Mari Okazaki

Neuropharmacology of control of respiratory rhythm and pattern in mature mammals

This review summarizes the current understanding of the neurotransmitters and neuromodulators that are involved, firstly, in respiratory rhythm and pattern generation, where glutamate plays an essential role in the excitatory mechanisms and glycine and gamma-aminobutyric acid mediate inhibitory postsynaptic effects, and secondly, in the transmission of input signals from the central and peripheral chemoreceptors and of motor outputs to respiratory motor neurons. Finally, neuronal mechanisms underlying respiratory modulations caused by respiratory depressants and excitants, such as general anesthetics, benzodiazepines, opioids, and cholinergic agents, are described.

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.

Glutamic acid decarboxylase-immunoreactivity of bulbar respiratory neurons identified by intracellular recording and labeling in rats.

To distinguish the GABAergic neuron in the ventral respiratory group (VRG) of rats, immunohistochemical staining of glutamic acid decarboxylase (GAD) was performed in neurons that had been individually identified by in vivo intracellular recording and labeling with neurobiotin. A total of five types of respiratory neurons were identified and labeled; augmenting inspiratory (aug-I, n=12), decrementing or early inspiratory (early-I, n=3), inspiration-expiration phase spanning or late inspiratory (late-I, n=3), decrementing expiratory or postinspiratory (PI, n=8), and augmenting or stage 2 expiratory (E2, n=3). In addition, expiration-inspiration phase-spanning or pre-inspiratory neurons (pre-I, n=2) were recorded, but not labeled. The membrane potential trajectory of each neuron type resembled that previously described in cat, suggesting a comparable neuronal organization between the two species. According to the axonal arborization, those labeled neurons were further classified as propriobulbar (6 aug-I, all early-I, all late-I, and 3 PI), bulbospinal (2 aug-I and all E2) and cranial-motor neurons (4 aug-I and 5 PI). GAD-immunoreactivity was consistently detected in the propriobulbar neurons, while it was not seen in cranial-motor and bulbospinal neurons. In addition, GAD-immunoreactive varicosities were found surrounding the somatic and dendritic surface of all labeled neurons. The present results illustrate that the propriobulbar types of early-I, aug-I, late-I and PI neurons are GABAergic inhibitory neurons and virtually all types of respiratory neurons receive GABAergic inputs in the rats VRG.

Biphasic effects of morphine on bulbar respiratory neuronal activities in decerebrate cats

To understand neuronal mechanisms underlying respiratory depression induced by morphine, membrane potential, input resistance and burst discharge in different types of respiratory neurons were recorded in decerebrate and vagotomized cats. Intravenous morphine (0.3-3.0 mg/kg) dose-dependently decreased the respiratory discharge in the phrenic and iliohypogastric nerves. The drug changed the respiratory frequency in a biphasic fashion, a transient increase (early phase) followed by a long-lasting decrease (late phase). During the early phase, the membrane was hyperpolarized throughout the respiratory cycle and the burst discharge was decreased in all types of respiratory neurons. During the late phase, the active phase depolarization and the inactive phase hyperpolarization were decreased, resulting in a decline of membrane potential fluctuations. Input resistance was decreased during the early phase and increased during the late phase. Iontophoresed (50-100 nA) morphine produced hyperpolarization of the membrane and a decrease in input resistance in respiratory neurons. This hyperpolarization remained unaltered after iontophoresed tetrodotoxin depressed the synaptic transmission. These effects of morphine were completely blocked by naloxone and beta-funaltrexamine, but not by naltrindole. The present results suggest that morphine depresses the respiratory neuronal activity through two different mechanisms, both of which are mediated by mu receptors.

GABAA receptor-mediated inspiratory termination evoked by vagal stimulation in decerebrate cats

To identify the GABAergic inhibitory mechanisms involved in inspiratory termination or off-switching (IOS), the effects of a specific enhancer of GABA(A) receptors, midazolam, and an antagonist, bicuculline, on vagally evoked inspiratory inhibitions and IOS were investigated in decerebrate cats. Stimulation of vagal afferents at late inspiration provoked either reversible inspiratory inhibition or IOS, depending on the stimulus intensity. Each response occurred at a constant latency (phase 1). The reversible response was triphasic, consisting of an early (phase 2) inhibition, a brief (phase 3) excitation and a late (phase 4) inhibition in the phrenic neurogram, and early (phase 2) IPSPs, brief (phase 3) EPSPs and late (phase 4) IPSPs in bulbar inspiratory (I) neurones. With an increasing stimulus intensity, phase 4 inhibitions were increased in amplitude and duration, leading to IOS. Midazolam (0.1 mg/kg i.v.) increased more selectively phase 4 IPSPs than phase 2 IPSPs in I neurones, and decreased the threshold for evoking IOS by producing an earlier and larger phase 4 IPSPs. Bicuculline (1.0 mg/kg i.v.) had an opposite effect. These results suggest that the late inhibitory response evoked by vagal stimulation in the I neuronal pool organizes an initial phase of IOS which is mediated by GABA(A) receptors.

Immunoreactivity for glutamic acid decarboxylase and N-methyl-D-aspartate receptors of intracellularly labeled respiratory neurons in the cat

In adult cats, immunofluorescence images of glutamic acid decarboxylase (GAD) and N-methyl-D-aspartate (NMDA) receptors were achieved in the ventral respiratory group (VRG) neurons, which had been individually identified by in vivo intracellular recording and labeling with neurobiotin. Among augmenting inspiratory (aug-I), postinspiratory (post-I), and augmenting expiratory (aug-E) neurons labeled, GAD-immunoreactivity was demonstrated only in those neurons that were not antidromically activated (NAA) by stimulation of the vagus nerve and the C2-C3 spinal cord. Substantial immunoreactivity for NMDA receptors was presented in virtually all types of neurons, but lesser reactivity in aug-E bulbospinal neurons. These results suggest that the aug-I, post-I, and aug-E types of NAA neurons are gamma-aminobutyric acid (GABA)ergic and that NMDA receptors distribute in lesser degree in aug-E bulbospinal neurons than in other types of VRG neurons.

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 CO2 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.

Mutual dependence of calcitonin-gene related peptide and acetylcholine release in neuromuscular preparations

To investigate the mutual dependence of calcitonin gene-related peptide (CGRP) and acetylcholine release, we examined the effect of a cholinesterase inhibitor neostigmine on the release of CGRP-like immunoreactivity in rat phrenic nerve-hemidiaphragm muscle preparation, and conversely, the effect of CGRP on [3H]acetylcholine release from motor nerve terminals loaded with [3H]choline in the same preparations of mice. Release of CGRP-like immunoreactivity was increased by electrical nerve stimulation (train of 40 pulses of 200 micros pulse duration and frequency of 50 Hz applied every 10 s) in the whole preparation but not in the segmental preparation containing the endplate region. Neostigmine (0.1-0.3 microM) enhanced the resting release of CGRP-like immunoreactivity in a concentration-dependent manner, whereas it depressed the nerve-evoked release of CGRP-like immunoreactivity. CGRP (1 microM) added to perfusate decreased nerve-evoked [3H]acetylcholine release. These results suggest that CGRP, which is released by electrical nerve stimulation or a cholinesterase inhibitor in intact skeletal muscles, negatively modulates nerve-evoked acetylcholine release.

Hypercapnic and hypoxic ventilatory responses in long-term streptozotocin-diabetic rats during conscious and pentobarbital-induced anesthetic states

To clarify the diabetes mellitus (DM)-associated changes in the respiratory neuronal control system, acute ventilatory responses to progressively increasing hypercapnia (6%) and hypoxia (10%) were compared between normal (N) and streptozotocin (60 mg/kg, i.v.) -DM rats for a long period up to 28 weeks. The same comparison was conducted during the anesthetic state induced with pentobarbital (35 mg/kg, i.p.). During the conscious state, basic ventilatory parameters, such as respiratory rate, tidal volume and minute ventilation, were not impaired in DM rats, but ventilatory responses to hypercapnia and hypoxia were reduced significantly at 16 weeks and later after streptozotocin injection. The reduced responses in DM rats were not recovered by insulin treatment (5-6 U/body, s.c., daily). During the anesthetic state, both hypoxic and hypercapnic responses were depressed more intensely in N rats than in DM rats, resulting in an equivalent level of the response in the two groups. The present study demonstrated that ventilatory responses to hypercapnia and hypoxia were reduced in a long-term DM condition. This may be derived from the impairment of the peripheral and central chemosensitivity. The reduction in ventilatory responses was exaggerated during the anesthetic state.

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.