Makito Iizuka

Influence of hypercapnic acidosis and hypoxia on abdominal expiratory nerve activity in the rat

We studied the influence of hypercapnic acidosis and hypoxia on the neural drive to abdominal muscles in anesthetized and decerebrate rats; this information is unavailable despite widespread use of the rat as an experimental model in respiratory physiology and neurobiology. To minimize confounding influences from receptors in the lungs and chest wall, the animals were vagotomized, paralyzed and mechanically ventilated, and electrical activity was recorded from abdominal muscle nerves. In anesthetized and decerebrate rats, both stimuli evoked steady, low amplitude expiratory discharge that persisted throughout the expiratory phase (E-all activity), but was inhibited during inspiration. We also observed late expiratory, high-amplitude bursts (E2 activity) superimposed on this steady activity, but only at the highest levels of respiratory drive. Hypoxia enhanced abdominal motor activity transiently, whereas hypercapnic acidosis caused a sustained increase in activity. Thus, both hypercapnic acidosis and hypoxia activate abdominal muscle motoneurons in the absence of phasic afferent inputs.

Development of the spatial pattern of 5-HT-induced locomotor rhythm in the lumbar spinal cord of rat fetuses in vitro

Developmental changes in the 5-hydroxytryptamine (5-HT)-induced locomotor rhythm were examined in isolated spinal cord preparations taken from fetal rats at embryonic day (E) 16.5, E18.5 and E20.5. Motor activity was recorded from L2/L3 and L5 ventral roots. Bath application of 5-HT evoked rhythmic bursts that were synchronized in all ventral roots studied at E16.5. At E18.5, 5-HT evoked rhythmic bursts that alternated between the two sides, while the bursts in the L2/L3 ventral root were synchronous with those in the ipsilateral L5 ventral root. At E20.5, 5-HT evoked alternate rhythmic bursts in L2/L3 and L5 ventral roots, representing activity in flexors and extensors, respectively. In the presence of strychnine, 5-HT induced rhythmic bursts that were synchronized in all ventral roots studied at E18.5 and E20.5, suggesting that the change in the pattern of rhythmic motor activity that occurs with age is due to the development of glycine-mediated inhibition.

Intercostal expiratory activity in an in vitro brainstem-spinal cord-rib preparation from the neonatal rat.

1 We examined whether expiratory activity can be observed when central chemoreceptors are activated by a decrement in the extracellular pH in an isolated brainstem‐spinal cord‐rib preparation from 0‐ to 3‐day‐old rats. Expiratory activity was defined as the burst activity that occurs in an internal intercostal muscle (IIM) during the silent period between the periodic inspiratory bursts in the C4 ventral root (which contains phrenic motor axons). 2 During perfusion with modified Krebs solution (26 mm HCO3−, 5% CO2, pH 7.4), there was no consistent activity in IIM, though rhythmic inspiratory motor activity always appeared in the C4 ventral root. 3 When the pH of the perfusate was lowered from about 7.4 to 7.1 by reducing [HCO3−] from 26 to 10 mm, the frequency of the C4 inspiratory rhythm increased, and rhythmic activity appeared in IIM. In most cases, the rhythmic burst in IIM started just after the cessation of the C4 inspiratory burst and coincided with movement of the ribs in a caudal direction. This intercostal expiratory burst was limited to the first half of the expiratory phase. 4 The coordinated reciprocal motor activity between the C4 ventral root and IIM changed to a largely overlapping pattern when strychnine (5–10 μm), a glycine receptor antagonist, was added to the perfusate. 5 These results suggest (i) that the neuronal mechanisms responsible for expiratory motor activity are preserved in this in vitro preparation and (ii) that the glycinergic inhibitory system plays an important role in the coordination between inspiratory and expiratory motor activity during respiration.

GABAA receptors mediate postnatal depression of respiratory frequency by barbiturates

We tested the hypothesis that barbiturates depress respiratory motor output by actions on the GABAA receptor. We examined the influence of pentobarbital sodium on nerve activity recorded from a fourth cervical (C4) ventral root (phrenic motoneuron output) in the in vitro brainstem-spinal cord preparation of neonatal rats aged 1-3 days. Bath application of pentobarbital slowed the respiratory rhythm but this effect could be reversed by drug washout or by simultaneous application of 8 microM bicuculline methiodide, a GABAA receptor antagonist. Pentobarbital up to a concentration of 80 microM (or 20 mg/l) did not change the magnitude of C4 nerve bursts. The GABAA receptor agonist muscimol evoked similar changes. The results support the hypothesis that respiratory depression by barbiturates is due to GABAA receptor-mediated inhibition, with the principal effects on rhythm generation. In the light of recent studies suggesting that GABAA receptors may be excitatory in the early neonatal period, we examined postnatal changes in the GABAergic slowing of respiratory rhythm. Stimulation of GABAA receptors slowed respiratory rhythm from the first postnatal day, with no change in efficacy over the first 3 days of life.

Respiration-related control of abdominal motoneurons

The abdominal muscles form part of the expiratory pump in cooperation with the other expiratory muscles, primarily the internal intercostal and triangularis sterni muscles. The discharge of abdominal muscles is divided into four main patterns: augmenting, plateau, spindle and decrementing. The patterns tend to be species-specific and dependent on the state of the central nervous system. Recent studies suggest that the abdominal muscles are more active than classically thought, even under resting conditions. Expiratory bulbospinal neurons (EBSN) in the caudal ventral respiratory group are the final output pathway to abdominal motoneurons in the spinal cord. Electrophysiological and anatomical studies indicated the excitatory monosynaptic inputs from EBSN to the abdominal motoneurons, although inputs from the propriospinal neurons seemed to be necessary to produce useful motor outputs. Respiration-related sensory modulation of expiratory neurons by vagal afferents that monitor the rate of change of lung volume and the end-expiratory lung volume (EELV) play a crucial role in modulating the drive to the abdominal musculature. Studies using in vitro and in situ preparations of neonatal and juvenile rats show bi-phasic abdominal activity, characterized by bursting at the end of expiration, a silent period during the inspiratory period, and another burst that occurs abruptly after inspiratory termination. Since the abdominal muscles rarely show these post-inspiratory bursts in the adult rat, the organization of the expiratory output pathway must undergo significant development alterations.

Rostrocaudal distribution of spinal respiratory motor activity in an in vitro neonatal rat preparation

The distribution of inspiratory and expiratory activities among rib-cage muscles was examined using isolated brainstem-spinal cord-rib preparations from neonatal rats. Expiratory activity was evoked by decreasing perfusate pH from 7.4 to 7.1. All internal intercostal muscles (IIMs) in the first to eleventh intercostal spaces showed expiratory bursts. Although the IIMs in the more caudal interspaces exhibited expiratory bursts for as long as the low pH solution was present in all preparations, the expiratory bursts obtained from the IIMs in the rostral interspaces gradually disappeared even under low pH conditions in about half the preparations, suggesting that the more caudal IIMs play the greater role in expiration. All thoracic ventral roots examined from T1VR-T11VR, but not T13VR, exhibited overt inspiratory bursts under normal pH conditions. Low pH solution induced additional expiratory bursts in all thoracic VRs. The ratio of the integral of the absolute electrical voltage during the expiratory phase to that during the inspiratory phase increased progressively and significantly from the rostral to the caudal interspaces. These results accord well with previous ones in mammals in vivo. Hence, the neuronal mechanisms necessary for a rostrocaudal gradient in spinal respiratory motor outputs seem to be preserved in this in vitro preparation.

GABAA and glycine receptors in regulation of intercostal and abdominal expiratory activity in vitro in neonatal rat

The roles played by GABAA and glycine receptors in inspiratory‐expiratory motor co‐ordination and in tonic inhibitory regulation of expiratory motor activity were studied using brainstem‐spinal cord (‐rib) preparations from neonatal rats. Inspiratory activity was recorded from the C4 ventral root. Expiratory activity in internal intercostal muscle, internal oblique muscle or T13 ventral root was evoked by a decrease in perfusate pH from 7.4 to 7.1 (i.e. from normal to low pH conditions) and was limited to the first part of the expiratory phase. Under low pH conditions, bath application of 10 μm bicuculline, a GABAA receptor antagonist, caused the inspiratory burst to overlap the expiratory burst in 2/7 preparations. Overlapping of the expiratory burst with the inspiratory burst was observed in 7/7 preparations made under 10 μm bicuculline. Furthermore, such preparations exhibited expiratory bursts under bicuculline‐containing normal pH conditions. Local application of 10 μm bicuculline to the brainstem under normal pH conditions evoked expiratory bursts, some of which overlapped the inspiratory bursts. Picrotoxin, another antagonist of the GABAA receptor, had similar effects. Under normal pH conditions, application of strychnine (0.2‐ 2.0 μm; a glycine receptor antagonist) to the brainstem did not evoke expiratory bursts. On subsequent application of strychnine‐containing low pH solution, expiratory bursts were evoked and some (0.5 μm) or all (2.0 μm) of these overlapped the inspiratory burst. Simultaneous application of picrotoxin and strychnine to the brainstem evoked expiratory bursts that overlapped the inspiratory bursts and a subsequent decrease in perfusate pH to 7.1 increased the frequency of the respiratory rhythm. It was a characteristic finding that the duration of the expiratory burst exceeded that of the inspiratory burst under control low pH conditions. This remained true during concurrent blockade of GABAA and glycine receptors. The results suggest that in the in vitro preparation from neonatal rats: (1) GABAA and glycine receptors within the brainstem play important roles in the co‐ordination between inspiratory and expiratory motor activity, (2) tonic inhibition via GABAA receptors, but not glycine receptors, plays a role in the regulation of expiratory motor activity and (3) inspiratory and expiratory burst termination is independent of both GABAA and glycine receptors.

Methylxanthines do not affect rhythmogenic preBötC inspiratory network activity but impair bursting of preBötC-driven motoneurons.

Clinical stimulation of preterm infant breathing with methylxanthines like caffeine and theophylline can evoke seizures. It is unknown whether underlying neuronal hyperexcitability involves the rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) in the brainstem or preBötC-driven motor networks. Inspiratory-related preBötC interneuronal plus spinal (cervical/phrenic) or cranial hypoglossal (XII) motoneuronal bursting was studied in newborn rat en bloc brainstem-spinal cords and brainstem slices, respectively. Non-respiratory bursting perturbed inspiratory cervical nerve activity in en bloc models at >0.25mM theophylline or caffeine. Rhythm in the exposed preBötC of transected en bloc preparations was less perturbed by 10mM theophylline than cervical root bursting which was more affected than phrenic nerve activity. In the preBötC of slices, even 10mM methylxanthine did not evoke seizure-like bursting whereas >1mM masked XII rhythm via large amplitude 1-10Hz oscillations. Blocking A-type γ-aminobutyric (GABAA) receptors evoked seizure-like cervical activity whereas in slices neither XII nor preBötC rhythm was disrupted. Methylxanthines (2.5-10mM), but not blockade of adenosine receptors, phosphodiesterase-4 or the sarcoplasmatic/endoplasmatic reticulum ATPase countered inspiratory depression by muscimol-evoked GABAA receptor activation that was associated with a hyperpolarization and input resistance decrease silencing preBötC neurons in slices. The latter blockers did neither affect preBötC or cranial/spinal motor network bursting nor evoke seizure-like activity or mask corresponding methylxanthine-evoked discharges. Our findings show that methylxanthine-evoked hyperexcitability originates from motor networks, leaving preBötC activity largely unaffected, and suggest that GABAA receptors contribute to methylxanthine-evoked seizure-like perturbation of spinal motoneurons whereas non-respiratory XII motoneuron oscillations are of different origin.

Abdominal expiratory muscle activity in anesthetized vagotomized neonatal rats

The pattern of respiratory activity in abdominal muscles was studied in anesthetized, spontaneously breathing, vagotomized neonatal rats at postnatal days 0–3. Anesthesia (2.0% isoflurane, 50% O2) depressed breathing and resulted in hypercapnia. Under this condition, abdominal muscles showed discharge late in the expiratory phase (E2 activity) in most rats. As the depth of anesthesia decreased, the amplitude of discharges in the diaphragm and abdominal muscles increased. A small additional burst frequently occurred in abdominal muscles just after the termination of diaphragmatic inspiratory activity (E1 or postinspiratory activity). Since this E1 activity is not often observed in adult rats, the abdominal respiratory pattern likely changes during postnatal development. Anoxia-induced gasping after periodic expiratory activity without inspiratory activity, and in most rats, abdominal expiratory activity disappeared before terminal apnea. These results suggest that a biphasic abdominal motor pattern (a combination of E2 and E1 activity) is a characteristic of vagotomized neonatal rats during normal respiration.

Respiratory activity in glossopharyngeal, vagus and accessory nerves and pharyngeal constrictors in newborn rat in vitro

1 Previously, in a brainstem‐spinal cord‐rib preparation from neonatal rats we demonstrated that a decrement in extracellular pH (from about 7.4 to 7.1) caused expiratory activity in an internal intercostal muscle (IIM) during the first half of the expiratory phase (Ea). As the initial step in finding nerves or muscles firing during the second half of the expiratory phase (Eb), the patterns of activity in the glossopharyngeal, vagus and accessory nerves were examined in the present study. 2 Since the emerging motor rootlets of these three nerves (> 20; collected into about 10 bundles before the jugular foramen) are distributed in a continuous fashion from rostral to caudal levels of the brainstem, visual identification was impossible. Therefore, antidromic compound action potentials evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) and the accessory nerve (XI) were recorded from the peripheral stumps of the various rootlets. Nerve rootlets could be categorised into rostral, intermediate and caudal groups (rostIX‐XI, intIX‐XI, caudIX‐XI). The rostIX‐XI rootlets showed their largest potential on IX stimulation, while the intIX‐XI and caudIX‐XI rootlets showed their largest potentials on CX stimulation. The intIX‐XI rootlets showed larger potentials on PhX and SLN stimulation than the caudIX‐XI rootlets. 3 Activity was recorded simultaneously from the central stumps of the rootlets in the above three groups. Most rootlets showed inspiratory bursts. Under low pH conditions, all representatives of group rostIX‐XI, most of intIX‐XI and about half of caudIX‐XI showed additional bursts during the Ea phase. Groups intIX‐XI and caudIX‐XI but not rostIX‐XI also showed discrete bursts during the Eb phase in some preparations. In general, expiratory activity was prominent in intIX‐XI. The spinal branch of XI showed no consistent respiratory activity. 4 Since the intIX‐XI rootlets showed Eb bursts and large antidromic potentials on stimulation of PhX and SLN (which innervate the inferior pharyngeal constrictor muscle (IPC)), electromyograms were recorded from the rostral and caudal parts of IPC (rIPC and cIPC). Under low pH conditions, cIPC showed bursts during the Ea and Eb phases, while rIPC showed bursts predominantly during the Eb phase. 5 These results indicate that recording from rIPC would be a useful way of examining the neuronal mechanisms responsible for Eb phase activity.