Articles in PresS. J Neurophysiol (November 26, 2014). doi:10.1152/jn.00487.2014

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HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity. Virginia E. Hawkins1*, Joanna M. Hawryluk1*, Ana C. Takakura2, Anastasios V. Tzingounis1, Thiago S. Moreira3, Daniel K. Mulkey1 1

Dept. of Physiology and Neurobiology, Univ. of Connecticut, Storrs, CT 06269, USA Dept. of Pharmacology, Univ. of Sao Paulo, Sao Paulo, SP, 05508, Brazil; 3 Dept. of Physiology and Biophysics, Univ. of Sao Paulo, Sao Paulo, SP, 05508, Brazil 2

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* V. H. and J. M. H. contributed equally to this work.

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Address of corresponding authors: Daniel K. Mulkey Dept. Physiology and Neurobiology University of Connecticut 75 N. Eagleville Rd. Unit 3156 Storrs, CT 06269 [email protected] 860 486 5700 860 486 3303

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ACKNOWLEDGMENTS This work was supported by funds from the National Institutes of Health Grants HL104101 (DKM) and NS073981 (AVT) and public funding from the São Paulo Research Foundation (FAPESP) grants 09/54888-7, 13/10573-8 (TSM) and 10/09776-3 (ACT).

Virginia E. Hawkins Dept. Physiology and Neurobiology University of Connecticut 75 N. Eagleville Rd. Unit 3156 Storrs, CT 06269 [email protected] 860 486 5572 860 486 3303

Abbreviated Title: Ih contributes to serotonergic modulation of breathing Keywords: retrotrapezoid nucleus, serotonin; 5-HT7, cAMP, Gs-coupled receptor Abstract Introduction Discussion Total including references Number of figures: Number of pages:

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Copyright © 2014 by the American Physiological Society.

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ABSTRACT

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Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H+-dependent

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drive to breathe and function as an integration center for the respiratory network, including

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serotonergic raphe neurons.

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chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward

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current. HCN channels are the molecular correlate of the hyperpolarization-activated inward

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current (Ih) and have a high propensity for modulation by serotonin. To investigate whether

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HCN channels contribute to basal activity and serotonergic modulation of RTN

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chemoreceptors, we characterize resting activity and effects of serotonin on RTN

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chemoreceptors in vitro, and on respiratory activity of anesthetized rats in the presence or

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absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs+) channels. We found in

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vivo that, bilateral RTN injections of ZD7288 increased respiratory activity, and in vitro HCN

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channel blockade increased activity of RTN chemoreceptors under control conditions but this

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was blunted by KCNQ channel inhibition. Furthermore, in vivo, unilateral RTN injection of

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XE991 plus ZD7288 eliminated the serotonin response and in vitro, serotonin-sensitivity was

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eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker).

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Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor

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blocker and mimicked by a 5-HT7-receptor agonist.

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depolarizing-shift in the voltage-dependent activation of Ih. These results suggest that HCN

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channels contribute to resting chemoreceptor activity, and that serotonin activates RTN

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chemoreceptors and breathing in part by a 5-HT7 receptor-dependent mechanism and

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downstream activation of Ih.

We recently showed that serotonergic modulation of RTN

In addition, serotonin caused a

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INTRODUCTION

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Central chemoreception is the mechanism by which the brain regulates breathing in response

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to changes in CO2/H+. A region of the brainstem called the retrotrapezoid nucleus (RTN) is

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an important site of chemoreception. Neurons (Wang et al. 2013) and astrocytes (Gourine et

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al. 2010) in this region sense changes in CO2/H+ to produce an integrated CO2/H+-dependent

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drive to breathe. Chemosensitive RTN neurons also integrate respiratory drive from other

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regions of the respiratory circuit such as serotonergic raphe neurons (Mulkey et al. 2007).

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Serotonin is a potent modulator of breathing (Richerson 2004; Corcoran et al. 2014),

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including at the level of the RTN, where serotonin has been shown to stimulate

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chemoreceptor activity in vitro and increase breathing in conscious and anesthetized animals

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(Hawryluk et al. 2012). In addition, disruption of serotonergic signaling has been shown to

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decrease the respiratory chemoreflex (Hodges et al. 2008; Ray et al. 2011) and may

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contribute to respiratory failure associated with sudden unexpected death in epilepsy

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(SUDEP) (Massey et al, 2014; Buchanan et al., 2014). Despite this critical physiological

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role, mechanisms by which serotonin activates RTN chemoreceptors have yet to be fully

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elucidated.

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Serotonergic signaling is mediated through seven receptor families (5-HT1-7), including Gi

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(5-HT1), Gq-(5HT2) and Gs- (5HT4,7) coupled receptors (Richter et al. 2003). In the RTN,

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serotonin sensitivity can be blocked with ketanserin (Mulkey et al. 2007), an antagonist for

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both 5-HT2 and 5-HT7 receptors (Beique et al. 2004; Jasper et al. 1997). Evidence also

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indicates that serotonergic modulation of RTN neurons is determined, in part, by inhibition of

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KCNQ channels (Hawryluk et al. 2012), most likely by a 5-HT2 Gq-coupled receptor

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mechanism (Mulkey et al. 2007; Delmas and Brown 2005). However, RTN chemoreceptors

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retain approximately half their serotonin sensitivity when KCNQ channels are blocked,

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suggesting involvement of other channels. Preliminary data suggests that when KCNQ

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channels are blocked, serotonin sensitivity of RTN neurons can be eliminated by inhibiting

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hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels (Hawryluk et al. 2012),

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which produce a hyperpolarization-activated inward current (Ih). HCN channels are known

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to be activated by cAMP (DiFrancesco and Totora 1991) and Gs-coupled receptor signaling

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(Ulens and Tytgat 2001).

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Therefore, we hypothesize that HCN channels contribute to

serotonergic modulation of RTN chemoreceptors.

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Here, we demonstrate the functional presence of both KCNQ and HCN channels in RTN

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chemoreceptors neurons. We show that inhibition of HCN channels increases ventilation in

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vivo and the firing rate of RTN chemoreceptors under control conditions, but not when Ca2+

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channels or other Cd2+-sensitive currents are blocked (Abbruzzese et al., 2010), suggesting

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that HCN channels dampen basal chemoreceptor excitability by a Cd+2-sensitive mechanism

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that may involve voltage-dependent Ca2+ channels, as described in other brain regions (Tsay

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et al. 2007). Inhibition of HCN channels also blunted serotonergic modulation of RTN

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neurons in vitro, and attenuated the ventilatory response to serotonin in vivo. During HCN

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and KCNQ blockade, serotonin failed to stimulate chemoreceptor activity or breathing,

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suggesting that coordinated activity of these channels is entirely responsible for serotonergic

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modulation of RTN chemoreceptors. Further, inhibition of adenylate cyclase blocked the Ih

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component of the serotonergic response in RTN chemoreceptors, suggesting involvement of a

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GS-coupled receptor signaling. Consistent with this, serotonin-mediated activation of RTN

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chemoreceptors was blocked with a 5-HT7-receptor blocker and mimicked by a 5-HT7-

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receptor activator. We also find that serotonin shifted the voltage-dependence of Ih

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activation to more depolarized potentials. These results build on our understanding of the

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mechanisms by which serotonin regulates breathing by identifying a novel role of HCN

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channels and 5-HT7 receptors in the serotonergic modulation of RTN chemoreceptors.

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Furthermore, considering that HCN and KCNQ are epilepsy associated ion channels and

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disruption of serotoninergic signaling may contribute to SUDEP, these results suggest that

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HCN and KCNQ channels are common substrates for respiratory dysfunction and epilepsy.

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METHODS

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Animals

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Animal use was in accordance with guidelines approved by the Universities of Connecticut

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and São Paulo Institutional Animal Care and Use Committees. The in vivo experiments were

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performed on male Wistar rats weighing 250-300 g (8-10 months old). In vitro experiments

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were performed on brain slices isolated from rat pups (7-12 days postnatal).

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In vivo preparation

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The surgical procedures and experimental protocols were done as described previously

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(Hawryluk et al. 2012). Briefly, general anesthesia was induced with 5% halothane in 100%

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O2. Artificial ventilation with 1.4-1.5% halothane in 100% O2 was maintained throughout

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surgery. Standard surgical procedures (bilateral vagotomy, arterial cannulation, phrenic nerve

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dissection, and dorsal transcerebellar access to the ventrolateral medulla oblongata) were

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used. After surgery, halothane was gradually replaced by urethane (1.2 g/kg, administered i.v.

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over 20 min). This initial dose was supplemented hourly and at least twice with additional

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urethane injections of 0.1 g/kg. After a total of 1.4-1.5 g/kg, the level of anesthesia was stable

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for the duration of the experiment (up to 4 hours after initial anesthetic crossover). Rats were

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ventilated with 100% O2 throughout the experiment and muscle relaxation was performed

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with pancuronium (1 mg/kg i.v.). Rectal temperature was maintained at 37°C, and end-tidal

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CO2 was monitored with a microcapnometer. The adequacy of anesthesia was continually

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monitored by testing for the absence of arterial pressure or phrenic nerve discharge (PND)

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responses to firm toe or tail pinch. After these criteria were satisfied, the muscle relaxant

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pancuronium was administered i.v. at an initial dose of 1 mg/kg, and the adequacy of the

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anesthesia was thereafter gauged by the lack of cardiorespiratory responses to a firm toe

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pinch.

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In vivo recordings of physiological parameters

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As described (Mulkey et al. 2004), mean arterial pressure (MAP) and PND were digitized

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with a Micro 1401 (Cambridge Electronic Design), stored on a computer, and processed off-

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line with Spike 2 software. Integrated PND (iPND) was obtained after rectification and

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smoothing (τ = 0.015 s) of the original signal, which was acquired with a 30-300 Hz

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bandpass filter. PND amplitude, frequency and the product of PND frequency and amplitude

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(i.e., the neural equivalent of minute ventilation) were expressed for each animal on a scale

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ranging from 0 (value during apnea) to 100 (value while breathing 10% CO2).

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Hypercapnia was produced by addition of pure CO2 to the 100% O2 supplied by artificial

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ventilation to increase the maximum end-expiratory CO2 to 9.5–10%. End-expiratory CO2

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was maintained for 5 min, and CO2 was removed. The level of CO2 required to stimulate

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breathing was identified by increasing inspired CO2 in a stepwise fashion (Δ 1%) from 2-3%

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up to 9-10% (5 min per step), and PND was measured during the last 30 s of each step when

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end-expiratory CO2 and PND appeared to have reached equilibrium. The CO2 PND threshold

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was determined as the % CO2 that first elicited PND. End-expiratory CO2 was measured by

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averaging the maximum values recorded from 50 consecutive breaths at the midpoint of the

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time interval sampled. One arbitrary unit represents the highest value of PND measured at

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steady state with end-expiratory CO2 set at 9.5–10%.

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Serotonin was injected by pressure (40–60 psi, 4 msec pulses, 50 nl in 3–5 sec) through glass

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pipettes (20 μm outside diameter) filled with 1 mM serotonin creatinine sulfate in a pH 7.3

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normal saline solution containing 1% (v/v) fluorescent microbeads (for histological

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verification of injection sites). The concentration of serotonin used in these experiments was

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based on the EC50 response elicited by serotonin injections into the hypoglossal nucleus of

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anesthetized rats (Fenik and Veasey 2003). The pipette tip was placed 200 μm ventral to the

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caudal edge of the facial motor nucleus under electrophysiological guidance as described

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below for drug injections. In all cases, correct injection was verified by post-mortem

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histological inspection of the location of fluorescent microbeads (Lumafluor, New City, NY).

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Histology

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At the end of each in vivo experiment, rats were deeply anesthetized with halothane and

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perfused through the heart with PBS, pH 7.4, followed by paraformaldehyde (4% in 0.1 M

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phosphate buffer, pH 7.4). The brains were removed and the medulla cut into 40-μm-thick

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coronal sections (Vibratome 1000S Plus). We confirmed that injection sites were located

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within the RTN by fluorescent visualization using an Axioskop 2 microscope (Carl Zeiss,

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Thornwood, NY). Sections from different brains were aligned with respect to a reference

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section, which was the most caudal section containing an identifiable cluster of facial motor

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neurons. This reference section was assigned a value of 11.6 mm caudal to bregma (bregma: -

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11.6 mm; Paxinos and Watson 1989).

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Brain slice preparation and slice-patch electrophysiology

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As described (Mulkey et al. 2004; Wenker et al. 2012b), neonatal rats (P7-12 days postnatal)

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were decapitated under ketamine/xylazine anesthesia, and transverse brainstem slices (300

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μm) were cut using a microslicer (DSK 1500E; Dosaka) in ice-cold substituted Ringer

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solution containing (in mM): 260 sucrose, 3 KCl, 5 MgCl2, 1 CaCl2, 1.25 NaH2PO4, 26

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NaHCO3, 10 glucose, and 1 kynurenic acid. Slices were incubated for ∼30 min at 37°C and

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subsequently at room temperature in normal Ringer solution (in mM): 130 NaCl, 3 KCl, 2

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MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. All solutions were equilibrated

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with 95% O2-5% CO2, extracellular pH 7.35.

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Individual slices containing the RTN were transferred to a recording chamber mounted on a

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fixed-stage microscope (Zeiss Axioskop FS) and perfused continuously (∼2 ml min−1) with

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normal Ringer solution bubbled with 95% O2-5% CO2. Hypercapnic solution was made by

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equilibrating normal Ringer solution with 15% CO2 balance O2 (pHo ~6.90). All recordings

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were made from neurons located within 100 µm of the ventral surface and below the caudal

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end of the facial motor nucleus using an Axopatch 200B patch-clamp amplifier, digitized

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with a Digidata 1322A A/D converter, and recorded using pCLAMP 10.0 software

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(Molecular Devices, Sunnyvale, CA). Recordings were obtained at room temperature

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(∼22°C) with electrodes pulled from borosilicate glass capillaries (Harvard Apparatus,

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Hamden, CT) on a two-stage puller (P89; Sutter Instrument) to a DC resistance of 5–7 MΩ

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when filled with an internal solution containing (in mM): 120 KCH3SO3; 4 NaCl; 1 MgCl2;

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0.5 CaCl2; 10 HEPES; 10 EGTA; 3 Mg-ATP; and 0.3 GTP-Tris (pH 7.2), electrode tips were

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coated with Sylgard 184 (Dow Corning Corporation, Midland, MI). All recordings of firing

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rate were performed using the cell-attached configuration and firing rate histograms were

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generated by integrating action potential discharge in 10 sec bins and plotted using Spike 5.0

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software. Whole cell current-clamp recordings were made using internal solution containing

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(in mM): 125 potassium gluconate; 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 4

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Mg-ATP; 0.3 Na-GTP; 0.1 EGTA; 10 2-Tris-phosphocreatine and 0.05% biocytin (pH 7.3).

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Cells were held at -60 mV and depolarizing steps from -100pA were applied (Δ +10 mV).

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Input resistance (Rin) was calculated from the voltage responses to hyperpolarizing steps.

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Initial spike frequency was measured following a 50 pA current injection and the rebound

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spike latency taken as the mean time to first action potential following hyperpolarizing steps

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(-100 to -70 pA). The sag ratio was expressed as [(1-∆Vss/∆Vmin) x 100%], where ∆Vss =

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resting membrane potential (RMP) - Vss, ∆Vmin = RMP – Vmin, RMP is the resting membrane

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potential, Vs is the steady state potential and Vmin is the initial minimum potential. Voltage-

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clamp recordings were made at a holding potential of -60 mV and in the presence of

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tetrodotoxin (TTX; 0.5 μM; Alomone Labs) to block action potentials and barium (1 mM) to

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block leak K+ channels.

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relationships were determined by applying hyperpolarizing voltage steps from -60 to -150

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mV for 1000 ms (Δ -10 mV). The maximal amplitude of Ih was quantified as the size of the

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time-dependent current during a step to -150 mV. The voltage dependence of Ih activation

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was obtained from tail currents measured at -70 mV following a series of hyperpolarizing

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steps; those data were normalized, plotted as a function of the initial step potential, and fitted

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to a Boltzmann function for calculation of voltage for half-maximal activation (V50) of Ih.

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For whole cell recordings series resistance (Ra) was typically 10% during an experiment.

Holding current, conductance, and current–voltage (I–V)

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Drugs

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All drugs were purchased from Tocris Bioscience unless otherwise stated. For in vivo

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experiments, the KCNQ channel blocker XE991 (Abcam Biochemicals, Cambridge, MA)

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and the HCN channel blocker ZD7288 (Sigma-Aldrich) were diluted to 50 µM in sterile

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saline (pH 7.4) and injected into the RTN using a single-barrel glass pipette (tip diameter of

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20 µm) connected to a pressure injector (Picospritzer III, Parker Hannifin Corp, Cleveland,

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OH). Since the RTN is located bilaterally and both structures contribute to the chemoreflex,

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to test for contributions of KCNQ and HCN channels to the CO2/H+-drive to breathe we

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injected XE991 or ZD7288 bilaterally. However, since unilateral RTN injection of serotonin

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can elicit a ventilatory response, we also tested contributions of KCNQ and HCN to this

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response by making same side injections of the blockers. For each injection we delivered a

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volume of 50 nl over a period of 5s. These glass pipettes also allowed for recordings of field-

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potential properties that were used to help direct the electrode tip to the desired site.

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Injections in the RTN region were guided by recordings of the facial field potential (Brown

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and Guyenet 1985), and were placed 250 μm below the lower edge of the field, 1.7 mm

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lateral to the midline, and 200 μm rostral to the caudal end of the field. Recordings were

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made on one side only; the second injection was made 1-2 min later at the same level on the

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contralateral side. We included a 2% dilution of fluorescent latex microbeads with all drug

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applications to mark the injection sites and verify the spread of the injections (Takaura and

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Moreira 2011; Takakura et al. 2011). For in vitro experiments, we bath-applied serotonin

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hydrochloride (5 µM, Sigma-Aldrich), XE991 (10 µM, Abcam Biochemicals) to block

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KCNQ channels, ZD7288 (50µM) or CsCl (2mM, Sigma-Aldrich) to block HCN channels,

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CdCl2 (100 µM, Sigma-Aldrich) to block voltage-gated calcium channels, synaptic block

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perfusate contained kynurenic acid (1 mM, Sigma-Aldrich) to block ionotropic glutamate

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receptors, bicuculline (10 µM, Sigma-Aldrich) to block GABAA receptors and strychnine (20

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µM, Sigma-Aldrich) to block glycine receptors, SQ22536 (100 µM) to inhibit adenylate

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cyclase activity, SB258719 (10 µM) to block 5-HT7 receptors, 5-carboxamidotryptamine (5-

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CT, 5 µM) to activate 5-HT7 as well as several Gi-coupled receptors (i.e., 5-HT1A/1B/1D/5),

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WAY100635 (0.1 µM) to block 5-HT1A receptors and the specific 5-HT7 agonist LP-44 (2

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uM).

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Statistical Analysis

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Data are reported as mean ± standard error of the mean. Statistical analysis was performed

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using GraphPad Prism version 3.0 software. Data was assessed for normality and the student

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t-test or one-way ANOVA followed by Newman-Keuls multiple-comparisons test were used

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as appropriate. The relevant values used for statistical analysis are provided in the results

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section.

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RESULTS

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This study consists of three sets of experiments designed to address the mechanistic

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underpinnings of serotonin-induced respiratory activity in the RTN. First, to determine if

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serotonin-mediated cardiorespiratory control within the RTN involves HCN channels, we

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examined breathing and blood pressure in anesthetized rats injected with serotonin into the

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RTN, either alone or after injection of KCNQ and/or HCN channel blockers.

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channels were blocked in a number of experiments to reveal the KCNQ independent

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component to the serotonin response. Second, to determine whether HCN channels regulate

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excitability and serotonergic modulation of RTN chemoreceptors we tested effects of

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blocking HCN channels, alone and during blockade of KCNQ channels, on RTN neuron

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activity (basal firing rate and serotonin-sensitivity) in the brain slice preparation.

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identify mechanism(s) underlying serotonergic modulation of HCN channels in RTN

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chemoreceptors, we assessed serotonin responses when adenylate cyclase activity is blocked,

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and tested the effects of subtype-specific serotonin receptor modulators on RTN

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chemoreceptor activity.

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effects of serotonin on amplitude and voltage-dependent activation of Ih.

KCNQ

Third, to

Furthermore, in voltage-clamp experiments, we determined the

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Here we focus on chemosensitive RTN neurons which have been shown to express the

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transcription factor Phox2b (Lazarenko et al. 2009; Stornetta et al. 2006). However, Phox2b

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is also expressed by other cells in relatively close proximity to the RTN, including

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catecholaminergic neurons (C1 and A5), facial motor neurons, and the superior salivatory

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nucleus (Kang et al. 2007). Therefore, we chose to functionally identify CO2/H+-sensitive

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RTN neurons based on their characteristic firing rate response to CO2/H+. Neurons were

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considered chemosensitive if they increased firing rate by ≥ 1.5 Hz following exposure to

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15% CO2. This level of CO2/H+ response is similar to what we, and others, have reported for

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chemosensitive RTN neurons in vitro (Mulkey et al. 2006; Ritucci et al. 2005; Wenker et al.

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2010). RTN neurons that did not exhibit this minimum response were considered non-

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chemosensitive and excluded from this study.

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HCN channels in the RTN contribute to basal activity and serotonergic modulation of respiratory drive

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modulation of RTN neurons (Hawryluk et al. 2012). Preliminary in vitro evidence also

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suggested that HCN channels are expressed in RTN neurons and contribute to serotonin

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responses (Hawryluk et al. 2012). To establish the functional significance of HCN channels

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in controlling breathing, we tested whether RTN injection of an HCN channel blocker

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(ZD7288) affects resting respiratory motor output of anesthetized rats.

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whether the ventilatory response to serotonin was altered by RTN injection of ZD7288. All

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injections (uni- or bilateral) were placed 250 µm below the facial motor nucleus and 200 µm

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rostral to the caudal end of this nucleus to target the region containing the highest density of

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CO2-sensitive RTN neurons (Mulkey et al. 2004; Takakura and Moreira 2011; Takakura et al.

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2011) (Figs. 1E and 2B).

Previous evidence indicates that KCNQ channels regulate activity and serotonergic

We then tested

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In anesthetized animals, we found that bilateral RTN injections of ZD7288 (50 µM, 50 nl

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each side) increased resting respiratory output as measured by a change in phrenic nerve

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discharge, PND. As described previously, bilateral injections of XE991 into the RTN

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increased the neural equivalent of minute ventilation (mvPND, product of PND amplitude

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and frequency) (Fig. 1B,E). Bilateral injections of ZD7288 to inhibit HCN channels also

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increased respiratory activity by 48 ± 7% (F(3,56) = 144.12, p 0.05). However,

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bilateral application of XE991 and/or ZD7288 shifted the CO2 threshold (i.e., the level of

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CO2 required to stimulate ventilatory output) from 5.3 ± 0.09 to 4.5 ± 0.1% (F(3,56) = 87.44, p

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< 0.01) (Fig. 1D). These data suggest that inhibition of KCNQ and HCN channels increases

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excitability of RTN chemoreceptors and consequently the respiratory system responses to

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changes in CO2.

We also found that the

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In agreement with previous studies (Hawryluk et al. 2012; Mulkey et al. 2007), unilateral

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RTN injection of serotonin (1 mM) increased PND amplitude by 37 ± 5% above baseline (F(3,

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29)=

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unilateral RTN injection of XE991 (50 μM, 50 nl) or the HCN blocker ZD7288 (50 µM, 50

340

nl) had no discernible effect on resting PND (F(3, 29)= 0.011, p > 0.05; Fig. 2A). However,

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injection with either channel blocker significantly blunted—and together essentially

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eliminated—serotonin modulation of respiratory activity (Figs. 2A, C).

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injection of serotonin after application of either XE991 (Fig. 2A1) or ZD7288 (Fig. 2A2)

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only increased PND amplitude 22 ± 2% and 29 ± 3% above baseline, respectively. This

129.83, p < 0.01; Figs. 2A, C). After respiratory activity returned to control levels,

For example,

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reflects a 40 ± 3% and 21 ± 2% decrease in serotonin responsiveness, respectively, relative to

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control conditions (F(3,29) = 167.74, p 0.05; Fig. 2A, summary data not shown), suggesting that

351

KCNQ and HCN channels are not involved in cardiovascular regulation at this level of the

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rostral ventrolateral medulla. These results demonstrate that KCNQ channels and HCN

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channels within the RTN serve as functional determinants of serotonergic modulation of

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respiratory drive.

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HCN channels contribute to both resting firing behavior and serotonergic modulation of RTN chemoreceptors in vitro

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characterized membrane potential responses to hyperpolarizing and depolarizing current steps

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under control conditions and during HCN channel blockade. In the whole-cell current-clamp

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configuration, chemosensitive RTN neurons have an Rin of 910 ± 83MΩ and exhibit a sag

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ratio of 32.3 ± 4.6 % in response to a -100 pA hyperpolarizing voltage step (Fig. 3A). At the

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end of the hyperpolarizing step chemosensitive RTN neurons produce a rebound spike with a

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latency of 109 ± 19 ms (Fig. 3A4). Bath application of ZD7288 (50µM) increased Rin to

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1196.0 ± 64.5 MΩ, eliminated the sag (to a sag ratio of only 3.1 ± 4.5 %) (T(5) = 5.96, p =

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0.0019; Fig. 3A2), and increased the rebound spike latency (T(5) = 3.68, p = 0.0142; Fig.

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3A4). We also found that the firing rate response to a depolarizing current injection (20 pA)

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was unaffected by ZD7288 (Fig. 3A3). This is not surprising considering that HCN channels

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are inhibited by depolarization. Furthermore, blocking KCNQ channels with XE991 (10µM)

371

caused a modest increase in Rin (T(4) = 4.71, p = 0.0093) but with no effect on the sag

To examine the contribution of HCN channels to activity of RTN chemoreceptors, we first

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potential or rebound spike latency (Figs. 3B).

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instantaneous spike frequency elicited by 20 pA current injections (Fig. 3B4). These results

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indicate that HCN and KCNQ channels regulate intrinsic electrical properties of

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chemosensitive RTN neurons.

In addition, XE991 also increased the

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Next, to determine whether HCN channels regulate excitability of RTN chemoreceptors, we

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tested the effects of HCN channel blockers (ZD7288 or Cs+) on basal activity in the rat

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brainstem slice preparation. We also used HCN channel blockers alone and in conjunction

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with XE991 to test for contributions of HCN channels to the serotonin response of RTN

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chemoreceptors. We found that under control conditions, inhibition of HCN channels with

382

ZD7288 (50 µM) or Cs+ (2 mM) increased chemoreceptor activity by 1.01 ± 0.17 Hz (T(16) =

383

5.93, p < 0.0001; Figs. 4A,B), suggesting that HCN channels can limit basal activity of RTN

384

chemoreceptors.

385

chemoreceptor activity (T(15) = 1.497, p = 0.1551) so data was pooled.

386

from other brain regions, HCN channels may inhibit neural activity by shunting synaptic

387

potentials (Stuart and Spruston 1998) or by influencing activity of other voltage-dependent

388

channels including KCNQ (George et al. 2009) and Ca2+ channels (Tsay et al. 2007). To

389

determine which of these possibilities may contribute to the inhibitory effects of HCN

390

channel on resting activity of RTN chemoreceptors, we characterized effects of HCN channel

391

blockade i) when excitatory and inhibitory receptors are blocked with a cocktail containing

392

kynurenic acid (1 mM), bicuculline (10 µM) and strychnine (20 µM) (i.e., synaptic

393

blockade); ii) when KCNQ channels were blocked with XE991, and iii) when voltage-gated

394

Ca2+ channels were blocked with Cd2+ (100 µM). We found that the firing rate response of

395

RTN chemoreceptors to HCN channel blockade (i.e., bath application of ZD7288 or Cs+) was

396

unaffected by synaptic blockade but was reduced to a change of 0.38 ± 0.16 Hz in XE991 and

Note that there was no difference in effects of ZD7288 and Cs+ on Based on evidence

15

397

eliminated (0.08 ± 0.06 Hz) in Cd2+ (F(3,39) = 7.13, p = 0.0006; Fig. 4A,B), suggesting that

398

HCN channels limit basal activity of RTN chemoreceptors indirectly by mechanisms

399

involving KCNQ and Cd+2-sensitive channels including Ca2+ channels.

400 401

As expected, bath application of serotonin (5 µM) increased activity of these cells by 1.18 ±

402

0.13 Hz (paired T(14) = 9.00, p < 0.001; Figs. 4A,C) and it did so in a reversible and

403

repeatable manner (ratio of the third serotonin response divided by the second response was

404

0.9 ± 0.1; Hawryluk et al. 2012). Bath application of XE991 (10 µM) to block KCNQ

405

channels increased baseline firing by 1.04 ± 0.43 Hz (paired T(10) = 5.42, p = 0.0003) and

406

blunted serotonin-responsiveness by 54.3 ± 11.9% (F(3,38) = 20.06, p < 0.0001; Figs. 4A,C), as

407

previously reported (Hawryluk et al. 2012). Application of ZD7288 (50µM) to block HCN

408

channels (with DC current injection to adjust baseline to control levels) reduced subsequent

409

serotonin response to an increase of only 0.57 ± 0.09 Hz (Fig. 4A,C). Notably, when both

410

KCNQ and HCN channels are blocked with XE991 and ZD7288 or Cs+ (with baseline

411

activity adjusted by DC current injection to near control levels), serotonin increased firing

412

rate less than under control conditions (Figs. 4A, C), i.e., serotonin increased activity by only

413

0.11 ± 0.04 Hz in XE991 plus ZD7288 and only 0.07 ± 0.17 Hz in XE991 plus Cs+ . When

414

the inhibitory contribution of HCN channel activity was blocked by Cd2+, exposure to

415

serotonin stimulated chemoreceptor activity by an amount similar to that of control

416

conditions (T(5) = 0.66; p = 0.5407; Figs 4A2,F), as expected for activation of an inward

417

depolarizing current. It should be noted that CO2/H+-sensitivity of RTN neurons was retained

418

when HCN channels were blocked with ZD7288 or Cs+ (Figs 4D, E). These results suggest

419

that both KCNQ and HCN channels are essential components of serotonergic modulation in

420

RTN chemoreceptor neurons. Collectively these results also suggest that the net contribution

421

of HCN channels to RTN chemoreceptor basal activity is to limit excitability indirectly by

16

422

influencing activity of KCNQ and Ca2+ channels.

423

presumably a select subset of HCN channels by serotonin can increase chemoreceptor

424

activity and contribute to the excitatory effects of serotonin on breathing.

However, targeted activation of

425 426 427 428 429

Serotonin activates Ih in RTN neurons by a Gs-coupled receptor and cAMP-dependent mechanism

430

KCNQ and HCN channels. HCN channels are known to be activated by Gs-coupled receptors

431

(Ulens and Tygat 2001) but inhibited by Gq-coupled receptor signaling (Lui et al. 2003),

432

indicating that HCN channel activity may be regulated by diverse signal transduction

433

pathways. Gs-coupled receptor signaling involves activation of adenylate cyclase and

434

increased cAMP production (Wettschureck and Offermanns 2005), and it is well known that

435

cAMP can directly bind HCN channels to facilitate Ih (DiFrancesco et al. 1991).

436

therefore tested the possibility that serotonin activates HCN channels by Gs-coupled receptor

437

signaling.

It is unlikely that a single serotonin receptor subtype mediates these divergent effects on

We

438 439

We first sought to determine if adenylate cyclase activity is required for serotonin-mediated

440

activation of Ih. To study serotonin modulation of HCN channels in relative isolation, we

441

performed these experiments in the presence of XE991 (10 µM) to block KCNQ channels.

442

As before, KCNQ channel blockade decreased the firing rate response to serotonin (control,

443

1.34 ± 0.21 Hz; XE991, 0.81 ± 0.22 Hz) (F(2,4) = 11.38, p = 0.0046; Figs. 5A, C). During

444

KCNQ channel blockade (and after baseline activity was adjusted by DC current injection to

445

near control levels) application of the adenylate cyclase inhibitor SQ22536 (100 µM)

446

increased the firing rate of RTN chemoreceptors by 0.80 ± 0.70 Hz (T(4) = 6.43, p = 0.0030;

447

Figs. 5A, B). However, when HCN channels are blocked with Cs+ (2 mM), bath application

448

of SQ22536 still increase in chemoreceptor activity by an amount similar to under control 17

449

conditions (N=3; data not shown), suggesting cAMP can modulate activity of RTN

450

chemoreceptors by mechanisms in addition to activation of HCN.

451

presence of SQ22536 inhibition of HCN with Cs+ increased RTN chemoreceptor activity by

452

an amount similar to under control conditions (N=4; data not shown), suggesting that cAMP

453

is not required for the maintenance of HCN channel activity under basal conditions. This

454

later finding is consistent with the possibility that the inhibitory contribution of HCN to basal

455

activity of RTN neurons is determined indirectly by the voltage dependent modulation of

456

KCNQ and Ca2+ channels. Nevertheless, in the presence of XE991 and SQ22536, exposure

457

to serotonin increased firing rate by only 0.25 ± 0.06 Hz (F(2,4) = 11.38, p = 0.0046; Figs. 5A,

458

C). These findings suggest that adenylate cyclase activity is required for the Ih component of

459

serotonergic modulation of chemosensitive RTN neurons.

Furthermore, in the

460 461

In other brain regions, cAMP-mediated activation of HCN channels results from a

462

depolarizing shift in voltage-dependent activation of Ih (DiFrancesco and Totora 1991). To

463

confirm this is the case for RTN chemoreceptors, we characterized effects of serotonin on the

464

maximum amplitude and voltage-dependent activation of Ih. Maximum Ih amplitude was

465

defined as the difference between instantaneous and steady-state currents during a -150 mV

466

step (represented in Fig 6A by dashed lines). In the whole-cell voltage-clamp configuration

467

(holding potential of -60 mV and in TTX [0.5 µM] to block action potentials), Ih was

468

detected by its characteristic time-dependent activation during hyperpolarizing voltage steps.

469

The maximum amplitude of Ih in RTN chemoreceptors was 64.55 ± 14.53 pA under control

470

conditions and 57.1 ± 12.5 pA during exposure to serotonin (Fig. 6B). In addition, bath

471

application of ZD7288 largely eliminated Ih (F(2,21) = 5.21, p = 0.0146; Fig. 6B). These

472

results suggest that serotonin does not affect maximum Ih in these cells.

473

18

474

To determine the effects of serotonin on the voltage dependency of Ih activation we measured

475

tail currents at a fixed potential of -70 mV under control conditions and after activating Ih

476

with a series of hyperpolarizing voltage steps. Normalized Ih tail currents were plotted as a

477

function of membrane potential during the initial hyperpolarizing steps and fitted with a

478

Boltzmann function to determine the half-activation voltage (V1/2) (Sirois et al. 2002; Wenker

479

et al. 2012a). The V1/2 for Ih activation averaged -120.1 ± 3.2 mV in control conditions and -

480

103.7 ± 6.0 mV in serotonin (T(4) = 2.35, p = 0.0394; Fig. 6C), indicating that serotonin

481

caused a depolarizing shift of ~16 mV in the voltage-dependent activation of Ih. These

482

results demonstrate Ih within RTN chemoreceptor neurons and strongly suggest that

483

serotonin activates HCN channels in the RTN by a Gs- and cAMP-dependent mechanism.

484 485

Several members of the serotonin receptor family are Gs-coupled including 5-HT4, 5-HT6

486

and 5-HT7. Of these, 5-HT7 receptors in particular are inhibited by ketanserin (Lesage et al.

487

1998; Shen et al. 1993). Since ketanserin was shown to block the serotonin responses of

488

chemosensitive RTN neurons (Mulkey et al. 2007), we considered the possibility that 5-HT7

489

receptors mediate the effects of serotonin on Ih. To test for involvement of 5-HT7 receptors,

490

we used 5-CT, which activates 5-HT7 as well as several Gi-coupled receptors (i.e., 5-

491

HT1A/1B/1D/5), and WAY100635, which selectively blocks 5-HT1A receptors (Beique et al.

492

2004). If exposure to 5-CT mimicked effects of serotonin on chemoreceptor activity under

493

control and WAY100635-exposed conditions by a Ih-dependent mechanism, this would

494

suggest that 5-HT7 receptors contribute to serotonin-mediated activation of HCN channels in

495

RTN neurons. Finally, we tested this by using the 5-HT7 specific antagonist SB258719, along

496

with the 5-HT7 receptor agonist LP-44.

497

19

498

We found that bath application of 5-CT (5 µM) increased chemoreceptor activity from 0.97 ±

499

0.37 Hz to 1.48 ± 0.41 Hz (T(12) = 7.02, p < 0.0001; Figs. 7A-B). This change in activity

500

(0.52 ± 0.07 Hz) was similar in magnitude to the serotonin response when KCNQ channels

501

were blocked (Fig. 4C). A second exposure to 5-CT, this time when HCN channels were

502

blocked with ZD7288, failed to significantly increase firing rate (with a rise of only 0.16 ±

503

0.05 Hz; F(4,27) = 7.83, p = 0.0003; Figs. 7A-B). Bath application of WAY100635 had no

504

effect on baseline activity (T(6) = 1.67, p = 0.1452) or firing-rate response to 5-CT (F(4,27) =

505

7.83, p = 0.0003; Fig. 7B), suggesting that 5-HT1A receptors do not exert direct or indirect

506

control (e.g., modulation of inhibitory input) over chemosensitive RTN neurons in vitro.

507

Application of a 5-HT7 receptor blocker (SB258719 10 µM) reduced the firing rate response

508

of RTN chemoreceptors to serotonin by approximately half (T(3) = 6.98 , p = 0.006, data not

509

illustrated)

510

Furthermore, the 5-HT7 agonist LP-44 (2 µM) induced a 0.77 ± 0.16 Hz increase in

511

chemoreceptor firing rate and this response was blocked by a 5-HT7 antagonist (SB258719;

512

10µM) (T(3) = 5.56, p = 0.0309; Fig. 7D).

513

receptors mediate the effects of serotonin on HCN channels in RTN neurons.

and

eliminated the 5-CT response (T(4) = 3.70, p =

0.0208; Fig. 7C).

Together, these results suggest that 5-HT7

514 515

DISCUSSION

516

The RTN is an important locus of respiratory control, as neurons in this region provide a

517

CO2/H+-dependent drive to breathe (Mulkey et al. 2004; Wang et al. 2013) and serve as a

518

point of convergence for other respiratory centers including the medullary raphe (Hawryluk

519

et al. 2012; Mulkey et al. 2007).

520

breathing, we are only just beginning to understand the ionic mechanisms that control their

521

resting membrane potential and response to neurotransmitters. Recent evidence indicates that

522

KCNQ channels in RTN neurons are key determinants of resting firing behavior and

Despite the importance of RTN chemoreceptors in

20

523

serotonergic modulation of breathing (Hawryluk et al. 2012). Here we show that HCN

524

channels are expressed in RTN chemoreceptor neurons and are a second essential regulator of

525

serotonin-mediated chemoreceptor function.

526

influence repetitive firing behavior of RTN chemoreceptors by depolarizing membrane

527

potential during hyperpolarization and shortening the rebound spike latency. We also show

528

that inhibition of HCN channels increased spontaneous activity, blunted serotonergic

529

modulation of RTN neurons in vitro, and attenuated the ventilatory response to serotonin in

530

vivo. The mechanism underlying serotonergic activation of HCN channels in RTN neurons

531

appears to involve adenylate cyclase activity and can be mimicked or blunted by drugs that

532

activate or block 5-HT7 receptors. Together with previous data, these results suggest that

533

serotonin regulates activity of RTN neurons by Gs-mediated activation of HCN channels and

534

Gq-mediated inhibition of KCNQ channels.

Specifically, we show that HCN channels

535 536

A limitation of our experimental approach is that our in vivo and in vitro conditions differ in

537

terms of animal age, temperature and CO2/H+ stimulus intensity. Age and temperature can

538

influence many physiological parameters including adenylate cyclase activity and HCN

539

channel kinetics, both of which are increased by warming from room to body temperature.

540

Therefore, it is possible that we underestimate the extent to which these channels contribute

541

to chemoreceptor activity. However, we have shown previously that chemosensitive RTN

542

neurons in slices from rat pups (recorded at room temperature) respond to raphe

543

neurotransmitters (e.g., serotonin) in a manner similar to chemosensitive RTN neurons in

544

anesthetized adult rats (Mulkey et al. 2007). Therefore, it is unlikely that fundamental

545

mechanisms underlying neurotransmitter modulation of these neurons (i.e., HCN channel

546

modulation) differs significantly with age or temperature. Although ZD7822 is a commonly

547

used HCN channel blocker, there is some evidence that it can also block Na+ and Ca2+

21

548

channels (Wu et al 2012). Therefore, to minimize potential off target effects, we also used a

549

second HCN channel block (Cs+) that chemically district and unlikely to produce common

550

off target effects. It should also be noted that Cd+2, in addition to blocking Ca2+-channels can

551

also block certain voltage-gated K+ channels (Abbruzzese et al., 2010). Therefore, additional

552

experiments are required to confirm involvement of Ca2+-channels. Lastly, we also

553

recognize that the level of CO2 used here to identify chemosensitive RTN neurons is high.

554

However, we consider this a minor issue because cells identified in vitro as chemoreceptors

555

using 15% CO2 appear to be of the same population of cells identified in vivo as RTN

556

chemoreceptors, i.e., located in the same region, are glutamatergic and express Phox2B

557

(Mulkey et al. 2004; Mulkey et al. 2007b)

558 559

HCN channels generate an inward current (Ih) at sub-threshold potentials that can have

560

opposing influences on neuronal excitability. For example, activation of Ih has been shown

561

to increase neuronal excitability by depolarizing membrane potential (Wenker et al. 2012a).

562

However, activation of Ih also inhibits neuronal activity by decreasing input resistance, as

563

well as by modulating activity of other voltage-dependent channels, e.g., Ih-mediated

564

depolarization can increase activity of KCNQ channels (George et al. 2009) and prolong

565

inactivation of Ca2+ channels (Tsay et al. 2007). Therefore, both activation and inhibition of

566

HCN channels may cause similar effects on neural activity. Furthermore, the net contribution

567

of Ih to RTN chemoreceptor activity likely depends on several factors including subcellular

568

HCN channel distribution and proximity to other voltage-gated ion channels and/or

569

neurotransmitter receptors. Here, we show in vitro that blocking HCN channels increased the

570

firing rate of RTN neurons under control conditions. Likewise, bilateral RTN injections of

571

ZD7288 increased basal respiratory activity in anesthetized rats. We also found in vitro that

572

the firing rate response of RTN chemoreceptors to HCN channel blockade was blunted by

22

573

XE991 and eliminated by Cd2+, suggesting that under resting conditions HCN channels limit

574

chemoreceptor activity by mechanisms involving KCNQ and Cd+2-sensitive channels

575

including Ca2+ channels. Consistent with a contribution of KCNQ channels to the inhibitory

576

effects of HCN channel blockade, we found in vivo that bilateral RTN injections of ZD7288

577

and XE991 increased respiratory output by an amount similar to either drug alone (i.e., the

578

responses were not additive). These results are consistent with evidence from other brain

579

regions that showed HCN-mediated depolarization can enhance activity of voltage-dependent

580

KCNQ channels and in doing so produced a net inhibitory influence on neuronal excitability

581

(George et al. 2009). An additional inhibitory effect of HCN channel activation has been

582

shown to result from prolonging Ca2+ channel inactivation (Tsay et al., 2007). Importantly,

583

the excitatory effect of serotonin on chemoreceptor activity was retained in Cd2+, as expected

584

for activation of an inward current. These results suggest that excitatory and inhibitory

585

effects of HCN channel activity can coexist in the same cells and can contribute to RTN

586

chemoreceptor activity by different mechanisms.

587 588

Serotonergic modulation of RTN chemoreceptor neurons appears to be intrinsic. For

589

example, previous (Mulkey et al. 2007a) and present results suggest that in the presence of

590

TTX (to block action potentials) RTN chemoreceptors respond to serotonin with a modest

591

decrease in holding current. Furthermore, we show here that RTN chemoreceptors exhibit a

592

characteristic Ih that was activated by serotonin, suggesting a direct effect of serotonin on

593

HCN channels in RTN chemoreceptors.

594

contribute to the mechanism of chemoreception (Gourine et al. 2010; Huckstepp et al. 2010;

595

Wenker et al. 2012), presumably by a TTX-independent mechanism, preliminary results

596

suggest that CO2/H+-sensitive RTN astrocytes do not exhibit a serotonin-sensitive current

597

(data not shown). However, there is some evidence that astrocytes in other brain regions

Although RTN astrocytes are also known to

23

598

express 5-HT receptors (Hirst et al. 1997; Miyazaki et al. 2013) and so it remains possible

599

that astrocytes influence serotonergic modulation of chemoreception at other levels of the

600

respiratory network.

601 602

The role we are attributing to HCN channels in chemosensitive RTN neurons differs from

603

that previously described for CO2-inhibited medullary neurons isolated from fetal rats, where

604

inhibition of HCN channels decreased resting activity and blocked CO2/H+-sensitivity

605

(Wellner-Kienitz and Shams 1998). The divergent roles of Ih in these cell types may be due

606

to developmental changes in the expression of HCN channel subunits as described in other

607

brainstem regions (Bayliss et al. 1994); however, this possibility requires further

608

investigation.

609 610

Our working model for serotonergic modulation of RTN chemoreceptor activity as

611

summarized in Figure 8. Ketanserin has been shown to block serotonin-stimulated activity of

612

RTN neurons (Mulkey et al. 2007), suggesting involvement of 5-HT2- and/or 5-HT7-

613

receptors. It is well known that 5-HT2 receptors are coupled to the phosphoinositol (i.e., Gq-

614

coupled) second messenger system, which can modulate ion channel function by depleting

615

plasma membrane levels of phosphatidylinositol 4,5-bisphosphate (PIP2) and by formation of

616

inositol 1,4,5-trisphosphate and diacylglycerol (Suh and Hille, 2002). Although not tested

617

directly in this study, it is likely that activation of 5-HT2 receptors and subsequent PIP2

618

depletion is the likely mechanism by which serotonin inhibits KCNQ channels in RTN

619

neurons (Hawryluk et al. 2012; Delmas and Brown 2005). It is also well established that 5-

620

HT7 receptors are coupled to trimeric Gs proteins and when activated stimulate adenylate

621

cyclase production of cAMP followed by activation of protein kinase A (PKA) (Matthys et

622

al. 2011).

We show that activation of HCN channels via this signaling pathway also

24

623

contributes to serotonergic modulation of RTN chemoreceptors. Although activation of each

624

of these pathways can independently increase neuronal activity, simultaneous activation of

625

both may antagonize downstream effects on KCNQ and HCN channels. For example, Gq-

626

mediated PIP2 depletion (Pian P et. al., 2006) and PKC activation (Lui et al., 2003) have been

627

shown to inhibit Ih whereas Gs-mediated activation of PKA may activate KCNQ channels in

628

smooth muscle cells (Simms et al. 1988). It is possible that coordinated activation of both Gs

629

and Gq cascades ensures a robust serotonin response, while cross-talk between pathways

630

prevents over-activation.

631 632

In summary, our data identify HCN channels as key determinants of both excitability and

633

serotonergic modulation of RTN chemoreceptor neurons and breathing. The mechanism by

634

which serotonin activates HCN channels in RTN chemoreceptor neurons involves Gs-

635

coupled receptor signaling via 5-HT7 receptors.

636 637

25

638

FIGURE LEGENDS

639

Figure 1.

640

ventilatory response to CO2 in anesthetized rats.

641

CO2 (etCO2), arterial pressure (AP) and integrated phrenic nerve discharge (iPND) show the

642

respiratory response to bilateral injections (arrows) of saline or HCN channel inhibitor

643

ZD7288 in the RTN. Under control conditions injections of ZD7288 (50 μM, 50 nl each

644

side) increased respiratory activity with an increase in mvPND (product of PND amplitude

645

and frequency) and significantly lowered PND CO2 threshold (D). However, CO2-

646

responsiveness was otherwise unaffected by application of ZD7288 in the RTN; lowering

647

etCO2 from to 3-4% inhibited respiratory output, and graded increases etCO2 up to 9-10%

648

increased mvPND by an amount similar to saline (control). B, Summary data plotted as

649

change in mvPND shows the effect of ZD7288 alone (50 μM) or in combination with KCNQ

650

channel inhibitor XE991 (50 μM) on resting respiratory activity (N= 7 animals). C, Summary

651

data showing CO2-induced changes in mvPND under control conditions, after injections of

652

ZD7288 or XE991 or after injections of ZD7288 + XE991 (N= 7 animals). D, Summary data

653

showing

654

level of CO2 required to stimulate PND activity (N= 7 animals). E, computer-assisted plots of

655

the center of the injection sites (coronal projection on the plane Bregma -11.6 and -11.3

656

mm; Paxinos and Watson 1989). Note that all injections were made in the caudal aspect of

657

the RTN where there is the highest density of chemosensitive RTN neurons. Abbreviations:

658

py, pyramids; Sp5, spinal trigeminal tract; VII, facial motor nucleus.

HCN channels in the RTN regulate resting breathing activity and the

that ZD7288

alone

or

in

combination

A, Traces of end expiratory

with

XE991

decreased

the

659 660

Figure 2.

661

respiratory drive in anesthetized rats. A1, traces of integrated phrenic nerve discharge

662

(iPND) and arterial pressure (AP) show effects of unilateral RTN injections of serotonin (5-

HCN channels in the RTN contribute to serotonergic modulation of

26

663

HT; 1 mM) on breathing and blood pressure under control conditions, after RTN injection of

664

XE991 (50 µM; A1) or ZD7288 (50 µM; A2) alone, and after injections of both XE991 and

665

the HCN channel blocker ZD7288 (A1). Inset, traces of iPND plotted on an expanded time

666

scale. Under control conditions RTN injection of serotonin increased PND amplitude and

667

decreased mean arterial pressure (MAP). Unilateral RTN injection of XE991 or ZD7288

668

decreased effects of serotonin on PND amplitude by ~40% and co-administration of ZD7288

669

with XE991 eliminated the serotonin response. Note the effect of serotonin on PND fully

670

recovered after 60 minutes. B, Computer-assisted plot shows the locations of injection sites

671

within the RTN (Bregma -11.6 mm). C, summary data (N = 6 animals) showing the effects

672

of serotonin on PND amplitude under control conditions, XE991 alone, ZD7288 alone and

673

XE991 plus ZD7288. *p < 0.05 versus control. Abbreviations: py, pyramids; RPa, raphe

674

pallidus; Sp5, spinal trigeminal tract; VII, facial motor nucleus.

675 676

Figure 3. Contributions of HCN and KCNQ channels to repetitive firing behavior of

677

chemosensitive RTN neurons. A1, membrane potential responses to a depolarizing step

678

(+20 pA) and hyperpolarizing current steps (-70 to -100pA, ∆10pA) under control conditions

679

and in ZD9288 (50µM, red). A2-A4, summary data (N=6) showing sag ratio (A2),

680

instantaneous spike frequency (ISF; A3) and rebound spike latency (A4) under control

681

conditions and in ZD7288. Note the characteristic HCN channel-dependent depolarizing sag

682

and short latency rebound spike. B1, voltage responses to a depolarizing step (+20 pA) and

683

hyperpolarizing current steps (-70 to -100pA, ∆10pA) under control conditions and in XE991

684

(10µM, blue). B2-B4, summary data (N=5) showing sag ratio (B2), ISF (B3) and rebound

685

spike latency (B4) under control conditions and in XE991 (10 µM, blue). Blocking KCNQ

686

channels increased ISF but did not affect the sag ratio or rebound spike latency.

27

687

Figure 4. HCN channels contribute to resting firing behavior and serotonergic

688

modulation of RTN chemoreceptors in vitro. A1, representative trace of firing rate from a

689

chemosensitive RTN neuron. Under control conditions exposure to serotonin (5-HT; 5 µM)

690

increased firing rate in a CO2 sensitive neuron. After washing out serotonin and returning to

691

a basal level of activity, bath application of XE991 (10 µM) also increased firing rate. In the

692

continued presence of XE991 (with baseline activity adjusted to near control levels by DC

693

current injection), a second exposure to serotonin only increased firing rate by approximately

694

half the control response.

695

firing rate by ~1 Hz. In the presence of XE991 plus ZD7288 (with baseline activity adjusted

696

to near control levels by DC current injection), a third exposure to serotonin had no

697

discernible effect on firing rate.

698

chemoreceptor to 5-HT and Cs+ under control conditions and in the presence of Cd2+ (100

699

µM). Note that Cd2+ eliminated the inhibitory effect of HCN channel blockade (reflected as

700

an increase in firing rate) but did not affect the serotonin response. B, summary data shows

701

the firing rate response of RTN chemoreceptors to HCN channel inhibition by ZD7288 or Cs+

702

(2 mM) under control conditions (N = 17 cells), in XE991 to block KCNQ channels (N = 12

703

cells), in synaptic blockers (N=7 cells), and in Cd2+ to block channels including voltage-gated

704

Ca2+ channels (N = 7 cells).

705

HCN channel inhibition. C, summary data shows the firing rate response to serotonin under

706

control conditions (N=16 cells), in XE991 alone (N = 13 cells), in ZD7288 alone (N = 4

707

cells) and in XE991 plus ZD7288 (N = 7 cells) or Cs2+ (2 mM; N = 6 cells). D, firing rate

708

trace shows that exposure to 15% CO2 increased activity of a chemosensitive RTN neuron by

709

similar amounts under control conditions and in the presence of ZD7288 (50 µM). E,

710

summary data (N = 6 cells) showing that CO2/H+-sensitivity of RTN neurons is fully retained

711

when HCN channels are blocked by ZD7288. F, summary data (N = 7 cells) shows that

Subsequent bath application of ZD7288 (50 µM) also increased

A2, firing rate trace shows the response of an RTN

Both XE991 and Cd2+ significantly reduced the responses to

28

712

serotonin responses were also retained during Ca2+ channel blockade. // marks 10 min time

713

breaks and ↓ indicates DC current injection. *p < 0.05, **p < 0.01, ***p < 0.001.

714 715

Figure 5. Serotonin activates HCN channels by a cAMP-dependent mechanism. A,

716

representative trace of firing rate from a chemosensitive RTN neuron. Under control

717

conditions exposure to serotonin (5-HT; 5 µM) increased firing in a CO2 sensitive neuron by

718

~1 Hz. After washout of serotonin, bath application of XE991 (10 µM) also increased firing

719

rate by ~1.2 Hz. In the continued presence of XE991 (with baseline activity adjusted to near

720

control levels by DC current injection), a second exposure to serotonin only increased firing

721

rate by ~0.5 Hz.

722

(100 µM) increased firing rate by ~0.8 Hz. In the presence of XE991 plus SQ22536 (with

723

baseline activity adjusted to near control levels by DC current injection), a third exposure to

724

serotonin no longer led to increased firing. B, summary data (N = 5 cells) of firing rate

725

responses to SQ22536 when KCNQ channels are blocked with XE991. C, summary data (N

726

= 5 cells) of firing rate responses to serotonin under control conditions, in XE991 alone and

727

in XE991 plus SQ22536. // marks 10 min time breaks and ↓ indicates DC current injection.

728

*p < 0.05, **p < 0.01.

Subsequent bath application of the adenylate cyclase inhibitor SQ22536

729 730

Figure 6.

731

Ih in RTN chemoreceptors. A, current responses to hyperpolarizing voltage steps recorded

732

under control conditions (A1), in serotonin (5 µM, A2), after washing serotonin for 5 min

733

(A3), and in ZD7288 (50 µM). Note that serotonin caused a modest decrease in holding

734

current (arrows indicate zero current levels). B, summary data (N=6 cells/ animals) showing

735

that serotonin did not increase maximal Ih amplitude, measured as the difference between

736

steady state and instantaneous currents at -150 mV (illustrated in A1). C, Ih activation curves

Serotonin causes a depolarizing shift in the voltage-dependent activation of

29

737

where tail currents were measured during a fixed step to -70 mV that followed each test pulse

738

(asterisks in A) and normalized data was fitted using the Boltzmann equation, under control

739

conditions and in serotonin. Serotonin causes a depolarizing shift in the voltage-dependent

740

activation of Ih (V1/2 values inset, N = 7). *p < 0.05, **p < 0.01.

741 742

Figure 7.

743

chemoreceptors. A, representative trace of firing rate from a chemosensitive RTN neuron

744

shows that under control conditions exposure to 5-carboxamidotryptamine (5-CT; 5 µM)

745

increased firing by ~0.4 Hz. As before, bath application of ZD7288 (50 µM) also increased

746

firing rate. A second exposure to 5-CT, this time in the presence of ZD7288 (with baseline

747

activity adjusted to near control levels by DC current injection), had no effect on firing rate.

748

B, trace of firing rate shows the response of an RTN chemoreceptor to 5-CT was blocked by

749

10 minute incubation in SB258719 (10 µM), a 5-HT7 receptor blocker. C, summary data (N

750

≥4 cells) showing the firing rate response to 5-CT under control conditions and in the

751

presence of ZD7288 alone, the 5-HT1A receptor antagonist WAY100635 (0.1µM) alone, or

752

the 5-HT7 receptor antagonist SB258719. Note that WAY100635 did not affect the response

753

to 5-CT, suggesting 5-HT1A receptors do not contribute to this response whereas 5-HT7

754

inhibition significantly reduced the serotonin response. D, average data (N = 3 cells) showing

755

the firing rate response of RTN chemoreceptors the 5-HT7 receptor agonist LP-44 (2µM)

756

under control conditions and in the presence of the 5-HT7 receptor antagonist SB258719. //

757

designates 10 min time breaks and ↓ indicates DC current injection. *p < 0.05, **p < 0.01,

758

***p < 0.001.

5-HT7 receptor activation mediates effects of serotonin

on RTN

759 760

Figure 8. Model depicting the molecular basis for serotonergic modulation of RTN

761

chemoreceptors.

Previous (Hawryluk et al., 2012) and present results show that serotonin

30

762

stimulates activity of RTN chemoreceptors by two independent but coordinated signaling

763

pathways: 5-HT2 mediated inhibition of KCNQ channels most likely by Gq signaling, and 5-

764

HT7-medaited activation of HCN channels by a mechanism involving activation of adenylate

765

cyclase and cAMP mediated depolarizing shift in the voltage-dependent activation of Ih. Our

766

evidence suggests that each of these signaling pathways can independently stimulate activity

767

of RTN chemoreceptors, and together their coordinated activity ensures a robust response to

768

serotonin. Note that our evidence also suggests that under basal conditions HCN channel

769

activity may indirectly modulate activity of voltage-dependent KCNQ and Cd+2-sensitive

770

channels including Ca2+ channels.

771

31

772

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