Cyclothiazide-induced persistent increase in respiratory-related activity in vitro

Abstract

Hypoglossal (XII) motoneurons (MNs) innervate the genioglossus muscle of the tongue, which plays an important role in maintaining upper airway patency, particularly during sleep, and modulating upper airway resistance. Discovering methods for inducing long-term increases in genioglossal motoneuronal excitability to AMPA-mediated drive may help in the development of therapeutics for upper airway motor disorders such as obstructive sleep apnoea. We show that the diuretic, anti-hypertensive, AMPA receptor modulator cyclothiazide (CTZ) induces a profound and long-lasting increase in the amplitude of respiratory-related XII nerve activity in rhythmically active neonatal rat medullary slices. Treatment of the slice with CTZ (90 μM) for 1 h increased the integrated XII ( XII) nerve burst amplitude to 262 ± 23% of pre-treatment control at 1 h post-treatment;much of this increase lasted at least 12 h. The amount of CTZ-induced facilitation (CIF) was dependent upon both CTZ dose and exposure time and was accompanied by a long-lasting increase in endogenous AMPA-mediated drive currents to XII MNs. CIF, however, is not a form of plasticity and does not depend on AMPA or NMDA receptor activation for its induction. Nor does it depend on coincident protein kinase A or C activity. Rather, measurement of mEPSCs along with mass spectrometric analysis of CTZ-treated slices indicates that the cause is prolonged bioavailability of CTZ. These results illustrate a latent residual capacity for potentiating AMPA-mediated inspiratory drive to XII MNs that might be applied to the treatment of upper airway motor deficits.

Figure 8. Estimated non-NMDA mEPSC probability density functions (PDFs)

PDFs for mEPSC peak amplitude (A) and interval (B) of untreated, CTZ-treated and CTZ-treated with 1 h wash slices. Insets show detail of PDF tail with enlarged y-axis. Comparison of distributions performed using piecewise bootstrap method (see Methods). *P < 0.05, ***P < 0.001.

Figure 1. Bath application of CTZ leads to long-lasting facilitation of endogenous inspiratory XII nerve bursts in neonatal rat medullary slice

A, ∫XII nerve burst amplitude and rate continue to increase in the presence of bath-applied CTZ for entire duration (∼2 h) of application. Arrows show periods of increased tonicity. Asterisk denotes period of tonicity on expanded timescale (right of main trace). B, protocol for experiments in C and Fig. 2. C, example traces showing the impact of bath application of CTZ (top), CX546 (middle) and DMSO (bottom) on ∫XII nerve burst amplitude and rate. Traces on expanded timescales below main traces show samples of ∫XII nerve bursts before and 1, 6 and 12 h post-treatment.

Figure 2. CTZ, but not CX546 or DMSO, leads to long-lasting facilitation of endogenous inspiratory ∫XII nerve burst amplitude

A–B, longitudinal data for effect of 1 h application of CTZ (90 μm, n= 5), CX546 (90 μm; n= 7) or DMSO (0.1%; n= 7) on normalized ∫XII nerve burst amplitude (A) and rate (B). Thick lines show group averages. Dotted lines show individual experiments. CTZ (squares), CX546 (diamonds), DMSO (triangles). Black bar above data shows timing and duration of treatment. C–D, comparisons for the effects of CTZ (squares) and CX546 (diamonds) vs. DMSO control (triangles) on normalized ∫XII nerve burst amplitude (C) and rate (D) at 1, 6 and 12 h post-treatment. Significance, assessed using difference tests following two-way RMANOVA, is as indicated in the figures. E–F, regressions assessing whether the effect of CTZ (90 μm; n= 11) on normalized ∫XII nerve burst amplitude at 1 h post-treatment vs. raw control ∫XII nerve burst amplitude (E) and normalized XII nerve burst rate at 1 h post-treatment vs. raw XII nerve burst rate (F) were significantly correlated.

Figure 3. Dose–response and exposure–response effects of CTZ on ∫XII nerve burst amplitude and rate 1 h post-treatment

A, dose–response for 1 h bath application of 3 μm, 9 μm, 30 μm or 90 μm CTZ (n= 5 for each concentration) on ∫XII nerve burst amplitude and rate. Concentration had significant effect on both amplitude (F(3,16) = 6.38, P < 0.01) and rate (F(3,16) = 6.50, P < 0.01) as determined by one-way ANOVA. B, exposure–response curves for 90 μm CTZ applied for 10 min (n= 6), 30 min (n= 6) or 1 h (n= 5). Exposure time had significant effect on ∫XII nerve burst amplitude (F(2,14) = 7.68, P < 0.01) but not rate (F(2,14) = 2.31, n.s.) as assessed by one-way ANOVA. In both panels, squares represent amplitude responses and circles represent rate responses. Large symbols show group averages and small symbols individual experiments. All measurements were taken at 1 h post-treatment.

Figure 4. Bath application of CTZ induces long-lasting increases in endogenous inspiratory drive to XII MNs

A, top trace shows effect of treating medullary slice with CTZ (90 μm) for 10 min. Expanded traces below show sample ∫XII nerve bursts and accompanying XII MN drive currents immediately prior to and 1 h post-treatment. Overlaid current traces to the right show average of 25 consecutive drive currents for each time point. B, comparison of normalized charge transfer of inspiratory drive currents in XII MNs before and 1 h post-treatment. Lines connect measurements from the same cell before and 1 h post-treatment. Significance tested using RM difference test (n= 5). C, regression showing high correlation between increases in XII MN drive currents and ∫XII nerve burst amplitude 1 h post-treatment.

Figure 5. CIF does not depend upon activation of AMPA or NMDA receptors during treatment with CTZ

A, sample traces showing the effects on ∫XII nerve activity after bath application of CNQX (10 μm) and APV (50 μm) (control slices, top trace) or CTZ (90 μm) in the presence of CNQX and APV (bottom trace). CNQX and APV were applied for 2.5 h. When CTZ was applied, it was applied for 1 h, 30 min after the start of CNQX and APV. This allowed CNQX and APV to take effect before CTZ and 1 h for the slices to be washed after CTZ before removing CNQX and APV. Black bars above traces illustrate the timing and duration of application. Transients resulting from electrostatic discharge during the slice silent periods have been removed. B, longitudinal data for all experiments run according to the protocols in A. CTZ had a significant effect (F(1,9) = 12.8, RMANOVA) on ∫XII nerve burst amplitude. In slices treated with CTZ (n= 6) ∫XII nerve burst amplitude was significantly greater than pre-treatment from 1 h post CNQX and d-APV (182 ± 6.8%, P < 0.001, RM difference test) to the end of the experiment 5 h post CNQX and d-APV (180 ± 25%, P < 0.001, RM difference test). ∫XII nerve burst amplitude in slices not treated with CTZ (n= 5) was neither facilitated nor depressed relative to pre-treatment (115 ± 21% 5 h post CNQX and d-APV, n.s., RM difference test). Slices receiving treatment with CTZ are marked by squares. Control slices (n= 5) are marked with triangles. Thick traces represent group means. Thin traces represent individual experiments. C, comparison of activity 1 h and 5 h post CNQX and d-APV in slices treated and not treated with CTZ. Significance was computed using difference tests that followed a two-way RMANOVA.

Figure 6. CIF is not PKA- or PKC-dependent

A, sample trace showing effects of bath application of chelerythrine (10 μm) and H89 (10 μm) on inspiratory ∫XII nerve activity. Last half hour of trace (during washout of H89 and chelerythrine) demonstrates failure of some slices not treated with CTZ to recover pre-treatment activity levels. (Transients resulting from perfusion noise have been removed.) B, comparison of ∫XII nerve burst amplitude (relative to pre-treatment control) between CTZ-treated (CTZ) and untreated (No CTZ) slices (n= 6 for both groups) 30 min post washout of H89 and chelerythrine, which was 1 h post the start of washout of CTZ, when CTZ was used. Symbols represent individual experiments and lines represent group averages. Significance assessed using difference test.

Figure 7. CTZ treatment of medullary slices leads to long-lasting increases in XII MN non-NMDA mEPSC amplitude and decay

A, sample mEPSCs from control cell (top), cell treated for 1 h with CTZ (90 μm; middle) and a cell treated with CTZ that was then washed for 1 h before recording (bottom). B–D, group data comparing average mEPSC peak amplitude (B), mEPSC decay time constant (C) and average mEPSC interval (D) for cells treated under 1 of the 3 conditions described in A (n= 6 for control cells and n= 7 for cells treated with CTZ and cells treated with CTZ and then washed for 1 h). Inset in B shows average of the average waveforms for each experiment under a given condition. Inset in C shows waveforms in B scaled to same peak value. Significance in B–D assessed using difference test. Individual symbols in B–D represent values for single experiments. Lines represent group averages.

Figure 9. Large quantities of CTZ remain trapped in medullary slice following wash with ACSF

Measurements made using liquid chromatography–tandem mass spectrometry. Slices treated with CTZ (90 μm) in 1 of 3 ways: 1 h, 1 h and washed for 1 h, 1 h and washed for 6 h (n= 5 for all groups). No significant difference among the three groups using one-way ANOVA or difference tests between groups. Individual symbols represent individual experiments. Lines represent group averages.