Differential roles of ionotropic glutamate receptors in canine medullary inspiratory neurons of the ventral respiratory group.

The relative roles of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in supplying excitatory drive to inspiratory (I) augmenting pattern neurons of the ventral respiratory group were studied in anesthetized, ventilated, paralyzed, and vagotomized dogs. Multibarrel micropipettes were used to record simultaneously single-unit neuronal activity and pressure microeject the NMDA antagonist, 2-amino-5-phosphonovalerate (AP5; 2 mM), the non-NMDA antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 0.25 mM), and an artificial cerebrospinal fluid vehicle. Ejected volume-rates were measured directly via meniscus level changes. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of the discharge patterns. Both AP5 and NBQX produced dose-dependent reductions in peak spontaneous I neuronal discharge frequency (Fn). The average (+/- SE) maximum reduction in peak Fn produced by AP5 was 69.1 +/- 4.2% and by NBQX was 47.1 +/- 3.3%. Blockade of both glutamate receptor subtypes nearly silenced these neurons, suggesting that their activity is highly dependent on excitatory synaptic drive mediated by ionotropic glutamate receptors. Differential effects were found for the two glutamatergic antagonists. AP5 produced downward, parallel shifts in the augmenting pattern of discharge, whereas NBQX reduced the slope of the augmenting discharge pattern. These results suggest that time-varying excitatory input patterns to the canine I bulbospinal neurons are mediated by non-NMDA glutamate receptors and that constant or tonic input patterns to these neurons are mediated by NMDA receptors.     

Respiratory responses to ionotropic glutamate receptor antagonists in the ventral respiratory group of the rabbit

Selective excitatory amino acid receptor antagonists acting on either N-methyl-D-aspartic acid (NMDA) or non-NMDA receptors were microinjected (30-50 nl) bilaterally into different subregions of the ventral respiratory group (VRG) of alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 or 10 mM) within the inspiratory portion of the VRG (iVRG) dose-dependently decreased the peak amplitude and rate of rise of phrenic nerve activity, without significant changes in respiratory timing. Decreases in respiratory frequency and peak phrenic amplitude up to apnoea were evoked by 20 mM D-AP5; phrenic nerve activity was restored transiently by hypoxic or hypercapnic stimulation during D-AP5-induced apnoea. Microinjections of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 or 20 mM) into the iVRG provoked less intense depressant respiratory effects. No significant respiratory responses were evoked by microinjections of these antagonists into more caudal VRG subregions. The results suggest that ionotropic glutamate receptors within the iVRG are involved mainly in the control of the intensity of inspiratory activity, with a major role played by NMDA receptors. Glutamate receptor antagonism in the iVRG does not seem to impair the basic mechanisms underlying respiratory rhythm generation.     

Physiological properties of late inspiratory neurons and their possible involvement in inspiratory off-switching in cats

To assess the functional significance of late inspiratory (late-I) neurons in inspiratory off-switching (IOS), membrane potential and discharge properties were examined in vagotomized, decerebrate cats. During spontaneous IOS, late-I neurons displayed large membrane depolarization and associated discharge of action potentials that started in late inspiration, peaked at the end of inspiration, and ended during postinspiration. Depolarization was decreased by iontophoresis of dizocilpine and eliminated by tetrodotoxin. Stimulation of the vagus nerve or the nucleus parabrachialis medialis (NPBM) also evoked depolarization of late-I neurons and IOS. Waves of spontaneous chloride-dependent inhibitory postsynaptic potentials (IPSPs) preceded membrane depolarization during early inspiration and followed during postinspiration and stage 2 expiration of the respiratory cycle. Iontophoresed bicuculline depressed the IPSPs. Intravenous dizocilpine caused a greatly prolonged inspiratory discharge of the phrenic nerve (apneusis) and suppressed late-inspiratory depolarization as well as early-inspiratory IPSPs, resulting in a small constant depolarization throughout the apneusis. NPBM or vagal stimulation after dizocilpine produced small, stimulus-locked excitatory postsynaptic potentials (EPSPs) in late-I neurons. Neurobiotin-labeled late-I neurons revealed immunoreactivity for glutamic acid decarboxylase as well as N-methyl-D-aspartate (NMDA) receptors. These results suggest that late-I neurons are GABAergic inhibitory neurons, while the effects of bicuculline and dizocilpine indicate that they receive periodic waves of GABAergic IPSPs and glutamatergic EPSPs. The data lead to the conclusion that late-I neurons play an important inhibitory role in IOS. NMDA receptors are assumed to augment and/or synchronize late-inspiratory depolarization and discharge of late-I neurons, leading to GABA release and consequently off-switching of bulbar inspiratory neurons and phrenic motoneurons.     

Positive feedback facilitation of external intercostal and phrenic inspiratory activity by pulmonary stretch receptors

Both in lightly pentobarbitone anesthetized and decerebrate cats increments in lung volume (V) during inspiration caused facilitation of inspiratory activity both in phrenic (Phr) and external intercostal (EI) motoneurons. This effect had low volume threshold, well below eupnoeic tidai volumes. It was readily reduced or abolished by small additional doses of pentobarbitone. This facilitatory effect appeared with considerably greater magnitude in El than in Phr. The response magnitude was linearly related to the corresponding increments in V? but not to increments in airflow (V?). Sustained elevation of V at zero V caused sustained facilitation of EI and Phr. This positive feedback facilitation which was similarly obtained in spontaneously breathing and paralysed cats occurred continuously with great regularity in every breath. It was abolished by bilateral vagotomy but could then be elicited by electrical stimulation of the central end of the vagus nerve at the same threshold strengths required to elicit a just detectable shortening of inspiratory duration. The results indicate that the slowly adapting pulmonary stretch receptors are responsible for this positive feedback facilitation prior to the negative feedback effect on the inspiratory ‘off-switch’ elicited by the same receptors. Clear distinctions are described between the reflex characteristics of this ‘low-threshold’ volume dependent facilitatory reflex and the ‘high-threshold’ transient excitatory reflex effects provoked by large and rapid inflations.     

N-methyl-D-aspartate (NMDA) receptors control respiratory off-switch in cat

Functionally active N-methyl-D-aspartate (NMDA) receptors on cat medullary respiratory neurones were revealed by local iontophoretic application of DL-2-amino-7-phosphonoheptanoic acid (AP7). Blockade of NMDA receptors by systemic administration of NMDA antagonists (MK-801, phencyclidine, ketamine, AP7) in vagotomized cats increased the duration of inspiration (Ti) without increasing expiration and caused an apneustic breathing pattern. The increase in Ti which followed systemic MK-801, was accompanied by a shift and complete reversal of early expiratory neuronal discharge in relation to phrenic nerve discharge.     

5-HT-1A receptor-mediated modulation of medullary expiratory neurones in the cat.

The involvement of the 5-HT-1A receptor in serotoninergic responses of stage 2 expiratory (E-2) neurones was investigated in pentobarbitone-anaesthetized, mechanically ventilated cats. The specific agonist of the 5-HT-1A receptor, 8-hydroxy-diproplaminotetralin (8-OH-DPAT), administered systemically or by ionophoresis directly on to the neurones, had a clear depressant effect. Administration of 8-OH-DPAT at doses of 10-50 micrograms kg-1 (I.V.) increased the membrane hyperpolarizations of E-2 neurones during the inspiratory and postinspiratory phases, and shortened their duration of activity in association with shortening of phrenic nerve activity. Discharges of E-2 neurones were also less intense. At doses of 50-90 micrograms kg-1, 8-OH-DPAT reduced or abolished inspiratory hyperpolarizations, and reduced expiratory depolarizations of membrane potential and discharge in parallel with inhibition of phrenic nerve discharges. The effects of the larger doses were reversed by I.V. injection of NAN-190, an antagonist at the 5-HT-1A receptor. Dose-dependent effects on the membrane potential and discharge of E-2 neurones, but not on phrenic nerve activity, were also seen by ionophoretic administration of 8-OH-DPAT on to E-2 neurones. At low currents, ejection of 8-OH-DPAT hyperpolarized the neurones without affecting the duration of inspiratory hyperpolarization and expiratory depolarization. This hyperpolarization depressed the intensity and the duration of expiratory discharges. Ejection with larger currents hyperpolarized the E-2 neurones further, and depressed expiratory depolarization leading to blockade of expiratory discharges. The effects on membrane potential were accompanied by decreased neuronal input resistance. This depressed the excitability of E-2 neurones as tested by discharge evoked by intracellular current injection. The amplitudes of action potentials decreased in parallel with the changes in input resistance. The effects were attributed to a postsynaptic effect of 8-OH-DPAT leading to a gradually developing inhibition by activation of 5-HT-1A receptors. Hyperventilatory apnoea depressed on-going synaptic activity and unmasked the effect of ionophoretically applied 8-OH-DPAT. The responses of the E-2 neurone were enhanced, as evidenced by increased membrane hyperpolarization and greater reduction of input resistance. Both responses faded appreciably, indicating receptor desensitization. The degree and rate of apparent desensitization depended on the dose/ejecting current. The greater sensitivity and faster desensitization to 8-OH-DPAT were attributed to the hyperventilatory alkalinization of the extracellular fluid, which might influence agonist binding to 5HT-1A receptors and/or receptor properties.     

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.     

Synaptic interactions between respiratory neurons during inspiratory on-switching evoked by vagal stimulation in decerebrate cats

To elucidate neuronal mechanisms underlying phase-switching from expiration to inspiration, or inspiratory on-switching (IonS), postsynaptic potentials (PSPs) of bulbar respiratory neurons together with phrenic nerve discharges were recorded during IonS evoked by vagal stimulation in decerebrate and vagotomized cats. A single shock stimulation of the vagus nerve applied at late-expiration developed an inspiratory discharge in the phrenic neurogram after a latency of 79+/-11 ms (n = 11). Preceding this evoked inspiratory discharge, a triphasic response was induced, consisting of an early silence (phase 1 silence), a transient burst discharge (phase 2 discharge) and a late pause (phase 3 pause). During phase 1 silence, IPSPs occurred in augmenting inspiratory (aug-I) and expiratory (E2) neurons, and EPSPs in postinspiratory (PI) neurons. During phase 2 discharge, EPSPs arose in aug-I neurons and IPSPs in PI and E2 neurons. These initial biphasic PSPs were comparable with those during inspiratory off-switching evoked by the same stimulation applied at late-inspiration. In both on- and off-switching, phase-transition in respiratory neuronal activities started to arise concomitantly with the phrenic phase 3 pause. These results suggest that vagal inputs initially produce a non-specific, biphasic response in bulbar respiratory neurons, which consecutively activates a more specific process connected to IonS.     

An analysis of synaptic transmission to motoneurones in the cat spinal cord using a new selective receptor blocker

We have investigated the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors on synaptic transmission in vivo between Ia primary afferents and cat spinal motoneurones using a selective non-N-methyl-d -aspartate (non-NMDA) receptor antagonist, GYKI 52466. Both microionophoretic and intravenous application of GYKI 52466 depressed the Ia excitatory post-synaptic potential (Ia EPSP) in a dose-dependent manner, without any apparent effect on membrane conductance or resting potential of the motoneurone. GYKI 52466 reduced selectively α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)- but not N-methyl-d -aspartate (NMDA)-induced depolarizations. Our results suggest that a large part of the Ia EPSP is mediated by AMPA receptors. The participation of other excitatory amino-acid receptors in the Ia EPSP is also discussed.     

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.     

Glutamate neurotransmission is not required for, but may modulate, hypoxic sensitivity of pre-Bötzinger complex in vivo

Focal hypoxia in the pre-B?tzinger complex (pre-B?tC) in vivo elicits excitation of inspiratory motor output by modifying the patterning and timing of phrenic bursts. Hypoxia, however, has been reported to enhance glutamate release in some regions of the brain, including the medullary ventral respiratory column; thus the pre-B?tC-mediated hypoxic respiratory excitation may result from, or be influenced by, hypoxia-induced activation of ionotropic glutamate [i.e., excitatory amino acid (EAA)] receptors. To test this possibility, the effects of focal pre-B?tC hypoxia [induced by sodium cyanide (NaCN)] were examined before and after blockade of ionotropic EAA receptors [using kynurenic acid (KYN)] in this region in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Before blockade of ionotropic EAA receptors, unilateral microinjection of NaCN (1 mM; 10-20 nl) into the pre-B?tC produced either phasic or tonic excitation of phrenic nerve discharge. Unilateral microinjection of KYN (50-100 mM; 40 nl) decreased the amplitude and frequency of basal phrenic nerve discharge; however, subsequent microinjection of NaCN, but not DL-homocysteic acid (DLH, a glutamate analog), still produced excitation of phrenic motor output. Under these conditions, the NaCN-induced excitation included frequency modulation (FM) of phasic phrenic bursts, and in many cases, augmented and/or fractionated phrenic bursts. These findings show that the hypoxia-sensing function of the in vivo pre-B?tC, which produces excitation of phrenic nerve discharge, is not dependent on activation of ionotropic glutamate receptors, but ionotropic glutamate receptor activation may modify the expression of the focal hypoxia-induced response. Thus these findings provide additional support to the concept of intrinsic hypoxic sensitivity of the pre-B?tC.     

Influence of hypercapnia and hypocapnia on bladder contractions and their respiratory consequences

Rhythmic contractions of the detrusor muscle, induced by gradual filling of the urinary bladder in decerebrate or anesthetized cats, are accompanied by decreased inspiratory activity in motor nerves to respiratory muscles, particularly those of the upper airway. We have examined the influence of hypercapnia and hypocapnia on these contractions and the accompanying activities of the phrenic and hypoglossal nerves in decerebrate, vagotomized, paralyzed and ventilated cats, some of which had denervated carotid chemoreceptors. Hypercapnia slowed, and then reversibly abolished bladder contractions in most animals, regardless of the state of the carotid chemoreceptors. Bladder contractions were well maintained in progressive hypocapnia, even at end-tidal CO(2) levels below the 'apneic' thresholds of the hypoglossal and phrenic activities. The reductions of the nerve activities in response to bladder contractions were not significantly altered by hypercapnia or hypocapnia. The abolition of bladder contractions by hypercapnia is unlikely to reflect a direct effect of CO(2) or H+ ion on the contractile mechanism of the detrusor muscle, but may be based on inhibition of stretch receptors in the bladder wall and/or an effect of CO(2) or H+ in or near the micturition centers in the brain stem.     

Opposing effects on the phrenic motor pathway attributed to dopamine-D1 and -D3/D2 receptor activation

Previous in vivo studies revealed that dopamine-D1-agonists elevate excitability of ventral respiratory column (VRC) neurons and increase discharge activity in the phrenic motor output through actions in the brainstem. In this in vivo study performed on pentobarbital-anesthetized cats, we show that D1-agonists (SKF-38393, dihydrexidine) given intravenously enhanced discharge activity in VRC inspiratory neurons and the phrenic nerve in two stages; discharge intensity first increased to a peak and then discharge duration increased. Cross-correlation analysis of VRC inspiratory neuron and phrenic nerve discharges showed that both stages increased strength of coupling between medullary inspiratory neurons and the phrenic motoneuron output. Intracellular recording and microiontophoresis experiments indicated that D1-agonists produced their stimulatory effects indirectly through actions on synaptic inputs to VRC inspiratory neurons. Because other laboratories have provided evidence that dopamine acting on other types of receptors depresses respiratory neuron excitability we tested the effects of piribedil, an agonist that activates receptors of the generally depressant D3/D2-dopamine receptor family, on phrenic nerve activity. Piribedil depressed phrenic nerve inspiratory discharge intensity, prolonged discharge duration, slowed burst frequency and slowed rate of action potential augmentation. The effects of piribedil were partially counteracted by intravenous injection of dihydrexidine. We propose that under normal, steady state conditions, D1-receptor-mediated excitatory modulation of phrenic motor output overrides D3/D2-receptor mediated inhibition.     

Effects of stimulation of phrenic afferent fibers on medullary respiratory neurons in cat

The effects of electrical stimulation of both cervical branches (C5 and C6) of the right phrenic nerve on medullary respiratory neuron activity were studied in anesthetized, spontaneously breathing cats. In 14 cats, the stimulation of the thin phrenic afferents had no effect on the inspiratory duration and evoked excitatory or inhibitory responses in only 3/86 inspiratory neurons tested. In 3 cats, the stimulation decreased the inspiratory duration and 26/26 inspiratory neurons showed a shortened discharge without modification of their discharge frequency. Although the effects of the stimulation were not analysed by averaging techniques, it is concluded that phrenic afferents do not exert an important control on the medullary respiratory neuron discharge.     

Characterization of L-homocysteate-induced currents in Purkinje cells from wild-type and NMDA receptor knockout mice

L-Homocysteic acid (HCA), an endogenous excitatory amino acid in the mammalian CNS, potently activates N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons. However, the responses to HCA in Purkinje cells, which lack functional NMDA receptors, have been largely unexplored: HCA may activate conventional non-NMDA receptors by its mixed agonistic action on both NMDA and non-NMDA receptors, or it may activate a novel non-NMDA receptor that has high affinity for HCA. To test these possibilities, we compared the responses to HCA in cultured Purkinje cells with those in hippocampal neurons by using the whole cell patch-clamp technique. To clearly isolate HCA responses mediated by non-NMDA receptors, we complemented pharmacological methods by using neurons from mutant mice (NR(-/-)) that lack functional NMDA receptors. A moderate dose of HCA (100 microM) induced substantial responses in Purkinje cells. These responses were blocked by non-NMDA receptor antagonists but were insensitive to NMDA receptor antagonists. HCA also activated responses mediated by non-NMDA receptors in both wild-type and NR1(-/-) hippocampal neurons. HCA responses in Purkinje cells had a pharmacological profile (EC(50) and Hill coefficient) very similar to that of non-NMDA receptor components of HCA responses in hippocampal neurons. Moreover, the amplitude of the non-NMDA receptor component of HCA responses was directly correlated with that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non-NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in Purkinje cells are similar to those in hippocampal neurons and that there is no distinct HCA-preferring receptor in Purkinje cells. We also found that in hippocampal neurons, the EC(50)s of HCA for non-NMDA receptors and for NMDA receptors were more similar than originally reported; this finding indicates that HCA is similar to other mixed agonists, such as glutamate. HCA responses may appear to be selective at NMDA receptors in cells that express these receptors, such as hippocampal neurons; in cells that express few functional NMDA receptors, such as Purkinje cells, HCA may appear to be selective at non-NMDA receptors.     

Activity of Brainstem Respiratory Neurones just before the Expiration-Inspiration Transition in the Rat

Inspiratory activity of the hypoglossal nerve (XIIn) often precedes that of the phrenic nerve (PHRn). By manipulating artificial respiration, this preceding activity (pre-I XIIn activity) can be lengthened or isolated prematurely (decoupled XIIn activity) without developing into overt PHRn-associated inspiratory bursts. We hypothesized that these pre-I and decoupled XIIn activities, collectively termed 'XIIn-w/o-PHRn activity', reflect certain internal states of the respiratory centre at the period just prior to the transition from the expiratory phase to the inspiratory phase. In decerebrate, neuromuscularly blocked and artificially ventilated rats, the firing properties of medullary respiratory neurones were examined during the period of the XIIn-w/o-PHRn activity. The majority of the inspiratory neurones examined could be classified into two types: one was active (XIIn-type) and the other was inactive (PHRn-type) during the XIIn-w/o-PHRn period. On the other hand, augmenting expiratory (E-AUG) neurones of the Bötzinger complex (BOT) and the caudal ventral respiratory group (VRG) fired intensively during this period. Their firing stopped at the onset of the overt inspiratory bursts in the XIIn and PHRn, suggesting that BOT E-AUG neurones inhibit PHRn-type, but not XIIn-type, inspiratory neurones. We hypothesize that XIIn-type inspiratory activity facilitates the phase change from expiration to inspiration, through activation of certain inspiratory neurones that inhibit the firing of BOT E-AUG neurones and generation of the overt inspiratory bursts in XIIn-type and PHRn-type inspiratory neurones.     

Respiratory control of hypoglossal motoneurones in the rat

In this study of adult and neonatal rats, we used cross-correlation analysis to detect synchronous neuronal events in hypoglossal and phrenic nerves to infer synaptic connections. We found evidence for the common excitation of medial and lateral hypoglossal motoneurones in 12 anaesthetized adult rats but not in 6 in vitro brainstem-spinal cord preparations. We did not find evidence for the common activation of phrenic and hypoglossal motoneurones in 23 adult and 10 neonatal rat preparations. We confirmed this negative result by demonstrating that 26 medullary inspiratory neurones activating phrenic motoneurones did not activate hypoglossal motoneurones in 23 adult decerebrate rats (except in one case). We also found that 15 B?tzinger expiratory neurones inhibiting phrenic motoneurones did not inhibit hypoglossal motoneurones. We conclude that: (1) motoneurones of the medial and lateral hypoglossal nerve branches receive inspiratory drive from a common premotor population in adult rats, but in neonatal rats adjacent nerve rootlets do not; (2) in both adult and neonatal rats phrenic premotor neurones do not monosynaptically excite hypoglossal motoneurones; (3) B?tzinger expiratory neurones that inhibit phrenic motoneurones do not inhibit hypoglossal motoneurones. We therefore suggest that the respiratory control of hypoglossal motoneurones is separate from that of phrenic motoneurones.     

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     

Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in vivo

Brainstem respiratory neurons innervate the hypoglossal motor nucleus which in turn transmits this respiratory drive signal to the genioglossus muscle of the tongue. The mechanism of this transmission is important to help maintain an open airspace for effective breathing, and is thought to rely almost exclusively on non-N-methyl-d-aspartate (non-NMDA) glutamate receptor activation during respiration. However those studies were performed in slices of medulla from neonatal animals in vitro which may have led to an underestimation of the contribution of NMDA glutamate receptors that may normally operate in intact preparations. The current study tests the hypothesis that both NMDA and non-NMDA receptors contribute to respiratory drive transmission at the hypoglossal motor nucleus in vivo. Experiments were performed in urethane-anesthetized and tracheotomized adult Wistar rats in which vagus nerves were either intact or sectioned. In the presence of augmented genioglossus activity produced by vagotomy, microdialysis perfusion of either an NMDA receptor antagonist (D-2-amino-5-phosphonovaleric acid, 0.001-10 mM) or a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione disodium salt, 0.001-1 mM) to the hypoglossal motor nucleus reduced respiratory-related genioglossus activity in a dose-dependent manner (P < 0.001) indicating that both NMDA and non-NMDA glutamate receptors are necessary for transmission of the respiratory drive signal to genioglossus muscle in vivo. Similar effects were observed in the vagus nerve intact rats. Further experiments demonstrated that each delivered antagonist had effects that were specific to its respective receptor. Regression analysis also revealed that the activity of both NMDA and non-NMDA receptors at the hypoglossal motor nucleus is related to levels of the prevailing respiratory drive. These results show that both NMDA and non-NMDA glutamate receptors at the hypoglossal motor nucleus are involved in transmission of the respiratory drive signal to genioglossus muscle in vivo.     

Pneumotaxic mechanisms in the non-human primate: effect of the N-methyl-D-aspartate (NMDA) antagonist ketamine

We tested the possible involvement of N-methyl-D-aspartate (NMDA) receptors in the central inspiratory-termination mechanism in non-human primates. Inspiratory bursts were recorded from the phrenic nerve in Macaca fascicularis monkeys paralyzed and ventilated by means of a servoventilator driven by the inspiratory discharge of the phrenic nerve. The central inspiratory termination mechanism was tested by withholding lung inflation. This transiently suppressed the vagal feedback from the lungs which produces inspiratory off-switching independent from the central mechanism. Under anaesthesia with ketamine, a potent NMDA antagonist, non inflation increased inspiratory time to 4s (1s with lungs inflated) whereas no such effect was observed during halothane anaesthesia. We conclude that the termination of inspiration in primates is controlled via central mechanisms in which NMDA receptors are involved.