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HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity - PubMed

  • ️Thu Jan 01 2015

HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity

Virginia E Hawkins et al. J Neurophysiol. 2015.

Abstract

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H(+)-dependent drive to breathe and function as an integration center for the respiratory network, including serotonergic raphe neurons. We recently showed that serotonergic modulation of RTN chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward current. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are the molecular correlate of the hyperpolarization-activated inward current (Ih) and have a high propensity for modulation by serotonin. To investigate whether HCN channels contribute to basal activity and serotonergic modulation of RTN chemoreceptors, we characterize resting activity and the effects of serotonin on RTN chemoreceptors in vitro and on respiratory activity of anesthetized rats in the presence or absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs(+)) channels. We found in vivo that bilateral RTN injections of ZD7288 increased respiratory activity and in vitro HCN channel blockade increased activity of RTN chemoreceptors under control conditions, but this was blunted by KCNQ channel inhibition. Furthermore, in vivo unilateral RTN injection of XE991 plus ZD7288 eliminated the serotonin response, and in vitro serotonin sensitivity was eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker). Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor blocker and mimicked by a 5-HT7-receptor agonist. In addition, serotonin caused a depolarizing shift in the voltage-dependent activation of Ih. These results suggest that HCN channels contribute to resting chemoreceptor activity and that serotonin activates RTN chemoreceptors and breathing in part by a 5-HT7 receptor-dependent mechanism and downstream activation of Ih.

Keywords: 5-HT7; Gs-coupled receptor; cAMP; retrotrapezoid nucleus; serotonin.

Copyright © 2015 the American Physiological Society.

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Figures

Fig. 1.
Fig. 1.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels in the retrotrapezoid nucleus (RTN) regulate resting breathing activity and the ventilatory response to CO2 in anesthetized rats. A: traces of end expiratory CO2 (etCO2), arterial pressure (AP), and integrated phrenic nerve discharge (iPND) show the respiratory response to bilateral injections (arrows) of saline or HCN channel inhibitor ZD7288 in the RTN. Under control conditions, injections of ZD7288 (50 μM, 50 nl each side) increased respiratory activity with an increase in mvPND (product of PND amplitude and frequency) and significantly lowered PND CO2 threshold (D). However, CO2 responsiveness was otherwise unaffected by application of ZD7288 in the RTN; lowering etCO2 from to 3 to 4% inhibited respiratory output, and graded increases etCO2 up to 9–10% increased mvPND by an amount similar to saline (control). B: summary data plotted as change in mvPND shows the effect of ZD7288 alone (50 μM) or in combination with KCNQ channel inhibitor XE991 (50 μM) on resting respiratory activity (n = 7 animals). C: summary data showing CO2-induced changes in mvPND under control conditions, after injections of ZD7288 or XE991 or after injections of ZD7288 + XE991 (n = 7 animals). D: summary data showing that ZD7288 alone or in combination with XE991 decreased the level of CO2 required to stimulate PND activity (n = 7 animals). E: computer-assisted plots of the center of the injection sites (coronal projection on the plane Bregma: −11.6 and −11.3 mm; 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. Sp5, spinal trigeminal tract; py, pyramids; VII, facial motor nucleus; au, arbitrary units. *P < 0.01.

Fig. 2.
Fig. 2.

HCN channels in the RTN contribute to serotonergic modulation of respiratory drive in anesthetized rats. A: traces of iPND and AP show effects of unilateral RTN injections of serotonin (5-HT; 1 mM) on breathing and blood pressure under control conditions, after RTN injection of XE991 (50 μM; A1) or ZD7288 (50 μM; A2) alone, and after injections of both XE991 and the HCN channel blocker ZD7288 (A1). Inset: traces of iPND plotted on an expanded time scale. Under control conditions RTN injection of serotonin increased PND amplitude and decreased mean arterial pressure (MAP). Unilateral RTN injection of XE991 or ZD7288 decreased effects of serotonin on PND amplitude by ∼40% and coadministration of ZD7288 with XE991 eliminated the serotonin response. Note the effect of serotonin on PND fully recovered after 60 min. B: computer-assisted plot shows the locations of injection sites within the RTN (Bregma: −11.6 mm). C: summary data (n = 6 animals) showing the effects of serotonin on PND amplitude under control conditions, XE991 alone, ZD7288 alone, and XE991 plus ZD7288. RPa, raphe pallidus. *P < 0.05 vs. control.

Fig. 3.
Fig. 3.

Contributions of HCN and KCNQ channels to repetitive firing behavior of chemosensitive RTN neurons. A1: membrane potential responses to a depolarizing step (+20 pA) and hyperpolarizing current steps (−70 to −100 pA, Δ10 pA) under control conditions and in ZD9288 (50 μM, red). A2–A4: summary data (n = 6) showing sag ratio (A2), instantaneous spike frequency (ISF; A3) and rebound spike latency (A4) under control conditions and in ZD7288. Note the characteristic HCN channel-dependent depolarizing sag and short latency rebound spike. B1: voltage responses to a depolarizing step (+20 pA) and hyperpolarizing current steps (−70 to −100 pA, Δ10 pA) under control conditions and in XE991 (10 μM, blue). B2-B4: summary data (n = 5) showing sag ratio (B2), ISF (B3), and rebound spike latency (B4) under control conditions and in XE991 (10 μM, blue). Blocking KCNQ channels increased ISF but did not affect the sag ratio or rebound spike latency. *P < 0.05, **P < 0.01.

Fig. 4.
Fig. 4.

HCN channels contribute to resting firing behavior and serotonergic modulation of RTN chemoreceptors in vitro. A1: representative trace of firing rate from a chemosensitive RTN neuron. Under control conditions exposure to serotonin (5-HT; 5 μM) increased firing rate in a CO2-sensitive neuron. After serotonin was washed out and returned to a basal level of activity, bath application of XE991 (10 μM) also increased the firing rate. In the continued presence of XE991 (with baseline activity adjusted to near control levels by DC current injection), a second exposure to serotonin only increased the firing rate by approximately half the control response. Subsequent bath application of ZD7288 (50 μM) also increased firing rate by ∼1 Hz. In the presence of XE991 plus ZD7288 (with baseline activity adjusted to near control levels by DC current injection), a third exposure to serotonin had no discernible effect on firing rate. A2: firing rate trace shows the response of an RTN chemoreceptor to 5-HT and Cs+ under control conditions and in the presence of Cd2+ (100 μM). Note that Cd2+ eliminated the inhibitory effect of HCN channel blockade (reflected as an increase in the firing rate) but did not affect the serotonin response. B: summary data show the firing rate response of RTN chemoreceptors to HCN channel inhibition by ZD7288 or Cs+ (2 mM) under control conditions (n = 17 cells), in XE991 to block KCNQ channels (n = 12 cells), in synaptic blockers (n = 7 cells), and in Cd2+ to block channels including voltage-gated Ca2+ channels (n = 7 cells). Both XE991 and Cd2+ significantly reduced the responses to HCN channel inhibition. C: summary data show the firing rate response to serotonin under control conditions (n = 16 cells), in XE991 alone (n = 13 cells), in ZD7288 alone (n = 4 cells), and in XE991 plus ZD7288 (n = 7 cells) or Cs2+ (2 mM; n = 6 cells). D: firing rate trace shows that exposure to 15% CO2 increased activity of a chemosensitive RTN neuron by similar amounts under control conditions and in the presence of ZD7288 (50 μM). E: summary data (n = 6 cells) showing that CO2/H+ sensitivity of RTN neurons is fully retained when HCN channels are blocked by ZD7288. F: summary data (N = 7 cells) shows that serotonin responses were also retained during Ca2+ channel blockade. // Marks 10-min time breaks and ↓ indicates DC current injection. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.
Fig. 5.

Serotonin activates HCN channels by a cAMP-dependent mechanism. A: representative trace of firing rate from a chemosensitive RTN neuron. Under control conditions exposure to serotonin (5-HT; 5 μM) increased firing in a CO2 sensitive neuron by ∼1 Hz. After washout of serotonin, bath application of XE991 (10 μM) also increased firing rate by ∼1.2 Hz. In the continued presence of XE991 (with baseline activity adjusted to near control levels by DC current injection), a second exposure to serotonin only increased firing rate by ∼0.5 Hz. Subsequent bath application of the adenylate cyclase inhibitor SQ22536 (100 μM) increased firing rate by ∼0.8 Hz. In the presence of XE991 plus SQ22536 (with baseline activity adjusted to near control levels by DC current injection), a third exposure to serotonin no longer led to increased firing. B: summary data (n = 5 cells) of firing rate responses to SQ22536 when KCNQ channels are blocked with XE991. C: summary data (n = 5 cells) of firing rate responses to serotonin under control conditions, in XE991 alone, and in XE991 plus SQ22536. // Marks 10-min time breaks and ↓ indicates DC current injection. *P < 0.05, **P < 0.01.

Fig. 6.
Fig. 6.

Serotonin causes a depolarizing shift in the voltage-dependent activation of hyperpolarization-activated inward current (Ih) in RTN chemoreceptors. A: current responses to hyperpolarizing voltage steps recorded under control conditions (A1), in serotonin (5 μM; A2), after being washed serotonin for 5 min (A3), and in ZD7288 (50 μM). Note that serotonin caused a modest decrease in holding current (arrows indicate zero current levels). B: summary data (n = 6 cells/animals) showing that serotonin did not increase maximal Ih amplitude, measured as the difference between steady-state and instantaneous currents at −150 mV (illustrated in A1). C: Ih activation curves where tail currents were measured during a fixed step to −70 mV that followed each test pulse (* in A) and normalized data were fitted using the Boltzmann equation, under control conditions and in serotonin. Serotonin causes a depolarizing shift in the voltage-dependent activation of Ih (V1/2 values inset, n = 7). *P < 0.05, **P < 0.01.

Fig. 7.
Fig. 7.

5-HT7 receptor activation mediates effects of serotonin on RTN chemoreceptors. A: representative trace of firing rate from a chemosensitive RTN neuron shows that under control conditions exposure to 5-carboxamidotryptamine (5-CT; 5 μM) increased firing by ∼0.4 Hz. As before, bath application of ZD7288 (50 μM) also increased firing rate. A second exposure to 5-CT, this time in the presence of ZD7288 (with baseline activity adjusted to near control levels by DC current injection), had no effect on firing rate. B: trace of firing rate shows the response of an RTN chemoreceptor to 5-CT was blocked by 10-min incubation in SB258719 (10 μM), a 5-HT7 receptor blocker. C: summary data (n ≥ 4 cells) showing the firing rate response to 5-CT under control conditions and in the presence of ZD7288 alone, the 5-HT1A receptor antagonist WAY100635 (0.1 μM) alone, or the 5-HT7 receptor antagonist SB258719. Note that WAY100635 did not affect the response to 5-CT, suggesting 5-HT1A receptors do not contribute to this response whereas 5-HT7 inhibition significantly reduced the serotonin response. D: average data (n = 3 cells) showing the firing rate response of RTN chemoreceptors the 5-HT7 receptor agonist LP-44 (2 μM) under control conditions and in the presence of the 5-HT7 receptor antagonist SB258719. // Marks 10-min time breaks and ↓ indicates DC current injection. *P < 0.05, **P < 0.01.

Fig. 8.
Fig. 8.

Model depicting the molecular basis for serotonergic modulation of RTN chemoreceptors. Previous (Hawryluk et al. 2012) and present results show that serotonin stimulates activity of RTN chemoreceptors by 2 independent but coordinated signaling pathways: 5-HT2-mediated inhibition of KCNQ channels most likely by Gq signaling, and 5-HT7-mediated activation of HCN channels by a mechanism involving activation of adenylate cyclase- and cAMP-mediated depolarizing shift in the voltage-dependent activation of Ih. Our evidence suggests that each of these signaling pathways can independently stimulate activity of RTN chemoreceptors and together their coordinated activity ensures a robust response to serotonin. Note that our evidence also suggests that under basal conditions HCN channel activity may indirectly modulate activity of voltage-dependent KCNQ and Cd+2-sensitive channels including Ca2+ channels.

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