Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties

Respiratory Physiology & Neurobiology 180 (2012) 323–330

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Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties Yonghua Chen a , Lili Hou a,b , Xujiao Zhou a , Dongying Qiu a,b , Wenjun Yuan c , Lei Zhu b , Jijiang Wang a,∗ a

The State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University Shanghai Medical College, China Fudan University Zhongshan Hospital, China c Department of Physiology and Neurobiology, Ning-xia Medical University, Yinchuan, China b

a r t i c l e

i n f o

Article history: Accepted 22 December 2011 Keywords: Airway Medulla oblongata Patch clamp Charybdotoxin Apamin Ion channel

a b s t r a c t This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratoryactivated neurons was blocked by apamin, a blocker of the small-conductance Ca2+ -activated K+ channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca2+ activated K+ channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The airway vagal nerves include the superior and inferior laryngeal nerves and the tracheobronchial branch of the vagus. These nerves dominate the control of airway function via modulating the activities of the tracheobronchial effectors including airway smooth muscles, submucosal secretory glands and the vasculature.

Abbreviations: ACSF, artificial cerebral spinal fluid; AHP, afterhyperpolarization; AP, action potential; AP5 , D-2-amino-5-phosphonovalerate; 4-AP, 4-aminopyridine; AVPNs, airway vagal preganglionic neurons; CbTx, charybdotoxin; cNA, compact portion of the nucleus ambiguus; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CNS, central nervous system; DMV, dorsal motor nucleus of the vagus; eNA, external formation of the nucleus ambiguus; EPSCs, excitatory postsynaptic currents; EPSPs, excitatory postsynaptic potentials; IPSCs, inhibitory postsynaptic currents; IPSPs, inhibitory postsynaptic potentials; LKCa , large-conductance Ca2+ -activated K+ channel; NA, nucleus ambiguus; NTS, the nucleus of the tractus; PAG, periaqueductal gray; pre-BötC, the pre-Bötzinger complex; RMP, resting membrane potential; SKCa , small-conductance Ca2+ -activated K+ channel; TTX, tetrodotoxin. ∗ Corresponding author at: The State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University Shanghai Medical College, 138 Yi-XueYuan Road, Shanghai 200032, China. Tel.: +86 21 54237857; fax: +86 21 64174579. E-mail address: [email protected] (J. Wang). 1569-9048/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2011.12.014

Abnormalities in airway parasympathetic pathways participate in some chronic airway diseases such as bronchial asthma and obstructive sleep apnea syndrome (Smith et al., 1998; Lee and Widdicombe, 2001; Lutz and Sulkowski, 2004; Kato, 2007; Loehrl, 2007). The airway vagal nerves originate from the airway vagal preganglionic neurons (AVPNs), which are the final common path connecting the central nervous system (CNS) with the airway. AVPNs project preganglionic nerves to the intrinsic tracheobronchial ganglia, whose postganglionic nerves innervate airway effectors (Baker et al., 1986; Maize et al., 1998; Dey, 2003). Previous retrograde tracing studies show that AVPNs are primarily located in three sites in the medulla: the compact portion of the nucleus ambiguus (cNA), the external formation of the nucleus ambiguus (eNA) and the dorsal motor nucleus of the vagus (DMV) (Haxhiu and Loewy, 1996; Kc et al., 2004; Atoji et al., 2005; Chen et al., 2007; Mazzone and McGovern, 2010). However, since AVPNs in the DMV are found to predominantly project to tracheobronchial secretory glands and blood vessels (Kalia and Mesulam, 1980; Haselton et al., 1992; Haxhiu et al., 1993; Haxhiu and Loewy, 1996), and stimulation of these neurons has little impact on airway resistance (Haselton et al., 1992; Kc et al., 2004), only AVPNs in the ventrolateral medulla

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are thought to be related to the control of airway smooth muscles. Furthermore, when tracers were directly applied to the laryngeal nerves or to the tracheobronchial branch of the vagus, retrogradely labeled AVPNs in the ventrolateral medulla were either exclusively within the cNA (Irnaten et al., 2001a,b; Barazzoni et al., 2005; Okano et al., 2006; Chen et al., 2007) or exclusively within the eNA (Kc et al., 2004), respectively. Therefore, the control of tracheobronchial smooth muscles is likely dominated by AVPNs in the eNA. In previous studies from our laboratory, AVPNs in the eNA were divided into inspiratory-activated and inspiratory-inhibited neurons (Chen et al., 2007; Qiu et al., 2011; Hou et al., in press). During the inspiratory phase, inspiratory-activated AVPNs receive bursting excitatory inputs while inspiratory-inhibited AVPNs receive bursting inhibitory inputs. However, the firing properties of AVPNs in the eNA have not been studied. Peripherally in the paratracheobronchial ganglion of cats, it is shown in vivo that some neurons burst during the inspiratory phase, and these “phasic” neurons primarily project to the tracheobronchial smooth muscles; others fire tonically during inspiratory intervals, and these “tonic” neurons primarily project to the intercartilaginous spaces (Mitchell et al., 1987). Postganglionic neurons with similar “phasic” or “tonic” firing properties are also proved both in humans and in animals in vitro; and they are proved to be different types of neurons with distinct intrinsic membrane properties (Baker, 1986; Lees et al., 1997; Myers, 1998; Myers et al., 1990). It is thus reasonable to conceive that the “phasic” postganglionic neurons are preferentially innervated by inspiratory-activated AVPNs; and the “tonic” postganglionic neurons by inspiratory-inhibited AVPNs. However, it is still unknown whether inspiratory-activated and inspiratory-inhibited AVPNs are intrinsically different or the same, just differentially modulated by inspiratory synaptic inputs. In the present study, AVPNs in the eNA were retrogradely labeled with a fluorescent tracer applied to the extrathoracic trachea and identified in brainstem slices with rhythmically bursting hypoglossal rootlets, and their electrophysiological properties were examined using patch-clamp technique. We aimed to test the hypothesis that inspiratory-activated and inspiratory-inhibited AVPNs are two types of neurons with different intrinsic membrane properties. 2. Materials and methods Ethical approval: A total of 58 newborn rats were used in this study. Animal procedures approved by the Ethical Committee of the Fudan University Shanghai Medical College (No. 20110307-060), and were in accordance with the recommendations in the ARRIVE Guidelines in the care and use of experimental animals. 2.1. Retrograde fluorescent labeling of AVPNs in the eNA Halothane, a volatile anesthetic, was dripped onto a cotton pad placed at the bottom of a glass box (5 cm × 5 cm × 5 cm). Threeto 5-day-old Sprague-Dawley rats (Shanghai Institute for Family Planning) were put in the box for 30 s with the lid closed. This procedure anesthetized the rats but kept their breathing at a relatively normal state. When the rat no longer responded to pinching the limbs, the body was surrounded by ice-water-filled plastic bags to decrease the body temperature and slow the heart rate. After the automatic breathing stopped (usually within 2 min), the animal was put on an ice-water-filled plastic bag in a supine posture, and fixed with gauze. A ventral midline incision was made in the neck to expose the extrathoracic trachea, and rhodamine (XRITC, Molecular Probes, 1% solution, 0.2–0.5 ␮L) was injected into the trachea adventitia between the fourth and the eighth tracheal cartilage with a glass pipette (tip diameter 30 ␮m), which was attached to a syringe through polyethylene tubing. The incision was rinsed with saline containing 5 mg mL−1 streptomycin sulfate and

50,000 U mL−1 penicillin, and sutured. In all instances, great care was taken to avoid unwanted spread of the dye onto neighboring tissues. The animals were heated with a thermo-pad to help recovery. During the entire surgery period (about 5 min), the body temperature of the animal was below 10 ◦ C and the animal had no automatic breathing or struggling. After the surgery, the animal usually resumed automatic breathing within 3 min and started moving freely within another 5 min. A single dose of morphine chloride (10 mg kg−1 ) was injected intraperitoneally to relief the pain after the surgery. The animals were allowed 36–48 h to recover. 2.2. Slice preparation The animals were anesthetized deeply with halothane and decapitated at the supracollicular level. The hindbrain was exposed, isolated and immerged in cold (4 ◦ C) artificial cerebral spinal fluid (ACSF) with the following composition (in mmol L−1 ): NaCl, 124; KCl, 3; KH2 PO4 , 1.2; CaCl2 , 2.4; MgSO4 , 1.3; NaHCO3 , 26; d-glucose, 10; and sucrose, 10. The solution was constantly bubbled with gas (95% O2 –5% CO2 ) and had a pH of 7.4. The cerebellum was removed from each brain and the brainstem was dissected under the aid of a dissection microscope. The brainstem was secured in the slicing chamber of a vibratome (Leica VT 1000S, Leica Microsystems, Wetzlar, Germany) filled with the same ACSF. The rostral end of the brainstem was set upwards and the dorsal surface was glued to an agar block facing the razor. The brainstem was sectioned serially at variable thicknesses (50–300 ␮m) in the transverse plane. The compact portion of the nucleus ambiguus (NA) was used as a landmark. Once it was visible under the microscope, a single 500–800-␮m-thick medullary slice with one to two hypoglossal rootlets retained in each lateral side was taken for experimentation. The thick medullary slice preparation, which contains the pre-Bötzinger complex (pre-BötC), local circuits for motor output generation and respiratory hypoglossal motor neurons, generates inspiratory-phase motor discharge in the hypoglossal nerves (Smith et al., 1991). The slice was transferred to a recording chamber and superfused with flowing ACSF (flow rate, 8–11 mL min−1 ). The rostral cutting plane of the slice was set upwards to allow fluorescent identification and patch-clamp recording of AVPNs in the eNA. The temperature was maintained at 23 ± 0.5 ◦ C, and the concentration of KCl in the ACSF was increased to 10 mmol L−1 to allow steady recording of the respiratory rhythm. 2.3. Electrophysiological recording Individual AVPN in the eNA was identified by the presence of the fluorescent tracer using an Olympus upright microscope (Olympus American Inc., Center Valley, PA, USA) through a 40× water immersion objective lens. The patch pipettes (2–4 M) were filled with a solution consisting of (in mmol L−1 ): K+ gluconate, 150; MgSO4 ·7H2 O, 2; CaCl2 , 0.1; HEPES, 10; EGTA, 1; K2 ATP, 2; and Na3 GTP, 0.1. The pH was adjusted to 7.2. The osmolality of the ACSF and the pipette solution was adjusted to 320 mOsm L−1 before use. In voltage clamp experiments, the membrane under the pipette tip was ruptured for whole cell configuration. Neurons with a stable membrane potential that was more negative than −40 mV were accepted for further study. The cells were normally clamped at −80 mV. Using this pipette solution and the holding voltage, the Cl− -mediated inhibitory synaptic currents were minimized and only excitatory synaptic events were detectable. However, when the holding voltage is switched to −50 mV, both excitatory synaptic events (inward) and inhibitory synaptic events (outward) can be detected. To obtain voltage-gated channel currents, 500 ms voltage steps from the holding potential of −80 mV to command potentials from −70 mV to +30 mV in 10 mV increments were tested. In some

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voltage clamp experiments, a 20 ms, 5 mV hyperpolarizing voltage was applied at an interval of 4 s for calculating the input resistance. In current clamp experiments, access was obtained by allowing nystatin to form pores in the cell membrane for a perforated patch configuration. Perforated patch access was monitored and experiments were performed only after a steady state access resistance was obtained (10–15 min after access). The pipette resistance and capacitance were not compensated either before or after gaining intracellular access. Resting membrane potential (RMP) and cell capacitance were measured 2 min after a stable recording was obtained. Current pulses from −0.2 to 0.25 nA with increment of 0.05 nA (1000 ms pulse duration) were delivered to evoke action potentials (APs). The rheobase was defined as the minimal current required to evoke an AP. AP voltage threshold was defined as the first point at which the rising rate of the AP’s rising phase exceeded 50 mV ms−1 . AP amplitude was measured between the AP threshold and the peak, and AP half-width was measured as the width at half-maximal spike amplitude. The patch-clamp signal was amplified with an Axopatch 700B amplifier (sampling frequency, 10 kHz; filter frequency, 1 kHz), digitized with a 1322A Digidata, and collected with the Clampex 9.2 software (Axon instruments, USA). The inspiratory bursts of the hypoglossal rootlets were recorded using a suction electrode, amplified with a BMA-931 bioamplifier (5 kHz sampling frequency; 10–1000 Hz bandpass; 50,000 times), and electronically integrated (time constant,  = 50 ms) with a MA-1000 Moving Averager (CWE Inc., PA, Ardmore, USA) before feeding into the computer.

ventrolateral vicinity (Fig. 1A), and the minority in the close ventral or ventromedial vicinity, of the cNA. Compared with the neurons in the cNA, which are typically spherical or oval, the labeled AVPNs in the eNA are bipolar or multipolar in shape and are generally larger in size (Fig. 1A). In each slice, usually 2–5 AVPNs, mostly in the eNA, could be fluorescently identified. Under voltage clamp, AVPNs with bursting inspiratory excitatory postsynaptic currents (EPSCs) were defined as inspiratoryactivated AVPNs (Fig. 1B), and those with bursting inspiratory inhibitory postsynaptic currents (IPSCs) at a −50 mV holding voltage were defined as inspiratory-inhibited AVPNs (Fig. 1C). In a collection of 164 fluorescence-labeled AVPNs, 120 (73.2%) are inspiratory-activated and 44 (26.8%) are inspiratory-inhibited. The majority of inspiratory-activated AVPNs (113 of 120, 94.2%) are located in the close ventrolateral vicinity, and the minority (7 of 120, 5.8%) in the close ventral or ventromedial vicinity, of the cNA. In contrast, the majority of inspiratory-inhibited AVPNs (40 of 44, 90.9%) are located in the close ventral or ventromedial vicinity, and the minority (4 of 44, 9.1%) in the close ventrolateral vicinity, of the cNA. Detailed distribution of the 164 AVPNs in the eNA is depicted in Fig. 1D. In 10 and 48 slices, 2 and 3 AVPNs were preliminarily identified as inspiratory-activated or inspiratory-inhibited, respectively. Some AVPNs with satisfactory recording were then used for further study of the electrophysiological properties. In each slice, antagonist drugs or channel blockers was tested only in one AVPN.

2.4. Drug application

Under current clamp, inspiratory-activated and inspiratoryinhibited AVPNs showed comparable cell capacitance, input resistance, AP amplitude and AP half-width (Table 1). However, compared with inspiratory-activated AVPNs, inspiratory-inhibited AVPNs exhibited significantly more positive resting membrane potential, more negative voltage threshold and lower rheobase (Table 1). Inspiratory-activated AVPNs examined under current clamp (n = 13) exclusively exhibited rhythmic inspiratory excitatory postsynaptic potentials (EPSPs). In the majority (11 of 13) of them, the EPSPs were superimposed by bursting APs (Fig. 2A). In these eleven rhythmically bursting inspiratory-activated AVPNs, six neurons also discharged sporadically during inspiratory intervals (Fig. 2A), while the others did not (not shown). The rhythmic depolarizing EPSPs, as well as the superimposed action potentials in inspiratoryactivated AVPNs, were abolished by combined application of CNQX (20 ␮mol L−1 ) and AP5 (50 ␮mol L−1 ). However, in the presence of CNQX and AP5 , injection of a depolarizing current (50–100 pA) caused repetitive firing with little frequency adaptation (Fig. 2B). Inspiratory-inhibited AVPNs examined under current clamp (n = 17) exclusively showed rhythmic inspiratory hyperpolarizing inhibitory postsynaptic potentials (IPSPs). Most of them (13 of 17) fired tonically during inspiratory intervals (Fig. 3A), while the others were silent throughout the respiratory cycle (not shown). The rhythmic hyperpolarization was blocked by focally applied strychnine (1 ␮mol L−1 ) and picrotoxin (20 ␮mol L−1 ) in combination (Fig. 3B). In the 13 inspiratory-inhibited AVPNs that fired tonically, eleven neurons became silent (Fig. 3C), and two neurons remained tonically active (not shown), after global application of CNQX and AP5 . In the presence of CNQX and AP5 , injection of a slight depolarizing current (0–50 pA) caused repetitive firing in inspiratory-inhibited AVPNs with little frequency adaptation (Fig. 3D). Both inspiratory-activated and inspiratory-inhibited AVPNs showed an afterhyperpolarization (AHP) after each action potential. In inspiratory AVPNs, the AHP was blocked by apamin (20 nmol L−1 ) (n = 4), a specific blocker of small-conductance Ca2+ activated K+ channel (SKCa ) (Fig. 2C and D). Apamin caused a slight

The drugs were normally used globally in the bath. Strychnine (1 ␮mol L−1 ) and picrotoxin (20 ␮mol L−1 ) were used to block glycine receptors and GABA receptors, respectively. 6Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 ␮mol L−1 ) and D-2-amino-5-phosphonovalerate (AP5 ; 50 ␮mol L−1 ) were used to block non-NMDA and NMDA glutamate receptors, respectively. Channel antagonists such as tetrodotoxin (TTX; 0.5 ␮mol L−1 ), 4-aminopyridine (4-AP; 10 mmol L−1 ), apamin (20 nmol L−1 ) and charybdotoxin (CbTx; 50 nmol L−1 ) were added to the solution immediately before use. In some inspiratory-inhibited AVPNs, strychnine (1 ␮mol L−1 ) and picrotoxin (20 ␮mol L−1 ) were focally applied to the recorded neuron using a puffer pipette positioned within a 10-␮m distance. All drugs were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.5. Data analysis Membrane currents were analyzed with the Clampfit 9.2 software (Axon instruments, USA). Data are presented as mean ± SEM. Individual measurements of the membrane properties in inspiratory-activated AVPNs and those in inspiratoryinhibited AVPNs were statistically compared with the independent Student’s t-test. The amplitude of the sodium currents evoked by different voltage steps was compared by one-way ANOVA followed by Bonferroni correction. At each depolarizing voltage, the lasting outward potassium current before and after 4-aminopiridine application was compared with the paired Student’s t-test. Significant difference was set at P < 0.05. 3. Results 3.1. Different distributing sites and density of retrogradely labeled inspiratory-activated and inspiratory-inhibited AVPNs within the eNA In the rostral cutting plane of the slices, the majority of fluorescence-labeled AVPNs in the eNA are located in the close

3.2. Electrophysiological properties of AVPNs under current clamp

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Fig. 1. Identification of AVPNs in the eNA. (A) Fluorescence-labeled AVPNs in the eNA (marked by star symbols), showing the location of these neurons in the ventrolateral vicinity of the cNA (marked by dash-line circles). Note that fluorescence-labeled AVPNs in the eNA are of bipolar or multipolar shapes; and the size is much larger compared with those within the cNA. Dash-line frames in the schematic drawings show the range from which photos were taken under infrared or fluorescent illumination. (B) Typical recording trace of an inspiratory-activated AVPN, showing the inspiratory bursts of EPSCs at a holding voltage of −80 mV. (C) Typical recording trace of an inspiratoryinhibited AVPN, showing the inspiratory bursts of IPSCs at a holdingvoltage of −50 mV. (D) Schematic drawing showing the relative distribution of 164 AVPNs in the eNA. () Inspiratory-activated neuron; (䊉) inspiratory-inhibited neuron. XII , integrated hypoglossal activities.

depolarization and increased the frequency of action potentials in inspiratory-activated AVPNs during the whole respiratory cycle (Fig. 2D). In inspiratory-inhibited AVPNs, the AHP was not altered by apamin (not shown), but was abolished by CbTx, a specific blocker of the large-conductance Ca2+ -activated K+ channel (LKCa ) at a concentration of 50 nmol L−1 (n = 4) (Fig. 3E and F). CbTx also caused a slight hyperpolarization and turned the action potential firing from tonic to sporadic in inspiratory-inhibited AVPNs (Fig. 3F). 3.3. Electrophysiological properties of AVPNs under voltage clamp Voltage steps from a holding voltage of −80 mV to −70 or −60 mV did not evoke any voltage dependent current in either inspiratory-activated or inspiratory-inhibited AVPNs. However, once the membrane potential was depolarized to levels more positive than −50 mV, voltage gated currents were activated in both types of AVPNs. The currents were composed of a rapidly activating and inactivating inward current, a transient outward current, and a lasting outward current (Figs. 4A and 5A). In 13

inspiratory-activated and five inspiratory-inhibited AVPNs tested, 0.5 ␮mol L−1 TTX completely blocked the inward current. A representative experiment in an inspiratory-activated AVPN is exampled in Fig. 4B. These results demonstrate that the rapid inward current component is caused by the opening of sodium channels in both types of neurons. Interestingly, within the voltage range of −50 to +30 mV, the evoked TTX-sensitive sodium current was not statistically different in the amplitude, in either inspiratory-activated AVPNs (P > 0.05, n = 13; one-way ANOVA followed by Bonferroni correction) or inspiratory-inhibited AVPNs (P > 0.05, n = 5; one-way ANOVA followed by Bonferroni correction). The current–voltage relationship of the TTX-sensitive sodium current in the two types of neurons is shown in Figs. 4C and 5B, respectively. Outward potassium currents, which consist of a fast transient and a long lasting component, were studied by depolarization of the membrane in the presence of TTX. The rapidly activating and inactivating outward transient was reversibly blocked by 4-AP (10 mmol L−1 ) in both types of neurons (Figs. 4D and 5C). Within the voltage range of −20 to +30 mV, the evoked long lasting outward current was significantly inhibited by 4-AP in inspiratory-activated AVPNs (P < 0.05,

Table 1 Membrane properties of inspiratory-activated and inspiratory-inhibited AVPNs. Neuronal types

n

Cm (pF)

RMP (mV)

Rin (M)

AP rheobase (pA)

AP VTH (mV)

AP amplitude (mV)

AP half-width (ms)

Inspiratory-activated Inspiratory-inhibited

8 8

112.5 ± 15.6 89.7 ± 12.6

−51.4 ± 3.5 −43.6 ± 3.7*

77.5 ± 2.7 79.4 ± 5.7

71.4 ± 10.1 42.9 ± 13.0*

−29.6 ± 1.3 −35.1 ± 0.7**

82.6 ± 3.6 75.9 ± 8.2

0.82 ± 0.04 0.83 ± 0.02

Values are mean ± SEM; Cm, cell capacitance; RMP, resting membrane potential; Rin , input resistance; AP, action potential; AP rheobase, current threshold of action potential; VTH, voltage threshold. Independent Student’s t-test. * P < 0.05. ** P < 0.01.

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Fig. 2. Electrophysiological properties of inspiratory-activated AVPNs under current clamp configuration. (A) An inspiratory-activated AVPN showed inspiratory rhythmic depolarizing EPSPs, on which action potential bursting were superimposed. (B) An inspiratory-activated AVPN was silent in the presence of CNQX and AP5 (bottom trace), and injection of a 50 pA (middle trace) or 100 pA (top trace) depolarizing current caused repetitive action potential discharge with little frequency adaptation. (C and D) In an inspiratory-activated AVPN apamin blocked the AHP (C), and slightly increased the frequency of the action potentials during both the inspiratory phase and the inspiratory intervals (D).

n = 6; paired Student’s t-test) (Fig. 4D and E), but was unaltered in inspiratory-inhibited AVPNs (P > 0.05, n = 6; paired Student’s t-test) (Fig. 5C). 4. Discussion There are several major new findings in the present study. First, inspiratory-activated and inspiratory-inhibited AVPNs are differentially distributed in the density and site preference within the eNA. Second, compared with inspiratory-activated AVPNs, inspiratory-inhibited AVPNs have significantly more positive resting membrane potential, more negative voltage threshold and lower rheobase. Third, both inspiratory-activated and inspiratoryinhibited AVPNs have an AHP after each action potential, but the types of the Ca2+ -activated potassium channels involved are different. Fourth, in both inspiratory-activated and inspiratoryinhibited AVPNs, depolarizing voltage steps evoked TTX-sensitive sodium currents, 4-AP-sensitive outward potassium transients and lasting outward potassium currents. However only in inspiratoryactivated AVPNs was the lasting outward potassium current partially sensitive to 4-AP. AVPNs are dissociated both anatomically and in functional control. AVPNs in the DMV have little impact on the airway resistance and are thought to be involved only in the control of airway mucosa and/or vasculatures (Kalia and Mesulam, 1980; Haselton et al.,

1992; Haxhiu et al., 1993; Haxhiu and Loewy, 1996; Kc et al., 2004). AVPNs in the ventrolateral medulla are involved in the functional control of all types of airway tissues: the smooth muscles, the submucosal glands, and the vasculatures (Haxhiu et al., 2000; Kc et al., 2004). In this confined ventrolateral medullary area, anatomical and functional dissociation of AVPNs is also indicated. On the anatomical aspect, AVPNs innervating different airway segments are located in different area: laryngeal-projecting AVPNs are exclusively within the cNA (Irnaten et al., 2001a,b; Barazzoni et al., 2005; Okano et al., 2006; Chen et al., 2007); and tracheobronchialprojecting AVPNs are exclusively within the eNA (Kc et al., 2004). On the functional aspect, both the AVPNs within the cNA and the AVPNs within the eNA include different neuronal types, which receive different phasic inspiratory synaptic inputs (Chen et al., 2007; Qiu et al., 2011; Hou et al., in press). The present study further found that even within the more localized eNA, inspiratoryactivated and inspiratory-inhibited AVPNs are distributed with different density and site preference: the majority of the labeled AVPNs are inspiratory-activated and are preferentially located in the ventrolateral vicinity of the cNA; and the minority of the labeled AVPNs are inspiratory-inhibited and are preferentially located in the ventromedial or ventral vicinity of the cNA. However, it must be acknowledged that in the present study, only a localized tracheal segment was injected with fluorescent dye and only the fluorescently labeled AVPNs located on the rostral surface of the slices

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Fig. 3. Electrophysiological properties of inspiratory-inhibited AVPNs under current clamp configuration. (A) An inspiratory-inhibited AVPN discharged tonically during inspiratory intervals and showed inspiratory rhythmic hyperpolarizing IPSPs. (B) The rhythmic hyperpolarizing IPSPs were blocked by focal application of picrotoxin and strychnine. (C) An inspiratory-inhibited AVPN was silent in the presence of CNQX and AP5 . (D) Injection of a 50 pA (middle trace) or 100 pA (top trace) depolarizing current caused repetitive action potential discharge in a typical inspiratory-inhibited AVPN. Note the repetitive discharge has little frequency adaptation. (E and F) CbTx blocked the AHP (E), and turned the action potential firing from tonic to sporadic in an inspiratory-inhibited AVPN (F).

were examined. The results may indicate, but may not be enough to fully reflect, the overall distribution of inspiratory-activated and inspiratory-inhibited AVPNs in the eNA. Under current clamp, inspiratory-inhibited AVPNs exhibited significantly more positive resting membrane potential, more negative voltage threshold and lower rheobase compared with inspiratory-activated AVPNs, which indicates that inspiratoryinhibited AVPNs are more excitable. The relatively higher excitability of inspiratory-inhibited AVPNs is consistent with their tonic firing pattern during inspiratory intervals; and the relatively lower excitability of inspiratory-activated AVPNs is consistent with their phasic inspiratory bursting pattern. In addition, the AHP in inspiratory-activated AVPNs was blocked by apamin; and that in inspiratory-inhibited AVPNs was blocked by CbTx, suggesting that the AHP in the two types of neurons is mediated by SKCa and LKCa , respectively. Under voltage clamp, the lasting outward potassium current evoked by depolarizing voltage steps was partially sensitive to 4-AP in inspiratory-activated AVPNs but was insensitive

to 4-AP in inspiratory-inhibited AVPNs, which indicates different types of voltage-dependent potassium channels in these two types of neurons. However, the functional significance of the differences in channel types in these two types of AVPNs is unclear and needs further investigation. These results for the first time supply electrophysiological evidence that inspiratory-activated and inspiratory-inhibited AVPNs in the eNA are intrinsically different, not just are located in different sites and/or receive different inspiratory synaptic inputs. Inspiratory-activated and inspiratory-inhibited AVPNs have different intrinsic electrophysiological properties and are distributed within the eNA with different density and site preference. These results further suggest that they might have different roles in the control of the airway, possibly via targeting at different types of the postganglionic neurons. Regretfully, neither the present study nor previous studies are able to answer whether the same AVPN is involved in the control of different target tissues (smooth muscles, submucosal glands and blood vessels) in the airway, or whether

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Fig. 5. Properties of depolarization-evoked voltage-gated currents in inspiratoryinhibited AVPNs. (A) The inward sodium current in a representative inspiratoryinhibited AVPN. (B) The L-shaped current–voltage relationship curve of the inward sodium current in inspiratory-inhibited AVPNs, showing that the amplitude of the current was not significantly different with the depolarizing voltages ranging from −50 to +30 mV (P > 0.05, n = 5). (C) The voltage-gated outward potassium currents evoked in the presence of TTX in a representative inspiratory-inhibited AVPN. Note the fast outward transient was blocked by 4-AP while the lasting component was not altered.

Fig. 4. Properties of the depolarization-evoked voltage-gated currents in inspiratory-activated AVPNs. (A) The inward sodium current in a representative inspiratory-activated AVPN. Inset, the stimulus waveform used. (B) 0.5 ␮mol L−1 TTX completely blocked the inward sodium current. (C) The L-shaped current–voltage relationship curve of the inward sodium current in inspiratory-activated AVPNs, showing that the amplitude of the current was not significantly different with the depolarizing voltages ranging from −50 to +30 mV (P > 0.05, n = 13). (D) The voltage-gated outward potassium currents evoked in the presence of TTX in a representative inspiratory-activated AVPN. Note TTX blocked the inward sodium current, and the outward potassium currents consist of a fast outward transient and a lasting

different target tissues are controlled by different groups of AVPNs. The eNA in the rostral cutting plane of the medullary slices in our study is consistent with the position of the pre-BötC, a primary region that may generate respiratory rhythm in mammals (Smith et al., 1991, 2000; Rekling and Feldman, 1998; Koshiya and Smith, 1999). Previous studies indicate that the cellular composition of the pre-BötC is complex, containing heterogeneous phenotypes of neurons including rhythm-generating pacemaker neurons (Smith et al., 2000; Del et al., 2001, 2002; Koizumi and Smith, 2008), hypoglossal (XII) premotoneurons (Koizumi et al., 2008), rhythmically active GABAergic and glycinergic inhibitory neurons (Kuwana et al., 2006; Winter et al., 2009). The results of the present study and our previous studies (Chen et al., 2007; Qiu et al., 2011; Hou et al., in press) further demonstrate that at least a subpopulation of the AVPNs is spatially located in the pre-BötC. In addition, previous studies indicate that AVPNs receive synaptic inputs from various brain regions including the ventral aspect of the medulla

component. The fast outward transient was blocked by 4-AP and the lasting component was inhibited by 4-AP. (E) The current–voltage relationship curves of the lasting component before and during application of 4-AP (n = 6). IK , the lasting component of potassium current. *P < 0.05; **P < 0.01; paired Student’s t-test.

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oblongata, the nucleus of the tractus, pons, ventrolateral part of periaqueductal gray matter, hypothalamus, and the central nucleus of amygdala (Haxhiu et al., 1993; Hadziefendic and Haxhiu, 1999). Considering the rhythmic inspiratory bursting of the excitatory and/or the inhibitory synaptic inputs of AVPNs, in all probability, these neurons also receive inputs from pacemaker neurons and/or interneurons related to the rhythmogenesis of respiration. In conclusion, the present study demonstrates that inspiratoryactivated and inspiratory-inhibited AVPNs within the eNA are differentially organized in the neuronal architecture, and have different intrinsic electrophysiological properties due to expression of different types of voltage-gated ion channels. These newly identified differences, together with the difference in the inspiratory synaptic control, might allow them to play different roles in the control of the airway, via targeting at different postganglionic neurons. Conflict of interest The authors affirm that there is no conflict that is relevant to the manuscript. Author contributions Yonghua Chen, Lili Hou and Xujiao Zhou: data acquisition, analysis and interpretation; Dongying Qiu, Wenjun Yuan, Weifang Rong and Lei Zhu: assistance in data acquisition and analysis; Jijiang Wang: experiment design, data analysis and paper writing. Acknowledgements This study was supported the Shanghai Natural Science Foundation grant 10ZR1402400 and the NSFC grant 81170019 to J. Wang, the NSFC grant 81030020 to Fengyan Sun and J. Wang, the Fudan University Development Grant for Young Investigators 08J07 to Y. Chen, and the NSFC grant 30900435 to Y. Chen. References Atoji, Y., Kusindarta, D.L., Hamazaki, N., Kaneko, A., 2005. Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study. Brain Res. 1031, 90–100. Baker, D.G., 1986. Parasympathetic motor pathways to the trachea: recent morphologic and electrophysiologic studies. Clin. Chest Med., 223–229 (review). Baker, D.G., McDonald, D.M., Basbaum, C.B., Mitchell, R.A., 1986. The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry. J. Comp. Neurol. 246, 513–526. Barazzoni, A.M., Clavenzani, P., Chiocchetti, R., Bompadre, G.A., Grandis, A., Petrosino, G., Costerbosa, G.L., Bortolami, R., 2005. Localisation of recurrent laryngeal nerve motoneurons in the sheep by means of retrograde fluorescent labelling. Res. Vet. Sci. 78, 249–253. Chen, Y., Li, M., Liu, H., Wang, J., 2007. The airway-related parasympathetic motoneurones in the ventrolateral medulla of newborn rats were dissociated anatomically and in functional control. Exp. Physiol. 92, 99–108. Del, N.C., Johnson, S.M., Butera, R.J., Smith, J.C., 2001. Models of respiratory rhythm generation in the pre-Botzinger complex. III. Experimental tests of model predictions. J. Neurophysiol. 86, 59–74. Del, N.C., Koshiya, N., Butera, R.J., Smith, J.C., 2002. Persistent sodium current, membrane properties and bursting behavior of pre-botzinger complex inspiratory neurons in vitro. J. Neurophysiol. 88, 2242–2250. Dey, R.D., 2003. Controlling from within: neurophysiological plasticity of parasympathetic airway neurons. Am. J. Physiol. Lung Cell Mol. Physiol. 284, L578–L580. Hadziefendic, S., Haxhiu, M.A., 1999. CNS innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus. J. Auton. Nerv. Syst. 76, 135–145. Haselton, J.R., Solomon, I.C., Motekaitis, A.M., Kaufman, M.P., 1992. Bronchomotor vagal preganglionic cell bodies in the dog: an anatomic and functional study. J. Appl. Physiol. 73, 1122–1129.

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