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

Abstract

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 microM 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 microM bicuculline. Furthermore, such preparations exhibited expiratory bursts under bicuculline-containing normal pH conditions. Local application of 10 microM 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 microM; 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 microM) or all (2.0 microM) 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.

Figure 1. Effects of 10 μm bicuculline on respiratory motor pattern under low pH conditions

A and B, records of discharges in right C4VR (R-C4VR), left and right IIM (L-IIM, R-IIM) and right L5VR (R-L5VR). Aa, bicuculline (10 μm) was applied at the beginning of the hatched bar. Ab–Ad, the portions of recording Aa above the horizontal bars displayed on an expanded time scale (note difference in time scale between Ac and Ab and Ad). During the initial phase of the activity evoked by bicuculline, alternating rhythmic bursts between L-IIM and R-IIM occurred (Ac). Arrowheads in Ad show representative large seizure-like discharges in L5VR. B, in 2/7 preparations, bath application of 10 μm bicuculline led to overlapping of IIM expiratory bursts with C4 inspiratory bursts. A and B were obtained from different preparations.

Figure 7. Effects of strychnine on the brainstem

A–D, records of discharges in C4VR, T13VR and L5VR. In each case, left and right panels show records obtained under normal and low pH conditions, respectively. Records were obtained in the absence (control) or presence of strychnine (0.5, 1.0 or 2.0 μm; in the rostral compartment of the chamber). All data were obtained from the same preparation and are shown in chronological order.

Figure 3. Respiratory activity in thoracic ventral roots

A and B, records of discharges in left and right C4VR, IIM and IOM (L-C4VR, R-C4VR L-IIM, R-IIM, L-IOM, R-IOM). Arrowhead in B shows point at which left T13VR was sectioned. C–E, third and fifth traces from top are records of discharges in left T9VR and T13VR (L-T9VR, L-T13VR). To obtain electrical activity from all motor axons in L-T9VR and L-T13VR, the whole ventral root was carefully incorporated into a glass suction electrode. A and C, records obtained under normal pH conditions; B and D, under low pH conditions; E, under high potassium conditions (40 mm KCl). All data were obtained from the same preparation.

Figure 2. Pattern of activity in a preparation isolated and set up under 10 μm bicuculline and normal pH conditions

A–E, records of discharges in C4VR, IIM, IOM and L5VR. The extreme left of recording A is from a time at which just 2 h had elapsed after the preparation was set up in the experimental chamber. Low pH solution was applied at the beginning of the hatched bar. B and C, the portions of recording A above the horizontal bars displayed on an expanded time scale. D, records obtained just after a 1 h wash out of 10 μm bicuculline by normal pH solution. E, records obtained under low pH conditions after the recording shown in D. F, the four respiratory bursts (indicated by arrowheads in A) displayed one above the other on an expanded time scale. In each pair, upper trace shows discharge in C4VR and lower trace discharge in IOM. G, the four respiratory bursts in E displayed one above the other on an expanded time scale. In the recordings obtained from C4VR in F and G and from the IOM in F, vertical gain was increased by a factor of two compared to the records in A–E.

Figure 5. Summary of the effects of bicuculline on I_end–E_onset

A, I_end–E_onset (see Methods) obtained from seven preparations (1–7) that were isolated and set up under bicuculline-containing normal pH conditions (○), and I_end–E_onset under low pH conditions after washout of bicuculline (×). I_end–E_onset values obtained under the two conditions are plotted adjacent to each other for each preparation. I_end–E_onset was measured for all pairs of C4 and IOM bursts that occurred during 40 min recording under bicuculline-containing normal and low pH conditions (20 min each). The total number of pairs is indicated under each column. I_end–E_onset was measured from 10 pairs of bursts under low pH conditions after washout (except in one preparation (no.1), in which only nine pairs occurred). B, I_end–E_onset obtained from eight preparations (1–8) in which bicuculline (10 μm) was applied to the brainstem under normal pH conditions for 12–28 min (○), and I_end–E_onset under low pH conditions after washout of bicuculline (×). I_end–E_onset was measured from all pairs of C4 and T13 bursts that occurred during the application of bicuculline, the number of pairs being indicated under each column. I_end–E_onset was measured from 10 pairs of bursts obtained under low pH conditions after wash out in each preparation. *P < 0.001, I_end–E_onset under bicuculline vs. that after washout in same preparation.

Figure 9. Effects of strychnine and picrotoxin on the brainstem

A–D, records of discharges in C4VR, T13VR and L5VR. A and B, records obtained under low and normal pH conditions, respectively; Ca and b, after local bath application of picrotoxin (100 μm) plus strychnine (5 μm) to the brainstem under normal pH conditions; D, after application of low pH solution to the brainstem in the presence of picrotoxin (100 μm) plus strychnine (5 μm). All data were obtained from the same preparation and are shown in chronological order.

Figure 4. Effects of 10 μm bicuculline on the brainstem under normal pH conditions

A–C, records of discharges in C4VR, T13VR and L5VR. Records obtained under normal pH conditions (A); after local bath application of 10 μm bicuculline to the brainstem under normal pH conditions (B); under low pH conditions after washout of bicuculline by normal pH solution for about 30 min (C). • in B indicate respiratory bursts in T13VR. All data were obtained from the same preparation and are shown in chronological order.

Figure 6. Effects of picrotoxin on the brainstem under normal pH conditions

A–C, records of discharges in C4VR, T13VR and L5VR. A, records obtained under normal pH conditions. B and C, records obtained after application of 20 and 100 μm picrotoxin, respectively, to the brainstem. Arrowhead in B shows an expiratory burst that occurred after the end of the C4 inspiratory burst. All data were obtained from the same preparation and are shown in chronological order.

Figure 8. Summary of the effects of application of strychnine to the brainstem on I_end–E_onset

Mean I_end–E_onset obtained from twenty pairs of C4 and T13 bursts under control and strychnine-containing low pH conditions at various strychnine concentrations (0.2, 0.3, 0.5, 1.0 and 2.0 μm). The same kind of symbol indicates values obtained from the same preparation. Error bar indicates ±S.D. *P < 0.05, **P < 0.01 and ***P < 0.001, for comparison with data obtained under control low pH conditions in the same preparation.

Figure 10. Summary of effects of strychnine and picrotoxin on various parameters

Each symbol indicates the mean of the data obtained from a given preparation and a line is drawn between data points obtained from the same preparation under different conditions. Where the data obtained showed a significant (P < 0.05) increase or decrease, this is indicated by + or -, respectively. * Significant difference between groups, P < 0.05. The group mean ±s.d. is given just above the label describing each condition.