Inspiratory Off-switch Research Articles

Distributed latent dynamics underlie breathing control in the brainstem during normal breathing, OIRD, and gasping

The neural control of breathing involves the coordinated activity of diverse populations of medullary neurons distributed along the rostrocaudal aspect of the ventrolateral medulla. Here, we use high‐density electrophysiological recordings (Neuropixels) to record spiking activity simultaneously from hundreds of neurons across the ventral respiratory column (VRC, including the Bötzinger and pre‐Bötzinger complexes) in anesthetized, freely breathing mice. Across sequential recordings, we have recorded from over 13,000 medullary neurons. We couple this with an optogenetic tagging approach to identify ChAT, Dbx1, Vgat, and Vglut2 positive neurons; and reconstruct the three dimensional location of each recorded neuron. We find diverse patterns of respiratory‐related neural activity throughout the medulla that tile the breathing phase such that discrete functional cell classes cannot be quantitatively defined. Moreover, while certain genotypes are biased towards particular anatomical locations or functional activity patterns, cells active during all parts of the respiratory phase are found across the VRC, and across all genotypes. We use dimensionality reduction techniques to show that the neural populations governing breathing follow rotational trajectories on low dimensional neural manifolds. Analyses of these trajectories indicate that the termination of inspiration (“inspiratory‐off switch”) may represent an attractor in the low‐dimensional neural space. Further, during opioid induced respiratory depression (OIRD), the network reconfigures, but the low‐dimensional trajectories of the neural population are preserved. Thus, patterns of neural coactivity remain the same, while breathing is slowed. Finally, we expose animals to hypoxia which reconfigures the respiratory network into gasping. The low‐dimensional neural activity no longer follows rotational trajectories. Instead, gasping is a ballistic, non‐rotational dynamic mode strikingly different than eupnea or OIRD. The transition to gasping is represented in the low dimensional trajectories by a gradual sequestering of the neural activity away from normal eupneic rotations. Together these data suggest that the VRC functions as a distributed and well‐coordinated population. The eupneic cycle begins with the inspiratory off‐switch and is maintained by the reciprocal interactions between inspiratory and expiratory neurons. This reciprocity is lost as the network transitions into gasping, a rhythm governed by fundamentally different mechanisms.

Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo.

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4–5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

Volumetric mapping of the functional neuroanatomy of the brainstem respiratory network in the perfused brainstem preparation of rats

One of the major goals of modern neuroscience is to understand the relationship between the functional neuroanatomy/connectivity of neural circuits and the behavior of an animal. The key challenge to addressing this goal with respect to control of breathing is the distributed nature of the respiratory network. While in cortical networks, short‐range connections (10–100 μm) out‐number long‐range connections (1+ mm), the opposite may be true of brainstem circuits. Specifically, anatomical tracing studies have identified many long‐range projections between brainstem respiratory nuclei that led to the concept that the brainstem respiratory network is compartmentally organized. Thus, measuring the functional neuroanatomy of the respiratory network—a task required to phenotype the functional role of neuronal populations identified in molecular mapping studies—requires monitoring the activity of respiratory neurons which may be spread over many millimeters.We hypothesized that the spatio‐temporal structure of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network in single preparations. To address our hypothesis, we developed an approach using silicon multi‐electrode arrays to record spontaneous LFPs from hundreds of electrode sites across the ponto‐medullary volume of the respiratory network while monitoring the respiratory motor pattern on phrenic and vagal nerves.Our results revealed the expression of rLFPs across the brainstem respiratory network. rLFPs were expressed selectively at the three transitions between respiratory phases: (1) from late‐expiration (E2) to inspiration (I), (2) from I to post‐inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions, rather than being equally distributed across the respiratory cycle. Spatially, the E2‐I (inspiratory on‐switch), and PI‐E2 transitions were localized to the ventral and dorsal respiratory groups, respectively, whereas the I‐PI (inspiratory off‐switch) transition was distributed across the ventral, dorsal and pontine respiratory groups. An independent component analysis (ICA) confirmed this spatio‐temporal organisation of rLFPs and identified a traveling wave of rLFPs that occurred at the I‐PI transition. Finally, a group‐wise ICA demonstrated that all preparations exhibited rLFPs with a similar temporal structure.Overall, under intact network conditions, our results confirm that inspiration is initiated by the pre‐Bötzinger complex, whereas post‐inspiration and late‐expiration depend on activity throughout the brainstem respiratory network. In conclusion, we have developed a general approach to volumetrically map spontaneous‐ or evoked‐respiratory network activity at the brainstem‐wide scale in single preparations to inform our understanding of the network mechanisms underlying the neural control of breathing.Support or Funding InformationThis work was supported by grants from the Australian Research Council (to MD), National Institutes of Health (to TED) and the Hartwell Foundation (to RFG).

Chapter 12 - Bidirectional Plasticity of Pontine Pneumotaxic Postinspiratory Drive: Implication for a Pontomedullary Respiratory Central Pattern Generator

The "pneumotaxic center" in the rostral dorsolateral pons as delineated by Lumsden nine decades ago is known to play an important role in promoting the inspiratory off-switch (IOS) for inspiratory-expiratory phase transition as a fail-safe mechanism for preventing apneusis in the absence of vagal input. Traditionally, the pontine pneumotaxic mechanism has been thought to contribute a tonic descending input that lowers the IOS threshold in medullary respiratory central pattern generator (rCPG) circuits, but otherwise does not constitute part of the rCPG. Recent evidence indicates that descending input from the Kölliker-Fuse nucleus (KFN) within the pneumotaxic center is essential for gating the postinspiratory phase of the three-phase respiratory rhythm to control the IOS in vagotomized animals. A critical question arising is whether such a descending pneumotaxic input from KFN that drives postinspiratory activity is tonic (null hypothesis) or rhythmic with postinspiratory phase modulation (alternative hypothesis). Here, we show that multifarious evidence reported in the literature collectively indicates that the descending pneumotaxic input may exhibit NMDA receptor-dependent short-term plasticity in the form of a biphasic neural differentiator that bidirectionally and phase-selectively modulates postinspiratory phase duration in response to vagal and peripheral chemoreceptor inputs independent of the responses in inspiratory and late-expiratory activities. The phase-selectivity property of the descending pneumotaxic input implicates a population of pontine early-expiratory (postinspiratory/expiratory-decrementing) neurons as the most likely neural correlate of the pneumotaxic mechanism that drives post-I activity, suggesting that the pontine pneumotaxic mechanism may be an integral part of a pontomedullary rCPG that underlies the three-phase respiratory rhythm.

M3-receptor activation counteracts opioid-mediated apneusis, but the apneusis per se is not necessarily related to an impaired M3 mechanism in rats

Aims Morphine slows the respiratory cycle due to a predominant prolongation of inspiration (apneusis) by postponing the spontaneous termination of inspiration (inspiratory off-switching). The present study investigates whether the morphine-induced apneusis results from impairment of cholinergic mechanisms in the central respiratory network. Main methods The efferent discharge was recorded from the phrenic nerve in artificially ventilated and anesthetized rats with vagotomy. All drugs were injected intravenously. Key findings The phrenic nerve displayed an augmenting discharge during inspiration and arrest of discharge during expiration in normal condition. Administration of morphine (0.3–10.0 mg/kg) dose-dependently provoked apneusis characterized by a long-lasting, plateau inspiratory discharge of the phrenic nerve. It shortened the expiratory duration. Subsequent administration of physostigmine (0.1 mg/kg) restored the morphine-induced apneusis to eupnea with a partial recovery of the augmenting inspiratory discharge. This modification of physostigmine was blocked by a non-specific muscarinic antagonist scopolamine (3.0 mg/kg), leading to re-prolongation of inspiration. A similar antagonism was affected by an antagonist of M3 cholinergic receptors, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 1.0 and 10.0 mg/kg) but not by an antagonist of M1 cholinergic receptors, pirenzepine (1.0 and 10.0 mg/kg). Significance These results demonstrate that the activation of endogenous M3 cholinergic mechanisms counteracts the morphine-induced apneusis.

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.

Neurokinin-1 Receptors Modulate the Excitability of Expiratory Neurons in the Ventral Respiratory Group

We studied the role of neurokinin-1 receptors (NK1-R) on the excitability of expiratory (E) neurons (tonic discharge, E(TONIC); augmenting, E(AUG); decrementing, E(DEC)) throughout the ventral respiratory group, including Bötzinger Complex (BötC) using extracellular single-unit recording combined with pressurized picoejection in decerebrate, arterially perfused juvenile rats. Responses evoked by picoejection of the NK1-R agonist, [Sar9-Met(O2)11]-substance P (SSP) were determined before and after the selective NK1-R antagonist, CP99,994. SSP excited 20 of 35 expiratory neurons by increasing the number of action potentials per burst (+33.7 +/- 6.5% of control), burst duration (+20.6 +/- 7.9% of control), and peak firing frequency (+16.2 +/- 4.8% of control; means +/- SE). Pretreatment with CP99,994 completely blocked SSP-evoked excitation in a subset of neurons tested, supporting the notion that SSP excitation was mediated through NK1-R activation. Because we had previously shown that E(AUG) neurons were crucial to locomotor-respiratory coupling (LRC), we reasoned that blockade of NK1-R would alter LRC by preventing somatic-evoked excitation of E(AUG) neurons. Blockade of NK1-Rs by CP99,994 in the BötC severely disrupted LRC and prevented somatic-evoked excitation of E(AUG) neurons. These findings demonstrate that LRC is dependent on endogenous SP release acting via NK1-Rs on E(AUG) neurons of the BötC. Taken together with our earlier finding that inspiratory off-switching by the Hering-Breuer Reflex requires endogenous activation of NK1-Rs through activation of NK1-Rs on E(DEC) neurons, we suggest that endogenous release of substance P in the BötC provides a reflex pathway-dependent mechanism to selectively modulate respiratory rhythm.

Role in the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons

Neurons of the pontine respiratory group (PRG) in the region of the nucleus parabrachialis medialis and the Kolliker-Fuse nucleus are believed to play an important role in promoting the inspiratory (I) off-switch (IOS). In decerebrate gallamine-paralyzed cats ventilated with a cycle-triggered pump system (lung inflation during the neural I phase), we studied the effects of vagal (V) afferent inputs on firing of I-modulated neurons (the most numerous population in PRG) and on I duration. The predominant V effect on unit activity was inhibitory, as shown by two types of test: (a) withholding of inflation during an I phase, which produced increase of unit firing and of its respiratory modulation (58/66 neurons in 14 cats), indicating disinhibition due to removal of phasic V input; (b) delivery of afferent V stimulus trains during a no-inflation I phase, which produced decrease of unit firing and of its respiratory modulation (20 neurons). In addition, application of V input during the neural expiratory (E) phase, which lengthened E phase duration, produced no effect on the neurons' firing, suggesting that the inhibition during I was presynaptic in origin. The results may be interpreted by the hypothesis that the medullary late-I presumptive IOS neurons receive excitatory inputs from the PRG I-modulated neurons as well as from V afferents.. With relatively strong V input, this pontine excitatory output is reduced by inhibition, whereas with relatively weak V input that excitatory output is increased due to reduction of inhibition. Thus the pontine and the V influences on the IOS can operate in a complementary manner dependent on state.

Synaptic mechanisms of inspiratory off-switching evoked by pontine pneumotaxic stimulation in cats.

To elucidate synaptic mechanisms and the involvement of N-methyl-D-aspartate (NMDA) receptors in inspiratory off-switching (IOS) evoked by the stimulation of the nucleus parabrachialis medialis (NPBM), excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were recorded from bulbar augmenting inspiratory (aug-I) and postinspiratory (PI) neurons in vagotomized cats. Stimulation of NPBM produced either transient inhibition or premature termination of inspiration (reversible or irreversible IOS), depending on the stimulus intensity. Each neuron displayed four-phasic postsynaptic responses during the reversible IOS, i.e. Phase 1 EPSPs, Phase 2 IPSPs, Phase 3 EPSPs and Phase 4 IPSPs in aug-I neurons, and Phase 1 plus 2 EPSPs, Phase 3 IPSPs and Phase 4 EPSPs in PI neurons. During the irreversible IOS, Phase 4 responses were replaced by sustained hyperpolarization in aug-I neurons and decrementing depolarization in PI neurons. Blockade of NMDA receptors by dizocilpine (0.3 mg kg(-1) i.v.) selectively increased Phase 4 potentials in both types of neurons and decreased the thresholds for evoking the irreversible IOS. The NPBM-induced responses had a pattern and time-course similar to those induced by vagal stimulation. The present results suggest that pneumotaxic and vagal inputs converge on the common IOS circuit, and the effectiveness of both inputs is modulated by NMDA receptors.

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.

Role of nucleus parabrachialis complex in inspiratory off-switch studied in isolated brainstem-spinal cord preparation from neonatal rat

The nucleus parabrachialis complex (NPB) of the pons is known as a respiratory modulating center. The NPB and the medullary respiratory center are mutually interconnected and vagus nerve afferents from pulmonary stretch receptors project to the NPB [1]. There is much evidence in in vivo preparations from adult mammals [2] that the NPB plays a crucial in the inspiratory off-switch. In the present study, we examined how the NPB participates in the inspiratory off-switch using brainstem-spinal cord preparations obtained from 0–4 days old rats. We hemi-sectioned the pons keeping one side intact. First, we examined the effects of NPB electrical stimulation on C4 ventral nerve inspiratory activity. The electrical stimulation (0.1 ms duration, 1–5 trains, 20 ms interval) applied at various times (100–300 ms after the onset of C4 inspiratory activity) induced a transient depression or termination in C4 inspiratory activity. This depression was stronger when the stimulation was applied near the end of inspiratory activity but less conspicuous when it was applied at around the peak of inspiratory activity. Thus, activation of the NPB neuronal elements could produce termination of inspiratory activity in this in vitro preparation similar to the in vivo preparation. Previous in vivo experiments in adult mammals suggested that an NMDA receptor- mediated off-switch mechanism is present in the inspiratory termination [3]. Then, we examined whether a similar mechanism is involved in the above-observed inspiratory off-switch in the in vitro preparation. It was shown that the inhibition of C4 inspiratory activity induced by the NPB stimulation was greatly reduced by perfusion of NMDA antagonists (MK-801, APV) and that the inhibition was blocked by perfusion of a GABAA-antagonist (Bicuculline). To determine the location of NMDA receptors responsible for this inhibition by the NPB stimulation, an NMDA-antagonist MK-801 (1 mM) was microinjected into the NPB using a glass capillary placed in the vicinity of the stimulation electrode. The injection of MK-801 reduced the inhibition of C4 inspiratory activity by the NPB stimulation, indicating that NMDA receptor-mediated processes take place within the NPB. Since neurons and/or neuron networks responsible for inspiratory off-switch are expected to exist in the NPB, we tried to find respiratory neurons and analyzed their distribution and firing patterns. Neurons were recorded from the cut-surface of the intact half of the pons using whole cell patch clamp method. We found several types of respiratory neurons: inspiratory, tonic inspiratory, expiratory and inspiratory-expiratory (I-E) neurons. The population of I-E neurons was the largest, being more than 50% of the recorded neurons. I-E neurons fired from middle of C4 inspiratory activity through the early expiratory phase and were inhibited at the early part of C4 inspiratory activity. Perfusion of MK-801 (10 M) decreased significantly the driving potential and burst duration of I-E neurons. In addition, this perfusion decreased also the amplitudes of EPSPs induced by vagal nerve stimulation in the I-E neurons. In conclusion, 1) the NPB is involved in the inspiratory off-switch in in vitro preparations, 2) NMDA receptors within the NPB mediate, at least partly, the NPB-related inspiratory off-switch, and 3) a relatively large population of I-E neurons of the NPB could be responsible for the NPB-related inspiratory termination.

Role of brainstem respiratory neuron types in phase-switching produced by afferent vagal stimulation.

A prominent feature of respiratory neural activity is the abrupt change of discharge at the phase transitions (IOS, inspiratory off-switch; EOS, expiratory off-switch). The observation of this nonlinearity has led to the search for specialized populations of brainstem neurons which change their activity immediately preceding a phase-switch. In this laboratory, we have used the method of producing perturbations of the respiratory cycle by means of phase-linked inputs and then comparing the temporal changes in the output (phrenic discharge) and in firing of different types of brainstem respiratory neurons. We here report the use of vagal (V) afferent inputs to study such responses.

Respiratory responses to pH of neonatal hamster and mouse in vitro compared with that of opossum

In the isolated CNS from neonatal opossum Monodelphis domestica, low pH superfusion increases both respiratory rate and amplitude. One unusual control mechanism in the isolated Monodelphis CNS is that the frequency of respiration depends primarily on changes in the duration of the expiratory rather than the inspiratory phase [1]. To reveal whether this is a characteristic common to other species, we analyzed in isolated hamster and mouse CNS the respiratory responses to pH in terms of variables for amplitude and for timing. Newborn Golden hamsters and CF1 mice (0-5 days old) were anesthetized with ether and cooled on ice. Their CNS were removed and decerebrated by pontobulbar transections. A vaseline seal at the level of the spinal cord segment C1 allowed selective superfusion of the brainstem. Basal conditions were obtained during brainstem superfusion with a continuos flow of 0.8 ml · min-1 artificial cerebrospinal fluid (aCSF) gassed with 95% O2 and 5% CO2, at 20-24°C (pH7.35-7.40). Fictive respiration was recorded from C3-C5 ventral roots with suction electrodes. Acidification (pH7.3 or lower) of the brainstem superfusion was attained by reducing the aCSF's bicarbonate final concentration. Acidification of the brainstem superfusion (from pH7.4-7.3) increased the frequency and decreased the amplitude of respiration in both hamster and mouse (P < 0.01, Wilcoxon test). A common feature of these results is that changes in rate are brought about solely by changes in the duration of the expiratory phase. The inspiratory phase remains constant in duration. Thus, cycle duration can be represented by a linear function exclusively depending on the expiratory duration. The slope of the straight line is very close to 1. These results indicate that the timing of the respiratory cycle in the neonatal hamster and mouse, as well as in the opossum, are controlled by an expiratory instead of an inspiratory off-switch. Increase in respiratory amplitude in response to chemical stimulation is a distinctive feature of the isolated CNS preparation obtained from newborn opossum.

Specific subnuclei of the nucleus tractus solitarius play a role in determining the duration of inspiration in the rat

Our previous data obtained in the cat suggest that the neurons of the ventrolateral subnucleus of the tractus solitarius (vlNTS) act as an inspiratory off-switch and terminate the inspiratory phase of the respiratory cycle (Berger et al., Eur. J. Pharmacol. 277 (1995) 195–208; Gillis et al., Neurosci. Abstr. 23 (1997) 725). The purpose of the present study was to determine whether inhibition of the region of the vlNTS of the rat using drugs that hyperpolarize, disfacilitate or block both axonal conduction and action potential generation would alter the inspiratory phase of the respiratory cycle. Experiments were conducted in anesthetized, vagotomized and spontaneously breathing rats while monitoring diaphragmatic electromyogram activity. Vagus nerves were sectioned in order to rule out prolongation of inspiration evoked by microinjection of agents into the vlNTS which block excitatory drive from lung afferent inputs. Bilateral microinjection of the inhibitory amino acid γ-aminobutyric acid (GABA) 25 nmol/45 nl produced an immediate prolongation of inspiratory duration (484±18 to 1291±84 ms) and an apneustic pattern of breathing. Other effects observed were a significant shortening of expiratory duration (778±36 to 432±38 ms), rise in blood pressure (83±4 to 108±6 mmHg) and a small but significant increase in heart rate (439±17 to 452±18 beats/min). Bilateral microinjection of the ionotropic glutamate receptor antagonist kynurenic acid (1 nmol) and the Na + channel blocker tetrodotoxin (10 pmol) into the region of the vlNTS consistently produced a similar prolongation of inspiratory duration and an apneustic pattern of breathing. These results support the hypothesis that neurons in the region of the vlNTS promote the transition from inspiration to expiration and function as part of the ‘Inspiratory Off Switch’.

Inspiration-promoting vagal reflex under NMDA receptor blockade in anaesthetized rabbits.

1. This study describes a novel vagal respiratory reflex in anaesthetized rabbits. In contrast to the well-known inspiratory (I) off-switching by vagal afferent excitation, this vagal reflex initiates and maintains the central I activity of phrenic nerve discharges in rabbits pre-treated with antagonists of N-methyl-D-aspartate-type excitatory amino acid receptors (NMDA-Rs). 2. Under NMDA-R blockade with either dizocilpine (0.025-0.3 mg kg-1), D-2-amino-5-phosphonopentanoic acid (AP5, 0.5-1 mg, i.c.v.) or ketamine (10 mg kg-1), vagal stimulation at low frequencies (5-40 Hz) during the I phase prevented or markedly delayed the spontaneous I termination. In contrast, stimulation of the same vagal afferent at the same intensity but at a higher frequency (100-160 Hz) during the I phase immediately terminated the I phase. 3. In non-vagotomized rabbits, maintaining the tidal volume at end-expiratory levels during the I phase prevented spontaneous I termination and maintained apneusis after NMDA-R blockade with dizocilpine. 4. Brief stimulation of vagal afferents at low frequency (5-40 Hz) during the expiratory (E) phase constantly initiated phrenic I discharge after NMDA-R block. 5. We conclude that low-frequency discharge of vagal pulmonary stretch receptor afferents, as when lung volume is near functional residual capacity, promotes central I activity under NMDA-R blockade.

NMDA receptor-mediated inspiratory off-switching in pneumotaxic-disconnected cats

The pneumotaxic center is thought to govern inspiratory off-switching (IOS), and blockade of N-methyl- d-aspartate (NMDA) receptors by dizocilpine impairs IOS causing apneusis. The present study is to examine whether the NMDA receptor-mediated IOS mechanism functions in the medullary respiratory network after disconnecting the pneumotaxic center. In decerebrate and vagotomized cats, the nucleus parabrachialis medialis (NPBM) and vagus nerves were stimulated to evoke IOS and a dorsal pontine transection was performed while the central respiratory activity was recorded in phrenic neurogram. The transection eliminated the NPBM-stimulated IOS but not the vagally evoked IOS, and developed two types of respiration; eupnea in 12 and apneusis in six out of 18 cats. Apneustic respiration was not changed into eupneic one by changing the end-tidal CO 2 level. In animals displaying eupnea after the transection, dizocilpine (0.3 mg/kg i.v.) produced apneusis, characterized by a prolonged inspiration, a shortened stage 1 expiration and an unchanged stage 2 expiration. Dizocilpine caused no further change in the apneustic pattern induced by the transection. The present results suggest that the medullary respiratory network is able to generate a eupneic respiration after disconnecting the pontine pneumotaxic center, and the NMDA mechanism plays an important role in the medullary respiratory network.

Activity of rat pump neurons is modulated with central respiratory rhythm

The firing properties of the second-order neurons of the slowly-adapting pulmonary stretch receptors called pump neurons (P-cells), were studied in Nembutal-anesthetized, paralyzed, and artificially-ventilated rats. Extracellular recording was made from single P-cells which were monosynaptically activated by electrical stimulation of the vagal afferents and fired in phase with lung inflations. In the majority of the P-cells examined (49/52), the firing was suppressed to various extents in the inspiratory phase: in effect, their firing was accentuated at the time of the inspiratory off-switch. This issue has never been reported in cat P-cells, on which relatively rich data have been accumulated. The present results suggest that rat P-cells do not only relay information from the lung stretch receptors but also integrate the central respiratory inputs.

Synaptic potentials in respiratory neurones during evoked phase switching after NMDA receptor blockade in the cat.

1. Blockade of NMDA receptors by dizocilpine impairs the inspiratory off-switch (IOS) of central origin but not the IOS evoked by stimulation of sensory afferents. To investigate whether this difference was due to the effects of different patterns of synaptic interactions on respiratory neurones, we stimulated electrically the superior laryngeal nerve (SLN) or vagus nerve in decerebrate cats before and after i.v. administration of dizocilpine, whilst recording intracellularly. 2. Phrenic nerve responses to ipsilateral SLN or vagal stimulation were: at mid-inspiration, a transient inhibition often followed by a brief burst of activity; at late inspiration, an IOS; and at mid-expiration, a late burst of activity. 3. In all neurones (n = 16), SLN stimulation at mid-inspiration evoked an early EPSP during phase 1 (latency to the arrest of phrenic nerve activity), followed by an IPSP in inspiratory (I) neurones (n = 8) and by a wave of EPSPs in post-inspiratory (PI) neurones (n = 8) during phase 2 (inhibition of phrenic activity). An EPSP in I neurones and an IPSP in PI neurones occurred during phase 3 (brief phrenic burst) following phase 2. 4. Evoked IOS was associated with a fast (phase 1) activation of PI neurones, whereas during spontaneous IOS, a progressive (30-50 ms) depolarization of PI neurones preceded the arrest of phrenic activity. 5. Phase 3 PSPs were similar to those occurring during the burst of activity seen at the start of spontaneous inspiration. 6. Dizocilpine did not suppress the evoked phrenic inhibition and the late burst of activity. The shapes and timing of the evoked PSPs and the changes in membrane potential in I and PI neurones during the phase transition were not altered. 7. We hypothesize that afferent sensory pathways not requiring NMDA receptors (1) terminate inspiration through a premature activation of PI neurones, and (2) evoke a late burst of phrenic activity which might be the first stage of the inspiratory on-switch.

Involvement of the motor trigeminal nucleus in respiratory phase-switching in the cat.

We investigated the hypothesis that the motor trigeminal nucleus, consisting of expiratory motoneurons, might be influential in termination of inspiration. We addressed the issue by comparing the effects on neural respiration of a reversible, unilateral, pharmacologic blockade of the motor trigeminal nucleus (5M), the medial parabrachial nucleus (PB), and of other nearby structures that are neutral for respiration in anesthetized, vagotomized, paralyzed, and ventilated cats. The blockade was achieved by microinjections of 2% xylocaine, laced with Pontamine Sky Blue to identify sites of injections, from the tip of a penetrating microelectrode. Integrated phrenic neurograms were recorded to quantify the time of neural inspiration (TI), expiration (TE), and the peak phrenic amplitude. We found that blockade of the 5M caused a pattern of apneustic respiration, consisting of a selective prolongation of inspiratory phases that were interrupted by short expiratory pauses. In contrast, blockade of the PB resulted in a prolongation of both TI and TE, which corresponds to a mere slowing of respiration. The results confirmed important functions of the rostral pons in ventilatory control but pointed to the 5M rather than PB as a structure underlying the inspiratory off-switch. We conclude that the motor trigeminal nucleus may have a part in the pontine pneumotaxic mechanism.

P-376 - Effects of dizocilpine on the peripherally evoked inspiratory off-switching in decerebrate cats

P-376 - Effects of dizocilpine on the peripherally evoked inspiratory off-switching in decerebrate cats