KCNQ channels determine serotonergic modulation of ventral surface chemoreceptors and respiratory drive

Abstract

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO(2)/H(+) changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we show in vitro and in vivo that KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 μm) increased basal activity and CO(2) responsiveness of RTN neurons in rat brain slices, whereas KCNQ channel activation (retigabine, 2-40 μm) silenced these neurons. Interestingly, we also find that KCNQ and apamin-sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO(2) responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance P or thyrotropin-releasing hormone, previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activity in vivo. Inhibition of KCNQ channels lowered the CO(2) threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders.

Figure 1.

KCNQ and SK channels differentially regulate RTN chemoreceptor activity in vitro. A, Left, Current traces show that RTN chemoreceptors exhibit a pronounced mAHP following depolarizing current injection (100 ms, 1nA) that could be reduced by apamin (100 nm) and XE991 (10 μm). Right, Summary data show peak mAHP amplitude in control, apamin, and XE991 (N = 7 cells/18 animals). B1, Left, Trace of firing rate from an RTN chemoreceptor shows the dose-dependent effects of retigabine (retig) on neuronal activity. Since RTN neurons have low basal activity, these experiments were performed in the continuous presence of 15% CO₂. Right, Summary data (N = 5 cells/ 10 animals) fit using the Michaelis–Menten equation shows that RTN chemoreceptors have a retigabine IC₅₀ of 0.6 μm ± 0.1 (coefficient standard error; R² = 0.964). B2, Firing rate trace from an RTN chemoreceptor shows that bath application of XE991 (2 μm) increased activity ∼1.2 Hz. In the continued presence of XE991, subsequent application of retigabine (40 μm) eliminated cell activity. C1, Firing rate trace shows the responses of an RTN chemoreceptor to 15% CO₂ under control conditions and in XE991. C2, Summary data (N = 4 cells/8 animals) fit using the Hill equation shows that XE991 increased activity at 5% CO₂ (i.e., baseline firing rate) and caused a left shift in the firing rate response to graded levels of CO₂, but with no change in slope. Note that exposure to 10 and 15% CO₂ significantly increased activity under control and in XE991. Asterisks designate a significance difference between control and in XE991 as determined by one-way repeated-measures ANOVA (p < 0.01). D, Responses of an RTN chemoreceptor to 15% CO₂ under control conditions and in apamin. Right, Summary graph (N = 6 cells/12 animals) showing lack of apamin effect on baseline activity and CO₂ sensitivity. Insets, Segments of membrane potential show effects of XE991 and apamin repetitive firing behavior. E, Firing rate trace shows CO₂ responsiveness under control conditions, in XE991 alone, and in XE991 plus apamin. Downward arrow (↓) designates DC current injection to approximate control level of activity. Insets, Segments of membrane potential show effects of XE991 alone and XE991 + apamin repetitive firing behavior. Right, Summary data (N = 10 cells/ 30 animals) show CO₂ responsiveness in XE991 alone and in XE991 plus apamin.

Figure 2.

KCNQ channels mediate serotonergic modulation of RTN chemoreceptors in vitro. A–C, Firing rate traces (left) and summary data (right) show responses of RTN chemoreceptors to 5 μm serotonin (5-HT; N = 7 cells/14 animals; A), TRH (200 nm; N = 8 cells/ 16 animals; B), and SP (0.5 μm; N = 4 cells/ 9 animals; C) under control conditions and in XE991 (10 μm). Summary graphs also show that apamin had no effect on firing rate response to 5-HT (N = 6 cells/14 animals), TRH (N = 7 cells/14 animals), or SP (N = 5 cells/10 animals). D, Left, Firing rate trace shows the response of a chemosensitive RTN neuron to TRH alone and in the presence of 5-HT. Summary data (right) show that RTN neurons can respond to 5-HT and TRH (N = 4 cells/8 animals) or SP (N = 3 cells/7 animals) independently of each other. Double slant bars (//) mark 10 min time breaks, and the downward arrow (↓) designates DC current injection.

Figure 3.

KCNQ channels in the RTN regulate resting and breathing activity and the ventilatory response to CO₂ and serotonin in anesthetized rats. A1, A2, Traces of end expiratory CO₂ (etCO₂), arterial pressure (AP), and integrated phrenic nerve discharge (∫PND) show the ventilatory response to bilateral injections (arrows) of saline (A1) or XE991 (A2) into the RTN. Under control conditions, injections of XE991 (50 μm, 30 nl each side) increased resting breathing activity as evidenced by an increase in mvPND (product of PND amplitude and frequency) and significantly lowered the PND CO₂ threshold from 5.3 ± 0.09% to 4.5 ± 0.1%. However, CO₂ responsiveness was otherwise unaffected by application of XE991 into the RTN; lowering etCO₂ from to 3–4% inhibited respiratory output, and graded increases etCO₂ up to 9–10% increased mvPND by an amount similar to that of saline controls. B, Summary data plotted as change in mvPND show the effect of XE991 (50 μm) on resting respiratory activity. C, Summary data showing CO₂-induced changes in mvPND under control conditions and after injections of XE991. D, Summary data showing that XE991 decreased the level of CO₂ required to stimulate PND activity. E, Summary data fit using the Hill equation shows that XE991 caused a left parallel shift in the CO₂ ventilatory response curve. Asterisks designate a significant difference between control and in XE991 (one-way repeated measures ANOVA, p < 0.01, N = 5 rats). F, Computer-assisted plots of the center of the injection sites (coronal projection on the plane; bregma, −11.6; Paxinos and Watson, 1989). Note that all injections were made in the caudal aspect of the RTN, where there is the highest density of chemosensitive RTN neurons. G, Traces of ∫PND, AP, and etCO₂ show that unilateral injection of XE991 (50 μm) into the RTN of an anesthetized rat decreased the normally robust excitatory effect of serotonin (5-HT) on PND amplitude. Injection of 5-HT (1 mm) caused a modest biphasic AP response that was unaffected by XE991. Inset, Traces of ∫PND show on an expanded time scale that injection of XE991 decreased effects of 5HT on PND amplitude with little effect on frequency. H, Computer-assisted plot of XE991 injection sites in the caudal RTN. I, Summary data (N = 5 animals) show that injection of XE991 into the RTN reversibly decreased effects of 5-HT on PND amplitude. Note the effect of serotonin on PND fully recovered after washing XE991 for ∼1 h. py, Pyramid; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bars, 1 mm.

Figure 4.

KCNQ channels in the RTN regulate the ventilatory response to exogenous serotonin in conscious awake rats. Whole-body plethysmography was used to measure respiratory rate (fR) and tidal volume (VT) in conscious, freely moving rats during exposure to hypercapnia (7% CO₂) after bilateral RTN injections of saline or XE991 (50 μm). A, Traces of arterial pressure, AP, and inspiratory (Insp) activity show respiratory activity after bilateral injections of saline or XE991 (50 μm) under control conditions and in response to 7% CO₂. B, Computer-assisted plot of XE991 injection sites in the RTN. py, Pyramid; VII, facial motor nucleus. Scale bar, 1 mm. C–E, Summary data (N = 6 animals) plotted as VT (Tidal Vol; C), fR (Resp freq; D), or minute ventilation (Min Ven; E) versus time show that RTN injections of XE991 had no effect on baseline breathing or the ventilatory response to CO₂ in conscious rats. F, Traces of AP and inspiratory activity show respiratory responses to injection of serotonin (1 mm) after unilateral injection of saline or XE991 (50 μm). G, Plot of 5-HT and XE991 injection sites in the RTN. Scale bar, 1 mm. H–J, Summary data (N = 6 animals) show that unilateral RTN injection of XE991 decreased exogenous 5-HT-induced (1 mm) increases in VT (H), fR (I), and minute ventilation (J).