurones) or to the vagal nerves (vagal neurones). Those neurones which could not be activated antidromically (NAA-neurones) by either procedure were subdivided into (inspiratory) R-neurones, which were monosynaptically excited by lung stretch receptor afferents, and into inspiratory and expiratory NAA-neurones, which did not receive a direct synaptic input, from these afferents.All types of neurone investigated revealed postsynaptic activity during both inspiration and expiration. The periods when synaptic activity was minimal were the periods of transition between respiratory phases.The input resistance of most respiratory neurones varied in parallel with the respiratory cycle. A drastic fall of the input resistance during expiration was observed in R-neurones and in some inspiratory vagal neurones. This was not seen in inspiratory bulbospinal neurones.In stable intracellular recordings, periodic postsynaptic inhibition was demonstrated in 52 of 53 respiratory neurones by IPSP reversal following chloride injection. Maximal membrane potential then was generally reached during one of the periods of respiratory phase transition. Reasons for the failure of others to demonstrate these IPSPs are presented and discrepancies between other findings and these are discussed. It is concluded that reciprocal inhibition between bulbar respiratory neurones does exist and is a general phenomenon.It is argued that reciprocal inhibition is the fundamental mechanism underlying respiratory gating of afferent inputs.The probable existence of recurrent inhibition is inferred from the changes in the pattern of membrane depolarization during the active period of neurones.Supported by the Deutsche Forschungsgemeinschaft     

Converse motor output of inspiratory bulbospinal premotoneurones during vomiting

Intracellular recordings of the activities of 10 inspiratory bulbospinal neurones of the medulla were performed in decerebrate cats. Fictive vomiting was induced by repetitive stimulation of the supra-diaphragmatic vagus nerves and was defined by series of synchronous large bursts in both the phrenic (inspiratory) and abdominal (expiratory) nerves. During these synchronous bursts the inspiratory bulbospinal neurones of both the dorsal and ventral respiratory groups were strongly hyperpolarized by chloride-dependent inhibitory postsynaptic potentials (IPSPs). We concluded that during vomiting the central pattern generator is inhibited, and that another pattern generator drives the spinal respiratory motoneurones.     

Bötzinger-complex, bulbospinal expiratory neurones monosynaptically inhibit ventral-group respiratory neurones in the decerebrate rat

Extracellularly recorded action potentials from 49 Bötzinger-complex, bulbospinal expiratory neurones were used as triggers to compute 162 spike-triggered averages (STAs) of intracellular potentials recorded from 167 respiratory neurones in the ventral respiratory group (VRG) near the obex in 15 vagotomized, paralysed, ventilated and decerebrated rats. All of the Bötzinger-complex expiratory neurones were antidromically activated from the ipsilateral border between the C2/C3 segments of the spinal cord and discharged only during the late part of expiration with an augmenting pattern. We found evidence for monosynaptic inhibitory post-synaptic potentials (IPSPs) in 74 (～44%) of the STAs computed using 34 (～69%) of the trigger neurones. For vagal motoneurones, IPSPs were found in 24 of the 53 STAs of expiratory motoneurones, but in none of the 12 STAs of inspiratory motoneurones. For inspiratory neurones, IPSPs were found in 23 of the 33 STAs of bulbospinal neurones and in 6 of the 26 STAs of not antidromically activated (NAA) neurones. For expiratory neurones, IPSPs were found in one of the two STAs of bulbospinal neurones and in 20 of the 36 STAs of NAA neurones. We conclude that Bötzinger-complex, bulbospinal expiratory neurones monosynaptically inhibit bulbospinal inspiratory neurones, expiratory vagal motoneurones and other unidentified inspiratory and expiratory neurones in the VRG of rats during the late part of expiration.     

Inspiratory on-switch evoked by mesencephalic stimulation: activity of medullary respiratory neurones

Summary The activity of medullary inspiratory and expiratory neurones was studied in urethan-chloralose anaesthetized cats during stimulus — evoked inspiratory phase (inspiratory on-switch). All neurones were characterized according to their axonal destination (i.e. bulbospinal neurones or vagal motoneurones) or the absence of such axonal projections (i.e. propriobulbar neurones), and to their location in the dorsal or ventral respiratory nuclei. 1. The inspiratory on-switch effects were elicited during expiration (E phase) by brief tetanic electrical stimulation (50 to 100 ms duration; 0.5 mA; 300 Hz) delivered to the mesencephalic periaqueductal central gray and the adjacent reticular formation. The evoked inspiratory effects observed on the phrenic nerve discharge consisted of: (i) an immediate response (latency 20 ± 5 ms) of stable duration related to the stimulus (primary response: Prim.R.), (ii) a delayed response (patterned response: Patt.R.) appearing after a latent period (silent phase: Sil.P.) of 100 ms maximal duration. The later the stimulus in the E phase, the longer was the duration of the Patt.R. (300 to 1000 ms). 2. The stimulation evoked an earlier activation of the inspiratory bulbospinal neurones (latency 12 ± 6 ms) than that obtained in the phrenic nerve (Prim.R.). Hence, the Prim.R. originated from the bulbospinal pathway and not from a pathway directly impinging on the motoneurones. Conversely during stimulation very few inspiratory propriobulbar neurones were activated and no expiratory neurone discharged. 3. During the phrenic Sil.P., 46% of the inspiratory bulbospinal neurones continued to discharge with a firing rate lower than that during the stimulus train, while most of the inspiratory propriobulbar and expiratory neurones were not active. 4. During the Patt.R. all inspiratory bulbospinal neurones discharged early and were strongly activated whatever the Patt.R. duration whereas the expiratory neurones were not active. Inspiratory propriobulbar neurones were either not recruited or recruited later, and the number of active neurones increased as the duration of the Patt.R. lengthened. 5. Our results suggest that the eliciting of the stimulus-evoked inspiration (Patt.R.) primarily depends on the activation of the inspiratory bulbospinal neurones. These neurones therefore would not only be the output neurones of the medullary respiratory centres, but they would serve other roles such as building up of the excitation in other respiratory neurones, thus acting as a component of the inspiratory ramp generator.Abbreviations Prim.R Primary response - Patt.R Patterned response - Sil.P Silent phase - I phase Inspiratory phase - E phase Expiratory phase - IBSN Inspiratory bulbospinal neurones - IPBN Inspiratory propriobulbar neurones - EBSN Expiratory bulbospinal neurones - EPBN Expiratory propriobulbar neurones - DRN Dorsal respiratory nucleus - VRN Ventral respiratory nucleus Supported by CNRS (LA 205 and ATP no 4188) and Fondation pour Ia recherche médicale     

Dopamine1 receptor agonists reverse opioid respiratory network depression, increase CO2 reactivity

In adult pentobarbital-anesthetized and unanesthetized decerebrate cats, the D(1)R agonists (6-chloro-APB, SKF-38393, dihydrexidine) given intravenously restored phrenic nerve and vagus nerve respiratory discharges and firing of bulbar post-inspiratory neurons after the discharges were abolished by the micro-opioid receptor agonist fentanyl given intravenously. Reversal of opioid-mediated discharge depression was prevented by the D(1)R antagonist SCH23390. Iontophoresis of the micro-opioid receptor agonist DAMGO depressed firing of medullary bulbospinal inspiratory neurons. Co-iontophoresis of SKF-38393 did not restore firing and had no effect on bulbospinal inspiratory neuron discharges when applied alone. The D(1)R agonists given intravenously prolonged and intensified phrenic nerve and bulbospinal inspiratory neuron discharges. They also increased reactivity to CO(2) by lowering the phrenic nerve apnea threshold and shifting the phrenic nerve-CO(2) response curve to lower et(CO(2)) levels. Intravenous fentanyl on the other hand decreased CO(2) reactivity by shifting the phrenic nerve apnea threshold and the response curve to higher et(CO(2)) levels. Fentanyl effects on reactivity were partially reversed by D(1)R agonists.     

A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm

In order to test whether glycinergic inhibition is essential for the in vivo respiratory rhythm, we analysed the discharge properties of neurones in the medullary respiratory network after blockade of glycine receptors in the in situ perfused brainstem preparation of mature wild type and oscillator mice with a deficient glycine receptor. In wild type mice, selective blockade of glycine receptors with low concentrations of strychnine (0.03-0.3 microM) provoked considerable changes in neuronal discharge characteristics: The cycle phase relationship of inspiratory, post-inspiratory and expiratory specific patterns of membrane potential changes was altered profoundly. Inspiratory, post-inspiratory and expiratory neurones developed a propensity for fast voltage oscillations that were accompanied by multiple burst discharges. These burst discharges were followed by "after-burst" hyperpolarisations that were capable of triggering secondary burst discharges. Blockade of glycine receptors and the "big" Ca2+-dependent K+-conductance by charybdotoxin (3.3 nM) resulted in loss of the respiratory rhythm, whilst only tonic discharge activity remained. In contrast, rhythmic activity was only weakened, but preserved after the "small" Ca2+-dependent activated K+ conductance was blocked with apamin (8 nM). Also low concentrations of pentobarbital sodium (6 mg/kg) abolished rhythmic respiratory activity after blockade of glycine receptors in the wild type mice and in glycine receptor deficient oscillator mice. The data imply that failure of glycine receptors provokes enhanced bursting behaviour of respiratory neurones, whilst the additional blockade of BKCa channels by charybdotoxin or with pentobarbital abolishes the respiratory rhythm.     

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.     

Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Bötzinger complex in the cat

Summary Axonal projections and synaptic connectivity of expiratory B?tzinger neurons with an augmenting firing pattern (Bot-Aug neurons) to neurons in the ipsilateral ventral respiratory group (VRG) were studied in anaesthetized cats. Antidromic mapping revealed extensive axonal arborizations of Bot-Aug neurons (24 of 45) to the rostral or caudal VRG, with some having arbors in both regions. Of 234 pairs of neurons studied with intracellular recording and spike-triggered averaging, monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked in 49/221 VRG neurons by 38/98 Bot-Aug neurons. The highest incidence of monosynaptic inhibition was found in inspiratory bulbospinal neurons (10 of 23 tested). Evidence was also found for monosynaptic inhibition, by a separate group of Bot-Aug neurons, of expiratory bulbospinal neurons (12/58), while excitatory postsynaptic potentials (EPSPs) were identified in another two of these neurons. In addition, monosynaptic IPSPs were recorded from 13 of 53 identified laryngeal motoneurons, and from 14 of 100 respiratory propriobulbar neurons. Presumptive disynaptic IPSPs were recorded from 11 of the 221 VRG neurons. We conclude that Bot-Aug neurons exert widespread inhibition on all major neuron categories in the ipsilateral VRG, and should be regarded as an important element in shaping the spatiotemporal output pattern of both respiratory motoneurons and premotor neurons.     

Responses of ventral respiratory neurones in the rat to vagus stimulation and the functional division of expiration.

In anaesthetized rats, extracellular and intracellular recordings were taken from 106 respiratory neurones in the intermediate region of the nucleus ambiguus. We observed unprovoked shortening of expiratory time accompanied, in all classes of respiratory neurone, by the elimination of the changes in membrane potential that were characteristic of stage II expiration. The demonstration of the elimination of stage II expiration in both the rat and cat strongly supports the functional division of expiration into stage I expiration (post-inspiration) and stage II expiration. In order to identify the neurones in the rat that receive inputs from vagal afferents and modulate the central respiratory rhythm, we examined whether any respiratory neurones responded to stimulation of the vagus nerve. Some post-inspiratory and stage II expiratory neurones responded. The short latency (< 2 ms) of four of the responses indicates that some vagal afferents act on post-inspiratory neurones via a disynaptic pathway. While repetitive stimulation of the vagus nerve could inhibit the phrenic rhythm, it appears that most inspiratory neurones in the intermediate region of the nucleus ambiguous complex are not directly involved in integrating the information from vagal afferents with the central respiratory rhythm.     

Selective actions of anesthetic agents on membrane potential trajectory in bulbar respiratory neurons of cats

The effects of two anesthetic agents, halothane and thiopental, on the membrane potential trajectory of respiratory-related neurons in the ventral respiratory group were investigated in decerebrate cats, of which the carotid sinus and vagal afferents were denervated. Infusion of halothane (2% for 90 s) depolarized the membrane in nearly half of the inspiratory (12/21), post-inspiratory (10/26) and expiratory (4/6) neurons and caused hyperpolarization in the rest of the population. Thiopental (2.5 mg/kg i.v.) produced depolarization in 11 inspiratory and 10 post-inspiratory neurons and hyperpolarization in 1 expiratory, 4 inspiratory and 7 postinspiratory neurons. In both hyperpolarized and depolarized neurons, reduction of the respiratory membrane potential fluctuations and an increase of input resistance were commonly observed. Both drugs depressed spontaneous firing in most of the neurons studied. An increase of firing was observed in 9 out of 47 depolarized cells. These two contrasting effects on the membrane potential trajectory occurred similarly in the known groups of respiratory neurons, but the response of a given cell was consistent for the two anesthetic agents. The present results demonstrate that the anesthetic drugs exert various influences on the ventral respiratory group neuron population in maintaining the membrane potential trajectory and discharge activity. This may reflect a functional heterogeneity in the bulbar respiratory network of neurons.     

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)     

Localization and properties of respiratory neurons in the rostral pons of the newborn rat

The distribution and discharge pattern of respiratory neurons in the ‘pneumotaxic center’ of the rostral pons in the rat has remained unknown. We performed optical recordings and whole-cell patch clamp recordings to clarify respiratory neuron activity in the rostral pons of a brainstem-spinal cord preparation from a newborn rat. Inspiratory nerve activity was recorded in the 4th cervical nerve and used as a trigger signal for optical recordings. Respiratory neuron activity was detected in the limited region of the rostral-lateral pons. The main active region was presumed to be primarily the Kölliker-Fuse nucleus. The location of respiratory neurons was further confirmed by Lucifer Yellow staining after conducting whole-cell recordings. From a membrane potential analysis of the respiratory neurons in the rostral pons, the respiratory neurons were divided into four types: inspiratory neuron (71.9%), pre-inspiratory neuron (5.3%), post-inspiratory neuron (19.3%), and expiratory neuron (3.5%). A noticeable difference between pontine and medullary respiratory neurons was that post-inspiratory neurons were more frequently encountered in the pons. Application of a μ-opioid agonist, [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, transformed the burst pattern of post-inspiratory neurons into that of pre-inspiratory neurons. The electrical stimulation of the sensory root of the trigeminal nerve induced three types of responses in 85% of pontine respiratory neurons: inhibitory postsynaptic potentials (42.7%), excitatory postsynaptic potentials (37.7%) and no response (15.1%). Our findings provide the first evidence in the rat for the presence of respiratory neurons in the rostral pons, with localization in the lateral region approximately overlapping with the Kölliker-Fuse nucleus.     

Microiontophoresis of flurazepam on inspiratory and postinspiratory neurons in the ventrolateral medulla of cats: an intracellular study in vivo

Effects of flurazepam on the periodic inhibitory postsynaptic potentials (IPSPs) and on the action of locally applied gamma-aminobutyric acid (GABA) were studied in bulbar respiratory neurons of decerebrate cats using concentric multibarrelled electrodes for intracellular recording and extracellular iontophoresis. Iontophoresis of flurazepam augmented spontaneous IPSPs and increased the hyperpolarization induced by GABA. Iontophoretic application of bicuculline suppressed the action of flurazepam. The reversal potential for spontaneous IPSPs was similar to that for the GABA-response. Intracellular Cl- injection shifted both the IPSP wave and the GABA response in a depolarizing direction. Flurazepam enhanced these depolarizing responses. These results suggest that GABA mediates the postsynaptic inhibition in bulbar respiratory neurons.     

Reflex prolongation of stage I of expiration

Experiments were performed on anesthetized cats to test the theory that the interval between phrenic bursts is comprised of two phases, stage I and stage II of expiration. Evidence that these represent two separate neural phases of the central respiratory rhythm was provided by the extent to which stage duration is controlled individually when tested by superior laryngeal, vagus and carotid sinus nerve stimulation. Membrane potential trajectories of bulbar postinspiratory neurons were used to identify the timing of respiratory phases.Stimulation of the superior laryngeal, vagus and carotid sinus nerves during stage I of expiration prolonged the period of depolarization in postinspiratory neurons without significantly changing the durations of either stage II expiratory or inspiratory inhibition, indicating a fairly selective prolongation of the first stage of expiration. Changes in subglottic pressure, insufflation of smoke into the upper airway, application of water to the larynx or rapid inflation of the lungs produced similar effects. Sustained tetanic stimulation of superior laryngeal and vagus nerves arrested the respiratory rhythm in stage I of expiration. Membrane potentials in postinspiratory, inspiratory and expiratory neurons were indicative of a prolonged postinspiratory period. Thus, such an arrhythmia can be described as a postinspiratory apneic state of the central oscillator. The effects of carotid sinus nerve stimulation reversed when the stimulus was applied during stage II expiration. This was accompanied by corresponding changes in the membrane potential trajectories in postinspiratory neurons.The results manifest a ternary central respiratory cycle with two individually controlled phases occurring between inspiratory bursts.     

Synaptic effects of intercostal tendon organs on membrane potentials of medullary respiratory neurons

1. Stimulation of intercostal muscle tendon organs or their afferent fibers reduces medullary inspiratory neuron activity, decreases motor output to inspiratory muscles, and increases the activity of expiratory laryngeal motoneurons. The present study examines the synaptic mechanisms underlying these changes to obtain information about medullary neurons that participate in the afferent limb of this reflex pathway. 2. Membrane potentials of medullary respiratory neurons were recorded in decerebrate paralyzed cats. Postsynaptic potentials (PSPs) elicited in these neurons by intercostal nerve stimulation (INS) were compared before and after intracellular iontophoresis of chloride ions. After chloride injection, the normal hyperpolarization caused by inhibitory (I) PSPs is "reversed" to depolarization. 3. In inspiratory neurons, reversal of IPSPs by chloride injection also reversed hyperpolarization produced by INS when applied during any portion of the respiratory cycle. This observation suggests that increased chloride conductance of the postsynaptic membrane mediated the inhibition. Further, it is very likely that the last-order interneuron in the afferent pathway must be excited by INS and alter inspiratory neuron activity via an inhibitory synapse. The linear relationship between the amplitude of the INS induced PSP and membrane potential of inspiratory neurons provided evidence that neurons in the afferent pathway are not respiratory modulated. 4. The membranes of expiratory vagal motoneurons and post-inspiratory neurons were depolarized by INS during all portions of the respiratory cycle before IPSP reversal. Reversal of IPSPs affected neither this depolarization of expiratory vagal motoneurons during stage I and II expiration nor that of post-inspiratory neurons during stage I expiration. Thus this depolarization probably resulted from synaptic excitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of rubrospinal tract and the adjacent mesencephalic reticular formation on the activity of medullary respiratory neurons and the phrenic nerve discharge in the rabbit

Suprapontine brain sites acting on the central respiratory system have been demonstrated to give rise to inspiratory as well as expiratory facilitatory effects. In the present study the inspiratory inhibitory effect which has been reported in the cat to be elicited consistently by electrical stimulation of the rubrospinal tract and the adjacent mesencephalic reticular formation was examined in the urethane-anaesthetized rabbit. Stimulation of these sites with single electrical shocks of moderate intensity induced a short latency (onset after 3.0 ms) transient (duration: 29 ms) inhibition of the phrenic nerve activity (PHR). Short volleys of stimuli applied in mid- to late-inspiration led to a premature off-switch of inspiration. The extracellularly recorded discharge activity of the different types of medullary respiration-related units (RRU) reflected these alterations, accordingly. Axonal connections of RRU with mesencephalic structures were evaluated. Examination of orthodromic responses of medullary RRU to stimulation of this pathway revealed that most bulbospinal inspiratory neurons (10 out of 13) were paucisynaptically inhibited after short latency (at least 1.2 ms). The conduction time from bulbospinal inspiratory neurons to the recording site of PHR was 1.6 ms. Thus, a disynaptic pathway — including bulbospinal inspiratory neurons — is suggested inducing inspiratory inhibition 3.0 ms after single shock midbrain stimulation. This inhibition results in disfacilitation of phrenic motoneurons. The fact that extensive electrolytic lesions of the pneumotaxic center in rostral pons did not abolish the observed inspiratory inhibitions excludes these structures from being involved. A direct pathway from the red nucleus and the adjacent reticular formation to phrenic nuclei of the spinal cord, however, can not be excluded from being involved in the demonstrated inspiratory inhibition. The described effects may play a role in behavioral or voluntary control of respiration.     

Inhibitory synaptic mechanisms regulating upper airway patency

The breathing cycle of vertebrates comprises three phases (inspiration, postinspiration and expiration) that are apparent in the activities generated in the ponto-medullary respiratory network. A large body of evidence now indicates that in adult mammals generation of this three-phase pattern is based on reciprocal synaptic inhibition between distinct subsets of respiratory neurones. This review summarises our recent experiments focused on the role of glycinergic inhibition in respiratory pattern formation: e.g. in co-ordinating the activity of spinal and cranial motor outputs that drive the ventilatory pump (thoracic and abdominal muscles) and adjust airflow by regulating laryngeal resistance (laryngeal abductors and adductors). We used arterially perfused in situ preparations of neonatal and mature rat and show that specific blockade of glycine receptors within the ponto-medullary network caused a severe disruption of the co-ordination of spinal and cranial motor outputs: postinspiratory neurones lose their characteristic inspiratory inhibition revealing excitatory synaptic drive coincident with inspiratory phrenic nerve activity. The resulting simultaneous discharge of inspiratory and postinspiratory neurones caused co-activation of both glottal abductors and adductors during neural inspiration. The latter resulted in a paradoxical inspiratory adduction of the vocal fold and severe disruption of the eupneic breathing pattern.The effect of blocking glycine receptors was the same in both mature and newborn rats suggesting that glycinergic inhibition is essential for co-ordinating cranial and spinal motor outputs from birth.     

Augmentation of muscle nociceptive respiratory reflex facilitation by vagal afferents

Vagal influence on the facilitation of phrenic neural activity during respiratory phase-locked, gastrocnemius muscle nerve nociceptive electrical stimulation was examined in anesthetized, glomectomized, paralyzed, and artificially ventilated cats. (1) In the vagi-intact state, respiratory reflex facilitation was characterized by a sharp rise in peak amplitude, maximum rate of rise or slope, and mean rate of rise of integrated phrenic nerve activity. This was greater during inspiratory phase-locked (T1-locked) muscle nerve electrical stimulation than during expiratory phase-locked (TE-locked) muscle nerve electrical stimulation. "Evoked post-inspiratory phrenic activity" during the early expiratory phase was also observed during TE-locked muscle nerve electrical stimulation. (2) Bilateral vagotomy significantly attenuated the respiratory facilitation during both T1- and TE-locked muscle nerve electrical stimulation. In particular, the "evoked post-inspiratory phrenic activity" during TE-locked muscle nerve electrical stimulation was also attenuated or almost completely abolished. (3) Conditioning electrical stimulation of the vagus nerve revealed facilitatory reflexes which co-exist with inspiratory inhibitory reflexes. (4) The "evoked post-inspiratory phrenic activity" during TE-locked muscle nerve electrical stimulation, which was attenuated or abolished after vagotomy, was restored after vagal T1-locked conditioning stimuli combined with TE-locked muscle nerve electrical stimulation. The results suggest that vagal facilitatory reflexes augment the respiratory reflex facilitation during muscle nociceptive stimulation.     

Anesthetic effects on synaptic transmission and gain control in respiratory control

All volatile and most intravenous general anesthetics currently in clinical use cause respiratory depression at concentrations suitable for surgery. While various in vitro studies have identified potential molecular targets, their contributions to respiratory depression are poorly understood. At surgical concentrations, anesthetics principally affect ligand-gated, rather than voltage-gated ion channels. Here we focus on anesthetic-induced effects on synaptic transmission in brainstem respiratory neurons. The spontaneous discharge patterns of canine respiratory bulbospinal premotor neurons in vivo depend principally on NMDA and non-NMDA receptor-mediated excitation, while GABAA receptors mediate gain modulation and silent-phase inhibition. Studies examining the effects of volatile anesthetics on synaptic neurotransmission to these neurons suggest a primary role for postsynaptic enhancement of GABAA receptor function, partly offset by a reduction in presynaptic inhibition and a presynaptic reduction in glutamatergic excitation. In studies involving canine inspiratory hypoglossal motoneurons in vivo, which are already strongly depressed by low concentrations (< 0.5 MAC) of volatile anesthetics, the role of acid-sensitive, two-pore domain K+ (TASK) channels was found to be minimal at these subanesthetic concentrations. Potentiation of GABAA receptor-mediated inhibition was suggested. These studies on canine respiratory neurons provide valuable insights into mechanisms of anesthetic depression within a respiratory control subsystem; future studies will be required to determine anesthetic effects on sources of respiratory drive, rhythm, and their control.