Role of synaptic inhibition in turtle respiratory rhythm generation

Abstract

In vitro brainstem and brainstem-spinal cord preparations were used to determine the role of synaptic inhibition in respiratory rhythm generation in adult turtles. Bath application of bicuculline (a GABA(A) receptor antagonist) to brainstems increased hypoglossal burst frequency and amplitude, with peak discharge shifted towards the burst onset. Strychnine (a glycine receptor antagonist) increased amplitude and frequency, and decreased burst duration, but only at relatively high concentrations (10-100 microM). Rhythmic activity persisted during combined bicuculline and strychnine application (50 microM each) with increased amplitude and frequency, decreased burst duration, and a rapid onset-decrementing burst pattern. The bicuculline-strychnine rhythm frequency decreased during mu-opioid receptor activation or decreased bath P(C)(O(2)). Synaptic inhibition blockade in the brainstem of brainstem-spinal cord preparations increased burst amplitude in spinal expiratory (pectoralis) nerves and nearly abolished spinal inspiratory activity (serratus nerves), suggesting that medullary expiratory motoneurons were mainly active. Under conditions of synaptic inhibition blockade in vitro, the turtle respiratory network was able to produce a rhythm that was sensitive to characteristic respiratory stimuli, perhaps via an expiratory (rather than inspiratory) pacemaker-driven mechanism. Thus, these data indicate that the adult turtle respiratory rhythm generator has the potential to operate in a pacemaker-driven manner.

Figure 8. Bicuculline-strychnine rhythm drives expiratory, but not inspiratory, spinal motoneurons

A, schematic drawing of a turtle brainstem-spinal cord preparation with the chamber partitioned into brainstem, rostral spinal, and caudal spinal compartments. B, suction electrodes attached to hypoglossal, pectoralis (expiratory), and serratus (inspiratory) nerves record bursts of respiratory activity. Traces of integrated respiratory activity on the three nerves are shown while the brainstem compartment was bathed with control solution (C), and with bicuculline and strychnine (50 μM each) (D). E, individual bursts from baseline recordings (thin lines labelled as ‘baseline’ with arrows) are superimposed onto bursts recorded during bicuculline-strychnine brainstem application (thick lines). All bursts were aligned with respect to the onset of hypoglossal activity (vertical dashed line). The sample tracings show that hypoglossal and pectoralis amplitudes were increased with the peak time occurring near the onset of activity, whereas serratus activity was nearly abolished. Hyp., hypoglossal; Pect., pectoralis; Serr., serratus.

Figure 1. Spontaneous respiratory motor activity produced by in vitro turtle brainstem preparations

A, turtle brainstems were isolated, placed in an in vitro recording chamber, and suction electrodes were attached to hypoglossal (XII) nerve rootlets to record respiratory activity. B, a sample recording trace shows rhythmic bursts of integrated respiratory activity.

Figure 2. Glycine receptor blockade alters hypoglossal burst amplitude, frequency, and duration

A, integrated hypoglossal bursts are shown following glycine receptor blockade with increasing strychnine concentrations (0-50 μM). Individual bursts shown on a faster time scale are aligned to the onset of hypoglossal burst onset (vertical dotted line; right-hand side). B-E: •, group data during drug application; ○, group baseline data (left) and washout data (right) in each graph. B, amplitude was not significantly altered until the strychnine concentration reached 50 μM. C, frequency tended to increase between 0.01-1.0 μM, and increased by > 100 % at 10–50 μM. D, duration decreased by 30–50 % at 1.0-50 μM. E, peak time was not significantly altered, although there was a tendency for a shift to a rapid onset-decrementing pattern at 10–50 μM. No strychnine effects were reversed during washout. *P < 0.05 relative to baseline. All data were derived from 13 brainstems except for the 50 μM (n= 7) and washout data (n= 4).

Figure 3. GABA_A receptor blockade alters burst amplitude, frequency, and pattern

A, traces of integrated hypoglossal respiratory motor output are shown with increasing bicuculline concentrations (0-50 μM). B-E, group data from 11 brainstems are shown with the same symbols as in Fig. 2. B, bicuculline tended to increase amplitude at 10 μM, and significantly increased amplitude by > 200 % at 50 μM. C, frequency was significantly increased at 10 and 50 μM by 4–8 bursts (10 min)⁻¹. D, burst duration was unaltered at all bicuculline concentrations, but there was a dose-dependent decrease in peak time (P < 0.05 at 10 and 50 μM), reflecting a shift to a rapid onset-decrementing pattern. E, all bicuculline effects were reversible following washout (n= 4).

Figure 4. GABA_B receptor blockade had no effect on respiratory activity

GABA_B receptor blockade with increasing concentrations of 2-hydroxysaclofen did not alter burst amplitude (A), frequency (B), duration (C), or peak time (D). Symbols as in Fig. 2.

Figure 5. Rhythmic activity persists during combined GABA_A and glycine receptor blockade

Bicuculline and strychnine (50 μM each) were applied to brainstems (n= 11) to block GABA_A and glycine receptors, respectively. A, a trace of integrated hypoglossal bursts from one brainstem shows that GABA_A and glycine receptor blockade caused an initial increase in frequency, followed by bursts superimposed on tonic activity. Afterwards, a high frequency rhythm emerged with large amplitude bursts. B, a comparison of a hypoglossal burst trace during baseline (1, thick line trace) with a hypoglossal burst trace after blocking GABA_A and glycine receptors (2, thin line trace) shows the shift to rapid onset-decrementing pattern of discharge. Group data show that amplitude (C) and frequency (D) increased significantly by > 100 % while duration (E) and peak time (F) decreased significantly. Data gathered over 30 min were averaged to give baseline; blockade value is the average of data gathered over 20 min.

Figure 7. Strychnine-bicuculline rhythm altered by μ-opioid receptor activation and low P_CO2

Brainstems were exposed to bicuculline and strychnine (50 μM each) for 1.5-2.0 h to establish a stable bicuculline-strychnine rhythm before DAMGO or low P_CO2 application. A and B, traces of integrated hypoglossal nerve activity produced during synaptic inhibition blockade by individual brainstems are shown. A, DAMGO (10 μM) application abolished rhythmic activity for 8.4 min, and decreased frequency by 19 ± 4 bursts (10 min)⁻¹ for the next 60 min. B, switching from standard solution containing bicuculline and strychnine (5 % CO₂; pH 7.39) to a similar solution bubbled with 1.2 % CO₂ (pH 7.94) decreased frequency by 33.7 ± 1.2 bursts (10 min)⁻¹ within 10 min. For both C and D, the frequency of the steady-state bicuculline-strychnine rhythm in control brainstems (•; n= 12) was 17 ± 2 bursts (10 min)⁻¹ at the zero time point, and remained within 17.4-18.5 bursts (10 min)⁻¹ for the next 80 min. All data were averaged in 20 min bins. C, continuous DAMGO application (10 μM, 80 min) reduced the steady-state bicuculline- strychnine frequency of 22.1 ± 2.4 bursts (10 min)⁻¹ by 19.4 ± 0.8 bursts (10 min)⁻¹ during 80 min application (○; n= 8). D, switching from 5 % to 1.2 % CO₂ (80 min) increased the steady-state bicuculline- strychnine frequency of 21.5 ± 1.8 bursts (10 min)⁻¹ at the zero time point by 7.6 ± 4.5 bursts (10 min)⁻¹ during the first 20 min before causing a decrease of 10.5 ± 0.8 bursts (10 min)⁻¹ during the next 60 min (○; n= 8). *P < 0.05 compared to the first 20 min for delta peak frequencies.

Figure 6. Activation of μ-opioid receptors abolishes respiratory activity

Application of DAMGO (a μ-opioid receptor agonist) had no effect at 0.001 μM, but decreased respiratory burst amplitude and frequency at 0.01-10 μM. A, amplitude was significantly decreased by 17 % at 0.01 μM and was decreased by ≈50 % at 1.0 and 10 μM (insufficient data available for statistical analysis), and following washout. Numbers in parentheses below symbols indicate the number of brainstems producing respiratory activity; otherwise the symbols are as in Fig. 2. B, frequency decreased at 0.01 μM and was abolished in 7/7 brainstems at 0.1 μM (P < 0.05), and 6/7 brainstems at 1.0 and 10 μM (P < 0.05). There were no significant changes in burst duration (C) or peak time (D).

Figure 9. Characteristics of rhythm during brainstem GABA_A and glycine receptor blockade

Group data for brainstem-spinal cord preparations (n= 6) under baseline conditions, and following blockade of GABA_A and glycine receptors in the brainstem. All data were averaged in 20 min bins. A, hypoglossal and pectoralis burst amplitude increased by 140 % and 260 %, respectively (P< 0.05), whereas serratus burst amplitude decreased by 80 % (P < 0.05). B, frequency increased significantly by nearly 7 bursts (10 min)⁻¹. C, duration decreased in all three nerves, but a significant decrease was observed only in hypoglossal bursts. Open bars indicate baseline data; black bars indicate data during blockade of brainstem GABA_A and glycine receptors. D, peak time significantly shifted from the middle to the beginning of the burst in hypoglossal and pectoralis nerves, whereas peak time significantly shifted from near the end of the burst to the middle of the burst for serratus nerves. E, burst onset, peak time, burst amplitude relative to baseline (horizontal dashed lines), and burst termination are graphed for baseline (○) and blockade of brainstem GABA_A and glycine receptors (•) for hypoglossal, pectoralis, and serratus. All timing data are graphed with respect to the onset of hypoglossal activity. Baseline amplitude is set at 1.0 arbitrary units (horizontal continuous lines); amplitude at burst onset and burst termination is set at zero (horizontal dashed lines). * Significant differences with respect to baseline in terms of timing only; significant amplitude differences have already been indicated in Fig. 9A.